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https://openalex.org/W2166292362	http://pure-oai.bham.ac.uk/ws/files/23735434/DNA_Bank_Manuscript_submitted_09052015.pdf	English	null	Establishing the UK DNA Bank for motor neuron disease (MND)	BMC genomic data	2,015	cc-by	8,562	Link to publication on Research at Birmingham portal Publisher Rights Statement: Eligibility for repository: Checked on 14/12/2015 Publisher Rights Statement: Eligibility for repository: Checked on 14/12/2015 General rights U l li Establishing the UK DNA Bank for motor neuron disease (MND) Smith, Lucy; Cupid, B. C.; Dickie, B. G M; Al-Chalabi, A.; Morrison, K. E.; Shaw, C. E.; Shaw, P. J. DOI: 10.1186/s12863-015-0236-6 License: Creative Commons: Attribution (CC BY) Document Version Peer reviewed version Citation for published version (Harvard): Smith, L, Cupid, BC, Dickie, BGM, Al-Chalabi, A, Morrison, KE, Shaw, CE & Shaw, PJ 2015, 'Establishing the UK DNA Bank for motor neuron disease (MND)', BMC Genetics, vol. 16, no. 1, 84. https://doi.org/10.1186/s12863-015-0236-6 Link to publication on Research at Birmingham portal Download date: 24. Oct. 2024 General rights l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. 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Oct. 2024 BMC Genetics Establishing the UK DNA bank for motor neuron disease (MND) --Manuscript Draft-- Manuscript Number: Full Title: Establishing the UK DNA bank for motor neuron disease (MND) Article Type: Methodology article Section/Category: Complex traits and quantitative genetics Funding Information: Wellcome Trust (070122/A/02/Z) Dame Professor Pamela J Shaw MND Association (Shaw/Nov02/6700-3) Dame Professor Pamela J Shaw Abstract: In 2003 the Motor Neurone Disease (MND) Association, together with The Wellcome Trust, funded the creation of a national DNA Bank specific for MND. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Manuscript Click here to download Manuscript: Establishing the UK DNA Bank for motor neurone disease April 2015.pdf Click here to view linked References download Manuscript: Establishing the UK DNA Bank for motor neurone disease April 2015.pdf view linked References Establishing the UK DNA Bank for motor neuron disease (MND) Smith L1, Cupid BC1, Dickie BGM1, #Al-Chalabi A2, #Morrison KE3, #Shaw CE2, #Shaw PJ4 corresponding author lucy.smith@mndassociation.org #Principal Investigators for the DNA Bank, listed in alphabetical order. 1Motor Neurone Disease Association, PO Box 246, Northampton, NN1 2PR, UK; belinda.cupid@mndassociation.org; brian.dickie@mndassociation.org 2NIHR Biomedical Research Unit in Dementia, Department of Clinical Neuroscience, King’s College London, London, SE5 8AF, UK; ammar.al-chalabi@kcl.ac.uk; chris.shaw@kcl.ac.uk 3Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK; k.morrison@bham.ac.uk 4Sheffield Institute for Translational Neuroscience, University of Sheffield, 385A Glossop Road, Sheffield, S10 2HQ, UK; pamela.shaw@sheffield.ac.uk 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 *corresponding author lucy.smith@mndassociation.org #Principal Investigators for the DNA Bank, listed in alphabetical order. 1Motor Neurone Disease Association, PO Box 246, Northampton, NN1 2PR, UK; General rights l li It was anticipated that the DNA Bank would constitute an important resource to researchers worldwide and significantly increase activity in MND genetic research. The DNA Bank houses over 3000 high quality DNA samples, all of which were donated by people living with MND, family members and non-related controls, accompanied by clinical phenotype data about the patients. Today the primary focus of the UK MND DNA Bank still remains to identify causative and disease modifying factors for this devastating disease. Corresponding Author: Lucy Smith, Ph.D MND Association Northampton, UNITED KINGDOM Corresponding Author Secondary Information: Corresponding Author's Institution: MND Association Corresponding Author's Secondary Institution: First Author: Lucy Smith, Ph.D First Author Secondary Information: Order of Authors: Lucy Smith, Ph.D Belinda Cupid Brian GM Dickie Ammar Al-Chalabi Christopher E Shaw Karen E Morrison Pamela J Shaw Order of Authors Secondary Information: Manuscript Classifications: 10.030: Epigenetics and chromosome biology; 10.040: Functional genetics; 20.100: Functional genomics; 20.120: Genetic screens; 20.340: Whole genome sequencing; 40.020: Genetic basis of human disease Opposed Reviewers: Garth Nicholson, Professor of medicine ANZAC Research Institute garth.nicholson@sydney.edu.au expert in the field, no competing interests with any author Markus Weber, Professor of Neurology Head Neuromuscular Diseases Unit, ALS Clinic Kantosspital St.Gallen markus weber@kssg ch expert in the field No competing interest Robert Swingler, Medicine Consultant, Ninewells Hospital, NHS Tayside, Dundee Expert in the field no conflict of interest Jochen Weishaupt, Professor Universitat Ulm jochen.weishaupt@uni-ulm.de expert in the field no conflict of interest Abstract Over 100 genes have now been implicated in the causation of MND [7]. No consistent environmental risk factor has been identified, although it is possible that such factors may trigger disease in genetically susceptible individuals, and therefore it is plausible that apparent sporadic cases of MND will be genetically determined to some degree [8, 9]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 An essential starting point for successful genetic research is access to high quality samples, accompanied by detailed clinical information. Large-scale gene sequencing and association studies need many thousands of samples to be screened such that results are statistically significant. Access to such samples had become a major obstacle in exploring the pathogenesis of MND and the concept of a MND DNA Bank was born. The objectives of the initial study were threefold: 1) To collect cohorts of patient, parent/sibling and control samples from sporadic and familial MND; 2) To collect clinical information in order to examine susceptibility traits in clinical subgroups of MND; 3) To make this resource available to the international research community and to foster collaboration between research teams, in order to identify genetic risk factors for MND. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Abstract In 2003 the Motor Neurone Disease (MND) Association, together with The Wellcome Trust, funded the creation of a national DNA Bank specific for MND. It was anticipated that the DNA Bank would constitute an important resource to researchers worldwide and significantly increase activity in MND genetic research. The DNA Bank houses over 3000 high quality DNA samples, all of which were donated by people living with MND, family members and non-related controls, accompanied by clinical phenotype data about the patients. Today the primary focus of the UK MND DNA Bank still remains to identify causative and disease modifying factors for this devastating disease. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 In 2003 the Motor Neurone Disease (MND) Association, together with The Wellcome Trust, funded the creation of a national DNA Bank specific for MND. It was anticipated that the DNA Bank would constitute an important resource to researchers worldwide and significantly increase activity in MND genetic research. The DNA Bank houses over 3000 high quality DNA samples, all of which were donated by people living with MND, family members and non-related controls, accompanied by clinical phenotype data about the patients. Today the primary focus of the UK MND DNA Bank still remains to identify causative and disease modifying factors for this devastating disease. Keywords: Motor Neurone Disease (MND), Amyotrophic Lateral Sclerosis (ALS), Biobank. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 eywords: Motor Neurone Disease (MND), Amyotrophic Lateral Sclerosis (ALS), Biobank. 1 1 Motor Neuron Disease (MND) is a fatal, rapidly progressive disease that affects the brain and spinal cord and which ultimately leads to respiratory failure around 2-5 years following symptom onset [1, 2]. Approximately 1 in 300 people develop MND but its prevalence is low, at about 6-8 in 100,000 because of short life expectancy [3]. There is no diagnostic test and treatment is largely palliative, with only one agent, riluzole, having a modest effect in extending survival. Genetic factors undoubtedly play a role in most cases of the disease, both in pathogenesis and rate of progression, with about 5-10% of all patients having a clear family history of MND and in some cases, frontotemporal dementia [4, 5, 6]. ORGANISATIONAL STRUCTURE OF THE UK MND DNA BANK 2 The UK MND DNA Bank was a collaborative project adopting a ‘Hub and Spoke’ model, with three regional 'Hub' centres linking with a total of 16 ‘Spoke' centres (Table 1). Samples were obtained from sporadic and familial MND patients attending MND clinics in the UK, their spouses (or other genetically unrelated controls) and blood relatives. Samples within the UK MND DNA Bank are housed at CIGMR Biobank, at the University of Manchester. In addition, as one of the Public Health England collections, the European Collection of Cell Cultures (ECACC) manages the transformation and storage of EBV-transformed lymphocytes derived from blood samples from participants providing an everlasting supply of DNA for the Bank. The MND Association’s Biomedical Research Advisory Panel (BRAP) oversee the governance and the strategic development of the DNA Bank, ensuring that samples are utilised in an appropriate fashion, and that any clinical information requested is appropriate for the proposed study. The Technical Access Committee (TAC) at CIGMR Biobank, determine sample requirements for the technology platform to be used, the quantity of sample required and ensure any leftover samples are returned or destroyed. All applications for access to the samples are judged on merit. In order to receive material and clinical information from the DNA Bank, all applicants must agree to the terms and conditions of sample use (see supplementary info). This specifies the user and specific purpose for which the samples and data are to be licensed, including standard terms as to the ownership, 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 SAMPLE COLLECTION, STORAGE AND QUALITY CONTROL ample collection began in 2003. All participants were over 18 years of age. In order to ensure that e patient cohort was representative of disease prognosis, patients must have experienced symptom nset (significant muscle weakness) on or after January 2002. All patients fulfilled El Escorial 3 3 criteria for probable or definite Amyotrophic Lateral Sclerosis (ALS) [10]. Patients presenting with Progressive Muscular Atrophy (PMA), Primary Lateral Sclerosis (PLS) or Progressive Bulbar Palsy (PBP) were also included in the study. Patients were recruited by consultant neurologists with a specialist interest in MND in participating centres. Patients participating in other clinical research projects were not excluded from the study. Blood samples were also collected from consenting partners/carers, providing some degree of matching in terms of age, education, environmental exposure and often ethnicity. Where patients presented with familial MND, blood samples were collected from family members for linkage analysis. Where patients presented with sporadic MND, where possible, blood samples were also collected from parents or from a parent and sibling, to give so-called ‘Trio samples’ increasing the amount of genetic information available for researchers. Informed consent to participate was sought from all patients, family members and controls. Ethical approval for the collection of samples and the creation of the UK MND DNA Bank was given by the Trent Research Ethics Committee in February 2003 ref MREC/02/4/107 and in July 2009, ref 09/HO405/32. Participants were provided with detailed information and contact details and could withdraw from the study at any time. The samples were pseudo-anonymised and an online clinical database was developed to facilitate data entry and collection by the research nurses and enable tracking of trends in clinical parameters such as symptom onset and presentation for data analysis. Storage and access to this data set is in accordance with the UK Data Protection Act 1998 [11]. Prior to 2010, DNA extraction from donated blood samples was carried out at individual Hub centres using the Nucleon BACC3 protocol (Amersham, UK). Extracted DNA was sent to CIGMR Biobank for long-term storage. On receipt, all DNA samples were run on 1% agarose gels alongside molecular weight markers of appropriate size to check integrity. SAMPLE COLLECTION, STORAGE AND QUALITY CONTROL An aliquot of untransformed PBLs was stored in liquid nitrogen for safekeeping, whilst the remaining PBLs were transformed using the Epstein Barr virus according to standard protocols [13]. The resulting lymphoblastoid cell lines were cryopreserved and are used to restock the DNA Bank when stock levels become low. 36 37 38 39 40 41 42 43 44 45 46 47 SAMPLE COLLECTION, STORAGE AND QUALITY CONTROL From August 2010, DNA extraction was carried out at CIGMR Biobank using automated robotic processing under ISO900:2000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 criteria for probable or definite Amyotrophic Lateral Sclerosis (ALS) [10]. Patients presenting with Progressive Muscular Atrophy (PMA), Primary Lateral Sclerosis (PLS) or Progressive Bulbar Palsy (PBP) were also included in the study. Patients were recruited by consultant neurologists with a specialist interest in MND in participating centres. Patients participating in other clinical research projects were not excluded from the study. Blood samples were also collected from consenting partners/carers, providing some degree of matching in terms of age, education, environmental exposure and often ethnicity. Where patients presented with familial MND, blood samples were collected from family members for linkage analysis. Where patients presented with sporadic MND, where possible, blood samples were also collected from parents or from a parent and sibling, to give so-called ‘Trio samples’ increasing the amount of genetic information available for researchers. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 4 DNA aliquots are stored in 2D bar coded tubes for sample tracking purposes. A relational database recorded the 2D barcodes associated with each patient/donor ID. All samples within the collection were screened for gender using PCR on presumed duplicate samples according to standard protocols. Samples with a mismatch between the expected gender as recorded in the patient information, and actual gender as confirmed by PCR, were rescreened using an alternative PCR method of gender identification based on the absence/presence of Alu sequence [12]. Any samples with a confirmed discrepancy were ring fenced from the collection and suspended from the in-house laboratory management system. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Peripheral blood lymphocytes (PBLs) were isolated from whole blood samples at ECACC using density gradient centrifugation. 4. THE UK MND DNA BANK In October 2012, at the end of the collection period, the UK MND DNA Bank comprised 3159 high quality DNA samples. Of these 1344 samples were taken from individuals diagnosed with sporadic MND (see figure 1A and 1B). There were 133 familial MND samples within the collection and a 5 further 500 samples taken from family members, including samples that form 28 parent trio sets and 27 sibling trio sets. The remaining 1085 samples were taken from controls. In line with population- based demographic for the disease [14] the breakdown of gender in the collection is around 60% male (Figure 1A). The average age of onset was approximately 62 years of age (Figure 1C). Each sample is accompanied by a minimum dataset of: age at which the samples were taken; gender; disease status; and where appropriate diagnostic certainty (El Escorial Status) and age of onset (calculated from date of birth and date of symptom onset). An extended dataset has been collected for as many participants as possible but it is not a complete dataset for the entire collection (see Table 2). In total 2653 frozen lymphoblastoid cell lines are held in storage at ECACC following a PBL transformation success rate of 97%. Of these, 1267 samples were generated from whole blood taken from patients with sporadic MND. 115 cell lines were generated from familial samples and the remaining 1058 cells lines have been established using blood samples obtained from control or family members (see Figure 1D). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Table 3 shows the success rates for PCRs performed on the DNA samples within the collection. The failure rate of the quality control assay was less than 1.5% suggesting that the quality of DNA within the collection is very high. The gender results from these assays were directly compared to the gender recorded for individuals on the clinical database. Where there was a discrepancy between the expected gender and that determined in the assay, patient clinical notes were rechecked. In the absence of a clerical error, samples were rescreened using both the original AMEL marker and an alternative gender marker, the Human ALU expansion [12]. 4. THE UK MND DNA BANK With researchers now encouraged to publish in an open access format as part of the DNA Bank governance, and to deposit data from sequencing projects within accessible databases such as ALSOD: the Amyotrophic Lateral Sclerosis Online Database [7] and European Genome-Phenome archive [29], the dissemination and discussion of results by the research community is ensured. In 2014 a proposal to perform whole genome sequencing on DNA samples from the UK MND DNA Bank as part of the international collaboration called Project MinE [30] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 The UK MND DNA Bank was designed to be available to the international research community. DNA samples from the bank represent an incident not prevalent population and are unlikely to be biased. The fundamental guarantee that any DNA Bank must be able to give is that it can provide high quality DNA samples with good integrity and accompanying high quality clinical data. DNA samples from the bank represent an incident not prevalent population and are unlikely to be biased. The fundamental guarantee that any DNA Bank must be able to give is that it can provide high quality DNA samples with good integrity and accompanying high quality clinical data. Understandably, a constraint of the DNA Bank is that the genomic DNA supply itself is limited and although cell lines have been established, the DNA from such cell lines may have sequence changes compared with the original genomic samples. This fact must be considered when choosing to use cell line derived DNA even if the DNA itself is of a high standard as demonstrated by the rigorous quality control assays in place. As part of the governance of the DNA Bank, the MND Association must ensure compliance with legal and regulatory requirements. The Association must also guarantee that the resource adheres to rigorous research standards and is used in the further understanding of motor neuron disease, this includes prioritising access to those parts of the DNA Bank that are limited in availability, clarifying intellectual property rights and disseminating the results that flow from it. 4. THE UK MND DNA BANK Sixty eight samples that continued to show a discrepancy between the expected gender and the assay gender were ring fenced from the collection and suspended from the laboratory management system. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 6 6 The UK MND DNA Bank was designed to be available to the international research community. DNA samples from the bank represent an incident not prevalent population and are unlikely to be biased. The fundamental guarantee that any DNA Bank must be able to give is that it can provide high quality DNA samples with good integrity and accompanying high quality clinical data. Understandably, a constraint of the DNA Bank is that the genomic DNA supply itself is limited and although cell lines have been established, the DNA from such cell lines may have sequence changes compared with the original genomic samples. This fact must be considered when choosing to use cell line derived DNA even if the DNA itself is of a high standard as demonstrated by the rigorous quality control assays in place. As part of the governance of the DNA Bank, the MND Association must ensure compliance with legal and regulatory requirements. The Association must also guarantee that the resource adheres to rigorous research standards and is used in the further understanding of motor neuron disease, this includes prioritising access to those parts of the DNA Bank that are limited in availability, clarifying intellectual property rights and disseminating the results that flow from it. To date more than twenty projects have withdrawn samples from the DNA Bank. DNA samples have been used in complex, technical protocols such as genotyping, gene sequencing and genome-wide association studies and numerous papers have been published or are in press [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28]. Importantly, projects using samples from the DNA Bank have directly led to the detection of several MND causing genes including C9orf72 and more recently Tub4A [15, 17, 22, 23 and 28]. 4. THE UK MND DNA BANK 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 7 To date more than twenty projects have withdrawn samples from the DNA Bank. DNA samples have been used in complex, technical protocols such as genotyping, gene sequencing and genome-wide association studies and numerous papers have been published or are in press [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28]. Importantly, projects using samples from the DNA Bank have directly led to the detection of several MND causing genes including C9orf72 and more recently Tub4A [15, 17, 22, 23 and 28]. With researchers now encouraged to publish in an open access format as part of the DNA Bank governance, and to deposit data from sequencing projects within accessible databases such as ALSOD: the Amyotrophic Lateral Sclerosis Online Database [7] and European Genome-Phenome archive [29], the dissemination and discussion of results by the research community is ensured. In 2014 a proposal to perform whole genome sequencing on DNA samples from the UK MND DNA Bank as part of the international collaboration called Project MinE [30] 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 7 7 was approved. This exciting project will allow Next Generation Sequencing data to be collected from DNA Bank samples and shared across research groups. The data will also confirm the accuracy of existing studies through imputation. It is hoped that sequencing DNA Bank samples will allow the identification of rare variants responsible for sporadic disease, continuously widening our knowledge about how genetic changes can contribute to MND. Ethical approval to extend the use of the cell lines beyond their original scope of providing an everlasting supply of DNA was granted in 2014 by the Derby- East Midlands Research Ethics Committee ref no. 14/EM/1088. This change in permission will potentially allow researchers to generate primary neuronal cultures and highly desirable induced pluripotent stem (iPS) cell lines from the cell lines stored at ECACC. The iPS cell lines could act as new disease models for drug screening and other potential treatments, as well as acting as tools for analysing downstream mechanisms involved in disease pathogenesis. 4. THE UK MND DNA BANK Clearly the role of the DNA Bank in the governance of such samples will be paramount; it is simply not enough to provide high quality samples, but following how those samples have been used and ensuring the results are disseminated and discussed is the only way to ensure research continues to move forward. The original scope of the DNA Bank was to make a quantal difference in our understanding of MND and it is well on the way to fulfilling this promise. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 8 Acknowledgments: This project was supported by a programme grant from the Motor Neurone Disease Association (grant ref 6700). Information regarding the DNA Bank, including the terms and conditions for sample use can be accessed on the MND Association website [31]. The creation of the lympoblastoid cell lines was supported by a grant from the Wellcome Trust (grant ref 070122/A/02/Z). Ethical approval for the creation of the UK MND DNA Bank was given by the Trent Research Ethics Committee in February 2003 ref MREC/02/4/107 and in July 2009, ref 09/HO405/32. Subsequent approval for the extended use of the cell lines within the DNA Bank was approved by the Derby- East Midlands Research Ethics Committee in Sept 5 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 8 2014, ref no. 14/EM/1088. We are grateful for the support of the Dementias and Neurodegenerative Disease Research Network (DeNDRoN). We would like to acknowledge our partners at the CIGMR Biobank (formerly BioBanking Solutions (BBS), UK DNA Bank Network (UDBN)) based at the University of Manchester and the European Cell Culture Collection at Public Health England. This project would now have been possible without the support of collaborators in the participating centres across the UK [31]. Thank you to all the people with MND and their families who participated in this project. 1 2 3 4 5 6 7 8 9 10 11 12 2014, ref no. 14/EM/1088. We are grateful for the support of the Dementias and Neurodegenerative Disease Research Network (DeNDRoN). 4. THE UK MND DNA BANK We would like to acknowledge our partners at the CIGMR Biobank (formerly BioBanking Solutions (BBS), UK DNA Bank Network (UDBN)) based at the University of Manchester and the European Cell Culture Collection at Public Health England. This project would now have been possible without the support of collaborators in the participating centres across the UK [31]. Thank you to all the people with MND and their families who participated in this project. References: 1. McDermott CJ and Shaw PJ. Diagnosis and management of motor neurone disease. BMJ 2008; 336: 658-662. doi: 10.1136/bmj.39493.511759.BE. 2. Chio A, Mora G, Calvo A, Mazzini L, Bottacchi E, Mutani R. Epidemiology of ALS in Italy: a 10- year prospective study. Neurology 2009; 72: 725-731. doi: 10.1212/01.wnl.0000343008.26874.d1. 3. Johnston CA, Stanton BR, Turner MR, Gray R, Blunt AH, Butt D, Ampong MA, Shaw CE, Leigh PN, Al-Chalabi A. ALS in an urban setting: a population based study of inner city London J. Neurology 2006; 253: 1642-43. 4. Mackenzie IR, Rademakers R, Neumann M. TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neurology 2010; 9: 995-1007. 5. Byrne S, Walsh C, Lynch C, Bede P, Elamin M, Kenna K, McLaughlin R, Hardiman O. Rate of familial ALS: a systematic review and metanalysis. J. Neurol Neurosurg Psychiatry 2011; 82: 623-627. doi: 10.1136/jnnp.2010.224501. 6. Byrne S, Heverin M, Elamin M, Bede P, Lynch C, Kenna K, Maclaughlin R, Walsh C, Al-Chalabi A, Hardiman O. Aggregation of neurologic and neuropsychiatric disease in amyotrophic lateral sclerosis kindreds: A population based case-control cohort study of familial and sporadic amyotrophic lateral sclerosis. Ann Neurol 2013; 74: 699-708. doi: 10.1002/ana.23969. 7. Lill CM, Abel O, Bertram L, Al-Chalabi A. Keeping up with the genetic discoveries in ALS: The ALSoD and ALSGene database. ALS 2011; 12: 238-249. doi: 10.3109/17482968.2011.584629. 8. Al-Chalabi A, Lewis CM. Modelling the effects of penetrance and family size on rates of sporadic and familial disease. Hum Hered 2011; 71: 281-88. doi: 10.1159/000330167. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 References: 1. McDermott CJ and Shaw PJ. Diagnosis and management of motor neurone disease. BMJ 2008; 336: 658-662. doi: 10.1136/bmj.39493.511759.BE. 15 16 17 18 19 2. Chio A, Mora G, Calvo A, Mazzini L, Bottacchi E, Mutani R. Epidemiology of ALS in Italy: a 10- year prospective study. Neurology 2009; 72: 725-731. doi: 10.1212/01.wnl.0000343008.26874.d1. 20 21 22 23 24 4. Mackenzie IR, Rademakers R, Neumann M. TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neurology 2010; 9: 995-1007. 31 32 33 34 35 5. Byrne S, Walsh C, Lynch C, Bede P, Elamin M, Kenna K, McLaughlin R, Hardiman O. Rate of familial ALS: a systematic review and metanalysis. J. Neurol Neurosurg Psychiatry 2011; 82: 623-627. doi: 10.1136/jnnp.2010.224501. 36 37 38 39 40 41 6. Byrne S, Heverin M, Elamin M, Bede P, Lynch C, Kenna K, Maclaughlin R, Walsh C, Al-Chalabi A, Hardiman O. Aggregation of neurologic and neuropsychiatric disease in amyotrophic lateral sclerosis kindreds: A population based case-control cohort study of familial and sporadic amyotrophic lateral sclerosis. Ann Neurol 2013; 74: 699-708. doi: 10.1002/ana.23969. 42 43 44 45 46 47 48 49 50 7. Lill CM, Abel O, Bertram L, Al-Chalabi A. Keeping up with the genetic discoveries in ALS: The ALSoD and ALSGene database. ALS 2011; 12: 238-249. doi: 10.3109/17482968.2011.584629. 51 52 53 54 55 8. Al-Chalabi A, Lewis CM. Modelling the effects of penetrance and family size on rates of sporadic and familial disease. Hum Hered 2011; 71: 281-88. doi: 10.1159/000330167. 6 7 8 9 9 9 9. 9. Factor-Litvak P, Al-Chalabi A, Ascherio A, Bradley W, Chío A, Garruto R, Hardiman O, Kamel F, Kasarskis E, McKee A, Nakano I, Nelson LM, Eisen A. Current pathways for epidemiological research in amyotrophic lateral sclerosis. Amyotroph Lateral Scler Frontotemporal Degener. 2013;14 Suppl 1:33-43. doi 10.3109/21678421.2013.778565. 10. Belsh JM. ALS diagnostic criteria of El Escorial Revisited: do they meet the needs of clinicians as well as researchers? Amyotroph Lateral Scler Other Motor Neuron Disord. 2000 Mar;1 Suppl 1:S57-60. 11. The Data Protection Act 1998, National Archives, http://www.legislation.gov.uk/ukpga/1998/29/contents. 12. Hedges DJ, Walker JA, Callinan PA, Shewale JG, Sinha SK, Batzer MA. Mobile element- based assay for human gender determination. Analytical Biochemistry 2003; 312: 77–79 13. Blick D, Cooper J, Baker N, Bracegirdle P, Biggins J, Burnet W. References: Generation of cell lines using Epstein-Barr Virus (EBV) transformation of small volumes of cryo-preserved whole blood and the use of bench-top flow cytometry to achieve high and reproducible success rates. http://www.nature.com/app_notes/nmeth/2011/111312/pdf/an8194.pdf 14. Alonso A, Logroscino G, Jick SS and Hernan MA. Incidence and lifetime risk of motor neuron disease in the United Kingdom: a population based study. Eur J Neurol 2009; 16: 745-51. 15. Shatunov A, Mok K, Newhouse S, Weale ME, Smith B, Vance C, Johnson L, Veldink JH, van Es MA van den Berg LH, Robberecht W, Van Damme P, Hardiman O, Farmer AE, Lewis CM, Butler AW, Abel O, Andersen PM, Fogh I, Silani V, Chiò A, Traynor BJ, Melki J, Meininger V, Landers JE, McGuffin P, Glass JD Pall H, Leigh PN, Hardy J, et al. Chromosome 9p21 in sporadic amyotrophic lateral sclerosis in the UK and seven other countries: a genome-wide association study. Lancet Neurol. 2010 Oct; 9(10):986-94. doi: 10.1016/S1474-4422(10)70197-6 16. Smith BN, Newhouse S, Shatunov A, Vance C, Topp S, Johnson L, Miller J, Lee Y, Troakes C, Scott KM, Jones A, Gray I, Wright J, Hortobágyi T, Al-Sarraj S, Rogelj B, Powell J, Lupton M, Lovestone S, Sapp PC, Weber M, Nestor PJ, Schelhaas HJ, Asbroek AA, Silani V, Gellera C, Taroni F, Ticozzi N, Van den Berg L, Veldink J, et al. The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder. Eur J Hum Genet. 2013 Jan; 21(1):102-8. doi: 10.1038/ejhg.2012.98. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10. Belsh JM. ALS diagnostic criteria of El Escorial Revisited: do they meet the needs of clinicians as well as researchers? Amyotroph Lateral Scler Other Motor Neuron Disord. 2000 Mar;1 Suppl 1:S57-60. 11. The Data Protection Act 1998, National Archives, http://www.legislation.gov.uk/ukpga/1998/29/contents. http://www.nature.com/app_notes/nmeth/2011/111312/pdf/an8194.pdf 10 17. Van Rheenen W, Diekstra FP, van Doormaal PT, Seelen M, Kenna K, McLaughlin R, Shatunov A, Czell D, van Es MA, van Vught PW, van Damme P, Smith BN, Waibel S, Schelhaas HJ, van der Kooi AJ, de Visser M, Weber M, Robberecht W, Hardiman O, Shaw PJ, Shaw CE, Morrison KE, Al-Chalabi A, Andersen PM, Ludolph AC, Veldink JH, van den Berg LH. H63D polymorphism in HFE is not associated with amyotrophic lateral sclerosis. Neurobiol Aging. 2013 May; 34(5):1517.e5-7. doi: 10.1016/j.neurobiolaging.2012.07.020. 1 2 3 4 5 6 7 8 9 10 11 12 17. Van Rheenen W, Diekstra FP, van Doormaal PT, Seelen M, Kenna K, McLaughlin R, Shatunov A, Czell D, van Es MA, van Vught PW, van Damme P, Smith BN, Waibel S, Schelhaas HJ, van der Kooi AJ, de Visser M, Weber M, Robberecht W, Hardiman O, Shaw PJ, Shaw CE, Morrison KE, Al-Chalabi A, Andersen PM, Ludolph AC, Veldink JH, van den Berg LH. H63D polymorphism in HFE is not associated with amyotrophic lateral sclerosis. Neurobiol Aging. 2013 May; 34(5):1517.e5-7. doi: 10.1016/j.neurobiolaging.2012.07.020. 1 2 3 4 5 6 7 8 9 10 11 12 11 Visser M, Weber M, Robberecht W, Hardiman O, Shaw PJ, Shaw CE, Morrison KE, Al-Chalabi A, Andersen PM, Ludolph AC, Veldink JH, van den Berg LH. H63D polymorphism in HFE is not associated with amyotrophic lateral sclerosis. Neurobiol Aging. 2013 May; 34(5):1517.e5-7. doi: 10.1016/j.neurobiolaging.2012.07.020. 18. Jones AR, Woollacott I, Shatunov A, Cooper-Knock J, Buchman V, Sproviero W, Smith B, Scott KM, Balendra R, Abel O, McGuffin P, Ellis CM, Shaw PJ, Morrison KE, Farmer A, Lewis CM, Leigh PN, Shaw CE, Powell JF, Al-Chalabi A. Residual association at C9orf72 suggests and alternative ALS causing hexanucelotide repeat. Neurbiol of Aging 2013; 34(9): 2234.e1-7. doi: 10.1016/j.neurobiolaging.2013.03.003. 19. Mok K, Laaksovirta H, Tienari PJ, Peuralinna T, Myllykangas L, Chiò A, Traynor BJ, Nalls MA, Gurunlian N, Shatunov A, Restagno G, Mora G, Nigel Leigh P, Shaw CE, Morrison KE, Shaw PJ, Al-Chalabi A, Hardy J, Orrell RW. Homozygosity analysis in amyotrophic lateral sclerosis. Eur J Hum Genet. 2013; 12:1429-35. doi: 10.1038/ejhg.2013.59. 20. Buchman VL, Cooper-Knock J, Connor-Robson N, Higginbottom A, Kirby J, Razinskaya OD, Ninkina N, Shaw PJ. (2013) Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridization. Mol Neurodegener. 2013 Apr 8; 8: 12. doi: 10.1186/1750-1326-8-12. http://www.nature.com/app_notes/nmeth/2011/111312/pdf/an8194.pdf 21. Ismail A, Cooper-Knock J, Highley JR, Milano A, Kirby J, Goodall E, Lowe J, Scott I, Constantinescu CS, Walters SJ, Price S, McDermott CJ, Sawcer S, Compston DA, Sharrack B, Shaw PJ. Concurrence of multiple sclerosis and amyotrophic lateral sclerosis in patients with hexanucleotide repeat expansions of C9ORF72. J Neurol Neurosurg Psychiatry. 2013; 84(1): 79-87. doi: 10.1136/jnnp-2012-303326. 22. Cooper-Knock J, Higginbottom A, Connor-Robson N, Bayatti N, Bury JJ, Kirby J, Ninkina N, Buchman VL, Shaw PJ. C9ORF72 transcription in frontotemporal dementia case with two expanded alleles. Neurology. 2013; 81(19):1719-21. doi: 10.1212/01.wnl.0000435295.41974.2e. 23. Fogh I, Ratti A, Gellera C, Lin K, Tiloca C, Moskvina V, Corrado L, Sorarù G, Cereda C, Corti S, Gentilini D, Calini D, Castellotti B, Mazzini L, Querin G, Gagliardi S, Del Bo R, Conforti FL, Siciliano G, Inghilleri M, Saccà F, Bongioanni P, Penco S, Corbo M, Sorbi S, Filosto M, Ferlini A, Di Blasio AM, 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 18. Jones AR, Woollacott I, Shatunov A, Cooper-Knock J, Buchman V, Sproviero W, Smith B, Scott KM, Balendra R, Abel O, McGuffin P, Ellis CM, Shaw PJ, Morrison KE, Farmer A, Lewis CM, Leigh PN, Shaw CE, Powell JF, Al-Chalabi A. Residual association at C9orf72 suggests and alternative ALS causing hexanucelotide repeat. Neurbiol of Aging 2013; 34(9): 2234.e1-7. doi: 10.1016/j.neurobiolaging.2013.03.003. 19. Mok K, Laaksovirta H, Tienari PJ, Peuralinna T, Myllykangas L, Chiò A, Traynor BJ, Nalls MA, Gurunlian N, Shatunov A, Restagno G, Mora G, Nigel Leigh P, Shaw CE, Morrison KE, Shaw PJ, Al-Chalabi A, Hardy J, Orrell RW. Homozygosity analysis in amyotrophic lateral sclerosis. Eur J Hum Genet. 2013; 12:1429-35. doi: 10.1038/ejhg.2013.59. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 22. Cooper-Knock J, Higginbottom A, Connor-Robson N, Bayatti N, Bury JJ, Kirby J, Ninkina N, 49 50 23. http://www.nature.com/app_notes/nmeth/2011/111312/pdf/an8194.pdf Fogh I, Ratti A, Gellera C, Lin K, Tiloca C, Moskvina V, Corrado L, Sorarù G, Cereda C, Corti S, 55 56 Gentilini D, Calini D, Castellotti B, Mazzini L, Querin G, Gagliardi S, Del Bo R, Conforti FL, Siciliano G, 58 59 11 Signorini S, Shatunov A, et al. A genome-wide association meta-analysis identifies a novel locus at 17q11.2 associated with sporadic amyotrophic lateral sclerosis. Hum Mol Genet 23(8): 2220-31. doi: 10.1093/hmg/ddt587. associated with sporadic amyotrophic lateral sclerosis. Hum Mol Genet 23(8): 2220-31. doi: 10.1093/hmg/ddt587. 24. Goris A, van Setten J, Diekstra F, Ripke S, Patsopoulos NA, Sawcer SJ; International Multiple Sclerosis Genetics Consortium, van Es M; Australia and New Zealand MS Genetics Consortium, Andersen PM, Melki J, Meininger V, Hardiman O, Landers JE, Brown RH Jr, Shatunov A, Leigh N, Al-Chalabi A, Shaw CE, Traynor BJ, Chiò A, Restagno G, Mora G, Ophoff RA, Oksenberg JR, Van Damme P, Compston A, Robberecht W, Dubois B, van den Berg LH, et al. No evidence for shared genetic basis of common variants in multiple sclerosis and amyotrophic lateral sclerosis. Hum Mol Genet. 2014; 23(7):1916-22.doi: 10.1093/hmg/ddt574. 25. Diekstra FP, Van Deerlin VM, van Swieten JC, Al-Chalabi A, Ludolph AC, Weishaupt JH, Hardiman O, Landers JE, Brown RH Jr, van Es MA, Pasterkamp RJ, Koppers M, Andersen PM, Estrada K, Rivadeneira F, Hofman A, Uitterlinden AG, van Damme P, Melki J, Meininger V, Shatunov A, Shaw CE, Leigh PN, Shaw PJ, Morrison KE, Fogh I, Chiò A, Traynor BJ, Czell D, Weber M, et al. C9orf72 and UNC13A are shared risk loci for amyotrophic lateral sclerosis and frontotemporal dementia: A genome-wide meta-analysis Ann Neurol 76(1):120-33. doi: 10.1002/ana.24198. 26. Savage AL, Wilm TP, Khursheed K, Shatunov A, Morrison KE Shaw PJ, Shaw CE, Smith B, Breen G, Al-Chalabi A, Moss D, Bubb VJ, Quinn JP. An evaluation of a SVA retrotransposon in the FUS promoter as a transcriptional regulator and its association to ALS. PLoS One. 2014; 9(6): e90833. doi: 10.1371/journal.pone.0090833. eCollection 2014. 27. Cooper-Knock J, Walsh MJ, Higginbottom A, Robin Highley J, Dickman MJ, Edbauer D, Ince PG, Wharton SB, Wilson SA, Kirby J, Hautbergue GM, Shaw PJ. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain 2014 Jul; 137 (Pt 7):2040-51. doi: 10.1093/brain/awu120. 28. http://www.nature.com/app_notes/nmeth/2011/111312/pdf/an8194.pdf Laboratory Technicians were also employed at each Hub Centre to assist in the preparation of sample collection packs for participating satellite centres, DNA extraction and final sample storage. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 http://www.nature.com/app_notes/nmeth/2011/111312/pdf/an8194.pdf Smith BN, Ticozzi N, Fallini C, Gkazi AS, Topp S, Kenna KP, Scotter EL, Kost J, Keagle P, Miller JW, Calini D, Vance C, Danielson EW, Troakes C, Tiloca C, Al-Sarraj S, Lewis EA, King A, Colombrita C, Pensato V, Castellotti B, de Belleroche J, Baas F, ten Asbroek AL, Sapp PC, McKenna-Yasek D, McLaughlin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 26. Savage AL, Wilm TP, Khursheed K, Shatunov A, Morrison KE Shaw PJ, Shaw CE, Smith B, Breen G, Al-Chalabi A, Moss D, Bubb VJ, Quinn JP. An evaluation of a SVA retrotransposon in the FUS promoter as a transcriptional regulator and its association to ALS. PLoS One. 2014; 9(6): e90833. doi: 35 36 37 38 39 40 41 10.1371/journal.pone.0090833. eCollection 2014. 12 RL, Polak M, Asress S, Esteban-Pérez J, et al. Exome-wide Rare Variant Analysis Identifies TUBA4A Mutations Associated with Familial ALS. Neuron. 2014 Oct 22; 84(2) :324-31. doi: 10.1016/j.neuron.2014.09.027. Epub 2014 Oct 22. 29. The European Genome-Phenome archive website: https://www.ebi.ac.uk/ega/home 30. The Project MinE website: https://www.projectmine.com/ 31. The MND Association Website: http://www.mndassociation.org/dnabank FIGURE LEGENDS Table 1: Hub and Spoke Model for Sample Collection The UK MND DNA Bank was a collaborative project adopting a ‘hub and spoke’ model with three regional ‘hub centres’ linking with a total of 16 ‘spoke centres’. These included hospitals that are part of the MND Association’s Care Centre Network and centres which form part of the Department of Health / NIHR Dementias and Neurodegenerative Diseases Research Network (DeNDRoN). The three Hub Centres were established at London (King’s College Hospital), Sheffield (Royal Hallamshire Hospital), and Birmingham (Queen Elizabeth Hospital). Recruitment to the study and sample collection was coordinated at the Hub centres by a DNA Bank Co-odinator based in London. Samples were obtained from sporadic and familial MND patients attending MND clinics in the UK, their spouses (or other genetically unrelated controls) and blood relatives. A DNA Bank research nurse was affiliated to each Hub centre to act as patient liaison, collect clinical information from patients, controls and family members, and take blood samples. Table 1: Hub and Spoke Model for Sample Collection The UK MND DNA Bank was a collaborative project adopting a ‘hub and spoke’ model with three regional ‘hub centres’ linking with a total of 16 ‘spoke centres’. These included hospitals that are part of the MND Association’s Care Centre Network and centres which form part of the Department of Health / NIHR Dementias and Neurodegenerative Diseases Research Network (DeNDRoN). The three Hub Centres were established at London (King’s College Hospital), Sheffield (Royal Hallamshire Hospital), and Birmingham (Queen Elizabeth Hospital). Recruitment to the study and sample collection was coordinated at the Hub centres by a DNA Bank Co-odinator based in London. Samples were obtained from sporadic and familial MND patients attending MND clinics in the UK, their spouses (or other genetically unrelated controls) and blood relatives. A DNA Bank research nurse was affiliated to each Hub centre to act as patient liaison, collect clinical information from patients, controls and family members, and take blood samples. Laboratory Technicians were also employed at each Hub Centre to assist in the preparation of sample collection packs for participating satellite centres, DNA extraction and final sample storage. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Figure 1: The UK MND DNA Bank Figure 1: The UK MND DNA Bank 13 The UK MND DNA Bank comprises 3159 high quality DNA samples. Of these 1344 samples were taken from individuals diagnosed with sporadic MND (see figure 1A and 1B). There were 133 familial MND samples within the collection and a further 500 samples taken from family members, including samples that form 28 parent trio sets and 27 sibling trio sets. The remaining 1085 samples were taken from controls. In line with previous findings, where MND has been diagnosed, the breakdown of gender in the collection is around 60% male (Figure 1A). The average age of onset was approximately 62 years of age (Figure 1C). In total 2653 frozen lymphoblastoid cell lines are held in storage at ECACC. Of these 1267 samples were generated from whole blood taken from patients with sporadic MND. 115 cell lines were generated from familial samples and the remaining 1058 cells lines have been established using blood samples obtained from control or family members (see Figure 1D). The UK MND DNA Bank comprises 3159 high quality DNA samples. Of these 1344 samples were taken from individuals diagnosed with sporadic MND (see figure 1A and 1B). There were 133 Table 2: Clinical information available from the UK MND DNA Bank Each sample withdrawn from the UK MND DNA Bank is accompanied by a minimum dataset of: age at which the samples were taken; gender; disease status; and where appropriate diagnostic certainty (El Escorial Status) and age of onset (calculated from date of birth and date of symptom onset). An extended dataset has been collected for as many participants as possible but it is not a complete dataset for the entire collection. The clinical information was collected by the Research Nurse using a brief clinical questionnaire. Identifying data was kept at each Hub centre in secure locations in accordance with the Data Protection Act. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Table 3: Quality Control PCR Assay fail rate 14 screened in a gender-based assay using the AMEL marker. Only 62 samples showed a continued discrepancy between the gender of the actual DNA sample and that stated in the clinical notes. screened in a gender-based assay using the AMEL marker. Only 62 samples showed a continued discrepancy between the gender of the actual DNA sample and that stated in the clinical notes. screened in a gender-based assay using the AMEL marker. Only 62 samples showed a continued discrepancy between the gender of the actual DNA sample and that stated in the clinical notes. Whilst this is still a low level of error for a collection of this size, the samples were ring fenced from the collection. Whilst this is still a low level of error for a collection of this size, the samples were ring fenced from the collection. Whilst this is still a low level of error for a collection of this size, the samples were ring fenced from the collection. 15 Table 1 Click here to download Table: Table 1.pdf Table 1 Click here to download Table: Table 1.pdf Table 1 : Hub and Spoke Model of Sample Collection Table 1 : Hub and Spoke Model of Sample Collection Figure 1: The UK MND DNA Bank Collection Click here to download Figure: Figure 1.pdf Figure 1: The UK MND DNA Bank Collection Click here to download Figure: Figure 1.pdf ND status and gender of samples held in the DNA bank 1B: Diagnostic certainty of samples within the DNA bank Age range of symptom onset within the DNA bank 1D: MND status and gender of cell line samples held at ECACC 1A: MND status and gender of samples held in the DNA bank 1C: Age range of symptom onset within the DNA bank 1A: MND status and gender of samples held in the DNA bank 1C: Age range of symptom onset within the DNA bank 1A: MND status and gender of samples held in the DNA ban 1C: Age range of symptom onset within the DNA bank 1B: Diagnostic certainty of samples within the DNA bank 1A: MND status and gender of samples held in the DNA bank 1C: Age range of symptom onset within the DNA bank 1B: Diagnostic certainty of samples within the DNA bank 1A: MND status and gender of samples held in the DNA bank 1A: MND status and gender of samples held in the DNA bank 1C: Age range of symptom onset within the DNA bank 1D: MND status and gender of cell line samples held at ECACC 1D: MND status and gender of cell line samples held at ECACC 1D: MND status and gender of cell line samples held at ECACC Figure 2 Figure 2 Click here to download Figure: Table 2.pdf Table 2 : Clinical information available from the UK MND DNA bank Table 2 : Clinical information available from the UK MND DNA bank Figure 3 Click here to download Figure: Table 3.pdf Figure 3 Figure 3 Figure 3 Click here to download Figure: Table 3.pdf Type of Assay No. samples screened % Assay fail Abi Identifilier Kit - AMEL Marker 768 1.30 Gender based PCR - AMEL marker 2750 0.62 Total number of individuals screened to confirm gender % Gender error across collection 3415 1.82 Table 3 : Quality Control PCR fail rates Type of Assay No. samples screened % Assay fail Abi Identifilier Kit - AMEL Marker 768 1.30 Gender based PCR - AMEL marker 2750 0.62 Total number of individuals screened to confirm gender % Gender error across collection 3415 1.82 Table 3 : Quality Control PCR fail rates Table 3 : Quality Control PCR fail rates Click here to download Supplementary Material: suplementary material_Terms and Conditions for Sample Use v2.1 April 2015.pdf
https://openalex.org/W4298034032	https://zenodo.org/records/3766176/files/A%20generalization%20of%20intrinsic%20geometry%20and%20its%20application%20to%20Hilbert's%206th%20problem%20-%20English%20Version.pdf	English	null	A generalization of intrinsic geometry and its application to Hilbert's 6th problem	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	53,487	Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) A generalization of intrinsic geometry and its application to Hilbert’s 6th problem Zhao-Hui Man the date of receipt and acceptance should be inserted later the date of receipt and acceptance should be inserted later Abstract The ﬁrst main work of this paper is to generalize intrinsic geometry. (1) Riemannian manifold is generalized to geometrical manifold. (2) The expression of Erlangen program is improved, and the concept of intrinsic geometry is generalized, so that Riemannian intrinsic geometry which is based on the ﬁrst fundamental form becomes a subgeometry of the generalized intrinsic geometry. The Riemannian geometry is thereby incorporated into the geometrical framework of improved Erlangen program. (3) The important concept of simple connection is discovered, which reﬂects more intrinsic properties of manifold than Levi-Civita connection. The second main work of this paper is to apply the generalized intrinsic geometry to Hilbert’s 6th problem at the most basic level. (1) It starts from an axiom and makes key principles, postu- lates and artiﬁcially introduced equations of fundamental physics all turned into theorems which automatically hold in intrinsic geometrical theory. (2) Intrinsic geometry makes gravitational ﬁeld and gauge ﬁeld uniﬁed essentially. Intrinsic geometry of external space describes gravi- tational ﬁeld, and intrinsic geometry of internal space describes typical gauge ﬁeld. They are uniﬁed into intrinsic geometry. (3) Intrinsic geometry makes gravitational theory and quantum mechanics have the same view of time and space and uniﬁed description of evolution. Keywords Erlangen program · geometrical manifold · intrinsic geometry · simple connection · Riemannian geometry · reference-system · time metric · actual evolution Mathematics Subject Classiﬁcation (2010) Primary 58A05, 51P05, 70A05 · Secondary 53C05, 53Z05 Contents Contents 0 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 Improved expression of Erlangen program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 Generalization of intrinsic geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.1 Geometrical manifold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2 Slack-tights and metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.3 Generalized intrinsic geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.4 Kernal geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zhao-Hui Man 10-2-1102, Hua Yang Nian Hua Bei Qu, Yinchuan, Ningxia, China Tel.: +86-15907366305 E-mail: shetcslion@163.com Contents 12 2.5 Riemannian geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.6 Universal geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.7 Simple connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 0 Introduction 2 Zhao-Hui Man 2.8 Summary of this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3 Intrinsic geometrical solution for Hilbert’s 6th problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.1 Axiom for Hilbert’s 6th problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.2 Mathematical treatment of time and space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.3 Mathematical treatment of evolution . . Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.3.1 Deﬁnition and coordinate form of evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.3.2 Evolution lemma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.3.3 Metric form of evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.3.4 Mathematical treatment of actual evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3.5 Mathematical treatment of actual evolution of potential ﬁeld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.3.6 Mathematical treatment of actual evolution of general charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.3.7 Intrinsic geometrical treatment of conservation of energy-momentum . . . . . . . . . . . . . . . . . . . . . . . . . 28 3.3.8 Two dual descriptions of gradient direction ﬁeld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contents . . . . 32 3.4 Intrinsic geometrical treatment of propagator and wave function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.5 Summary of this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4 Intrinsic geometrical treatment of inversion transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 5 Intrinsic geometrical treatment of classical spacetime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 5.1 Existence and uniqueness of submanifold with classical spacetime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 5.2 Intrinsic geometrical treatment of gravitational ﬁeld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 5.3 Two coordinate representations of intrinsic geometrical property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 5.4 Geometrical treatment of classical spacetime evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 5.5 Intrinsic geometrical treatment of Legendre transformation and equation of motion . . . . . . . . . . . . . . . . . Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 0 Introduction (i) Intrinsic geometry. Contents . . . . . 46 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation . . . . . . . . . . . . . . . . . . . . . . . . . . 47 6 Several intrinsic geometrical properties in 5-dimensional case . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 7 Several intrinsic geometrical properties in 6-dimensional case . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 8 Several intrinsic geometrical properties in 8-dimensional case . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 9 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 2.8 Summary of this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3 Intrinsic geometrical solution for Hilbert’s 6th problem . . . . . . . . . . . . . . . . . . . . . . . . Contents . . . . . . . . . . . . . . . 17 3.1 Axiom for Hilbert’s 6th problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.2 Mathematical treatment of time and space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.3 Mathematical treatment of evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.3.1 Deﬁnition and coordinate form of evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.3.2 Evolution lemma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.3.3 Metric form of evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.3.4 Mathematical treatment of actual evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3.5 Mathematical treatment of actual evolution of potential ﬁeld . . . . . . . . . . . . . . . Contents . . . . . . . . . . . . . . . 24 3.3.6 Mathematical treatment of actual evolution of general charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.3.7 Intrinsic geometrical treatment of conservation of energy-momentum . . . . . . . . . . . . . . . . . . . . . . . . . 28 3.3.8 Two dual descriptions of gradient direction ﬁeld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3.4 Intrinsic geometrical treatment of propagator and wave function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.5 Summary of this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4 Intrinsic geometrical treatment of inversion transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 5 Intrinsic geometrical treatment of classical spacetime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 5.1 Existence and uniqueness of submanifold with classical spacetime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 5.2 Intrinsic geometrical treatment of gravitational ﬁeld . . . . . . . . . . . . . . . . . . . . . . . . Contents . . . . . . . . . . . . . . 39 5.3 Two coordinate representations of intrinsic geometrical property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 5.4 Geometrical treatment of classical spacetime evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 5.5 Intrinsic geometrical treatment of Legendre transformation and equation of motion . . . . . . . . . . . . . . . . . . . . . . 46 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation . . . . . . . . . . . . . . . . . . . . . . . . . . 47 6 Several intrinsic geometrical properties in 5-dimensional case . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 7 Several intrinsic geometrical properties in 6-dimensional case . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 8 Several intrinsic geometrical properties in 8-dimensional case . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 9 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 References . . . . . . . . . . . . . . . . . . . . . . . . . . (i) Intrinsic geometry. In 1827, Friedrich Gauss created the theory of intrinsic geometry of surface in his article Disquisitiones Generales Circa Superﬁcies Curves, and essentially uniﬁed the Euclidean geom- etry and various non-Euclidean geometries at that time. In 1854, Bernhard Riemann generalized Gauss’s ideas of intrinsic geometry to high dimensions in his speech Ueber Die Hypothesen, Welche Der Geometrie Zu Grunde Liegen, and expressed the ﬁrst fundamental form with met- ric tensor. In Riemannian geometry, all values of intrinsic geometrical properties are totally determined by metric tensor. However, in this paper it is discovered that the traditional practice that characterizing intrinsic geometry with metric tensor is not necessarily able to cover all the intrinsic properties of manifold. It has been known for a long time that the coeﬃcients of metric tensor surely remain unchanged when orthogonal transformations eﬀect on semi-metric [14, 19]. Although there have been many studies on semi-metric, and some non-mathematical researches have shown advantages of semi-metric [13, 14, 53], however their mathematical meanings have not been studied suﬃciently. Some articles treat semi-metric either as an alternative expression form of metric, or as an insigniﬁcant mathematical substitution [14]. And there is an article [11] which has noticed that semi-metric causes some indications beyond Riemannian geometry, but it argues that a well-done semi-metric theory should not cause such indications. We must see that the mathematical signiﬁcance of semi-metric has not yet been fully revealed. It is noticed that a traditional intrinsic geometrical property is an invariant under identical transformations of metric, and we can also speak of it as an invariant under orthogonal transfor- mations of semi-metric. It indicates that two diﬀerent properties under two diﬀerent orthogonal transformations of semi-metric cannot be distinguished by the traditional intrinsic geometry. If A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 3 we take diﬀerent orthogonal transformations of semi-metric at diﬀerent points of a Riemannian manifold, the Riemannian manifold remains unchanged, and these orthogonal transformations just exactly constitute a local gauge transformation. That is to say, Riemannian geometry is still not exquisite enough. Therefore, there exists a kind of more extensive intrinsic geometry with Riemannian geometry as its subgeometry. In order to understand the generalized concept of intrinsic geometry deﬁned later more conveniently, the intuition of intrinsic geometry must be concisely explained here in a new way diﬀerent from the perspective of metric of traditional intrinsic geometry. Fig. (i) Intrinsic geometry. 1 The intuition of intrinsic geometry of curve Fig. 1 The intuition of intrinsic geometry of curve First, consider the case of one-dimension, that is the intuition of intrinsic geometry of curve. As shown in Fig.1, select a curve L in the plane rectangular coordinate system. Project the coordinates of y axis onto L continuously and uniformly, and then onto x axis. First, consider the case of one-dimension, that is the intuition of intrinsic geometry of curve. As shown in Fig.1, select a curve L in the plane rectangular coordinate system. Project the coordinates of y axis onto L continuously and uniformly, and then onto x axis. In this way, the original continuous and uniform coordinate distribution becomes a continuous but ununiform distribution via L as a medium, thus we obtain an interval S with some ununiform distribution shown in the right ﬁgure of Fig. 1. This is actually the intuition of intrinsic geometry of curve L. It can be said that curve S is curve L in intrinsic geometry. uch an intuition of intrinsic geometry can be described strictly in the following way. Such an intuition of intrinsic geometry can be described strictly in the following way Let S be a one-dimensional manifold, which is homeomorphic to an Euclidean straight line. Take two coordinate charts (S, x) and (S, y) on S such that we have a coordinate relation y = y (x). Let S be a one-dimensional manifold, which is homeomorphic to an Euclidean straight line. Take two coordinate charts (S, x) and (S, y) on S such that we have a coordinate relation y = y (x). As shown in the above ﬁgure, at every point of S, it shows a kind of intuition reﬂecting the degree of slackness and tightness of coordinate distribution of y axis in x axis. Such a degree of slackness and tightness can be strictly described by dy dx. Then the one-dimensional manifold S given the degree of slackness and tightness dy dx is the curve L deﬁned in way of intrinsic geometry. The case of two-dimensional surface is similar. The intuition of intrinsic geometry of surface z = e−(x2+y2) in the left ﬁgure of Fig.2 can be shown by the degree of slackness and tightness of coordinate net u1, u2 in coordinate system x1, x2 at each point of the right ﬁgure of Fig.2. (i) Intrinsic geometry. This degree of slackness and tightness can be strictly described by ∂uk ∂xi (i, k = 1, 2). It can be said that the degree of slackness and tightness ∂uk ∂xi of the right ﬁgure deﬁnes the surface of the left ﬁgure in way of intrinsic geometry. The coordinate net in this ﬁgure shows a special solution of 4 Zhao-Hui Man Fig. 2 The intuition of intrinsic geometry of surface u1, u2 such that            u1 x1, x2 = x1 p (x1)2 + (x2)2 Z √ (x1)2+(x2)2 0 q 1 + 4ρ2e−2ρ2dρ, u2 x1, x2 = x2 p (x1)2 + (x2)2 Z √ (x1)2+(x2)2 0 q 1 + 4ρ2e−2ρ2dρ. Fig. 2 The intuition of intrinsic geometry of surface Fig. 2 The intuition of intrinsic geometry of surface Fig. 2 The intuition of intrinsic geometry of surface u1, u2 such that            u1 x1, x2 = x1 p (x1)2 + (x2)2 Z √ (x1)2+(x2)2 0 q 1 + 4ρ2e−2ρ2dρ, u2 x1, x2 = x2 p (x1)2 + (x2)2 Z √ (x1)2+(x2)2 0 q 1 + 4ρ2e−2ρ2dρ. These above are intuitive descriptions of two simple cases about one and two dimensions, emphasizing the central role of the degree of slackness and tightness ∂uk ∂xi determined by two coordinate systems in reﬂecting the intuition of intrinsic geometry. In this way, the general concept of intrinsic geometry will be deﬁned strictly in this paper. Such a generalized intrinsic geometry is worth studying. It is discovered in this paper, that the geometrical properties in such a geometry can just exactly reﬂect the physical properties of elementary particles in the Standard Model, which cannot be described by the traditional intrinsic geometry. With such a generalization, it is not only gravitational ﬁeld but also gauge ﬁeld, that can be described by intrinsic geometry, and further more the whole fundamental physics will be uniﬁed in the intrinsic geometry. Therefore, it will naturally give a solution for Hilbert’s 6th problem. (ii) Hilbert’s 6th problem. In the above sense, fundamental physical theories in history are too abstract and lack of concrete mathematical constructions. For examples: (1) In electrodynamics, the relationship between mechanics and electromagnetics can be established just only by Lorentz force equation FFF = q (EEE + vvv × BBB). However, various variables in the formula are all abstract vectors and scalar, which are lack of concrete mathematical constructions. For example, EEE and BBB can only be distinguished ontologically, but as two abstract vectors there is no diﬀerence of mathematical connotation between them. As a result, the Lorentz force equation can just only be artiﬁcially introduced and regarded as a principle and cannot become a theorem automatically. (1) In electrodynamics, the relationship between mechanics and electromagnetics can be established just only by Lorentz force equation FFF = q (EEE + vvv × BBB). However, various variables in the formula are all abstract vectors and scalar, which are lack of concrete mathematical constructions. For example, EEE and BBB can only be distinguished ontologically, but as two abstract vectors there is no diﬀerence of mathematical connotation between them. As a result, the Lorentz force equation can just only be artiﬁcially introduced and regarded as a principle and cannot become a theorem automatically. (2) After the establishment of quantum mechanics [3–5,8,9,58–63], the quantum ﬁeld theory established. The suggestion of Yang-Mills theory [73] eventually led to Glashow-Weinberg- Salam’s uniﬁed theory of weak electricity [20, 29, 38, 41–43, 45, 57, 66], quantum chromody- namics [2,6,21,24,25,27,31–33,56,65] and various great uniﬁed theories [10,23,26,49–51]. Not long ago, Yue-Liang Wu brought gravitational ﬁeld into the framework of QFT in inertial system [68–72]. However, such ﬁelds are still abstractly deﬁned functions and lack of concrete mathematical construction. Both Hamiltonian function and Lagrangian function are abstract objects, because ﬁeld functions composing them are abstract. On one hand, physics describes gauge ﬁeld with abstract concept of connection on a ﬁbre bundle, but without giving concrete mathematical constructions to the connections. On the other hand, the spinor ﬁeld, which is composed of several complex-valued functions, is sometimes used to refer to a charged lepton ﬁeld, and sometimes a neutrino ﬁeld. It is not clear in physics that how to distinguish ﬁeld function of charged lepton and ﬁeld function of neutrino by mathematical constructions. (ii) Hilbert’s 6th problem. The purpose of Hilbert’s 6th problem is to axiomatize the physics. Theoretical physics at the most basic level is an important aspect about it. The unity of the physical world has always been a belief held by many people. The history of theoretical physics is a process that the unity expands step by step. In this paper, it is essentially regarded as a process that the concept of geometry expands step by step. (1) From Newtonian mechanics to special relativity [16], and then to general relativity [17,18], it is a process that ﬂat Riemannian geometry expands to general Riemannian geometry. (1) From Newtonian mechanics to special relativity [16], and then to general relativity [17,18], it is a process that ﬂat Riemannian geometry expands to general Riemannian geometry. (2) Gauge ﬁeld theory actually expresses a kind of geometry that cannot be described by Riemannian geometry. It is usually described by abstract connections on abstract ﬁbre bundles, however, that is not concrete enough. In the perspective of concrete constructivity, the generalization of intrinsic geometry in this paper makes the concept of geometry expand further more, thereby Riemannian geometry and gauge ﬁeld geometry can be uniformly described by the generalized intrinsic geometry. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 5 Besides the intrinsic geometry, the solution at the most basic level for Hilbert’s 6th problem also depends on a constructivity method. There are two approaches to develop mathematical theory, one is the approach of concrete constructivity based on set theory, the other is the approach of abstract structure based on category theory. Although the eﬀectiveness of these two research approaches is the same, without either of them, the cognition to this mathematical intuition is not complete. For example, consider the concept of real number. From the approach of abstract structure, some conventions as the connotation of abstract structure are combined to form axiomatized deﬁnition of real number, i.e. a real number is an abstract element in the complete archimedean ordered ﬁeld. From the approach of concrete constructivity, natural numbers are constructed from empty set, then integers and rational numbers are constructed, and then irrational numbers are constructed via Dedekind cut to form the real number set. Such two concepts of real number deﬁned in two approaches reﬂect the same mathematical intuition. And such two theories of real number provide a complete cognition for the intuition of real number. (iii) General deﬁnition of geometry. In order to generalize intrinsic geometry, we have to make a few improvements to the expression form of Erlangen program. It is mainly based on the consideration of the following two issues. (1) In history, there appeared two diﬀerent approaches to unify Euclidean geometry and non Euclidean geometries. One is from Gauss and Riemann, that is to say, Gauss-Bonet theorem distinguishes geometries with angle sum of a triangle. The other is the Erlangen program [46] proposed by Felix Klein in 1872, which distinguishes geometries with transformation group. However, Riemannian geometry was regarded as one that cannot be incorporated into the framework of Erlangen’s program, so these two approaches still fail to associate clearly. (2) Based on the idea of Erlangen program, starting from the second half of the 20th century, theoretical physics began to emphasize the notion of symmetry and research it with the concept of group extensively. It is right, but easy to cause a kind of misunderstanding, that is, symmetry and group are regarded as equivalent things. (2) Based on the idea of Erlangen program, starting from the second half of the 20th century, theoretical physics began to emphasize the notion of symmetry and research it with the concept of group extensively. It is right, but easy to cause a kind of misunderstanding, that is, symmetry and group are regarded as equivalent things. In fact, the essential idea of symmetry is the invariance under transformations, and the essential idea of group is the relationship between transformations. The former is a geometrical property, and the latter is an algebraic property. Therefore, symmetry and group should not be confused. In order to deal with the above two issues and to generalize intrinsic geometry, the expression form of Erlangen program will be improved in this paper. The original idea of this improvement has been referred to in literatures [15, 52], but they have not expressed this idea as a strict deﬁnition of general concept of geometry in an explicit form. Such a deﬁnition will be given below. (ii) Hilbert’s 6th problem. (3) Early Kaluza-Klein theory [44, 47, 48] and later string theory as well as superstring theory [1,12,28,30,34–37,39,40,55,64,67] attempted to provide a uniﬁed explanation of this problem in high dimensional space. But for Hilbert’s 6th problem, they still cannot be regarded as success. 6 Zhao-Hui Man The details of the above theories will not be discussed here. What should be emphasized is that we will supply concrete mathematical constructions for the above various physical concepts from the perspective of intrinsic geometry, so that theoretical physics can be axiomatized at the most basic level. It will start from a unique basic axiom, and strictly deduce the framework of physics, and turn principles, postulates, and artiﬁcially introduced equations into theorems, so as to give a kind of eﬀective solution for Hilbert’s 6th problem at the most basic level. 1 Improved expression of Erlangen program Deﬁnition 1.1. Let C be a set and ∼a relation of equivalence. The classiﬁcation C/ ∼is called a geometry on C with respect to ∼. Let t : C →C be a transformation. If ∀[c] ∈C/ ∼, ∀c ∈[c] such that t(c) ∈[c], we say t is an equivalent transformation with respect to ∼. The totality T of all equivalent transformations on C with respect to ∼is called an equivalent transformation set on C with respect to ∼. Evidently, ∼and T are mutually determined, therefore, C/ ∼can also be denoted by C/T. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 7 ∀[c] ∈C/T is called a geometrical object. ∀a ∈[c] is called a geometrical instance of [c]. Denote S ≜S c∈C c, each element in S is called a point, each subset of S is called a geometrical ﬁgure, and (S, T) is called a kind of geometrical theory. ∀[c] ∈C/T is called a geometrical object. ∀a ∈[c] is called a geometrical instance of [c]. Denote S ≜S c∈C c, each element in S is called a point, each subset of S is called a geometrical ﬁgure, and (S, T) is called a kind of geometrical theory. Let H be a set, and let a map h : C →H satisfy ∀c1, c2 ∈C, c1 ∼c2 ⇔h(c1) = h(c2). Then h induces a map ˜h : C/ ∼→H, [c] 7→h(c). Each of h and ˜h is called a geometrical property on C. The image of h and ˜h in H is called the value of geometrical property. Suppose there are two relations of equivalence ∼a and ∼b on C. If ∼a⊂∼b, we say geometry C/ ∼a is larger than C/ ∼b, and C/ ∼b smaller than C/ ∼a, we also say C/ ∼b is a subgeometry of C/ ∼a. Remark 1.1. The above deﬁnition is equivalent to the traditional expression of Erlangen pro- gram. It is remarkable that it characterizes geometry with a relation of equivalence, but not a group. Why a new deﬁnition should be adopted? Because it is very inconvenient to describe geometry in the traditional form of Erlangen program in the case where a group is diﬃcult to expressed in an explicit form due to its complicated form or uncertain structure. 1 Improved expression of Erlangen program But if we ﬁnd a certain condition to deﬁne a relation of equivalence, according to the above new deﬁnition we are able to deﬁne our required geometry without specifying group structure. Thereby it will be convenient for our study, such as what Discussion 6.1 says. In addition, Deﬁnition 2.3.2 , Deﬁnition 2.4.2 , Deﬁnition 2.5.2 and section 2.6 are also treated in such a way. In the past, Erlangen program was used to deal with groups with simple structure. The corresponding geometry was conﬁned to either local of the manifold or homogeneous manifold such as constant curvature manifold. The Riemannian geometry was not incorporated into the framework of Erlangen program in traditional way. By contrast, based on the expression form of this paper, the deﬁnition of geometry can completely incorporate Riemannian geometry into the framework of improved Erlangen program, see Discussion 2.6.2 . To say the least, if the group structure has to be emphasized, the following additional deﬁnition is needed. The elements in equivalent transformation set T naturally imply a group structure with respect to composite operation of maps. The group T is called the tranformation group of geometry C/T, and C/T is called the geometry of group T. Therefore, the group structure exists on the equivalent transformation set naturally, and it is not necessary to make explicit requirements in the deﬁnition of geometry as the traditional form of Erlangen program. Suppose transformation group T1 acting on S1 and transformation group T2 acting on S2 are isomorphic. (S1, T1) and (S2, T2) are called the same kind of geometrical theory. If T1 is a proper subgroup of T2, we say T1 is smaller than T2, and T2 larger than T1. Evidently, the smaller the group, the larger the geometry; conversely, the larger the group, the smaller the geometry. Deﬁnition 1.2. On any set C, there must be a special geometry, which has only one equivalence class, that is C itself. This geometry is called a universal geometry. The set C is the only geometrical object in universal geometry, and it is called a universal geometrical object. Each geometrical property in universal geometry is called a universal geometrical property, and also called a geometrical invariant on C. Each universal geometrical property with its unique value is called a geometrical identity on C. Zhao-Hui Man 8 2.1 Geometrical manifold A diﬀerential manifold M with a reference-system f is called a geometrical manifold given shape by f, and denoted by (M, f). A diﬀerential manifold M with a reference-system f is called a geometrical manifold given shape by f, and denoted by (M, f). 2.1 Geometrical manifold Deﬁnition 2.1.1. Let M be a D-dimensional connected smooth real manifold. ∀p ∈M, take a coordinate chart (Up, φUp) on a neighborhood Up of p. They constitute a coordinate covering {(Up, φUp)}p∈M, which is called a point-by-point covering. φUp is called a coordinate frame on neighborhood Up of p. For the sake of simplicity, below Up is denoted by U, and φUp is denoted by φU. For any two coordinate frames φU and ψU on neighborhood U of point p, if fp ≜φU ◦ψ−1 U : ψU(U) →φU(U) is a smooth homeomorphism, fp is called a (local) reference-system on neighborhood U of point p, where ψU is the basis coordinate frame of fp, and φU is the performance coordinate frame of fp. For any local reference-systems fp and gp at p, if the coordinate frames of fp and the coordinate frames of gp are C∞-compatible, we say fp and gp are C∞-compatible. The totality of the local reference-systems that are mutually C∞-compatible is called a (local) reference-system space, denoted by REFp(U) or REFp. The totality of all the local reference-systems with ψU as the basis coordinate frame is denoted by REFp(U, ψU). The totality of all the local reference-systems with ψU as the basis coordinate frame is denoted by REFp(U, ψU). The totality of all the local reference-systems with φU as the performance coordinate frame is denoted by REFp(φU, U). The totality of all the local reference-systems with φU as the performance coordinate frame is denoted by REFp(φU, U). Deﬁnition 2.1.2. Denote REF ≜ S p∈M REFp, where ∀p, q ∈M all the elements in REFp and REFq are C∞-compatible. Deﬁnition 2.1.2. Denote REF ≜ S p∈M REFp, where ∀p, q ∈M all the elements in REFp and REFq are C∞-compatible. REFq are C∞-compatible. If the map f : M →REF, p 7→f(p) ∈REFp satisﬁes that the slack-tights BA M and CM A in deﬁnition 2.2.2 are all smooth real functions on M, f is called a reference-system on M. The totality of all reference-systems which are mutually C∞-compatible on M is denoted by REFM. If the map f : M →REF, p 7→f(p) ∈REFp satisﬁes that the slack tights BM and CA in deﬁnition 2.2.2 are all smooth real functions on M, f is called a reference-system on M. The totality of all reference-systems which are mutually C∞-compatible on M is denoted by REFM. Deﬁnition 2.1.3. ∀p ∈M, ∀ρU ◦ψ−1 U ∈REFp(U) induces two (local) reference-system trans- formations Deﬁnition 2.1.3. ∀p ∈M, ∀ρU ◦ψ−1 U ∈REFp(U) induces two (local) reference-system trans- formations    LρU◦ψ−1 U :REFp(ψU, U) →REFp(ρU, U), ψU ◦φ−1 U 7→(ρU ◦ψ−1 U ) ◦(ψU ◦φ−1 U ) = ρU ◦φ−1 U , RρU◦ψ−1 U :REFp(U, ρU) →REFp(U, ψU), φU ◦ρ−1 U 7→(φU ◦ρ−1 U ) ◦(ρU ◦ψ−1 U ) = φU ◦ψ−1 U .    LρU◦ψ−1 U :REFp(ψU, U) →REFp(ρU, U), ψU ◦φ−1 U 7→(ρU ◦ψ−1 U ) ◦(ψU ◦φ−1 U ) = ρU ◦φ−1 U , RρU◦ψ−1 U :REFp(U, ρU) →REFp(U, ψU), φU ◦ρ−1 U 7→(φU ◦ρ−1 U ) ◦(ρU ◦ψ−1 U ) = φU ◦ψ−1 U . ∀f ∈REFM, ∀p ∈M, suppose Lf(p) and Rf(p) are induced by f(p). The maps Lf : p 7→ Lf(p) ≜Lf(p) and Rf : p 7→Rf(p) ≜Rf(p) are called reference-system transformations on manifold M. ∀f ∈REFM, ∀p ∈M, suppose Lf(p) and Rf(p) are induced by f(p). The maps Lf : p 7→ Lf(p) ≜Lf(p) and Rf : p 7→Rf(p) ≜Rf(p) are called reference-system transformations on manifold M. Remark 2.1.1. Suppose there is a reference-system f on manifold M. Construct reference- system e in the following way: ∀p ∈M, on neighborhood U of point p, take the basis coordinate frame of f(p) as the basis coordinate frame of e(p), and take the same basis coordinate frame 9 A generalization of intrinsic geometry and its application to Hilbert’s 6th problem of f(p) as the performance coordinate frame of e(p). Thus, reference-system transformation Lf sends e to f just exactly. of f(p) as the performance coordinate frame of e(p). Thus, reference-system transformation Lf sends e to f just exactly. 2.2 Slack-tights and metrics Deﬁnition 2.2.1. For convenience, some index symbols have to be speciﬁed. In the absence of a special declaration, the indices used below are valued in the following range: Deﬁnition 2.2.1. For convenience, some index symbols have to be speciﬁed. In the absence of a special declaration, the indices used below are valued in the following range: (1) for basis coordinate frame (U, ξ), indices A, B, C, D, E = 1, 2, · · · , D, such as ξA; (1) for basis coordinate frame (U, ξ), indices A, B, C, D, E = 1, 2, · · · , D, such as ξA; (2) for performance coordinate frame (U, x), indices M, N, P, Q, R = 1, 2, · · · , D, such as xM. (2) for performance coordinate frame (U, x), indices M, N, P, Q, R = 1, 2, · · · , D, such as xM. Deﬁnition 2.2.2. Let (M, f) be a geometrical manifold. ∀p ∈M, on a neighborhood Up of point p, let the coordinate representation of local reference-system f(p) be ξA = ξA(xM), xM = xM(ξA). Their derivative functions        bA M : Up →R, q 7→bA M(q) ≜∂ξA ∂xM (q), cM A : Up →R, q 7→cM A (q) ≜∂xM ∂ξA (q), on Up are called the degrees of slackness and tightness of f(p), or slack-tights for short. If it is needed to emphasize f(p) explicitly, bA M and cM A can be denoted by (bf(p))A M and (cf(p))M A . D ﬁ t ki d f th l f ti if ld M on Up are called the degrees of slackness and tightness of f(p), or slack-tights for short. If it is needed to emphasize f(p) explicitly, bA M and cM A can be denoted by (bf(p))A M and (cf(p))M A . Deﬁne two kinds of smooth real functions on manifold M: Deﬁne two kinds of smooth real functions on manifold M: Deﬁne two kinds of smooth real functions on manifold    BA M : M →R, p 7→BA M(p) ≜(bf(p))A M(p) CM A : M →R, p 7→CM A (p) ≜(cf(p))M A (p) , then BA M and CM A are called the slack-tights of reference-system f on manifold M. then BA M and CM A are called the slack-tights of reference-system f on manifold M. Discussion 2.2.1. Corresponding to the two coordinate frames of f(p), the tangent space Tp at point p has two sets of natural bases ∂ ∂ξA p , ∂ ∂xM p ∈Tp, and the cotangent space T ∗ p also has two sets of natural bases dξA p , dxM p ∈T ∗ p . Thus, on Up we have (bf(p))A M = * ∂ ∂xM p , dξA p + , (cf(p))A M = * ∂ ∂ξA p , dxM p + . Therefore, ∀p ∈M we have Therefore, ∀p ∈M we have BA M(p) = * ∂ ∂xM p , dξA p + (p), CM A (p) = * ∂ ∂ξA p , dxM p + (p). which can concisely be denoted by BA M = ∂ ∂xM , dξA , CM A = ∂ ∂ξA , dxM . Deﬁnition 2.2.3. Denote Deﬁnition 2.2.3. Denote Deﬁnition 2.2.3. Denote Deﬁnition 2.2.3. Denote εMN = εMN= εM N ≜    1, M = N 0, M ̸= N , δAB = δAB= δA B ≜    1, A = B 0, A ̸= B . 10 Zhao-Hui Man Let (M, f) be a geometrical manifold. ∀p ∈M, let U be a neighborhood of point p. (1) The coordinate frames (U, ξA) and (U, xM) of f(p) respectively inherit metric tensor ﬁelds (1) The coordinate frames (U, ξA) and (U, xM) of f(p) respectively inherit metri ordinate frames (U, ξA) and (U, xM) of f(p) respectively inherit metric tensor ﬁelds    g ≜δABdξA ⊗dξB = gMNdxM ⊗dxN h ≜εMNdxM ⊗dxN = hABdξA ⊗dξB ,    gMN = δABbA MbB N hAB = εMNcM A cN B . If it is needed to emphasize f(p) explicitly, g and h can be expressed as gf(p) and hf(p), then gMN and hAB can be expressed as (gf(p))MN and (hf(p))AB. If it is needed to emphasize f(p) explicitly, g and h can be expressed as gf(p) and hf(p), then gMN and hAB can be expressed as (gf(p))MN and (hf(p))AB. (2) On manifold M, deﬁne smooth real functions    GMN : M →R, p 7→GMN(p) ≜(gf(p))MN(p) HAB : M →R, p 7→HAB(p) ≜(hf(p))AB(p) ,    ∆AB : M →R, p 7→∆AB(p) ≜δAB EMN : M →R, p 7→EMN(p) ≜εMN . Thus on the entire manifold M, two metric tensor G and H are constructed, the local restrictions of which can be expressed as Thus on the entire manifold M, two metric tensor G and H are constructed, the local restrictions of which can be expressed as    G = ∆ABdξA ⊗dξB = GMNdxM ⊗dxN H = EMNdxM ⊗dxN = HABdξA ⊗dξB ,    GMN = ∆ABBA MBB N HAB = EMNCM A CN B . Deﬁnition 2.2.4. Denote dξA ≜hABdξB, dxM ≜gMNdxN, which induce ∂ ∂ξA and ∂ ∂xM in tangent space, such that D ∂ ∂ξB , dξA E = δB A and D ∂ ∂xN , dxM E = εN M. Denote Deﬁnition 2.2.4. Denote dξA ≜hABdξB, dxM ≜gMNdxN, which induce ∂ ∂ξA and ∂ ∂xM in tangent space, such that D ∂ ∂ξB , dξA E = δB A and D ∂ ∂xN , dxM E = εN M. Deﬁnition 2.2.3. Denote Denote          cMA ≜∂xM ∂ξA bAM ≜∂ξA ∂xM ,        cAM ≜∂xM ∂ξA bMA ≜∂ξA ∂xM ,        ¯bM A ≜∂ξA ∂xM ¯cA M ≜∂xM ∂ξA . Deﬁne the following tensors: Deﬁne the following tensors: Deﬁne the following tensors:    g ≜δABdξA ⊗dξB = gMNdxM ⊗dxN = gMNdxM ⊗dxN h ≜εMNdxM ⊗dxN = hABdξA ⊗dξB = hABdξA ⊗dξB ,        x ≜δAB ∂ ∂ξA ⊗ ∂ ∂ξB = xMN ∂ ∂xM ⊗ ∂ ∂xN = xMN ∂ ∂xM ⊗ ∂ ∂xN y ≜εMN ∂ ∂xM ⊗ ∂ ∂xN = yAB ∂ ∂ξA ⊗ ∂ ∂ξB = yAB ∂ ∂ξA ⊗ ∂ ∂ξB , where    gMN = δABbA MbB N gMN = δABbAMbBN ,    hAB = εMNcM A cN B hAB = εMNcMAcNB ,    xMN = δABcAMcBN xMN = δABcM A cN B ,    yAB = εMNbMAbNB yAB = εMNbA MbB N . Proposition 2.2.1. gMN = xMN, gMN = xMN, hAB = yAB, hAB = yAB. Proof. Proof. (1) gMP bC MbD P = δCD ⇒gMNbC MbD NcP CcQ D = δCDcP CcQ D ⇒gPQ = xPQ. (1) gMP bC MbD P = δCD ⇒gMNbC MbD NcP CcQ D = δCDcP CcQ D ⇒gPQ = xPQ. xMN ≜δABcAMcBN ⇒gPMxMNgNQ = gPMδABcAMcBNgNQ = δAB(cAMgPM)(cBNgNQ) = δABcP AcQ B = gPQ ⇒gPMxMN = εP N ⇒xMN = gMN. (2) hABcP AcQ B = εPQ ⇒hABcP AcQ BbC P bD Q = εPQbC P bD Q ⇒hCD = yCD. yAB ≜εMNbMAbNB ⇒hCAyABhBD = hCAεMNbMAbNBhBD = εMN(bMAhCA)(bNBhBD) = εMNbC MbD N = hCD ⇒hCAyAB = δC B ⇒yAB = hAB. ⊓⊔ A B N (2) hABcP AcQ B = εPQ ⇒hABcP AcQ BbC P bD Q = εPQbC P bD Q ⇒hCD = yCD. Q Q yAB ≜εMNbMAbNB ⇒hCAyABhBD = hCAεMNbMAbNBhBD = εMN(bMAhCA)(bNBhBD) = εMNbC MbD N = hCD ⇒hCAyAB = δC B ⇒yAB = hAB. ⊓⊔ 11 A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 11 Deﬁnition 2.3.3. Intrinsic transformation. (1) Local intrinsic transformation. [fp] induces equivalence classes L[fp] : [hp] 7→[fp] ◦[hp] and R[fp] : [kp] 7→[kp] ◦[fp] of Lfp : hp 7→fp ◦hp and Rfp : kp 7→kp ◦fp. Then we say L[fp] and R[fp] are local intrinsic transformations. (1) Local intrinsic transformation. [fp] induces equivalence classes L[fp] : [hp] 7→[fp] ◦[hp] and R[fp] : [kp] 7→[kp] ◦[fp] of Lfp : hp 7→fp ◦hp and Rfp : kp 7→kp ◦fp. Then we say L[fp] and R[fp] are local intrinsic transformations. (2) Intrinsic transformation on manifold. [f] induces equivalence classes L[f] : [h] 7→[f] ◦[h] and R[f] : [k] 7→[k] ◦[f] of Lf : h 7→f ◦h and Rf : k 7→k ◦f. Then we say L[f] and R[f] are intrinsic transformations of geometrical manifolds, or general gauge transformations, see Proposition 5.6.2 for reasons. Discussion 2.3.1. Intrinsic transformation group. Deﬁnition 2.3.1. Intrinsic geometry of reference-system. Deﬁnition 2.3.1. Intrinsic geometry of reference-system. (1) Intrinsic geometry of local reference-system. (1) Intrinsic geometry of local reference-system. ∀fp, gp ∈REFp(U), let slack-tights of fp and gp be (bf)A M and (bg)A M respectively. Deﬁne a relation of equivalence ∼=, such that fp ∼= gp if and only if ∀q ∈U, (bf)A M(q) = (bg)A M(q). Thus, REFp(U)/ ∼= is called the intrinsic geometry on REFp(U), where the geomet- rical object [fp] is called the core of fp. Deﬁne a relation of equivalence ∼=, such that fp ∼= gp if and only if ∀q ∈U, (bf)A M(q) = (bg)A M(q). Thus, REFp(U)/ ∼= is called the intrinsic geometry on REFp(U), where the geomet- rical object [fp] is called the core of fp. (2) Intrinsic geometry of reference-system on manifold. ∀f, g ∈REFM, let slack-tights of f and g be (Bf)A M and (Bg)A M respectively. Deﬁne a relation of equivalence ≡, such that f ≡g if and only if ∀p ∈M, f(p) ∼= g(p). Thus, the geometry REFM/ ≡is called the strict intrinsic geometry on REFM. Deﬁne a relation of equivalence ≡, such that f ≡g if and only if ∀p ∈M, f(p) ∼= g(p). Thus, the geometry REFM/ ≡is called the strict intrinsic geometry on REFM. Deﬁne a relation of equivalence ∼=, such that f ∼= g if and only if ∀p ∈M, (Bf)A M(p) = (Bg)A M(p). Thus, the geometry REFM/ ∼= is called the intrinsic geometry on REFM, where the geometrical object [f] is called the core of f. Due to the one to one correspondence between maps [f] ≜[p 7→f(p)] and p 7→[f(p)], the core of f can also be expressed as [f] : p 7→[f(p)]. 2.3 Generalized intrinsic geometry Deﬁnition 2.3.1. Intrinsic geometry of reference-system. Deﬁnition 2.3.2. Intrinsic geometry on geometrical manifold. Deﬁnition 2.3.2. Intrinsic geometry on geometrical manifold. The totality of all the geometrical manifolds on M is denoted by M(M). Deﬁne a relation of equivalence ∼= of geometrical manifolds, such that (M, f) ∼= (M, g) if and only if f ∼= g. The geometry C(M) ≜M(M)/ ∼= is called the intrinsic geometry on geometrical manifolds, where geometrical object (M, [f]) is called an intrinsic geometrical manifold given shape by [f]. According to Deﬁnition 1.1, the value of each intrinsic geometrical property is completely depends on the core of reference-system, and thereby depends on the slack-tights BA M or CM A . Deﬁnition 2.3.4. The general linear group GL(M) is called intrinsic transformation group, or general gauge transformation group. Specially, for the case that the transformation groups at diﬀerent points of manifold are isomorphic to each other, the general linear group GL(D, R) is called homogeneous intrinsic transformation group. Furthermore: Let S be a subgroup of GL(D, R). If f satisﬁes the following two conditions: (1) ∀p ∈ M, [f(p)] ∈S; (2) for any subgroup T of S, ∃q ∈M, [f(q)] /∈T; then we say objects determined by f, such as f, (M, [f]), L[f], R[f], etc., are generated by group S. As the equivalent transformation set, the totality of all intrinsic transformations generated by group S is used to deﬁne a relation of equivalence ∼S, so we can deﬁne a geometry M(M)/ ∼S, which is called the geometry generated by group S, also denoted by M(M)/S. Let us deﬁne three important subgeometries of intrinsic geometry in the following three sections, which are kernal geometry, Riemannian geometry and universal geometry. Discussion 2.3.1. Intrinsic transformation group. (1) Locally, on the neighborhood of any point p on manifold M, the slack-tights bA M or cM A of a reference-system constitute a D-order invertible square matrix. The intrinsic geometry REFp(U)/ ∼= is isomorphic to general linear group GL(D, R). (2) On manifold M, an intrinsic transformation sends an intrinsic geometrical manifold to another intrinsic geometrical manifold. The intrinsic geometry REFM/ ∼= is isomorphic to general linear group GL(M) ≜` p∈M GL(D, R)p, where ` is disjoint union. 12 Zhao-Hui Man Suppose S is a subgroup of GL(M). The group structure of S may be complicated and its description may be cumbersome, it is because transformation groups at various points are diﬀerent from each other in general. According to the original form of Erlangen program, geometry is dependent on group, that is to say, if group structure does not described clearly, geometry could not be established. This is an important reason why in history Riemannian geometry was not incorporated into the framework of Erlangen program. It indicates that if not adopting the concept of geometry of section 1 , we are not able to unify the viewpoints of Riemannian geometry and Erlangen program. Therefore, in order to conveniently study geometry of manifold, it is not necessary to specify detail informations of structure of transformation group, but we can take constraints for slack-tights, and thereby construct a relation of equivalence about reference-systems, so that we can study geometry by the concepts of section 1 , just like what we do in Deﬁnition 2.3.2 , Deﬁnition 2.4.2 , Deﬁnition 2.5.2 and section 2.6 . 2.4 Kernal geometry Deﬁnition 2.4.1. Let k be a reference-system on manifold M, and its slack-tights BA M are constants. We say L[k] and R[k] induced by k are ﬂat transformations of reference-systems. If det[BA M] = 1 is satisﬁed as well, we say L[k] and R[k] are unimodular ﬂat transformations of reference-systems, or global gauge transformations. Deﬁnition 2.4.2. Let there be intrinsic geometrical manifolds (M, [f]) and (M, [g]). Deﬁne rela- tion of equivalence ≃, such that [f] ≃[g] and (M, [f]) ≃(M, [g]) if and only if there exists a ﬂat transformation F[k] such that F[k]([f]) = [g], where F[k] represents L[k] or R[k]. The corresponding equivalence classes are denoted by \|f\| and (M, \|f\|) respectively, and \|f\| is called the kernal of f. The geometry C(M)/ ≃is called the kernal geometry on geometrical manifolds, where the geometrical object (M, \|f\|) is called a kernal geometrical manifold. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 13 Specially, if F[k] is a unimodular ﬂat transformation, C(M)/ ≃is called a regular kernal geometry. Remark 2.4.1. Kernal geometry is a subgeometry of the intrinsic geometry of Deﬁnition 2.3.2 . It can be understood intuitively as follows. Consider Fig.1 of introduction section. Fix axis and scale, and rotate the entire curve L by an angle. The intrinsic geometrical curve S′ now is diﬀerent from the intrinsic geometrical curve S before, but the major bending characteristics remain unchanged under the rotation. These bending characteristics are described by various regular kernal geometrical properties of [S]. 2.5 Riemannian geometry Deﬁnition 2.5.1. Let k be a reference-system on manifold, and its slack-tights BA M satisfy ∆ABBA MBB N = EMN. Then L[k] and R[k] induced by k are called orthogonal transformations of reference-systems. Deﬁnition 2.5.2. Let there be intrinsic geometrical manifolds (M, [f]) and (M, [g]), and their slack-tights be (Bf)A M and (Bg)A M. Deﬁne relation of equivalence ≃O, such that [f] ≃O [g] and (M, [f]) ≃O (M, [g]) if and only if there exists an orthogonal transformation F[k] such that F[k]([f]) = [g], where F[k] represents L[k] or R[k]. The equivalence classes are denoted by [f]O and (M, [f]O) respectively. We say the geometry C(M)/ ≃O is Riemannian geometry, and a geometrical object (M, [f]O) is a Riemannian manifold. Proposition 2.5.1. [f] ≃O [g] if and only if (Gf)MN = (Gg)MN. P f W d t id th f F R d F L ti l Proposition 2.5.1. [f] ≃O [g] if and only if (Gf)MN = (Gg)MN. Proposition 2.5.1. [f] ≃O [g] if and only if (Gf)MN = (Gg)MN. Proof. We need to consider the cases of F[k] = R[k] and F[k] = L[k], respectively. Proof. We need to consider the cases of F[k] = R[k] and F[k] = L[k], respectively. (1) Suppose R[k] is a transformation induced by reference-system k, such that R[k]([f]) = [g]. Let the slack-tights of [f] be (Bf)A M and (Cf)M A , and the slack-tights of [k] be (Bk)A′ A and (Ck)A A′. Then the slack-tights of [g] are (Bg)A′ M = (Bk)A′ A (Bf)A M and (Cg)M A′ = (Ck)A A′(Cf)M A . (1) Suppose R[k] is a transformation induced by reference-system k, such that R[k]([f]) = [g]. Let the slack-tights of [f] be (Bf)A M and (Cf)M A , and the slack-tights of [k] be (Bk)A′ A and (Ck)A A′. Then the slack-tights of [g] are (Bg)A′ M = (Bk)A′ A (Bf)A M and (Cg)M A′ = (Ck)A A′(Cf)M A . 2.5 Riemannian geometry We notice that: (1) Deﬁnition 2.5.2 tells us that Riemannian geometry is a subgeometry of intrinsic geometry of Deﬁnition 2.3.2 . (1) Deﬁnition 2.5.2 tells us that Riemannian geometry is a subgeometry of intrinsic geometry of Deﬁnition 2.3.2 . (2) Proposition 2.5.1 indicates that Deﬁnition 2.5.2 is consistent with the traditional deﬁnition of Riemannian manifold. (2) Proposition 2.5.1 indicates that Deﬁnition 2.5.2 is consistent with the traditional deﬁnition of Riemannian manifold. Hence, the intrinsic geometry of Deﬁnition 2.3.2 is larger than Riemannian geometry, and geometrical manifold is a more fundamental concept than Riemannian manifold. According to the viewpoint of Riemannian geometry, the ultimate origin of its geometrical property is metric. According to the viewpoint of geometrical manifold, the geometrical property has more basic origin, which ultimately boils down to reference-system and its slack-tights BA M or CM A . Hence, the intrinsic geometry of Deﬁnition 2.3.2 is larger than Riemannian geometry, and geometrical manifold is a more fundamental concept than Riemannian manifold. According to the viewpoint of Riemannian geometry, the ultimate origin of its geometrical property is metric. According to the viewpoint of geometrical manifold, the geometrical property has more basic origin, which ultimately boils down to reference-system and its slack-tights BA M or CM A . (1) In history, the slack-tights is called a semimetric in traditional theory of Riemannian geometry. Physicists noticed long ago [14,53] that when researching interactions between gravi- tational ﬁeld and elementary particles, especially problems about spinor ﬁeld, it can be described only by adopting semimetric representation, and it does not work by using metric representa- tion. However, they did not realize that it means the connotation of traditional intrinsic geometry needs to be generalized. (2) On one hand, it can be seen from Deﬁnition 2.2.3 that the slack-tights on geometrical manifold determines the metric on Riemannian manifold. On the other hand, even when the coeﬃcients of metric tensors of two geometrical manifolds are completely the same, their slack-tights are not necessarily the same. These two aspects indicate that the theory of intrinsic geometry on geometrical manifold has richer geometrical properties than the traditional theory of intrinsic geometry on Riemannian manifold. (3) It is important that there exists a concept of simple connection on geometrical manifold, see section 2.7 . If simple connection vanishes, Levi-Civita connection must vanish too. But if Levi-Civita connection vanishes, the simple connection does not necessarily vanish. 2.5 Riemannian geometry Therefore, simple connection reﬂects more intrinsic properties of manifold than Levi-Civita connection. These properties just exactly can be used to describe the characteristics of gauge ﬁelds. 2.5 Riemannian geometry According to Deﬁnition 2.2.3 , the metric tensor of [f] is (Gf)MN = ∆AB(Bf)A M(Bf)B N, thereby the metric tensorof [g] is (Gg)MN = ∆A′B′(Bg)A′ M(Bg)B′ N = ∆A′B′ (Bk)A′ A (Bf)A M (Bk)B′ B (Bf)B N = According to Deﬁnition 2.2.3 , the metric tensor of [f] is (Gf)MN = ∆AB(Bf)A M(Bf)B N, thereby the metric tensorof [g] is (Gg)MN = ∆A′B′(Bg)A′ M(Bg)B′ N = ∆A′B′ (Bk)A′ A (Bf)A M (Bk)B′ B (Bf)B N = ∆A′B′(Bk)A′ A (Bk)B′ B (Bf)A M(Bf)B N.Hence, (Gf)MN = (Gg)MN ifandonlyif ∆A′B′(Bk)A′ A (Bk)B′ B = ∆AB, i.e. R[k] is orthogonal. (2) Suppose L[k] is a transformation induced by reference-system k, such that L[k]([f]) = [g]. Let the slack-tights of [f] be (Bf)A M and (Cf)M A , and the slack-tights of [k] be (Bk)M M′ and (Ck)M′ M . Then the slack-tights of [g] are (Bg)A M′ = (Bk)M M′(Bf)A M and (Cg)M′ A = (Ck)M′ M (Cf)M A . (2) Suppose L[k] is a transformation induced by reference-system k, such that L[k]([f]) = [g]. Let the slack-tights of [f] be (Bf)A M and (Cf)M A , and the slack-tights of [k] be (Bk)M M′ and (Ck)M′ M . Then the slack-tights of [g] are (Bg)A M′ = (Bk)M M′(Bf)A M and (Cg)M′ A = (Ck)M′ M (Cf)M A . According to Deﬁnition 2.2.4 , we consider tensors (Hf)AB and (Hg)AB.(Hf)AB = (Yf)AB = EMN(Bf)A M(Bf)B N, (Hg)AB = EM′N′(Bg)A M′(Bg)B N′ = EM′N′ (Bk)M M′(Bf)A M (Bk)N N′(Bf)B N = EM′N′(Bk)M M′(Bk)N N′ (Bf)A M(Bf)B N.Hence, (Hf)AB = (Hg)AB ifandonlyif EM′N′(Bk)M M′(Bk)N N′ = EMN, i.e. L[k] is orthogonal. According to Deﬁnition 2.2.4 , we consider tensors (Hf)AB and (Hg)AB.(Hf)AB = (Yf)AB = EMN(Bf)A M(Bf)B N, (Hg)AB = EM′N′(Bg)A M′(Bg)B N′ = EM′N′ (Bk)M M′(Bf)A M (Bk)N N′(Bf)B N = EM′N′(Bk)M M′(Bk)N N′ (Bf)A M(Bf)B N.Hence, (Hf)AB = (Hg)AB ifandonlyif EM′N′(Bk)M M′(Bk)N N′ = EMN, i.e. L[k] is orthogonal. [ ] Then due to (Hf)AB = (Hg)AB ⇔(Gf)MN = (Gg)MN ⇔(Gf)MN = (Gg)MN, the proposition is true. ⊓⊔ [ ] Then due to (Hf)AB = (Hg)AB ⇔(Gf)MN = (Gg)MN ⇔(Gf)MN = (Gg)MN, the proposition is true. ⊓⊔ ⊓⊔ Zhao-Hui Man 14 Discussion 2.5.1. 2.6 Universal geometry Discussion 2.6.1. Let there be geometrical manifolds (M, f) and (M, g). Deﬁne relation of equivalence ∼, such that (M, f) ∼(M, g) if and only if there exists F[k] such that F[k]([f]) = [g], where F[k] represents L[k] or R[k]. In fact such a transformation always exists, which is L[g◦f−1] or R[f−1◦g]. Therefore, M(M) becomes the only equivalence class in the geometry ˜ M(M) ≜M(M)/ ∼. It makes ˜ M(M) the universal geometry on geometrical manifolds. Discussion 2.6.2. Make a summary for these subgeometries of intrinsic geometry. On geomet- rical manifold: Discussion 2.6.2. Make a summary for these subgeometries of intrinsic geometry. On geomet- rical manifold: (1) An intrinsic geometrical property is an invariant under identical intrinsic transformation of reference-systems. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 15 (2) A kernal geometrical property is an invariant under ﬂat transformation of reference- systems. (2) A kernal geometrical property is an invariant under ﬂat transformation of reference- systems. (2) A kernal geometrical property is an invariant under ﬂat transformation of reference- systems. (3) A Riemannian geometrical property is an invariant under orthogonal transformation of reference-systems. (3) A Riemannian geometrical property is an invariant under orthogonal transformation of reference-systems. (4) A universal geometrical property is an invariant under arbitrary transformation of reference-systems. (4) A universal geometrical property is an invariant under arbitrary transformation of reference-systems. (5) Let e be the unit element of GL(M). According to Remark 1.1, {e} as the transformation group of intrinsic geometry is the smallest transformation group, and GL(M) as the transfor- mation group of universal geometry is the largest transformation group. In other words, on geometrical manifold, intrinsic geometry is the largest geometry, and universal geometry is the smallest geometry. (5) Let e be the unit element of GL(M). According to Remark 1.1, {e} as the transformation group of intrinsic geometry is the smallest transformation group, and GL(M) as the transfor- mation group of universal geometry is the largest transformation group. In other words, on geometrical manifold, intrinsic geometry is the largest geometry, and universal geometry is the smallest geometry. 2.7 Simple connection Discussion 2.7.1. Let Γ M NP be smooth real functions on manifold M. ∀p ∈M, dxM and ∂ ∂xM are natural basis vector ﬁelds in coordinate frame (U, xM) of local reference-system f(p). Consider the restriction of smooth real functions Γ M NP on U, aﬃne connection can be expressed as: D ∂ ∂xN ≜Γ M NP dxP ⊗ ∂ ∂xM , DdxN ≜−Γ N MP dxP ⊗dxM. (1) (1) In order to enable aﬃne connection to describe intrinsic geometry, Γ M NP should be deﬁned as the ones dependent on slack-tights BA M or CM A , such as Levi-Civita connection In order to enable aﬃne connection to describe intrinsic geometry, Γ M NP should be deﬁned as the ones dependent on slack-tights BA M or CM A , such as Levi-Civita connection Γ M NP ≜1 2GMQ ∂GNQ ∂xP + ∂GPQ ∂xN −∂GNP ∂xQ , or other forms. or other forms. According to Deﬁnition 2.7.1 , the original simple connection and the one applied transformation Ltp are respectively According to Deﬁnition 2.7.1 , the original simple connection and the one applied transformation Ltp are respectively (Γf)M NP ≜1 2CM A ∂BA N ∂xP + ∂BA P ∂xN ! , (Γf)M′ N′P ′ ≜1 2CM′ A ∂BA N′ ∂xP ′ + ∂BA P ′ ∂xN′ ! . Calculate the local transformation relation of the simple connection on U: (Γf)M′ N′P ′ ≜1 2CM′ A ∂BA N′ ∂xP ′ + ∂BA P ′ ∂xN′ ! = 1 2cM′ M CM A ∂ bN N′BA N ∂xP ′ + ∂ bP P ′BA P ∂xN′ ! = 1 2cM′ M CM A ∂bN N′ ∂xP ′ BA N + bN N′ ∂BA N ∂xP ′ + ∂bP P ′ ∂xN′ BA P + bP P ′ ∂BA P ∂xN′ ! = 1 2cM′ M CM A bN N′ ∂BA N ∂xP ′ + bP P ′ ∂BA P ∂xN′ ! + 1 2cM′ M CM A ∂bN N′ ∂xP ′ BA N + ∂bP P ′ ∂xN′ BA P ! = 1 2cM′ M CM A ∂BA N ∂xP + ∂BA P ∂xN ! bN N′bP P ′ + 1 2cM′ M ∂bM N′ ∂xP ′ + ∂bM P ′ ∂xN′ ! = (Γf)M NP cM′ M bN N′bP P ′ + cM′ M ∂bM N′ ∂xP ′ . This result is consistent with the general relation of local tansformation of aﬃne connection. So it has been proved that the simple connection Γ M NP ≜1 2CM A ∂BA N ∂xP + ∂BA P ∂xN is indeed an aﬃne connection, and evidently it is torsion-free. ⊓⊔ ⊓⊔ Proposition 2.7.2. Suppose there are reference-systems g and k on manifold M. Let the slack- tights of g be BA M and CM A , the slack-tights of k be BM M′ and CM′ M , and the slack-tights of g′ ≜Lk(g) be BA M′ = BA MBM M′ and CM′ A = CM A CM′ M . Then let the simple connections of (M, g), (M, k) and (M, g′) be (Γg)M NP, (Γk)M′ N′P ′ and (Γg′)M′ N′P ′, respectively. Thus, Proposition 2.7.2. Suppose there are reference-systems g and k on manifold M. or other forms. Levi-Civita connection is the unique torsion-free and metric-compatible connection, but it is not fundamental enough. On one hand it is because Levi-Civita connection cannot describe the intrinsic properties determined by BA M and CM A when GMN are all constants, on the other hand Levi-Civita connection is not simple enough. The torsion-free condition is very helpful to simplify theoretical form, but the metric- compatible condition restricts the further simpliﬁcation of connection form. We consider that the metric-compatible condition DG = 0 was introduced to establish the intuition of Levi-Civita parallel displacement, but it is not the condition that more general concept of parallel displace- ment must rely on. Therefore, in order to simplify connection furthermore, it can be imagined that the torsion-free condition remains and the metric-compatible condition is given up. A nice choice is to adopt the following deﬁnition. Deﬁnition 2.7.1. Let there be an aﬃne connection D, which is expressed as equation (1) on performance coordinate frame (U, xM). If the connection coeﬃcients are deﬁned as Γ M NP ≜1 2CM A ∂BA N ∂xP + ∂BA P ∂xN ! = 1 2 BA N ∂CM A ∂xP + BA P ∂CM A ∂xN ! , (2) (2) D is called a simple connection. 16 Zhao-Hui Man Proposition 2.7.1. The simple connection is indeed an aﬃne connection. Proposition 2.7.1. The simple connection is indeed an aﬃne connection. Proof. Let there be a reference-system f on manifold M. And let there be a local reference- system tp : xM′ = xM′(xM), which induces a reference-system transformation Ltp sending f(p) : xM = xM(ξA) to h(p) ≜tp ◦f(p) : xM′ = xM′(ξA), where (U, ξA) is the common basis coordinate frame of f(p) and h(p). They can be expressed as a diagram: (U, ξA) (U, xM′) (U, xM) f(p) h(p) tp (U, ξA) (U, xM′) (U, xM) f(p) h(p) tp (U, ξA) (U, xM′) (U, xM) f(p) h(p) tp Let the slack-tights of tp be bM M′ ≜∂xM ∂xM′ , cM′ M ≜∂xM′ ∂xM . M Let the slack-tights of f be BA M and CM A . For the restriction of them on U, the slack-tights applied transformation Ltp are Let the slack-tights of f be BA M and CM A . For the restriction of them on U, the slack-tights applied transformation Ltp are BA M′ = bM M′BA M, CM′ A = cM′ M CM A . 2.8 Summary of this section emannian manifold is generalized to geometrical manifold. 1. Riemannian manifold is generalized to geometrical manifold. 1. Riemannian manifold is generalized to geometrical manifold. 2. Intrinsic geometry is generalized, so that kernal geometry, Riemannian geometry and universal geometry are all subgeometries of intrinsic geometry. 2. Intrinsic geometry is generalized, so that kernal geometry, Riemannian geometry and universal geometry are all subgeometries of intrinsic geometry. 3. The important concept of simple connection is deﬁned, which is just exactly the key to describe elementary particles and gauge ﬁelds from the perspective of intrinsic geometry. 3. The important concept of simple connection is deﬁned, which is just exactly the key to describe elementary particles and gauge ﬁelds from the perspective of intrinsic geometry. By applying the generalized intrinsic geometry, we can obtain an eﬀective constructivity method for Hilbert’s 6th problem, which may be used to unify elementary frameworks of theoretical physics. ΓMNP + ΓNPM + ΓPMN = ΛMNP + ΛNPM + ΛPMN. ΓMNP + ΓNPM + ΓPMN = ΛMNP + ΛNPM + ΛPMN. (2) It is evident that when GMN are all constants, Levi-Civita connection must be zero, and the corresponding Riemannian curvature tensor also must be zero. Meanwhile, simple connection is however not necessarily zero, and the corresponding Riemannian curvature tensor is also not necessarily zero. It indicates that simple connection reﬂects more intrinsic bending-properties of manifold than Levi-Civita connection. or other forms. Let the slack- tights of g be BA M and CM A , the slack-tights of k be BM M′ and CM′ M , and the slack-tights of g′ ≜Lk(g) be BA M′ = BA MBM M′ and CM′ A = CM A CM′ M . Then let the simple connections of (M, g), (M, k) and (M, g′) be (Γg)M NP, (Γk)M′ N′P ′ and (Γg′)M′ N′P ′, respectively. Thus, (Γg′)M′ N′P ′ = (Γg)M NP CM′ M BN N′BP P ′ + (Γk)M′ N′P ′. (3) (3) A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 17 Proof. Calculate the following smooth functions of M on an arbitrary coordinate neighb (Γg′)M′ N′P ′ ≜1 2CM′ A ∂BA N′ ∂xP ′ + ∂BA P ′ ∂xN′ ! = 1 2CM′ M CM A ∂ BN N′BA N ∂xP ′ + ∂ BP P ′BA P ∂xN′ ! = 1 2CM′ M CM A ∂BN N′ ∂xP ′ BA N + BN N′ ∂BA N ∂xP ′ + ∂BP P ′ ∂xN′ BA P + BP P ′ ∂BA P ∂xN′ ! = 1 2CM′ M CM A BN N′ ∂BA N ∂xP ′ + BP P ′ ∂BA P ∂xN′ ! + 1 2CM′ M CM A ∂BN N′ ∂xP ′ BA N + ∂BP P ′ ∂xN′ BA P ! = 1 2CM′ M CM A ∂BA N ∂xP + ∂BA P ∂xN ! BN N′BP P ′ + 1 2CM′ M ∂BM N′ ∂xP ′ + ∂BM P ′ ∂xN′ ! = (Γg)M NP CM′ M BN N′BP P ′ + (Γk)M′ N′P ′. = (Γg)M NP CM′ M BN N′BP P ′ + (Γk)M′ N′P ′. ⊓⊔ Remark 2.7.1. Simple connection proposed by this paper has two evident properties. (1) Denote ΓMNP ≜GMM′Γ M′ NP, ΛMNP ≜1 2 ∂GNM ∂xP + ∂GP M ∂xN −∂GNP ∂xM then it is easy to verify Remark 2.7.1. Simple connection proposed by this paper has two evident properties. Remark 2.7.1. Simple connection proposed by this paper has two evident properties. (1) Denote ΓMNP ≜GMM′Γ M′ NP, ΛMNP ≜1 2 ∂GNM ∂xP + ∂GP M ∂xN −∂GNP ∂xM then it is easy to verify Remark 2.7.1. Simple connection proposed by this paper has two evident properties. (1) Denote ΓMNP ≜GMM′Γ M′ ΛMNP ≜1 ∂GNM + ∂GP M −∂GNP then it is easy to verify (1) Denote ΓMNP ≜GMM′Γ M′ NP, ΛMNP ≜1 2 ∂GNM ∂xP + ∂GP M ∂xN −∂GNP ∂xM then it is easy t ΓMNP = 1 2δABBB M ∂BA N ∂xP + ∂BA P ∂xN ! , ΓMNP + ΓNPM + ΓPMN = ΛMNP + ΛNPM + ΛPMN. 3 Intrinsic geometrical solution for Hilbert’s 6th problem 3.1 Axiom for Hilbert’s 6th problem We have to abstract and separate out physical connotations, so as to focus on mathematical constructions. A convenient way is to adopt the following axiom. There is only one axiom, and we do not need any more. 18 Zhao-Hui Man The fundamental axiom. Physical reality should be cognized by using the concept of reference-system on geometrical manifold. Such a concept of reference-system is strictly deﬁned in Deﬁnition 2.1.2 . This axiom has an evident corollary as below, which can be called the principle of universal relativity. Corollary. Universal physical property should be cognized by using universal geometrical property of geometrical manifold. Such a concept of universal geometrical property is strictly deﬁned in Deﬁnition 1.2 and section 2.6 . In the following sections, universal physical properties, such as time metric, space metric, evolution, etc., will be strictly deﬁned as some universal geometrical properties of geometrical manifold. Other main conclusions, postulates and equations of fundamental physics can also be strictly deﬁned, constructed and proved in pure mathematical sense. 3.2 Mathematical treatment of time and space Deﬁnition 3.2.1. On a neighborhood U of point p, according to Deﬁnition 2.2.3 , there are dξ0 and dx0 deﬁned on two coordinate frames (U, ξA) and (U, xM) of f(p) as    (dξ0)2 ≜δABdξAdξB = gMNdxMdxN, (dx0)2 ≜εMNdxMdxN = hABdξAdξB. (4) (4) peak of dξ0 and dx0 as total space metrics of (U, ξA) and (U, xM), or as time metrics. We speak of dξ0 and dx0 as total space metrics of (U, ξA) and (U, xM), or as time metrics. On geometrical manifold (M, f), dξ0 and dx0 deﬁned as On geometrical manifold (M, f), dξ0 and dx0 deﬁned as    (dξ0)2 ≜∆ABdξAdξB = GMNdxMdxN (dx0)2 ≜EMNdxMdxN = HABdξAdξB (5) (5) (5) are called total space metrics or time metrics of M. Remark 3.2.1. Such a new treatment about the concept of time in this paper is very important. It will make the two evolution notions of gravitational theory and quantum mechanics become uni- ﬁed and coordinated. In this way, time metric reﬂects the total evolution in the total-dimensional space, while a speciﬁc spatial metric reﬂects a partial evolution in a speciﬁc direction. Deﬁnition 3.2.2. Let P and N be closed submanifolds of manifold M = P×N. Denote r ≜dim P. Let s, i = 1, · · · , r and a, m = r +1, · · · , D. Select some proper coordinate frames {ξA} and {xM} such that on P there are coordinate frames {ξs} and {xi} inherited from M, and on N there are coordinate frames {ξa} and {xm} inherited from M. Correspondingly, two subspace metrics can be deﬁned on the coordinate neighborhoods on P and N respectively:            (dξ(P))2 ≜ r X s=1 (dξs)2 = δstdξsdξt (dx(P))2 ≜ r X i=1 (dxi)2 = εijdxidxj ,              (dξ(N))2 ≜ D X a=r+1 (dξa)2 = δabdξadξb (dx(N))2 ≜ D X m=r+1 (dxm)2 = εmndxmdxn . X m=r+1 For convenience, N is called a submanifold of internal space and P is called a submanifold of external space. dξ(N) and dx(N) are called propertime metrics. 19 A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 3.3.1 Deﬁnition and coordinate form of evolution Deﬁnition 3.3.1.1. Let there be two reference-systems f and g on manifold M, if ∀p ∈M, f(p) ≜φU ◦ψ−1 U and g(p) ≜φU ◦ρ−1 U have the same performance coordinate frame φU, namely it can be intuitively expressed as a diagram ψU(U) f(p) −−→φU(U) g(p) ←−−ρU(U), we say f and g motion relatively and interact mutually, and also say that f evolves in g, or f evolves on geometrical manifold (M, g). Meanwhile, g evolves in f, or say g evolves on (M, f). Deﬁnition 3.3.1.2. Let there be a one-parameter group of diﬀeomorphisms φX : M × R →M acting on M. φX determines a smooth tangent vector ﬁeld X on M. If X is nonzero everywhere, we say φX is a set of evolution paths on M, and X is an evolution direction ﬁeld on M. Let T ⊆R be an interval. Suppose a smooth map Lp : T →M constitutes a regular submanifold of M, and there exists a smooth tangent vector ﬁeld X such that Lp is on the orbit φX,p(t) of φX through p. Now the map Lp is called an evolution path through p, or path for short. The tangent vector d dt ≜[Lp] = X(p) is called an evolution direction at p. If it does not need to emphasize the point p, Lp can be denoted by L concisely. Deﬁnition 3.3.1.3. ∀p ∈M, let the coordinate representations of f(p) ≜φU ◦ψ−1 U be Deﬁnition 3.3.1.3. ∀p ∈M, let the coordinate representations of f(p) ≜φU ◦ψ−1 U be xM = xM(ξA), ξA = ξA(xM), xM = xM(ξA), ξA = ξA(xM), ions of g(p) ≜φU ◦ρ−1 U be xM = xM(ζA), ζA = ζA(xM). xM = xM(ξA), ξA = ξA(xM), nd the coordinate representations of g(p) ≜φU ◦ρ−1 U be xM = xM(ζA), ζA = ζA(xM). Then let time metrics on (U, φU), (U, ψU), (U, ρU) be dx0, dξ0, dζ0, respectively Then let time metrics on (U, φU), (U, ψU), (U, ρU) be dx0, dξ0, dζ0, respectively. 3.3.2 Evolution lemma Deﬁnition 3.3.2.1. Let L be a path on manifold M, ∀p ∈L. Suppose Tp(M) and Tp(L) are the tangent spaces at p on M and L respectively, and T ∗ p (M) and T ∗ p (L) are the cotangent spaces. ∀p ∈L, the regular imbedding π : L →M, q 7→q induces tangent map and cotangent map    π∗\|p : Tp(L) →Tp(M), [γL] 7→[π ◦γL], π∗\|p : T ∗ p (M) →T ∗ p (L), df 7→d(f ◦π). (8) (8) Evidently, restricting on L, the tangent map is an injection, and the cotangent map is a surjection. ∀d dtL ∈Tp(L), ∀df ∈T ∗ p (M), denote Evidently, restricting on L, the tangent map is an injection, and the cotangent map is a surjection. ∀d dtL ∈Tp(L), ∀df ∈T ∗ p (M), denote d dt ≜π∗\|p d dtL ∈Tp(M), dfL ≜π∗\|p (df) ∈T ∗ p (L). (9) (9) We say d dt and d dtL are equivalent, df and dfL are homomorphic. They are denoted by d dt ∼= d dtL , df ≃dfL. (10) (10) The above locally deﬁned concepts can also be applied to the entire manifold, and furthermore, they can be transplanted without hindrance to any-order tensor product space generated by tangent bundle and cotangent bundle, such as: The above locally deﬁned concepts can also be applied to the entire manifold, and furthermore, they can be transplanted without hindrance to any-order tensor product space generated by tangent bundle and cotangent bundle, such as: (1) If d dt ∼= d dtL, then ∀df we say df⊗d dt and df⊗d dtL are equivalent, denoted by df⊗d dt ∼= df⊗d dtL. (2) If df ≃dfL, then ∀d dt we say df ⊗d dt and dfL ⊗d dt are homomorphic, denoted by df ⊗d dt ≃dfL ⊗d dt. (1) If d dt ∼= d dtL, then ∀df we say df⊗d dt and df⊗d dtL are equivalent, denoted by df⊗d dt ∼= df⊗d dtL. (2) If df ≃dfL, then ∀d dt we say df ⊗d dt and dfL ⊗d dt are homomorphic, denoted by df ⊗d dt ≃dfL ⊗d dt. Proposition 3.3.2.1. If d dt ∼= d dtL and df ≃dfL, then D d dt, df E = D d dtL , dfL E . d d Proof. Then we obtain the coordinate form of evolution of f: Then we obtain the coordinate form of evolution of f:    ξA = ξA(xM) = ξA(x0) ξ0 = ξ0(x0) ,    xM = xM(ξA) = xM(ξ0) x0 = x0(ξ0) . (7) (7) 3.3.2 Evolution lemma 3.3.1 Deﬁnition and coordinate form of evolution Each path Lp is a 1-dimensional regular submanifold of M, therefore on open set UL ≜U ∩Lp there exist coordinate neighborhoods (UL, φUL) ,(UL, ψUL) and (UL, ρUL) such that the regular imbedding π : Lp →M, q 7→q (6) π : Lp →M, q 7→q π : Lp →M, q 7→q (6) induces coordinate maps and parameter equations induces coordinate maps and parameter equations          ψU ◦π ◦ψ−1 UL : R →RD, (ξ0) 7→(ξA), ξA = ξA(ξ0) φU ◦π ◦φ−1 UL : R →RD, (x0) 7→(xM), xM = xM(x0) ρU ◦π ◦ρ−1 UL : R →RD, (ζ0) 7→(ζA), ζA = ζA(ζ0) which satisfy D P A=1 dξA dξ0 2 = 1, D P M=1 dxM dx0 2 = 1, D P A=1 dζA dζ0 2 = 1.          ψU ◦π ◦ψ−1 UL : R →RD, (ξ0) 7→(ξA), ξA = ξA(ξ0) φU ◦π ◦φ−1 UL : R →RD, (x0) 7→(xM), xM = xM(x0) ρU ◦π ◦ρ−1 UL : R →RD, (ζ0) 7→(ζA), ζA = ζA(ζ0) dξA dξ0 2 = 1, D P dxM dx0 2 = 1, D P dζA dζ0 2 = 1.          ψU ◦π ◦ψ−1 UL : R →RD, (ξ0) 7→(ξA), ξA = ξA(ξ0) φU ◦π ◦φ−1 UL : R →RD, (x0) 7→(xM), xM = xM(x0) ρU ◦π ◦ρ−1 UL : R →RD, (ζ0) 7→(ζA), ζA = ζA(ζ0) which satisfy D P A=1 dξA dξ0 2 = 1, D P M=1 dxM dx0 2 = 1, D P A=1 dζA dζ0 2 = 1. 20 Zhao-Hui Man Deﬁnition 3.3.1.4. f(p) = φU ◦ψ−1 U and f−1(p) = ψU ◦φ−1 U on U induce reference-systems on Deﬁnition 3.3.1.4. f(p) = φU ◦ψ−1 U and f−1(p) = ψU ◦φ−1 U on U induce reference-systems on UL: UL:    fL(p) ≜φUL ◦ψ−1 UL, x0 = x0(ξ0) f−1 L (p) ≜ψUL ◦φ−1 UL, ξ0 = ξ0(x0) .   f ( ) φ ψUL (ξ ) f−1 L (p) ≜ψUL ◦φ−1 UL, ξ0 = ξ0(x0) .  f−1 L (p) ≜ψUL ◦φ−1 UL, ξ0 = ξ0(x0) .  hen we obtain the coordinate form of evolution of f: 3.3.2 Evolution lemma The tangent vectors d dt and d dtL are respectively deﬁned as equivalence classes [γ] and [γL] of parameter curves, the cotengent vectors df and dfL are respectively deﬁned as equivalence classes [f] and [fL] of smooth functions, which satisfy γ = π ◦γL, fL = f ◦π. Hence, d dt, df = d dtL , dfL ⇔⟨[γ], [f]⟩= ⟨[γL], [fL]⟩⇔d(f ◦γ) dt = d(fL ◦γL) dt , where f ◦γ = f ◦(π ◦γL), fL ◦γL = (f ◦π) ◦γL. f ◦γ = f ◦(π ◦γL), fL ◦γL = (f ◦π) ◦γL. f ◦γ = f ◦(π ◦γL), fL ◦γL = (f ◦π) ◦γL. Evidently, f ◦γ = fL ◦γL, which makes D d dt, df E = D d dtL , dfL E true. Evidently, f ◦γ = fL ◦γL, which makes D d dt, df E = D d dtL , dfL E true. ⊓⊔ A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 21 Deﬁnition 3.3.2.2. ∀p ∈L, on coordinate neighborhood UL of point p, deﬁne bA 0 ≜dξA dx0 , b0 0 ≜dξ0 dx0 , cM 0 ≜dxM dξ0 , c0 0 ≜dx0 dξ0 , εM 0 ≜dxM dx0 = b0 0cM 0 = bA 0 cM A , δA 0 ≜dξA dξ0 = c0 0bA 0 = cM 0 bA M. They determine the following smooth functions on the entire L:    BA 0 : L →R, p 7→BA 0 (p) ≜(bf(p))A 0 (p) CM 0 : L →R, p 7→CM 0 (p) ≜(cf(p))M 0 (p) ,    B0 0 : L →R, p 7→B0 0(p) ≜(bf(p))0 0(p) C0 0 : L →R, p 7→C0 0(p) ≜(cf(p))0 0(p) . For convenience, if no confusion, still using notations ε and δ, we also have smooth functions: For convenience, if no confusion, still using notations ε and δ, we also have smooth functions: εM 0 ≜B0 0CM 0 = BA 0 CM A , δA 0 ≜C0 0BA 0 = CM 0 BA M. Deﬁne dξ0 ≜dx0 dξ0 dx0 and dx0 ≜dξ0 dx0 dξ0, which induce d dξ0 and d dx0, such that D d dξ0 , dξ0 E = 1, D d dx0 , dx0 E = 1. 3.3.2 Evolution lemma On UL we also deﬁne Deﬁne dξ0 ≜dx0 dξ0 dx0 and dx0 ≜dξ0 dx0 dξ0, which induce d dξ0 and d dx0, such that D d dξ0 , dξ0 E = 1, D d dx0 , dx0 E = 1. On UL we also deﬁne ¯b0 A ≜dξA dx0 , ¯b0 0 ≜dξ0 dx0 , ¯c0 M ≜dxM dξ0 , ¯c0 0 ≜dx0 dξ0 , ¯ε0 M ≜dxM dx0 = ¯b0 0¯c0 M = ¯b0 A¯cA M, ¯δ0 A ≜dξA d¯ξ0 = ¯c0 0¯b0 A = ¯c0 M¯bM A . ¯b0 A ≜dξA dx0 , ¯b0 0 ≜dξ0 dx0 , ¯c0 M ≜dxM dξ0 , ¯c0 0 ≜dx0 dξ0 , ¯ε0 M ≜dxM dx0 = ¯b0 0¯c0 M = ¯b0 A¯cA M, ¯δ0 A ≜dξA d¯ξ0 = ¯c0 0¯b0 A = ¯c0 M¯bM A . They determine the following smooth functions on the entire L:    ¯B0 A : L →R, p 7→¯B0 A(p) ≜(¯bf(p))0 A(p) ¯C0 M : L →R, p 7→¯C0 M(p) ≜(¯cf(p))0 M(p) ,    ¯B0 0 : L →R, p 7→¯B0 0(p) ≜(¯bf(p))0 0(p) ¯C0 0 : L →R, p 7→¯C0 0(p) ≜(¯cf(p))0 0(p) . ¯ε0 M ≜¯B0 0 ¯C0 M = ¯B0 A ¯CA M, ¯δ0 A ≜¯C0 0 ¯B0 A = ¯C0 M ¯BM A . Proposition 3.3.2.2. (Evolution lemma). Let L be a path on manifold M. Suppose there are tangent vector ﬁelds wM ∂ ∂xM , ¯wM ∂ ∂xM and cotangent vector ﬁelds wMdxM, ¯wMdxM on M, and there are tangent vector ﬁelds w0 d dx0 , ¯w0 d dx0 and cotangent vector ﬁelds ∀w0dx0, ¯w0dx0 on L. Then the following conclusions hold:      wM ∂ ∂xM ∼= w0 d dx0 ⇔wM = w0εM 0 wMdxM ≃w0dx0 ⇔εM 0 wM = w0 ,      ¯wM ∂ ∂xM ∼= ¯w0 d dx0 ⇔¯wM = ¯w0¯ε0 M ¯wMdxM ≃¯w0dx0 ⇔¯ε0 M ¯wM = ¯w0 . Proof. The following locally discussion can also be applied to the entire manifold. 1. Consider the case that basis vectors are dxM and ∂ ∂xM . For tangent vector, Proof. The following locally discussion can also be applied to the entire manifold. Proof. The following locally discussion can also be applied to the entire manifold. 1. Consider the case that basis vectors are dxM and ∂ ∂xM . 1. For tangent vector, π∗ d dx0 = dxM dx0 ∂ ∂xM ⇔dxM dx0 ∂ ∂xM ∼= d dx0 ⇔¯ε0 M ∂ ∂xM ∼= d dx0 ⇔¯w0¯ε0 M ∂ ∂xM ∼= ¯w0 d dx0 Because the tangent map is an injection, then ¯wM ∂ ∂xM ∼= ¯w0 d dx0 ⇔¯wM = ¯w0¯ε0 M. Because the tangent map is an injection, then ¯wM ∂ ∂xM ∼= ¯w0 d dx0 ⇔¯wM = ¯w0¯ε0 M. For cotangent vector, dxM ≃¯ε0 Mdx0 ⇒¯wMdxM ≃¯ε0 M ¯wMdx0, then ¯wMdxM ≃¯w0dx0 ⇔ ¯ε0 ¯M ¯0 ⊓⊔ For cotangent vector, dxM ≃¯ε0 Mdx0 ⇒¯wMdxM ≃¯ε0 M ¯wMdx0, then ¯wMdxM ≃¯w0dx0 ⇔ ¯ε0 M ¯wM = ¯w0. ⊓⊔ Deﬁnition 3.3.3.1. On UL deﬁne Deﬁnition 3.3.3.1. On UL deﬁne g00 ≜dx0 dx0 = b0 0b0 0, g00 ≜dx0 dx0 = c0 0c0 0, h00 ≜dξ0 dξ0 = c0 0c0 0, h00 ≜dξ0 dξ0 = b0 0b0 0. They determine the following smooth functions deﬁned on L: They determine the following smooth functions deﬁned on L: G00 ≜B0 0B0 0, G00 ≜C0 0C0 0, H00 ≜C0 0C0 0, H00 ≜B0 0B0 0. Proposition 3.3.3.1. On L, d dx0 = G00 d dx0 , d dξ0 = H00 d dξ0 . Proposition 3.3.3.1. On L, d dx0 = G00 d dx0 , d dξ0 = H00 d dξ0 . Proof. At any point, tangent vector d dx0 can be expanded as d dx0 = X d dx0 about basis d dx0, and tangent vector d dξ0 can be expanded as d dξ0 = Y d dξ0 about basis d dξ0. p , dx0 dx0 , dξ0 dξ0 Proof. At any point, tangent vector d dx0 can be expanded as d dx0 = X d dx0 about basis d dx0, and tangent vector d dξ0 can be expanded as d dξ0 = Y d dξ0 about basis d dξ0. d dx0 , dx0 = 1 ⇔ X d dx0 , g00dx0 = 1 ⇔Xg00 = 1 ⇔X = 1 g00 = g00 ⇒ d dx0 = g00 d dx0 , d dξ0 , dξ0 = 1 ⇔ Y d dξ0 , h00dξ0 = 1 ⇔Y h00 = 1 ⇔Y = 1 h00 = h00 ⇒ d dξ0 = h00 d dξ0 . This local conclusion can be applied to the entire path, so d dx0 = G00 d dx0 and d dξ0 = H00 d dξ0 hold This local conclusion can be applied to the entire path, so d dx0 = G00 d dx0 and d dξ0 = H00 d dξ0 hold L This local conclusion can be applied to the entire path, so d dx0 = G00 d dx0 and d dξ0 = H00 d dξ0 hold on L. ⊓⊔ Proposition 3.3.3.2. On L, H00 = HABδA 0 δB 0 , G00 = GMNεM 0 εN 0 . Proof. On a neighborhood UL of any point on L, Proposition 3.3.3.2. On L, H00 = HABδA 0 δB 0 , G00 = GMNεM 0 εN 0 . Proof. On a neighborhood UL of any point on L, Proof. 3.3.2 Evolution lemma Consider the case that basis vectors are dxM and ∂ ∂xM . For tangent vector, π∗ d dx0 = dxM dx0 ∂ ∂xM ⇔dxM dx0 ∂ ∂xM ∼= d dx0 ⇔εM 0 ∂ ∂xM ∼= d dx0 ⇔w0εM 0 ∂ ∂xM ∼= w0 d dx0 . Because the tangent map is an injection, then wM ∂ ∂xM ∼= w0 d dx0 ⇔wM = w0εM 0 . ∂x dx For cotangent vector, dxM ≃εM 0 dx0 ⇒wMdxM ≃εM 0 wMdx0, then wMdxM ≃w0dx0 ⇔ εM 0 wM = w0. For cotangent vector, dxM ≃εM 0 dx0 ⇒wMdxM ≃εM 0 wMdx0, then wMdxM ≃w0dx0 ⇔ εM 0 wM = w0. 2. Consider the case that basis vectors are dxM and ∂ ∂xM . 2. Consider the case that basis vectors are dxM and ∂ ∂xM . 22 Zhao-Hui Man For tangent vector, Deﬁnition 3.3.3.1. On UL deﬁne On a neighborhood UL of any point on L,        hABδA 0 δB 0 = εMNcM A cN B δA 0 δB 0 = εMN dxM dξ0 dxN dξ0 = dx0 dξ0 dx0 dξ0 = h00 gMNεM 0 εN 0 = δABbA MbB NεM 0 εN 0 = δAB dξA dx0 dξB dx0 = dξ0 dx0 dξ0 dx0 = g00 . ⊓⊔ So H00 = HABδA 0 δB 0 and G00 = GMNεM 0 εN 0 hold on the entire L. So H00 = HABδA 0 δB 0 and G00 = GMNεM 0 εN 0 hold on the entire L. ⊓⊔ Proposition 3.3.3.3. The following left-hand formulas are true on L. When D > 1, the right-hand formulas are in general false on L. Proposition 3.3.3.3. The following left-hand formulas are true on L. When D > 1, the right-hand formulas are in general false on L.                          XMN ∂ ∂xM ⊗ ∂ ∂xN ∼= X00 d dx0 ⊗ d dx0 , XMN ∂ ∂xM ⊗ ∂ ∂xN ∼= X00 d dx0 ⊗ d dx0 , Y AB ∂ ∂ξA ⊗ ∂ ∂ξB ∼= Y 00 d dξ0 ⊗d dξ0 , YAB ∂ ∂ξA ⊗ ∂ ∂ξB ∼= Y00 d dξ0 ⊗d dξ0 .                GMNdxM ⊗dxN ≃G00dx0 ⊗dx0, GMNdxM ⊗dxN ≃G00dx0 ⊗dx0, HABdξA ⊗dξB ≃H00dξ0 ⊗dξ0, HABdξA ⊗dξB ≃H00dξ0 ⊗dξ0.                GMNdxM ⊗dxN ≃G00dx0 ⊗dx0, GMNdxM ⊗dxN ≃G00dx0 ⊗dx0, HABdξA ⊗dξB ≃H00dξ0 ⊗dξ0, HABdξA ⊗dξB ≃H00dξ0 ⊗dξ0. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 23 Proof. Due to Proposition 3.3.3.2 and evolution lemma, the left-hand formulas hold evidently. Now consider the right-hand ones. On UL when D > 1, xMN cannot be expressed as the form like yεM 0 εN 0 . Deﬁnition 3.3.3.1. On UL deﬁne Otherwise, let xMN = yεM 0 εN 0 , then the following two conclusions contradict with each other:        dxMdxM = xMNdxMdxN = (yεM 0 εN 0 )dxMdxN = ydxMdxMdxNdxN dx0dx0 = yg00dxMdxM ⇒y = 1 g00 = g00, D = xMNxMN = (yεM 0 εN 0 )gMN = y(gMNεM 0 εN 0 ) = yg00 ⇒y = D g00 = Dg00. ⊓⊔ ⊓⊔ Remark 3.3.3.1. Due to the above proposition and section 2.2 , we know that although tensors G and X have relations GMN = XMN and GMN = XMN and tensors H and Y have relations HAB = YAB and HAB = Y AB, when considering evolution, G and H have better properties than X and Y. Remark 3.3.3.1. Due to the above proposition and section 2.2 , we know that although tensors G and X have relations GMN = XMN and GMN = XMN and tensors H and Y have relations HAB = YAB and HAB = Y AB, when considering evolution, G and H have better properties than X and Y. 3.3.4 Mathematical treatment of actual evolution Deﬁnition 3.3.4.1. Let Vn be the totality of n-order tensor ﬁelds generated by tangent vector bundle and cotangent vector bundle on M. Then suppose T ≜t n ∂ ∂x ⊗dx o ∈Vn, where smooth real functions t represent the coeﬃcients of tensor T. The regular imbedding π : L →M induces tL ≜t ◦π : L →R and TL ≜tL n ∂ ∂x ⊗dx o . (1) Let D be aﬃne connection, and denote tL;0 ≜t;QεQ 0 , then the absolute diﬀerential of T and TL can be expressed as        DT ≜Dt ⊗ ∂ ∂x ⊗dx ≜t;QdxQ ⊗ ∂ ∂x ⊗dx , DLTL ≜DLtL ⊗ ∂ ∂x ⊗dx ≜tL;0dx0 ⊗ ∂ ∂x ⊗dx , where Dt ≜t;QdxQ, DLtL ≜tL;0dx0. where Dt ≜t;QdxQ, DLtL ≜tL;0dx0. (2) Gradient operator ∇is called the actual evolution. The absolute gradient of T and TL can be expressed as (2) Gradient operator ∇is called the actual evolution. The absolute gradient of T and TL can be expressed as (2) Gradient operator ∇is called the actual evolution. The absolute gradient of T and TL can be expressed as (2) Gradient operator ∇is called the actual evolution. The absolute gradient of T and TL can be expressed as        ∇T ≜∇t ⊗ ∂ ∂x ⊗dx ≜t;Q ∂ ∂xQ ⊗ ∂ ∂x ⊗dx , ∇LTL ≜∇LtL ⊗ ∂ ∂x ⊗dx ≜tL;0 d dx0 ⊗ ∂ ∂x ⊗dx , where ∇t ≜t;Q ∂ ∂xQ , ∇LtL ≜tL;0 d dx0. The gradient direction (ﬁeld) ∇t is called the actual evolution direction (ﬁeld) of T. The integral curve of ∇t is called the gradient line or actual evolution path of T. Proposition 3.3.4.1. DT ≃DLTL if L is an arbitrary path. ∇T ∼= ∇LTL if and only if L is the gradient line of T. Proof. According to deﬁnition, the ﬁrst conclusion is evident. Now consider the second conclu- sion. Proof. According to deﬁnition, the ﬁrst conclusion is evident. Now consider the second conclu- sion. 24 Zhao-Hui Man (1) The suﬃciency. Let L be the gradient line of T. ∀p ∈L, the gradient direction at p is t;Q ∂ ∂xQ ∈Tp(M). Because tangent map is injection, there exists a unique X d dx0 ∈Tp(L) such that t;Q ∂ ∂xQ ∼= X d dx0. Applying evolution lemma we obtain (1) The suﬃciency. Let L be the gradient line of T. ∀p ∈L, the gradient direction at p is t;Q ∂ ∂xQ ∈Tp(M). Because tangent map is injection, there exists a unique X d dx0 ∈Tp(L) such that t;Q ∂ ∂xQ ∼= X d dx0. Applying evolution lemma we obtain t;Q = X dxQ dx0 L , dxQ ≃dxQ dx0 L dx0, thereby thereby t;QdxQ ≃X dxQ dx0 L dxQ dx0 L dx0. According to Deﬁnition 3.3.1.3 , (dξ0)2 = D P A=1 (dξA)2 holds on UL, i.e. dx0dx0 = dxQdxQ. Substitute it into the above formula, then we obtain t;QdxQ ≃Xdx0. Due to evolution lemma, X = t;Q dxQ dx0 = tL;0, and then ∇T ∼= ∇LTL hold. (2) The necessity is evident. In fact, ∀p ∈L, suppose tangent vector t;0 d dx0 on L makes ∇T ∼= ∇LTL hold, so t;Q ∂ ∂xQ ∼= t;0 d dx0. (2) Gradient operator ∇is called the actual evolution. The absolute gradient of T and TL can be expressed as And suppose ∀p ∈L, XQ ∂ ∂xQ ∼= t;0 d dx0. Because tangent map is injection, XQ = t;Q, which indicates that XQ ∂ ∂xQ can only be the gradient direction. Additionally, p is arbitrary on L, hence L can only be the gradient line, not other paths. ⊓⊔ Deﬁnition 3.3.4.2. According to evolution lemma, ∇T ∼= ∇LTL if and only if t;Q = tL;0¯ε0 Q or t;Q = tL;0εQ 0 . These two equations are called the actual evolution equations of T. Deﬁnition 3.3.4.3. Suppose there is a tensor product U ≜uQdxQ ⊗ n ∂ ∂x ⊗dx o , such that the system of 1-order non-homogeneous linear equations t;Q = uQ has a unique solution t. Then ∇t satisﬁes ∂ d uQdxQ ≃u0dx0, uQ ∂ ∂xQ ∼= u0 d dx0 . We say the actual evolution direction ∇t = uQ ∂ ∂xQ is determined by uQdxQ. Remark 3.3.4.1. Now for any universal geometrical property deﬁned in form of tensor product on geometrical manifold, we are able to study its actual evolution in way of absolute gradient. Then two important gradient directions will be discussed. One is the actual evolution of potential ﬁeld of reference-system itself. The other is the case that general charge of one reference-system evolves in another reference-system. They are both attributed to geometrical properties of geometrical manifold. 3.3.5 Mathematical treatment of actual evolution of potential ﬁeld Deﬁnition 3.3.5.1. Suppose f evolves in g, that is, ∀p ∈M, (U, ξA) f(p) −−→(U, xM) g(p) ←−−(U, ζA). We will always take the following notations in coordinate frame (U, xM). (1) Let the connection of geometrical manifold (M, f) be ΛM NP, and the connection of (M, g) be Γ M NP. Colon ":" is used to express the absolute derivative on (M, f), and semicolon ";" is used to express the absolute derivative on (M, g), such as uQ:P = ∂uQ ∂xP + uHΛQ HP , uQ;P = ∂uQ ∂xP + uHΓ Q HP . 25 A generalization of intrinsic geometry and its application to Hilbert’s 6th problem We also call ΛM NP the potential ﬁeld of f, and Γ M NP the potential ﬁeld of g, or say potential for short. In order to describe intrinsic geometry, we must adopt aﬃne connection dependent on slack-tights. It can be either Levi-Civita connection or simple connection of Deﬁnition 2.7.1 . Because the scope of applicability of simple connection is larger than Levi-Civita connection, we suppose ΛM NP and Γ M NP are both simple connections. (2) Let the coeﬃcients of Riemannian curvature of (M, f) be KM NPQ, and the coeﬃcients of Riemannian curvature of (M, g) be RM NPQ, then (2) Let the coeﬃcients of Riemannian curvature of (M, f) be KM NPQ, and the coeﬃcients of Riemannian curvature of (M, g) be RM NPQ, then          KM NPQ ≜ ∂ΛM NQ ∂xP −∂ΛM NP ∂xQ + ΛH NQΛM HP −ΛH NP ΛM HQ, RM NPQ ≜ ∂Γ M NQ ∂xP −∂Γ M NP ∂xQ + Γ H NQΓ M HP −Γ H NP Γ M HQ. (3) The values of indices of internal space and external space are taken according to Deﬁnition 5.1.1 . (3) The values of indices of internal space and external space are taken according to Deﬁnition 5.1.1 . Discussion 3.3.5.1. Considerevolutionof f.Denote ρM N0 ≜KM NPQ :P εQ 0 ,thenwehave KM NPQ :P dxQ ≃ ρM N0dx0 in an arbitrary direction. Discussion 3.3.5.1. Considerevolutionof f.Denote ρM N0 ≜KM NPQ :P εQ 0 ,thenwehave KM NPQ :P dxQ ≃ ρM N0dx0 in an arbitrary direction. Specially,wehave KM NPQ :P ∂ ∂xQ ∼= ρM N0 d dx0 inthegradientdirectiondeterminedby KM NPQ :P dxQ. 3.3.5 Mathematical treatment of actual evolution of potential ﬁeld Now due to Proposition 3.3.2.2 , we obtain the actual evolution equation KM NPQ :P = ρM N0¯ε0 Q. De- note jM NQ ≜ρM N0¯ε0 Q, then we obtain KM NPQ :P = jM NQ, (11) (11) which is called the general Yang-Mills ﬁeld equation of f. Then due to Proposition 3.3.4.1 we directly obtain the following theorem. which is called the general Yang-Mills ﬁeld equation of f. Then due to Proposition 3.3.4.1 we directly obtain the following theorem. Proposition 3.3.5.1. (Evolution theorem of general gauge ﬁeld ). ρM N0 and jM NQ are both intrinsic geometrical properties of (M, f). Equation KM NPQ :P = jM NQ holds if and only if its evolution direction ﬁeld is the gradient direction ﬁeld determined by KM NPQ :P dxQ. Deﬁnition 3.3.5.2. Besides ρM N0, there are also ρM0 N ≜G00ρM N0, ρMN0 ≜GMM′ρM′ N0 and ρMN 0 ≜ G00ρMN0. We call each of them a general charge, or charge for short. 3.3.6 Mathematical treatment of actual evolution of general charge Discussion 3.3.6.1. Without loss of generality, on geometrical manifold (M, g) we can discuss the actual evolution of charge tensor F0 ≜ρMN 0dxM ⊗dxN of f in way of absolute gradient. For the sake of simplicity, denote ρMN 0 by ρMN concisely. The absolute diﬀerential of F0 on (M, g) is DF0 ≜DρMN ⊗dxM ⊗dxN, where DρMN ≜ ρMN;RdxR, and D is the simple connection of (M, g). The absolute gradient of F0 on (M, g) is ∇F0 ≜∇ρMN ⊗dxM ⊗dxN, where ∇ρMN ≜ ρMN;R ∂ ∂xR. 26 Zhao-Hui Man ue to Proposition 3.3.4.1 , we directly obtain the following theorem. Due to Proposition 3.3.4.1 , we directly obtain the following theorem. Due to Proposition 3.3.4.1 , we directly obtain the following theorem. Proposition 3.3.6.1. (General charge evolution theorem) The following relations are true on geometrical manifold (M, g) if and only if evolution direction ﬁeld of ρMN is the gradient direction ﬁeld ∇ρMN.      ρMN;RdxR ≃ρMN;0dx0 ρMN;R ∂ ∂xR ∼= ρMN;0 d dx0 ,      ρMN ;RdxR ≃ρMN ;0dx0 ρMN ;R ∂ ∂xR ∼= ρMN ;0 d dx0 , (12) (12) which are called charge evolution equations. which are called charge evolution equations. which are called charge evolution equations. which are called charge evolution equations. which are called charge evolution equations. eﬁnition 3.3.6.1. For more convenience, further abbreviate the notation ρMN to ρ. Deﬁnition 3.3.6.1. For more convenience, further abbreviate the notation ρMN to (1) Call E0 ≜ρ;0 ≜ρ;R¯ε0 R and E0 ≜ρ;0 ≜ρ;RεR 0 the total energy or total mass of ρ. (2) Call pR ≜ρ;R and pR ≜ρ;R the momentum of ρ. (3) Call H0 ≜ dρ dx0 and H0 ≜ dρ dx0 the canonical energy of ρ. (4) Call P R ≜ ∂ρ ∂xR and PR ≜ ∂ρ ∂xR the canonical momentum of ρ. (5) Call V 0 ≜E0 −H0 and V0 ≜E0 −H0 the potential energy of interaction. (6) Call V R ≜pR −P R and VR ≜pR −PR the momentum of interaction. ) Call E0 ≜ρ;0 ≜ρ;R¯ε0 R and E0 ≜ρ;0 ≜ρ;RεR 0 the total energy or total mass of ρ. (1) Call E0 ≜ρ;0 ≜ρ;R¯ε0 R and E0 ≜ρ;0 ≜ρ;RεR 0 the total energy or total mass of ρ. (2) Call pR ≜ρ;R and pR ≜ρ;R the momentum of ρ. (3) Call H0 ≜ dρ dx0 and H0 ≜ dρ dx0 the canonical energy of ρ. (4) Call P R ≜ ∂ρ ∂xR and PR ≜ ∂ρ ∂xR the canonical momentum of ρ. (5) Call V 0 ≜E0 −H0 and V0 ≜E0 −H0 the potential energy of interaction. (6) Call V R ≜pR −P R and VR ≜pR −PR the momentum of interaction. Proposition 3.3.6.2. If and only if evolution direction of ρ evolving in g is the gradient direction ∇ρ, equation Proposition 3.3.6.2. If and only if evolution direction of ρ evolving in g is the gradient direction ∇ρ, equation ∇ρ, equation E0E0 = pRpR holds, which is called the general energy-momentum equation of ρ. Proof. According to Proposition 3.3.6.1 , gradient direction is equivalent to      E0dx0 ≃pRdxR E0 d dx0 ∼= pR ∂ ∂xR ,      E0dx0 ≃pRdxR E0 d dx0 ∼= pR ∂ ∂xR . (13) (13) Then we obtain the directional derivative in gradient direction: Then we obtain the directional derivative in gradient direction: Then we obtain the directional derivative in gradient direction: E0 d dx0 , E0dx0 = pR ∂ ∂xR , pMdxM , M, or E0E0 = pRpR. ⊓⊔ i.e. G00E0E0 = GRMpRpM, or E0E0 = pRpR. i.e. G00E0E0 = GRMpRpM, or E0E0 = pRpR. i.e. G00E0E0 = GRMpRpM, or E0E0 = pRpR. ⊓⊔ ⊓⊔ ⊓⊔ Proposition 3.3.6.3. The following equations Proposition 3.3.6.3. The following equations pR = E0 dxR dx0 , pR = E0 dxR dx0 (14) (14) hold if and only if the evolution direction of ρ is the gradient direction ∇ρ. hold if and only if the evolution direction of ρ is the gradient direction ∇ρ. Proof. According to Proposition 3.3.6.1 , gradient direction is equivalent to pR ∂ ∂xR ∼= E0 d dx0 and pR ∂ ∂xR ∼= E0 d dx0. Then due to evolution lemma we immediately obtain pR = E0 dxR dx0 and pR = E0 dxR dx0 . ⊓⊔ Remark 3.3.6.1. In the gradient direction, the conclusion of this proposition is consistent with the traditional p = mv. Remark 3.3.6.1. In the gradient direction, the conclusion of this proposition is consistent with the traditional p = mv. 27 A generalization of intrinsic geometry and its application to Hilbert’s 6th problem Deﬁnition 3.3.6.2. Let L be the totality of paths from a to b. And suppose Lρ ∈L, and the evolution parameter x0 satisﬁes ta ≜x0(a) < x0(b) ≜tb. The functional sρW Lρ ≜ Z Lρ Dρ = Z tb ta E0dx0 = Z tb ta pRdxR is called general evolution quantity of ρ. is called general evolution quantity of ρ. Proposition 3.3.6.4. (Extreme value theorem of general evolution quantity) Lρ is the gradient line of ρ if and only if δsρW Lρ = 0. 3.3.7 Intrinsic geometrical treatment of conservation of energy-momentum The most general abstract theory about conserved quantity is the Neother’s theorem. However, it is not enough to just only content with abstract point of view. In consideration of that a conserved quantity is a geometrical property, we need to study its concrete construction in way of intrinsic geometry. 3.7.1. Denote Proposition 3.3.6.4. (Extreme value theorem of general evolution quantity) Lρ is the gradient line of ρ if and only if δsρW Lρ = 0. The directional derivative ⟨X, Dρ⟩= ρ;0 cos θ, where θ is the included angle between evolution direction X and the gradient direction. Then ⟨δX, Dρ⟩= ρ;0δ cos θ = −ρ;0 sin θδθ. Now When b →a, δdsρW = ⟨δX, Dρ⟩dx0. The directional derivative ⟨X, Dρ⟩= ρ;0 cos θ, where θ is the included angle between evolution direction X and the gradient direction. Then ⟨δX, Dρ⟩= ρ;0δ cos θ = −ρ;0 sin θδθ. Now δdsρW = −ρ;0 sin θδθdx0. δdsρW = −ρ;0 sin θδθdx0. Hence, for general ρ, δdsρW = 0 if and only if sin θ = 0, namely evolution direction at this point is exactly the gradient direction (take the positive direction without loss of generality). Take integration from a to b, then δ R tb ta dsρW = 0 if and only if evolution direction at each point of Lρ is the gradient direction of ρ. In other words, δsρW = 0 if and only if Lρ is the gradient line of ρ. ⊓⊔ ⊓⊔ Remark 3.3.6.2. In the Minkowski coordinate frame deﬁned later, evolution parameter x0 will be changed to ˜xτ, then there still exists a concept of gradient direction. Correspondingly, the evolution quantity R tb ta E0dx0 will present as R τb τa ˜mτdτ, where ˜mτ is rest-mass. Thus, the principle of least action will become a theorem, no longer as a principle. Proposition 3.3.7.1. The following two equations hold: (1)[ρFPQ] = [ρEPQ]; (2)[ρFPQ] −[ρBPQ] = ρMH,P Γ H NQ −ρMH,QΓ H NP + ρHN,P Γ H MQ −ρHN,QΓ H MP . (1)[ρFPQ] = [ρEPQ]; (2)[ρFPQ] −[ρBPQ] = ρMH,P Γ H NQ −ρMH,QΓ H NP + ρHN,P Γ H MQ −ρHN,QΓ H MP . f Deﬁnition 3.3.7.1. Denote        [ρΓG] ≜∂ρ ∂xG −ρ;G ≜∂ρMN ∂xG −ρMN;G = ρMHΓ H NG + ρHNΓ H MG, [ρΓ0] ≜dρ dx0 −ρ;0 ≜dρMN dx0 −ρMN;0 = ρMHΓ H N0 + ρHNΓ H M0.    [ρΓ Q] ≜GGQ[ρΓG], [ρΓ 0] ≜G00[ρΓ0].        [ρBPQ] ≜ρMH ∂Γ H NQ ∂xP −∂Γ H NP ∂xQ ! + ρHN ∂Γ H MQ ∂xP −∂Γ H MP ∂xQ ! , [ρRPQ] ≜ρMHRH NPQ + ρHNRH MPQ.      [ρFPQ] ≜∂[ρΓQ] ∂xP −∂[ρΓP ] ∂xQ , [ρEPQ] ≜[ρΓQ];P −[ρΓP ];Q. Proposition 3.3.7.1. The following two equations hold: Proposition 3.3.7.1. The following two equations hold: Proposition 3.3.6.4. (Extreme value theorem of general evolution quantity) Lρ is the gradient line of ρ if and only if δsρW Lρ = 0. line of ρ if and only if δsρW Lρ = 0. Proof. Let the parameter equation of Lρ be xR = xR(x0), ta ⩽x0 ⩽tb, xR = xR(x0), ta ⩽x0 ⩽tb, and let the parameter equation of Lρ + δLρ be xR = xR(x0) + δxR(x0), ta ⩽x0 ⩽tb, δxR(ta) = δxR(tb) = 0. Let the unit tangent vector on Lρ at any x0 be Let the unit tangent vector on Lρ at any x0 be X ≜π∗ d dx0 ≜dxR dx0 x0 ∂ ∂xR = εR 0 x0 ∂ ∂xR , and let the unit tangent vector on Lρ + δLρ be X + δX ≜d xR + δxR dx0 x0 ∂ ∂xR = dxR dx0 + δdxR dx0 ! x0 ∂ ∂xR = εR 0 x0 + δεR 0 x0 ∂ ∂xR . Then consider the variation of sρW (Lρ). Then consider the variation of sρW (Lρ). Then consider the variation of sρW (Lρ). Then consider the variation of sρW (Lρ). ∆sρW (Lρ) = ∆ Z Lρ pRεR 0 dx0 = Z Lρ+δLρ pRεR 0 dx0 − Z Lρ pRεR 0 dx0 = Z Lρ+δLρ ρ;RεR 0 dx0 − Z Lρ ρ;RεR 0 dx0 = Z Lρ+δLρ ⟨X, Dρ⟩dx0 − Z Lρ ⟨X, Dρ⟩dx0 = Z tb ta D X + δX, Dρ xR + δxRE dx0 − Z tb ta D X, Dρ xRE dx0 = Z tb ta * X + δX, Dρ(xR) + ∂Dρ(xR) ∂xM δxM + o(δx) + dx0 − Z tb ta D X, Dρ(xR) E dx0 = Z tb ta ⟨X + δX, Dρ⟩+ X + δX, ∂Dρ ∂xM δxM dx0 − Z tb ta ⟨X, Dρ⟩dx0 + o (δx) = Z tb ta ⟨δX, Dρ⟩+ X, ∂Dρ ∂xM δxM dx0 + o (δx) = Z tb ta (⟨δX, Dρ⟩+ ⟨X, δDρ⟩) dx0 + o (δx) = Z tb ta ⟨δX, Dρ⟩dx0+ Z tb ta δDρ + o (δx) t = Z tb ta (⟨δX, Dρ⟩+ ⟨X, δDρ⟩) dx0 + o (δx) = Z tb ta ⟨δX, Dρ⟩dx0+ Z tb ta δDρ + o (δx) t = Z tb ta ⟨δX, Dρ⟩dx0 + o (δx) . = Z tb ta ⟨δX, Dρ⟩dx0 + o (δx) . δsρW = Z tb ta ⟨δX, Dρ⟩dx0. 28 Zhao-Hui Man 28 Zhao-Hui Man When b →a, δdsρW = ⟨δX, Dρ⟩dx0. Proof. = ρMH Γ H NQ;P −Γ H NP ;Q + ρHN Γ H MQ;P −Γ H MP ;Q + ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP − ρMHΓ H GP Γ G NQ −ρMHΓ H GQΓ G NP − ρHNΓ H GP Γ G MQ −ρHNΓ H GQΓ G MP = ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP + ρMH Γ H NQ;P −Γ H NP ;Q + Γ H GQΓ G NP −Γ H GP Γ G NQ + ρHN Γ H MQ;P −Γ H MP ;Q + Γ H GQΓ G MP −Γ H GP Γ G MQ = ρMH P Γ H ρMH QΓ H + ρHN P Γ H ρHN QΓ H + ρMH ∂Γ H NQ ∂Γ H NP ! ! = ρMH,P Γ H NQ −ρMH,QΓ H NP + ρHN,P Γ H MQ −ρHN,QΓ H MP + [ρBPQ] = ∂ ∂xP ρMHΓ H NQ + ρHNΓ H MQ − ∂ ∂xQ ρMHΓ H NP + ρHNΓ H MP = ∂[ρΓQ] ∂xP −∂[ρΓP ] ∂xQ = [ρFPQ]. = ∂[ρΓQ] ∂xP −∂[ρΓP ] ∂xQ = [ρFPQ]. ⊓⊔ Proposition 3.3.7.2. The following two equations hold: Proposition 3.3.7.2. The following two equations hold: Proposition 3.3.7.2. The following two equations hold: Proof. = ρMH Γ H NQ;P −Γ H NP ;Q + ρHN Γ H MQ;P −Γ H MP ;Q + ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP − ρMHΓ H GP Γ G NQ −ρMHΓ H GQΓ G NP − ρHNΓ H GP Γ G M = ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP = ρMH Γ H NQ;P −Γ H NP ;Q + ρHN Γ H MQ;P −Γ H MP ;Q + ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP − ρMHΓ H GP Γ G NQ −ρMHΓ H GQΓ G NP − ρHNΓ H GP Γ G MQ −ρHNΓ H GQΓ G MP = ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP + ρMH Γ H NQ;P −Γ H NP ;Q + Γ H GQΓ G NP −Γ H GP Γ G NQ + ρHN Γ H MQ;P −Γ H MP ;Q + Γ H GQΓ G MP −Γ H GP Γ G MQ = ρMH,P Γ H NQ −ρMH,QΓ H NP + ρHN,P Γ H MQ −ρHN,QΓ H MP + ρMH ∂Γ H NQ ∂xP −∂Γ H NP ∂xQ ! + ρHN ∂Γ H MQ ∂xP −∂Γ H MP ∂xQ ! = ρMH,P Γ H NQ −ρMH,QΓ H NP + ρHN,P Γ H MQ −ρHN,QΓ H MP + [ρBPQ] = ∂ ∂xP ρMHΓ H NQ + ρHNΓ H MQ − ∂ ∂xQ ρMHΓ H NP + ρHNΓ H MP = ∂[ρΓQ] ∂ P −∂[ρΓP ] ∂ Q = [ρFPQ]. Proof. [ρEPQ] = [ρΓQ];P −[ρΓP ];Q = ρMHΓ H NQ + ρHNΓ H MQ ;P − ρMHΓ H NP + ρHNΓ H MP ;Q = ρMH Γ H NQ;P −Γ H NP ;Q + ρHN Γ H MQ;P −Γ H MP ;Q + ρMH;P Γ H NQ −ρMH;QΓ H NP + ρHN;P Γ H MQ −ρHN;QΓ H MP = ρMH Γ H NQ;P −Γ H NP ;Q + ρHN Γ H MQ;P −Γ H MP ;Q + ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP − ρMGΓ G HP Γ H NQ −ρMGΓ G HQΓ H NP − ρGNΓ G HP Γ H MQ −ρGNΓ G HQΓ H MP A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 29 A generalization of intrinsic geometry and its application to Hilbert’s 6th problem = ρMH Γ H NQ;P −Γ H NP ;Q + ρHN Γ H MQ;P −Γ H MP ;Q + ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP − ρMHΓ H GP Γ G NQ −ρMHΓ H GQΓ G NP − ρHNΓ H GP Γ G MQ −ρHNΓ H GQΓ G MP = ρMH,P Γ H NQ + ρHN,P Γ H MQ − ρMH,QΓ H NP + ρHN,QΓ H MP + ρMH Γ H NQ;P −Γ H NP ;Q + Γ H GQΓ G NP −Γ H GP Γ G NQ + ρHN Γ H MQ;P −Γ H MP ;Q + Γ H GQΓ G MP −Γ H GP Γ G MQ = ρMH,P Γ H NQ −ρMH,QΓ H NP + ρHN,P Γ H MQ −ρHN,QΓ H MP + ρMH ∂Γ H NQ ∂xP −∂Γ H NP ∂xQ ! + ρHN ∂Γ H MQ ∂xP −∂Γ H MP ∂xQ ! Proposition 3.3.7.2. The following two equations hold: According to Proposition 3.3.7.2 , ∂pP ∂xQ −∂pQ ∂xP −[ρFPQ] = 0. Substitute equation (2) of Proposition 3.3.7.1 into this one, then we obtain ∂xQ ∂ Proposition 3.3.7.1 into this one, then we obtain ∂pP ∂xQ −∂pQ ∂xP − ρMH,P Γ H NQ −ρMH,QΓ H NP − ρHN,P Γ H MQ −ρHN,QΓ H MP = [ρBPQ] ⇔∂ρMN;P ∂xQ −∂ρMN;Q ∂xP − ρMH,P Γ H NQ −ρMH,QΓ H NP − ρHN,P Γ H MQ −ρHN,QΓ H MP = [ρBPQ] ⇔ ∂ρMN;P ∂xQ −ρMH,P Γ H NQ −ρHN,P Γ H MQ − ∂ρMN;Q ∂xP −ρMH,QΓ H NP −ρHN,QΓ H MP = [ρBPQ] ⇔ ∂ρMN;P ∂xQ −ρMH,P Γ H NQ −ρHN,P Γ H MQ −ρMN;HΓ H PQ − ∂ρMN;Q ∂xP −ρMH,QΓ H NP −ρHN,QΓ H MP −ρMN;HΓ H QP + ρMN;H Γ H PQ −Γ H QP = [ρBPQ] ⇔ρMN;P;Q −ρMN;Q;P = [ρBPQ] ⇔pP;Q −pQ;P −[ρBPQ] = 0. Consider π∗pP;QdxQ −pQ;P dxQ −[ρBPQ]dxQ. Consider π∗pP;QdxQ −pQ;P dxQ −[ρBPQ]dxQ. π∗: pP;QdxQ 7→pP;Q dxQ dx0 dx0 = pP;0dx0, π∗: pQ;P dxQ 7→pQ;P dxQ dx0 dx0 =   pQ dxQ dx0 ! ;P −pQ dxQ dx0 ! ;P  dx0 = E0;P dx0 −pQεQ 0;P dx0, dx π∗: pQ;P dxQ 7→pQ;P dxQ dx0 dx0 =   pQ dxQ dx0 ! ;P −pQ dxQ dx0 ! ;P  dx0 = E0;P dx0 −pQεQ 0 π∗: [ρBPQ]dxQ 7→[ρBPQ]dxQ dx0 dx0 = [ρBPQ]εQ 0 dx0. π∗: [ρBPQ]dxQ 7→[ρBPQ]dxQ dx0 dx0 = [ρBPQ]εQ 0 dx0. π∗: [ρBPQ]dxQ 7→[ρBPQ]dxQ dx0 dx0 = [ρBPQ]εQ 0 dx0. π∗: [ρBPQ]dxQ 7→[ρBPQ]dxQ dx0 dx0 = [ρBPQ]εQ 0 dx0. Then pP;0dx0 −E0;P dx0 + pQεQ 0;P dx0 −[ρBPQ]εQ 0 dx0 = 0, ﬁnally pP;0 −E0;P + pQεQ 0;P −[ρBPQ]εQ 0 = 0. ⊓⊔ Proposition 3.3.7.4. With torsion-free connection, the following three equations hold: Proposition 3.3.7.4. With torsion-free connection, the following three equations hold: Proposition 3.3.7.4. With torsion-free connection, the following three equations hold: (1)pP;Q −pQ;P −[ρRPQ] = 0; (2)pP;0 −E0;P + pQεQ 0;P −[ρRPQ]εQ 0 = 0; . (3)[ρBPQ] = [ρRPQ]. Proof. The covariant derivatives of Proof. The covariant derivatives of Proof. The covariant derivatives of Proof. Proposition 3.3.7.2. The following two equations hold: (1)∂pP ∂xQ −∂pQ ∂xP −[ρFPQ] = 0; (2)dpP dx0 −∂E0 ∂xP + pQ ∂εQ 0 ∂xP −[ρFPQ]εQ 0 = 0. Proof. According to Deﬁnition 3.3.6.1 , Proof. According to Deﬁnition 3.3.6.1 , ∂PP ∂xQ −∂PQ ∂xP = 0 ⇔∂pP ∂xQ −∂pQ ∂xP + ∂[ρΓP ] ∂xQ −∂[ρΓQ] ∂xP = 0 ⇔∂pP ∂xQ −∂pQ ∂xP −[ρFPQ] = 0. Consider π∗ ∂pP ∂xQ dxQ −∂pQ ∂xP dxQ −[ρFPQ]dxQ . π∗: ∂pP ∂xQ dxQ 7→∂pP ∂xQ dxQ dx0 dx0 = dpP dx0 dx0, ∂PP ∂xQ −∂PQ ∂xP = 0 ⇔∂pP ∂xQ −∂pQ ∂xP + ∂[ρΓP ] ∂xQ −∂[ρΓQ] ∂xP = 0 ⇔∂pP ∂xQ −∂pQ ∂xP −[ρFPQ] = 0. Consider π∗ ∂pP ∂xQ dxQ −∂pQ ∂xP dxQ −[ρFPQ]dxQ . ∂PP ∂xQ −∂PQ ∂xP = 0 ⇔∂pP ∂xQ −∂pQ ∂xP + ∂[ρΓP ] ∂xQ −∂[ρΓQ] ∂xP = 0 ⇔∂pP ∂xQ −∂pQ ∂xP −[ρFPQ] = 0. Consider π∗ ∂pP ∂xQ dxQ −∂pQ ∂xP dxQ −[ρFPQ]dxQ . π∗: ∂pP ∂xQ dxQ 7→∂pP ∂xQ dxQ dx0 dx0 = dpP dx0 dx0, π∗: ∂pQ ∂xP dxQ 7→∂pQ ∂xP dxQ dx0 dx0 = ∂ pQ dxQ dx0 ∂xP dx0 −pQ ∂ ∂xP dxQ dx0 ! dx0 = ∂E0 ∂xP dx0 −pQ ∂εQ 0 ∂xP dx0, d Q π∗: ∂pP ∂xQ dxQ 7→∂pP ∂xQ dxQ dx0 dx0 = dpP dx0 dx0, π∗: ∂pQ ∂xP dxQ 7→∂pQ ∂xP dxQ dx0 dx0 = ∂ pQ dxQ dx0 ∂xP dx0 −pQ ∂ ∂xP dxQ dx0 ! dx0 = ∂E0 ∂xP dx0 −pQ ∂εQ 0 ∂xP dx0, ! π∗: [ρFPQ]dxQ 7→[ρFPQ]dxQ dx0 dx0 = [ρFPQ]εQ 0 dx0. Th π∗: [ρFPQ]dxQ 7→[ρFPQ]dxQ dx0 dx0 = [ρFPQ]εQ 0 dx0. π∗: [ρFPQ]dxQ 7→[ρFPQ]dxQ dx0 dx0 = [ρFPQ]εQ 0 dx0. π∗: [ρFPQ]dxQ 7→[ρFPQ]dxQ dx0 dx0 = [ρFPQ]εQ 0 dx0. Then dpP dx0 dx0 −∂E0 ∂xP dx0 + pQ ∂εQ 0 ∂xP dx0 −[ρFPQ]εQ 0 dx0 = 0, ﬁnally dpP dx0 −∂E0 ∂xP + pQ ∂εQ 0 ∂xP −[ρFPQ]εQ 0 = 0. ⊓⊔ ⊓⊔ Proposition 3.3.7.3. With torsion-free connection, the following two equations hold: (1)pP;Q −pQ;P −[ρBPQ] = 0; (2)pP;0 −E0;P + pQεQ 0;P −[ρBPQ]εQ 0 = 0. 30 Zhao-Hui Man Proof. According to Proposition 3.3.7.2 , ∂pP ∂xQ −∂pQ ∂xP −[ρFPQ] = 0. Substitute equation (2) of Proposition 3 3 7 1 into this one then we obtain Proof. According to Proposition 3.3.7.2 , ∂pP ∂xQ −∂pQ ∂xP −[ρFPQ] = 0. Substitute equation (2) of Proposition 3.3.7.1 into this one, then we obtain Proof. Proposition 3.3.7.2. The following two equations hold: The covariant derivatives of pP ≜ρ;P ≜ρMN;P = ρMN,P −ρMHΓ H NP −ρHNΓ H MP are pP;Q = ρMN;P;Q = ρMN;P,Q −ρMH;P Γ H NQ −ρHN;P Γ H MQ −ρMN;HΓ H PQ, pQ;P = ρMN;Q;P = ρMN;Q,P −ρMH;QΓ H NP −ρHN;QΓ H MP −ρMN;HΓ H QP . A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 31 Substract them: pP;Q −pQ;P = ρMN;P,Q −ρMN;Q,P + ρMH;QΓ H NP −ρMH;P Γ H NQ + ρHN;QΓ H MP −ρHN;P Γ H MQ + ρMN;HΓ H QP −ρMN;HΓ H PQ = ρMN;P,Q −ρMN;Q,P + ρMH;QΓ H NP −ρMH;P Γ H NQ + ρHN;QΓ H MP −ρHN;P Γ H MQ = ρMN,P −ρMHΓ H NP −ρHNΓ H MP ,Q − ρMN,Q −ρMHΓ H NQ −ρHNΓ H MQ ,P + ρMH,Q −ρMGΓ G HQ −ρGHΓ G MQ Γ H NP − ρMH,P −ρMGΓ G HP −ρGHΓ G MP Γ H NQ + ρHN,Q −ρHGΓ G NQ −ρGNΓ G HQ Γ H MP − ρHN,P −ρHGΓ G NP −ρGNΓ G HP Γ H MQ = ρMHΓ H NQ ,P + ρHNΓ H MQ ,P − ρMHΓ H NP ,Q + ρHNΓ H MP ,Q + ρMH,Q −ρMGΓ G HQ −ρGHΓ G MQ Γ H NP − ρMH,P −ρMGΓ G HP −ρGHΓ G MP Γ H NQ + ρHN,Q −ρHGΓ G NQ −ρGNΓ G HQ Γ H MP − ρHN,P −ρHGΓ G NP −ρGNΓ G HP Γ H MQ = ρMHΓ H NQ,P + ρHNΓ H MQ,P − ρMHΓ H NP,Q + ρHNΓ H MP,Q + −ρMGΓ G HQΓ H NP −ρGHΓ G MQΓ H NP − −ρMGΓ G HP Γ H NQ −ρGHΓ G MP Γ H NQ + −ρHGΓ G NQΓ H MP −ρGNΓ G HQΓ H MP − −ρHGΓ G NP Γ H MQ −ρGNΓ G HP Γ H MQ = ρMH(Γ H NQ,P −Γ H NP,Q + Γ H GP Γ G NQ −Γ H GQΓ G NP ) + ρHN(Γ H MQ,P −Γ H MP,Q + Γ H GP Γ G MQ −Γ H GQΓ G MP ) = ρMHRH NPQ + ρHNRH MPQ = [ρRPQ]. Substract them: pP;Q −pQ;P = ρMN;P,Q −ρMN;Q,P + ρMH;QΓ H NP −ρMH;P Γ H NQ + ρHN;QΓ H MP −ρHN;P Γ H MQ + ρMN;HΓ H QP −ρMN;HΓ H PQ = ρMN;P,Q −ρMN;Q,P + ρMH;QΓ H NP −ρMH;P Γ H NQ + ρHN;QΓ H MP −ρHN;P Γ H MQ = ρMN,P −ρMHΓ H NP −ρHNΓ H MP ,Q − ρMN,Q −ρMHΓ H NQ −ρHNΓ H MQ ,P + ρMH,Q −ρMGΓ G HQ −ρGHΓ G MQ Γ H NP − ρMH,P −ρMGΓ G HP −ρGHΓ G MP Γ H NQ + ρHN,Q −ρHGΓ G NQ −ρGNΓ G HQ Γ H MP − ρHN,P −ρHGΓ G NP −ρGNΓ G HP Γ H MQ H H H H = ρMN;P,Q −ρMN;Q,P + ρMH;QΓ H NP −ρMH;P Γ H NQ + ρHN;QΓ H MP −ρHN;P Γ H MQ = ρMN,P −ρMHΓ H NP −ρHNΓ H MP ,Q − ρMN,Q −ρMHΓ H NQ −ρHNΓ H MQ ,P + ρMH,Q −ρMGΓ G HQ −ρGHΓ G MQ Γ H NP − ρMH,P −ρMGΓ G HP −ρGHΓ G MP Γ H NQ + ρHN,Q −ρHGΓ G NQ −ρGNΓ G HQ Γ H MP − ρHN,P −ρHGΓ G NP −ρGNΓ G HP Γ H MQ + ρMH,Q ρMG HQ ρGH MQ NP ρMH,P ρMG HP ρGH MP NQ + ρHN,Q −ρHGΓ G NQ −ρGNΓ G HQ Γ H MP − ρHN,P −ρHGΓ G NP −ρGNΓ G HP Γ H MQ = ρMHΓ H NQ ,P + ρHNΓ H MQ ,P − ρMHΓ H NP ,Q + ρHNΓ H MP ,Q + ρMH,Q −ρMGΓ G HQ −ρGHΓ G MQ Γ H NP − ρMH,P −ρMGΓ G HP −ρGHΓ G MP Γ H NQ + ρHN,Q −ρHGΓ G NQ −ρGNΓ G HQ Γ H MP − ρHN,P −ρHGΓ G NP −ρGNΓ G HP Γ H MQ = ρMHΓ H NQ ,P + ρHNΓ H MQ ,P − ρMHΓ H NP ,Q + ρHNΓ H MP ,Q + ρMH,Q −ρMGΓ G HQ −ρGHΓ G MQ Γ H NP − ρMH,P −ρMGΓ G HP −ρGHΓ G MP Γ H NQ + ρHN,Q −ρHGΓ G NQ −ρGNΓ G HQ Γ H MP − ρHN,P −ρHGΓ G NP −ρGNΓ G HP Γ H MQ = ρMHΓ H NQ,P + ρHNΓ H MQ,P − ρMHΓ H NP,Q + ρHNΓ H MP,Q + −ρMGΓ G HQΓ H NP −ρGHΓ G MQΓ H NP − −ρMGΓ G HP Γ H NQ −ρGHΓ G MP Γ H NQ + −ρHGΓ G NQΓ H MP −ρGNΓ G HQΓ H MP − −ρHGΓ G NP Γ H MQ −ρGNΓ G HP Γ H MQ + −ρMGΓ G HQΓ H NP −ρGHΓ G MQΓ H NP − −ρMGΓ G HP Γ H NQ −ρGHΓ G MP Γ H NQ + −ρHGΓ G NQΓ H MP −ρGNΓ G HQΓ H MP − −ρHGΓ G NP Γ H MQ −ρGNΓ G HP Γ H MQ = ρMH(Γ H NQ,P −Γ H NP,Q + Γ H GP Γ G NQ −Γ H GQΓ G NP ) + ρHN(Γ H MQ,P −Γ H MP,Q + Γ H GP Γ G MQ −Γ H GQΓ G MP ) = ρMHRH NPQ + ρHNRH MPQ = [ρRPQ]. Substract them: That is pP;Q −pQ;P −[ρRPQ] = 0. And compare it with equation (1) of Proposition 3.3.7.3 , then [ρBPQ] = [ρRPQ] is obtained. Finally, due to equation (2) of Proposition 3.3.7.3 , pP;0 − E0;P + pQεQ 0;P −[ρRPQ]εQ 0 = 0 holds. ⊓⊔ Deﬁnition 3.3.7.2. According to the above propositions, equations Deﬁnition 3.3.7.2. According to the above propositions, equations      FP ≜dpP dx0 = ∂E0 ∂xP −pQ ∂εQ 0 ∂xP + [ρFPQ]εQ 0 , fP ≜pP;0 = E0;P −pQεQ 0;P + [ρRPQ]εQ 0 . are called the general Lorentz force equations, and the intrinsic geometrical properties FP and fP are called general force on ρ. are called the general Lorentz force equations, and the intrinsic geometrical properties FP and fP are called general force on ρ. Proposition 3.3.7.5. Suppose there is a tensor YMN satisﬁes YMN ;M = −G00(E0;N −pQεQ 0;N + [ρRNQ]εQ 0 ) −¯ε0 ;M M pN, and let WMN ≜E0¯ε0 M ¯ε0 N, then in gradient direction of ρ, intrinsic geo- metrical property TMN ≜WMN + YMN satisﬁes TMN ;M = 0, TMN ;M = 0, which can be called the conservation of energy-momentum of ρ. 3.3.8 Two dual descriptions of gradient direction ﬁeld Discussion 3.3.8.1. For any two non-vanishing smooth tangent vector ﬁelds X and Y on manifold M, let LY be the Lie derivative operator induced by the one-parameter group of diﬀeomorphisms φY corresponding to Y . According to a well-known theorem [7], Lie derivative equation [X, Y ] = LY X holds. On one hand, suppose H is the ﬁeld of unit tangent vector in gradient directions of ρ, and φH is the one-parameter group of diﬀeomorphisms corresponding to H, and the the parameter of φH is x0. The Lie derivative equation induced by φH is [X, H] = LHX. Lie derivative operator LH and tangent vector ﬁeld d dx0 are both uniquely determined by H, so it can be denoted that d dx0 X ≜LHX. Thus, [X, H] = LHX becomes [X, H] = d dx0 X. On the other hand, the regular imbedding of path induces H ∼= HL. Then according to Proposition 3.3.2.1 , for any smooth function f, equation ⟨H, df⟩= ⟨HL, dfL⟩holds. Notice that HL and d dx0 are the same, hence we have Hf = d dx0 fL. In a word, LH and HL are both uniquely determined by the gradient direction H. Due to the above discussion, we immediately obtain the following proposition. Proposition 3.3.8.1. Let H be the ﬁeld of unit tangent vector in gradient directions of ρ, for any X and any f, equations [X, H] = d dx0 X, Hf = d dx0 fL (15) (15) hold if and only if d dx0 is the ﬁeld of unit tangent vector in gradient directions of ρ. hold if and only if d dx0 is the ﬁeld of unit tangent vector in gradient directions of ρ Deﬁnition 3.3.8.1. Equation [X, H] = d dx0 X is called the general Heisenberg equation. Equa- tion Hf = d dx0 fL is called the general Schrödinger equation. Discussion 3.3.8.2. Both the two equations describe gradient direction ﬁeld. H not only can be taken as the gradient direction determined by Dρ, but can also taken as the gradient direction of any other geometrical property. According to Deﬁnition 3.3.4.1 , gradient operator is a universal geometrical property of geometrical manifold, so these two equations remain unchanged under arbitrary tranformation of reference-systems. These two equations reﬂect two descriptions of the same geometrical property by two mutually dual linear spaces, which are tangent bundle and cotangent bundle. which can be called the conservation of energy-momentum of ρ. Proof. According to Proposition 3.3.6.3 , E0¯ε0 N = pN in gradient direction of ρ. Then Proof. According to Proposition 3.3.6.3 , E0¯ε0 N = pN in gradient direction of ρ. Then WMN ;M = ¯ε0 MpN ;M = pN ;M ¯ε0 M + ¯ε0 ;M M pN = pN ;0 + ¯ε0 ;M M pN = −YMN ;M, Thus, (WMN + YMN);M = 0, that is TMN ;M = 0. WMN ;M = ¯ε0 MpN ;M = pN ;M ¯ε0 M + ¯ε0 ;M M pN = pN ;0 + ¯ε0 ;M M pN = −YMN ;M, Thus, (WMN + YMN);M = 0, that is TMN ;M = 0. WMN ;M = ¯ε0 MpN ;M = pN ;M ¯ε0 M + ¯ε0 ;M M pN = pN ;0 + ¯ε0 ;M M pN = −YMN ;M, M M WMN ;M = ¯ε0 MpN ;M = pN ;M ¯ε0 M + ¯ε0 ;M M pN = pN ;0 + ¯ε0 ;M M pN = −YMN ;M, ⊓⊔ 32 Zhao-Hui Man 3.3.8 Two dual descriptions of gradient direction ﬁeld From real-valued evolution equations on tangent bundle and cotangent bundle, we can surely deduce complex-valued evolution equations on operator space and state space, such as section 5.6 . What they describe is none other than gradient direction. It is true both for wave function and ﬁeld function. We can say that the value of gradient direction is determined by intrinsic geometry, and it is independent of either real-valued form or complex-valued form. The eﬀectivenesses of describing intrinsic geometry with complex-valued form and real-valued form are the same. In a word, there is no need to be constrained on theoretical forms. Intrinsic geometry and gradient direction are the very essences which should be grasped. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 33 The only necessity of using complex form is that it is the most convenient for describing the coherent superposition of propagator. However, it is a diﬀerent problem from that of this section and it will be speciﬁcally discussed in the next section. In order to achieve the purpose of clarifying concepts, it is beneﬁcial to separate the two equations here from the construction of coherent superposition of propagators of the next section. 3.4 Intrinsic geometrical treatment of propagator and wave function Discussion 3.4.1. Intrinsic geometry relies on reference-system on manifold, hence: (1) It is impossible for the coordinate of a single point to reﬂect the full picture of intrinsic geometrical shape of manifold. (1) It is impossible for the coordinate of a single point to reﬂect the full picture of intrinsic geometrical shape of manifold. (2) It is also impossible for a single gradient direction to reﬂect the full picture of intrinsic geometrical shape of manifold. In order to describe the intrinsic geometrical shape of (M, g), it is meaningful to study distribution of gradient directions of ρ on (M, g). Intuitively: In order to describe the intrinsic geometrical shape of (M, g), it is meaningful to study distribution of gradient directions of ρ on (M, g). Intuitively: ) If g is trivial, the gradient direction ﬁeld of ρ distributes uniformly on the ﬂat (M, g). (2) If g is non-trivial, the intrinsic geometrical shape of (M, g) eﬀects the distribution of gradient directions of ρ. Deﬁnition 3.4.1. Let ρ be a geometrical property determined by f, then ρ is a universal geomet- rical property on (M, g), and H ≜∇ρ is a gradient direction ﬁeld of ρ on (M, g). Deﬁnition 3.4.1. Let ρ be a geometrical property determined by f, then ρ is a universal geomet- rical property on (M, g), and H ≜∇ρ is a gradient direction ﬁeld of ρ on (M, g). Let T be the totality of ﬂat transformations deﬁned in section 2.4 . ∀T ∈T, the ﬂat transformation T : f 7→Tf induces a transformation T∗: ρ 7→T∗ρ. Denote HT ≜∇(T∗ρ), \|ρ\| ≜{ρT ≜T∗ρ\|T ∈T} and \|H\| ≜{HT \|T ∈T}. Let T be the totality of ﬂat transformations deﬁned in section 2.4 . ∀T ∈T, the ﬂat transformation T : f 7→Tf induces a transformation T∗: ρ 7→T∗ρ. Denote HT ≜∇(T∗ρ), \|ρ\| ≜{ρT ≜T∗ρ\|T ∈T} and \|H\| ≜{HT \|T ∈T}. Let φH be the one-parameter group of diﬀeomorphisms corresponding to H, the parameter of which is x0. ∀a ∈M, suppose φH,a is the orbit through point a. Without loss of generality, let a = φH,a(0). We say φ\|H\|,a ≜{φX,a\|X ∈\|H\|} is a system of gradient lines of \|ρ\| through a. ∀t ∈R+, we say φ\|H\|,a(t) ≜{φX,a(t)\|X ∈\|H\|} is the evolution image of a at x0 = t. Let φH be the one-parameter group of diﬀeomorphisms corresponding to H, the parameter of which is x0. 3.4 Intrinsic geometrical treatment of propagator and wave function Further- more, ∀t ∈R+, we say the measure P φ\|OΩ\|,a (t) = P g−1 ∗∗ φ\|HΩ\|,a (t) is the distribution of gradient directions of \|ρ\| on φ\|HΩ\|,a(t), or the evolution distribution of \|ρ\| at time x0 = t after starting from a in directions \|HΩ\|. Due to T ∼= GL(D, R), let Ωbe a certain neighborhood of T, with respect to the topology of GL(D, R). Now at the starting point a, we say \|HΩ(a)\| is a neighborhood of HT (a), and \|OΩ(a)\| ≜g−1 ∗(\|HΩ(a)\|) is a neighborhood of OT (a) ≜g−1 ∗(HT (a)). When Ωis suﬃciently small, \|HΩ(a)\| and \|OΩ(a)\| are both suﬃciently small, and ∀t ∈R+, φ\|HΩ\|,a(t) and φ\|OΩ\|,a(t) are also suﬃciently small. Concretely, when Ω→T, HT = lim Ω→T \|HΩ\|, HT (a) = lim Ω→T \|HΩ(a)\|, and the evolution image φ\|HΩ\|,a(t) of a at t will approach to a point bT ≜φHT ,a(t) = lim \|HΩ(a)\|→HT (a) φ\|HΩ\|,a(t). The limit wa (bT ) ≜dVOT dVHT ≜lim Ω→T P φ\|OΩ\|,a (t) P φ\|HΩ\|,a (t) = lim Ω→T P g−1 ∗∗ φ\|HΩ\|,a (t) P φ\|HΩ\|,a (t) (16) (16) is called the distribution density of gradient directions at time x0 = t after starting from a in direction HT, or distribution density of evolution. Remark 3.4.2. Radon-Nikodym theorem [54] guarantees the existence of such a limit. And evidently such a distribution density is an intrinsic geometrical property of (M, g). For any two points a and b on manifold M, it anyway makes sense to discuss the gradient line of \|ρ\| from a to b. It is because even if the gradient line of ρ starting from a does not pass through b, it just only needs to carry out a certain ﬂat transformation T deﬁned in section 2.4 to obtain a ρ′ ≜T∗ρ so that the gradient line of ρ′ starting from a can just exactly pass through b. Intuitively, when \|ρ\| takes two diﬀerent initial directions of motion, \|ρ\| presents as ρ and ρ′, respectively. Deﬁnition 3.4.3. (Evolutor) ∀a, b ∈M, if ∃ρ′ ∈\|ρ\| such that a and b are both on the gradient line L(b, a) of ρ′, then we say L(b, a) is a gradient line of \|ρ\|. According to Deﬁnition 3.3.6.2 , let sL(b, a) be the evolution quantity on L(b, a). 3.4 Intrinsic geometrical treatment of propagator and wave function Denote rL(b, a) ≜ p wa(b), RL(b, a) ≜rL(b, a)eisL(b,a), which are both intrinsic geometrical properties of (M, g). We say RL(b, a) is the evolutor of \|ρ\| on L(b, a). Deﬁnition 3.4.4. (Propagator) Let L(b, a) be the totality of gradient lines of \|ρ\| from a to b. ∀L(b, a) ∈L(b, a), let RL(b, a) be the evolutor of \|ρ\| on L(b, a). The intrinsic geometrical property K(b, a) ≜ X L∈L(b,a) RL(b, a) (17) (17) is called the propagator of \|ρ\| from a to b. 3.4 Intrinsic geometrical treatment of propagator and wave function ∀a ∈M, suppose φH,a is the orbit through point a. Without loss of generality, let a = φH,a(0). We say φ\|H\|,a ≜{φX,a\|X ∈\|H\|} is a system of gradient lines of \|ρ\| through a. ∀t ∈R+, we say φ\|H\|,a(t) ≜{φX,a(t)\|X ∈\|H\|} is the evolution image of a at x0 = t. ∀Ω⊆T, \|HΩ\| ≜{HT \|T ∈Ω} is a subset of \|H\|, and φ\|HΩ\|,a ≜{φX,a\|X ∈\|HΩ\|} is a subset of φ\|H\|,a. Correspondingly, ∀t ∈R+, φ\|HΩ\|,a(t) ≜{φH,a(t)\|X ∈\|XΩ\|} is a subset of φ\|H\|,a(t). ∀a ∈M, the restrictions of \|H\| and \|HΩ\| at a are denoted by \|H(a)\| ≜{HT (a)\|T ∈T} and \|HΩ(a)\| ≜{HT (a)\|T ∈Ω}, respectively. Remark 3.4.1. When t = 0, intuitively, the gradient directions \|H(a)\| of \|ρ\| start from a and point to all directions around a uniformly. If (M, g) is not ﬂat, when evolving to a certain time t > 0, the distribution of gradient directions on φ\|H\|,a(t) is no longer as uniform as they are around a. The following deﬁnition precisely characterizes such a distribution, which thereby reﬂect the intrinsic geometrical shape in a new approach. Deﬁnition 3.4.2. (Evolution distribution). Let transformation Lg−1 act on g, then we obtain the trivial e ≜Lg−1(g). Now (M, g) is sent to a ﬂat (M, e), and the gradient direction ﬁeld \|H\| of \|ρ\| on (M, g) is sent to a gradient direction ﬁeld \|O\| of \|ρ\| on (M, e). Correspondingly, φ\|H\|,a(t) is Zhao-Hui Man 34 sent to φ\|O\|,a(t). In a word, Lg−1 induces the following two maps: sent to φ\|O\|,a(t). In a word, Lg−1 induces the following two maps: g−1 ∗ : \|H\| →\|O\|, g−1 ∗∗: φ\|H\|,a →φ\|O\|,a. g−1 ∗ : \|H\| →\|O\|, g−1 ∗∗: φ\|H\|,a →φ\|O\|,a. ∀\|HΩ\| ⊆\|H\|, denote \|OΩ\| ≜g−1 ∗ (\|HΩ\|) ⊆\|O\| and φ\|OΩ\|,a ≜g−1 ∗∗ φ\|HΩ\|,a ⊆φ\|O\|,a. Further- more, ∀t ∈R+, we say the measure P φ\|OΩ\|,a (t) = P g−1 ∗∗ φ\|HΩ\|,a (t) is the distribution of gradient directions of \|ρ\| on φ\|HΩ\|,a(t), or the evolution distribution of \|ρ\| at time x0 = t after starting from a in directions \|HΩ\|. ∀\|HΩ\| ⊆\|H\|, denote \|OΩ\| ≜g−1 ∗ (\|HΩ\|) ⊆\|O\| and φ\|OΩ\|,a ≜g−1 ∗∗ φ\|HΩ\|,a ⊆φ\|O\|,a. is called the propagator of \|ρ\| from a to b. The propagator K(a, q) is an intrinsic geometrical property of (M, g), so the above deﬁned wave function ψ is also an intrinsic geometrical property of (M, g). Remark 3.4.5. The propagator K(a, q) is an intrinsic geometrical property of (M, g), so the above deﬁned wave function ψ is also an intrinsic geometrical property of (M, g). is called the propagator of \|ρ\| from a to b. is called the propagator of \|ρ\| from a to b. Remark 3.4.3. Abstractly, propagator is deﬁned as the Green function of evolution equation. Concretely, propagator still needs a constructive deﬁnition. One method is to construct with A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 35 Feynman path integral [22] R xb xa eiSDx(t), which is expressed in form of functional integral. However, until now the functional integral has strict deﬁnition just in some special cases, but the strict deﬁnition in general case is still an unsolved problem. Feynman path integral [22] R xb xa eiSDx(t), which is expressed in form of functional integral. However, until now the functional integral has strict deﬁnition just in some special cases, but the strict deﬁnition in general case is still an unsolved problem. Deﬁnition 3.4.4 reduces the scope of summation to the totality of gradient lines from a to b. If (M, g) is ﬂat, \|ρ\| has a unique gradient line from a to b, but it is not unique for general (M, g). Remark 3.4.4. As the simplest example, consider the propagator of free particle. In this case (M, g) is ﬂat. In sense of Remark 3.4.1 , the system of gradient lines of \|ρ\| starting from a spreads uniformly in all directions around a. No matter where b is, wa(b) is identically equal to 1. Then for a ﬁxed b, the evolutor on gradient line L(b, a) is RL(b, a) = rL(b, a)eisL(b,a) = p wa(b)eisL(b,a) = eisL(b,a). Because there is only one element in L(b, a), the propagator is K(b, a) = RL(b, a) = eisL(b,a). Of course, if normalizing on wavefront, there would be a coeﬃcient of normalization. Deﬁnition 3.4.5. (Wave function). Let a be a point on geometrical manifold (M, g). ∀a0 ∈M, d(a, a0) is the geodesic distance between a0 and a. Denote Σa(a0) ≜{q ∈M\|d(a, q) = d(a, a0)}. If function ψ : M →C satisﬁes both the following two conditions, then ψ is called a wave function of \|ρ\| on (M, g). (1) ∃r > 0 such that lim d(a,a0)→r ψ(a0) = 0, or lim d(a,a0)→∞ψ(a0) = 0. (1) ∃r > 0 such that lim d(a,a0)→r ψ(a0) = 0, or lim d(a,a0)→∞ψ(a0) = 0. (2) ∀a0, a ∈M, (2) ∀a0, a ∈M, ψ(a) = Z Σa(a0) K(a, q)ψ(q)dσq. ψ(a) = Z Σa(a0) K(a, q)ψ(q)dσq. Remark 3.4.5. 4 Intrinsic geometrical treatment of inversion transformation 4 Intrinsic geometrical treatment of inversion transformation 4 Intrinsic geometrical treatment of inversion transformation In this section, indices of internal and external space are taken values according to Deﬁnition 5.1.1 . In this section, indices of internal and external space are taken values according to Deﬁnition 5.1.1 . Deﬁnition 4.1. Let the local coordinate representation of reference-system k be x′j = −δj i xi, x′n = δn mxm, then we say P ≜L[k] : xi →−xi, xm →xm is parity inversion. Let the local coordinate representation of reference-system h be x′j = δj i xi, x′n = −δn mxm, then we say C ≜ L[h] : xi →xi, xm →−xm is charge conjugate inversion. In addition, denote T0 : x0 →−x0, which is called time coordinate invesion. Deﬁnition 4.2. Reviewing Deﬁnition 3.2.1 , without loss of generality, positive or negative sign of metric, which marks two opposite directions of evolution, is independent of positive or negative sign of coordinate. Let N be a closed submanifold of M, and its metric be dx(N). The transformation T (N) 0 : dx(N) →−dx(N) is called a single inversion of space metric on N. Specially, when N = M, T (M) 0 : dx0 →−dx0 is called a single inversion of time metric. Denote the totality of closed submanifolds of M by B(M), and denote T (M) ≜Q B∈B(M) T (B) 0 . We say T (M) is total inversion of metrics. Deﬁnition 4.3. T ≜T (M)T0 is called time inversion. The joint transformation of total inversion of coordinates CPT0 and total inversion of metrics T (M) is called space-time inversion, that is CPT0T (M) = CPT. Remark 4.1. Summerize the above deﬁnitions, then we have: Remark 4.1. Summerize the above deﬁnitions, then we have: CPT0 : xR →−xR, x0 →−x0, dxR →dxR, dx0 →dx0, T (M) : xR →xR, x0 →x0, dxR →−dxR, dx0 →−dx0, CPT : xR →−xR, x0 →−x0, dxR →−dxR, dx0 →−dx0. Proposition 4.1. Consider CPT on g. Denote s ≜ Z L Dρ, and DP eis ≜ ∂ ∂xP + i[ρΓP ] eis, then: (1) CPT : Dρ →Dρ, (2) CPT : DP eis →−DP eis. Proof. (1) On one hand, for CP : xR →−xR we have CP : ∂ ∂xR →− ∂ ∂xR. According to the deﬁnition of simple connection we obtain CP : Γ M NP →−Γ M NP. In consideration of that ρ is determined by f, so ρ is required to remain unchanged under transformations of g. Due to pR ≜ ∂ρ ∂xR + [ρΓP ] we know CP : pR →−pR. On the other hand, according to the deﬁnition of T we immediately obtain T : dxR →−dxR. In summary, we have CPT : pRdxR → (−pR)(−dxR) that is CPT : Dρ →Dρ Proposition 4.1. Consider CPT on g. Denote s ≜ Z L Dρ, and DP eis ≜ ∂ ∂xP + i[ρΓP ] eis, then: (1) CPT D D (2) CPT D is D is Proposition 4.1. Consider CPT on g. Denote s ≜ Z L Dρ, and DP eis ≜ ∂ ∂xP + i[ρΓP ] eis, then: (1) CPT D D (2) CPT D is D is Proposition 4.1. Consider CPT on g. Denote s ≜ Z L Dρ, and DP eis ≜ ∂ ∂xP + i[ρΓP ] eis, then: (1) CPT : Dρ →Dρ, (2) CPT : DP eis →−DP eis. Proof. (1) On one hand, for CP : xR →−xR we have CP : ∂ ∂xR →− ∂ ∂xR. According to the deﬁnition of simple connection we obtain CP : Γ M NP →−Γ M NP. In consideration of that ρ is determined by f, so ρ is required to remain unchanged under transformations of g. Due to pR ≜ ∂ρ ∂xR + [ρΓP ] we know CP : pR →−pR. On the other hand, according to the deﬁnition of T we immediately obtain T : dxR →−dxR. In summary, we have CPT : pRdxR → (−pR)(−dxR), that is CPT : Dρ →Dρ. Proposition 4.1. Consider CPT on g. 3.5 Summary of this section 1. This section proposes an intrinsic geometrical solution for Hilbert’s 6th problem at the most basic level, that is, starting from an axiom, based on intrinsic geometry, the theoretical framework at the most basic level of physics is deduced in sense of pure mathematics. 2. Postulates that have a status as principle in physics are theorems that hold automatically in intrinsic geometry, such as Yang-Mills ﬁeld equation, Lorentz force equation, energy-momentum equation, conservation law of energy-momentum, gravitational ﬁeld equation(see Discussion 5.4.4 ), least action principle, Schrödinger equation, Heisenberg equation, Dirac equation(see section 5.6 ), etc., which in intrinsic geometry are no longer necessary to be regarded as principles and postulates. In addition, this section adopts general coordinate form, the evolution parameter of which is x0. In section 5.2 , the Minkowski coordinate will be constructed, the evolution parameter of which is xτ. It has to be emphasized that no matter what coordinate forms are adopted, their geometrical essences are the same. Intrinsic geometrical properties in some special cases will be discussed in the following sections. 36 Zhao-Hui Man 5.1 Existence and uniqueness of submanifold with classical spacetime 5.1 Existence and uniqueness of submanifold with classical spacetime Deﬁnition 5.1.1. Suppose M = P × N, D ≜dimM and r ≜dimP = 3. According to Deﬁnition 3.2.2 , we have a submanifold of external space P and a submanifold of internal space N. P inherits coordinate {ξs}{xi} from M, and N inherits coordinate {ξa}{xm} from M. The values of indices are speciﬁed as follows. (1) Total indices of basis coordinate frame ξ are A, B, C, D = 1, 2, · · · , D. Total indices of performance coordinate frame x are M, N, P, Q = 1, 2, · · · , D. (1) Total indices of basis coordinate frame ξ are A, B, C, D = 1, 2, · · · , D. Total indices of performance coordinate frame x are M, N, P, Q = 1, 2, · · · , D. (2) External indices of ξ are s, t, u, v = 1, 2, · · · , r. Internal indices of ξ are a, b, c, d = r + 1, r + 2, · · · , D. (3) External indices of x are i, j, k, l = 1, 2, · · · , r. Internal indices of x are m, n, p, q = r + 1, r + 2, · · · , D. (4) Regular indices of ξ are S, T, U, V = 1, 2, · · · , r, τ. Minkowski indices of ξ are α, β, γ, δ = 0, 1, 2, · · · , r. (5) Regular indices of x are I, J, K, L = 1, 2, · · · , r, τ. Minkowski indices of x are µ, ν, ρ, σ = 0, 1, 2, · · · , r. Deﬁnition 5.1.2. Let there be a smooth tangent vector ﬁeld X on (M, f). If ∀p ∈M, X(p) = bA ∂ ∂ξA p = cM ∂ ∂xM p satisﬁes that ba are not all zero and cm are not all zero, then we say X is internal-directed. p internal-directed. Proposition 5.1.1. Suppose M = P × N and X is a smooth tangent vector ﬁeld on M. Fix a point o ∈M. Remark 4.1. Summerize the above deﬁnitions, then we have: Due to T : dxR →−dxR, then T : pRdxR →−pRdxR, that is A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 37 T : Dρ →−Dρ, hence T : s →−s. Moreover, T : DP eis →DP e−is = DP (eis)∗. In sum- mary, we have CPT : DP eis →−DP (e−is)∗= −DP eis. ⊓⊔ ⊓⊔ Remark 4.2. The above proposition gives the intrinsic geometrical origin of CPT invariance. In addition, in physics there is a complex conjugation in time inversion about wave function T : ψ(x, t) →ψ∗(x, −t), the mathematical orgin of which can essentially be attributed to Deﬁnition 4.1 , Deﬁnition 4.2 and Deﬁnition 4.3 . In fact we have the following proposition, where the coordinate ˜xµ is Minkowski coordinate deﬁned in section 5.2 such that ˜xi = xi, ˜x0 = x0, and Minkowski metric satisﬁes (d˜xτ)2 = (d˜x0)2 −P(d˜xi)2, ˜mτ ≜˜ρ;τ. Remark 4.2. The above proposition gives the intrinsic geometrical origin of CPT invariance. In addition, in physics there is a complex conjugation in time inversion about wave function T : ψ(x, t) →ψ∗(x, −t), the mathematical orgin of which can essentially be attributed to Deﬁnition 4.1 , Deﬁnition 4.2 and Deﬁnition 4.3 . In fact we have the following proposition, where the coordinate ˜xµ is Minkowski coordinate deﬁned in section 5.2 such that ˜xi = xi, ˜x0 = x0, and Minkowski metric satisﬁes (d˜xτ)2 = (d˜x0)2 −P i (d˜xi)2, ˜mτ ≜˜ρ;τ. Proposition 4.2. Denote S ≜ Z L ˜mτd˜xτ and ψ(˜xi, ˜x0) ≜f(˜xi, ˜x0)eiS, then T : ψ(˜xi, ˜x0) → ψ∗(˜xi, −˜x0). ψ∗(˜xi, −˜x0). Proof. According to Deﬁnition 4.3 we know T : ˜mτ →˜mτ, d˜xτ →−d˜xτ, ˜x0 →−˜x0, therefore T : ˜mτd˜xτ →−˜mτd˜xτ, then T : S →−S, f(˜xi, ˜x0) →f(˜xi, −˜x0), and furthermore T : f(˜xi, ˜x0)eiS →f(˜xi, −˜x0)e−iS, that is T : ψ(˜xi, ˜x0) →ψ∗(˜xi, −˜x0). ⊓⊔ Proof. According to Deﬁnition 4.3 we know T : ˜mτ →˜mτ, d˜xτ →−d˜xτ, ˜x0 →−˜x0, therefore T : ˜mτd˜xτ →−˜mτd˜xτ, then T : S →−S, f(˜xi, ˜x0) →f(˜xi, −˜x0), and furthermore T : f(˜xi, ˜x0)eiS →f(˜xi, −˜x0)e−iS, that is T : ψ(˜xi, ˜x0) →ψ∗(˜xi, −˜x0). ⊓⊔ Remark 4.1. Summerize the above deﬁnitions, then we have: Denote s ≜ Z L Dρ, and DP eis ≜ ∂ ∂xP + i[ρΓP ] eis, then: (1) CPT : Dρ →Dρ, (2) CPT : DP eis →−DP eis. Proof. (1) On one hand, for CP : xR →−xR we have CP : ∂ ∂xR →− ∂ ∂xR. According to the deﬁnition of simple connection we obtain CP : Γ M NP →−Γ M NP. In consideration of that ρ is determined by f, so ρ is required to remain unchanged under transformations of g. Due to pR ≜ ∂ρ ∂xR + [ρΓP ] we know CP : pR →−pR. On the other hand, according to the deﬁnition of T we immediately obtain T : dxR →−dxR. In summary, we have CPT : pRdxR → (−pR)(−dxR), that is CPT : Dρ →Dρ. Due to pR ≜ ∂ρ ∂xR + [ρΓP ] we know CP : pR →−pR. On the other hand, according to the deﬁnition of T we immediately obtain T : dxR →−dxR. In summary, we have CPT : pRdxR → (−pR)(−dxR), that is CPT : Dρ →Dρ. (2) On one hand, in consideration of CP : ∂ ∂xP + i[ρΓP ] →− ∂ ∂xP −i[ρΓP ], and due to s ≜ Z L Dρ = Z L pRdxR we know CP : s →−s, hence CP : ∂ ∂xP + i[ρΓP ] eis → − ∂ ∂xP −i[ρΓP ] e−is, that is CP : DP eis →−DP e−is. On the other hand, ∂ ∂xP + i[ρΓP ] and pR are both independent of metrics, therefore ∂ ∂xP + i[ρΓP ] and pR both remain unchanged under transformation T. Due to T : dxR →−dxR, then T : pRdxR →−pRdxR, that is (2) On one hand, in consideration of CP : ∂ ∂xP + i[ρΓP ] →− ∂ ∂xP −i[ρΓP ], and due to s ≜ Z L Dρ = Z L pRdxR we know CP : s →−s, hence CP : ∂ ∂xP + i[ρΓP ] eis → − ∂ ∂xP −i[ρΓP ] e−is, that is CP : DP eis →−DP e−is. On the other hand, ∂ ∂xP + i[ρΓP ] and pR are both independent of metrics, therefore ∂ ∂xP + i[ρΓP ] and pR both remain unchanged under transformation T. 5.1 Existence and uniqueness of submanifold with classical spacetime If X is internal-directed, then: (1) There exist a unique (r + 1)-dimensional regular submanifold γ : ˜ M →M, p 7→p and a unique smooth tangent vector ﬁeld ˜X on ˜ M such that: (i) P × {o} is a closed submanifold of 38 Zhao-Hui Man ˜ M, (ii) tangent map γ∗: T( ˜ M) →T(M) satisﬁes that ∀q ∈˜ M, γ∗: ˜X(q) 7→X(q). Such an ˜ M is called a submanifold with classical spacetime determined by X through o. ˜ M, (ii) tangent map γ∗: T( ˜ M) →T(M) satisﬁes that ∀q ∈˜ M, γ∗: ˜X(q) 7→X(q). Such an ˜ M is called a submanifold with classical spacetime determined by X through o. (2) Let φX be the one-parameter group of diﬀeomorphisms on M corresponding to X, and φ ˜ X be the one-parameter group of diﬀeomorphisms on ˜ M corresponding to ˜X. Thus, we have φ ˜ X = φX\| ˜ M. (2) Let φX be the one-parameter group of diﬀeomorphisms on M corresponding to X, and φ ˜ X be the one-parameter group of diﬀeomorphisms on ˜ M corresponding to ˜X. Thus, we have φ ˜ X = φX\| ˜ M. Proof. Step 1: construction of ˜ M. We can deﬁne a closed submanifold P × {o} on M through o via parameter equation xm = xm o . Then let φX : M × R →M be the one-parameter group of diﬀeomorphisms corresponding to X. Restrict φX to P × {o} and we obtain Proof. Step 1: construction of ˜ M. We can deﬁne a closed submanifold P × {o} on M through o via parameter equation xm = xm o . Then let φX : M × R →M be the one-parameter group of diﬀeomorphisms corresponding to X. Restrict φX to P × {o} and we obtain φX\|P×{o} : P × {o} × {t} 7→P ′ × {o′}, where points o and o′ are on the same orbit Lo ≜φX,o. P × {o} and P ′ × {o′} are both homeomorphic to P. If we do not distinguish P and P ′, we have where points o and o′ are on the same orbit Lo ≜φX,o. P × {o} and P ′ × {o′} are both homeomorphic to P. If we do not distinguish P and P ′, we have φX\|P×{o} : P × {o} × R →P × Lo. 5.1 Existence and uniqueness of submanifold with classical spacetime Then consider all of such {o} on the entire orbit Lo, and we obtain a map Then consider all of such {o} on the entire orbit Lo, and we obtain a map φX\|P×Lo : P × Lo × R →P × Lo. Denote ˜ M ≜P × Lo, then φX\| ˜ M : ˜ M × R →˜ M constitutes a one-parameter group of diﬀeomorphisms on ˜ M. Step 2: constructions of γ : ˜ M →M and ˜X. Because X is internal-directed, the restriction of X to Lo ≜φX,o : R →M is non-vanishing everywhere and Lo is an injection. The image set of Lo can also be denoted by Lo. ∀t ∈R, q ≜φX,o(t) ∈Lo, we can deﬁne a closed submanifold Nq on M through q via parameter equation xi = xi q, and Nq is homeomorphic to N. Due to the one-to-one correspondence between q and Nq, Lo →N is a regular imbedding. Furthermore: of Lo can also be denoted by Lo. ∀t ∈R, q ≜φX,o(t) ∈Lo, we can deﬁne a closed submanifold Nq on M through q via parameter equation xi = xi q, and Nq is homeomorphic to N. Due to the one-to-one correspondence between q and Nq, Lo →N is a regular imbedding. Furthermore: (1) γ : P × Lo →P × N is a regular imbedding, that is, γ : ˜ M →M. Hence the tangent map γ∗: T( ˜ M) →T(M) is an injection. Therefore, the smooth tangent vector ˜X which satisﬁes ∀q ∈˜ M, γ∗: ˜X(q) 7→X(q) is uniquely deﬁned by X via γ−1 ∗. (1) γ : P × Lo →P × N is a regular imbedding, that is, γ : ˜ M →M. Hence the tangent map γ∗: T( ˜ M) →T(M) is an injection. Therefore, the smooth tangent vector ˜X which satisﬁes ∀q ∈˜ M, γ∗: ˜X(q) 7→X(q) is uniquely deﬁned by X via γ−1 ∗. (2) We notice that o ∈˜ M, hence Lo ≜φX,o is an orbit of φX\| ˜ M. In consideration of that Lo uniquely determines γ, and γ uniquely determines γ∗, and γ−1 ∗ uniquely determines ˜X, so ﬁnally ˜X is uniquely determined by φX\| ˜ M. Thus, we have φ ˜ X = φX\| ˜ M. 5.2 Intrinsic geometrical treatment of gravitational ﬁeld Proposition 5.2.1. Let ˜ M be the submanifold with classical spacetime determined by X on (M, f), and L be a path on an orbit of φ ˜ X of ˜ M. ∀p ∈L, suppose on neighborhood U that f(p) : ξA = ξA(xM) = ξA(x0), ξ0 = ξ0(x0). Thus: (1) There exists a unique local reference-system ˜f(p) on ˜U ≜U ∩˜ M such that Thus: (1) There exists a unique local reference-system ˜f(p) on ˜U ≜U ∩˜ M such that ˜f(p) : ξU = ξU(xK) = ξU(x0), ξ0 = ξ0(x0) and (dxτ)2 = D P m=r+1 (dxm)2, (dξτ)2 = D P a=r+1 (dξa)2. (2) The above coordinate frames ( ˜U, ξU) and ( ˜U, xK) of ˜f(p) uniquely determine coordinate frames ( ˜U, ˜ξα) and ( ˜U, ˜xµ), such that (2) The above coordinate frames ( ˜U, ξU) and ( ˜U, xK) of ˜f(p) uniquely determine coordinate frames ( ˜U, ˜ξα) and ( ˜U, ˜xµ), such that frames ( ˜U, ˜ξα) and ( ˜U, ˜xµ), such that ˜f(p) : ˜ξα = ˜ξα (˜xµ) = ˜ξα (˜xτ) , ˜ξτ = ˜ξτ (˜xτ) and coordinates ξ = ξ , ξ = ξ , ξ = ξ , x = x , x = x , x = x . Proof. (1) According to the proof of previous proposition, if L is on an orbit of φ ˜ X, L is a regular submanifold of N. Let the metrics on N be (dxτ)2 = D P m=r+1 (dxm)2 and (dξτ)2 = D P a=r+1 (dξa)2. Review Deﬁnition 3.3.1.3 and we know the regular imbedding π : L →N, q 7→q induces parameter equations xm = xm τ (xτ) and ξa = ξa τ (ξτ) of L. 5.1 Existence and uniqueness of submanifold with classical spacetime In summary of (1) and (2), it also indicates that ˜ M is determined by X, therefore it is uni In summary of (1) and (2), it also indicates that ˜ M is determined by X, therefore it is unique. ⊓⊔ ⊓⊔ Remark 5.1.1. (1) ˜ M is not independent of M, but determined by smooth tangent vector ﬁeld X on M. Remark 5.1.1. (1) ˜ M is not independent of M, but determined by smooth tangent vector ﬁeld X on M. Remark 5.1.1. (1) ˜ M is not independent of M, but determined by smooth tangent vector ﬁeld X on M. X on M. (2) ˜ M is a regular submanifold of M, so not all intrinsic geometrical properties of M can be inherited by ˜ M. (3) The correspondence between ˜X and the restriction of X to ˜ M is one-to-one. For conve- nience, we later will not distinguish the notations X and ˜X on ˜ M, but uniformly denote them by X. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 39 (4) An arbitrary path ˜L : T →˜ M, t 7→p on ˜ M uniquely corresponds to a path L ≜γ ◦˜L : T →M, t 7→p on M. Evidently the image sets of L and ˜L are the same, that is, L(T) = ˜L(T). For convenience, we later will not distinguish the notations L and ˜L on ˜ M, but uniformly denote them by L. Deﬁnition 5.2.1. ˜f is called a classical spacetime reference-system on ˜ M, and ( ˜ M, ˜f) is called a gravitational manifold. ( ˜U, ξU) and ( ˜U, xK) are called regular coordinate frames on ( ˜ M, ˜f), meanwhile ( ˜U, ˜ξα) and ( ˜U, ˜xµ) are called Minkowski coordinate frames on ( ˜ M, ˜f). Deﬁnition 5.2.1. ˜f is called a classical spacetime reference-system on ˜ M, and ( ˜ M, ˜f) is called a gravitational manifold. ( ˜U, ξU) and ( ˜U, xK) are called regular coordinate frames on ( ˜ M, ˜f), meanwhile ( ˜U, ˜ξα) and ( ˜U, ˜xµ) are called Minkowski coordinate frames on ( ˜ M, ˜f). Remark 5.2.1. ˜f is uniquely determined by f, and ˜f encapsulates the internal space of f. Although ( ˜ M, ˜f) can reﬂect intrinsic geometrical properties of external space of (M, f), it cannot totally reﬂect intrinsic geometrical properties of internal space of (M, f). There is a further illustration in Discussion 5.4.2 . In addition, ˜f presents locally as ˜f(p), so the mathematical origin of the principle of equiv- alence has been able to be attributed to Deﬁnition 2.1.2 , Deﬁnition 2.2.2 and Deﬁnition 5.2.1 . Of course its physical connotation can only be endowed by the fundamental axiom of section 3.1 . Besides, with respect to the gravitational ﬁeld equation, see Discussion 5.4.4 . In addition, ˜f presents locally as ˜f(p), so the mathematical origin of the principle of equiv- alence has been able to be attributed to Deﬁnition 2.1.2 , Deﬁnition 2.2.2 and Deﬁnition 5.2.1 . Of course its physical connotation can only be endowed by the fundamental axiom of section 3.1 . Besides, with respect to the gravitational ﬁeld equation, see Discussion 5.4.4 . It is well-known that there is no logically strict deﬁnition of inertial system in physics. However, now we can give a strict deﬁnition to inertial system from the perspective of intrinsic geometry. It is a reference-system, not a coordinate frame. Deﬁnition 5.2.2. Suppose we have a geometrical manifold ( ˜ M, ˜g). F˜g is a transformation induced by ˜g. (1) According to Discussion 5.3.2 , if d˜ζτ = d˜xτ, then we must have ˜Gµν = ˜ ∆αβ ˜Bα µ ˜Bβ ν = ˜Eµν. Thus, we say ˜g is orthogonal, F˜g is an orthogonal transformation, and ( ˜ M, ˜g) is an isotropic spacetime. For convenience, d˜ζτ and d˜xτ are uniformly denoted by dτ. (1) According to Discussion 5.3.2 , if d˜ζτ = d˜xτ, then we must have ˜Gµν = ˜ ∆αβ ˜Bα µ ˜Bβ ν = ˜Eµν. Thus, we say ˜g is orthogonal, F˜g is an orthogonal transformation, and ( ˜ M, ˜g) is an isotropic spacetime. 5.2 Intrinsic geometrical treatment of gravitational ﬁeld Then substitute them into f(p) and we obtain ξA = ξA xM = ξA x0 ⇔    ξu = ξu xk, xm τ (xτ) = ξu L x0 ξa τ (ξτ) = ξa xk, xm τ (xτ) = ξa L x0 ⇔    ξu = ξu xk, xm τ (xτ) = ξu L x0 ξτ = (ξa τ )−1 ◦ξa xk, xm τ (xτ) = (ξa τ )−1 ◦ξa L x0 ⇔    ξu = ξu τ xk, xτ = ξu L x0 ξτ = ξτ xk, xτ = ξτ L x0 ⇔ξU = ξU(xK) = ξU L (x0), abbreviated to ξU = ξU(xK) = ξU(x0). (2) As same as the above, we also obtain xK = xK(ξU) = xK(ξ0). The relation between two parameters x0 and xτ of L can be expressed as xτ = xτ L(ξ0(x0)), and the relation between two parameters ξ0 and ξτ can be expressed as ξτ = ξτ L(x0(ξ0)). Deﬁnition 5.2.1. ˜f is called a classical spacetime reference-system on ˜ M, and ( ˜ M, ˜f) is called a gravitational manifold. ( ˜U, ξU) and ( ˜U, xK) are called regular coordinate frames on ( ˜ M, ˜f), meanwhile ( ˜U, ˜ξα) and ( ˜U, ˜xµ) are called Minkowski coordinate frames on ( ˜ M, ˜f). For convenience, d˜ζτ and d˜xτ are uniformly denoted by dτ. (2) If the slack-tights ˜Bα µ and ˜Cµ α of ˜g are constants on ˜ M, then we say ˜g is ﬂat, F˜g is a ﬂat transformations, and ( ˜ M, ˜g) is a ﬂat spacetime. (2) If the slack-tights ˜Bα µ and ˜Cµ α of ˜g are constants on ˜ M, then we say ˜g is ﬂat, F˜g is a ﬂat transformations, and ( ˜ M, ˜g) is a ﬂat spacetime. (3) If ˜g is both orthogonal and ﬂat, then we say ˜g is an inertial-system, F˜g is a Lorentz transformations, and the isotropic and ﬂat ( ˜ M, ˜g) is Minkowski spacetime. (3) If ˜g is both orthogonal and ﬂat, then we say ˜g is an inertial-system, F˜g is a Lorentz transformations, and the isotropic and ﬂat ( ˜ M, ˜g) is Minkowski spacetime. (3) If ˜g is both orthogonal and ﬂat, then we say ˜g is an inertial-system, F˜g is a Lorentz transformations, and the isotropic and ﬂat ( ˜ M, ˜g) is Minkowski spacetime. Proposition 5.2.2. Let L be a path on ˜ M, and ˜U be a coordinate neighborhood. Denote ˜UL ≜ ˜U ∩L, and c ≜ dxi/dx0. If ∀q ∈˜UL, tangent vector [L] ∈Tq(M) is not internal-directed, then: (i) we have c = 1 on path ˜UL. (ii) c = 1 remains unchanged under orthogonal transformation. (iii) c = 1 remains unchanged under Lorentz transformation. Proof. In regular coordinate frame ( ˜U, xK), ˜UL can be described by equations xi = xi(x0) and xτ = const about parameter x0. We notice that xτ = const, so there does not exist an equation of ˜UL with respect to parameter ˜xτ in Minkowski coordinate frame ( ˜U, ˜xµ). Therefore, we always have d˜xτ = dxτ = 0 on ˜UL. Thus, c = dxi/dx0 = ±dxi/ p (dxi)2 + (dxτ)2 = ±dxi/dxi = 1. According to Deﬁnition 5.2.2 , an orthogonal transformation satisﬁes d˜ζτ = d˜xτ = 0, hence Proposition 5.2.2. Let L be a path on ˜ M, and ˜U be a coordinate neighborhood. Denote ˜UL ≜ ˜U ∩L, and c ≜ dxi/dx0. If ∀q ∈˜UL, tangent vector [L] ∈Tq(M) is not internal-directed, then: (i) we have c = 1 on path ˜UL. (ii) c = 1 remains unchanged under orthogonal transformation. (iii) c = 1 remains unchanged under Lorentz transformation. Proof. 5.2 Intrinsic geometrical treatment of gravitational ﬁeld Substitute them into ˜f(p), then            ξu = ξu xk, xτ L ξ0 x0 = ξu L x0 (xτ L)−1 (xτ) ξτ L x0 ξ0 = ξτ xk, xτ L ξ0 x0 = ξτ L x0 (xτ L)−1 (x ξ0 ξτ L −1 (ξτ) = (xτ L)−1 (xτ) ξU = ξU(xK) = ξU(x0) ⇔            ξu = ξu xk, xτ L ξ0 x0 = ξu L x0 (xτ L)−1 (xτ) ξτ L x0 ξ0 = ξτ xk, xτ L ξ0 x0 = ξτ L x0 (xτ L)−1 (xτ) ξ0 ξτ L −1 (ξτ) = (xτ L)−1 (xτ) (18) (18) 40 Zhao-Hui Man ⇔            ξu = ξu xk, xτ L ξ0 x0 = ξu L x0 (xτ L)−1 (xτ) ξ0 = ξ0 (ξτ L)−1 ξτ xk, xτ L ξ0 x0 = (xτ L)−1 (xτ) ξτ = ξτ L x0 (xτ L)−1 (xτ) , abbreviated to          ξu = ˜ξu(xk, x0) = ˜ξu L(x ξ0 = ˜ξ0(xk, x0) = ˜ξ0 L(xτ ξτ = ˜ξτ(xτ) Denote ˜ξs ≜ξs, ˜ξτ ≜ξτ, ˜ξ0 ≜ξ0, ˜xi ≜xi, ˜xτ ≜xτ, ˜x0 ≜x0, hence we have ˜ξα = ˜ξα (˜xµ) = ˜ξα L (˜xτ) , ˜ξτ = ˜ξτ (˜xτ), abbreviated to ˜ξα = ˜ξα (˜xµ) = ˜ξα (˜xτ) , ˜ξτ = ˜ξτ (˜xτ). ⊓⊔ Denote ˜ξs ≜ξs, ˜ξτ ≜ξτ, ˜ξ0 ≜ξ0, ˜xi ≜xi, ˜xτ ≜xτ, ˜x0 ≜x0, hence we have ˜ξα = ˜ξα (˜xµ) = ˜ξα L (˜xτ) , ˜ξτ = ˜ξτ (˜xτ), abbreviated to ˜ξα = ˜ξα (˜xµ) = ˜ξα (˜xτ) , ˜ξτ = ˜ξτ (˜xτ). ⊓⊔ Deﬁnition 5.2.1. ˜f is called a classical spacetime reference-system on ˜ M, and ( ˜ M, ˜f) is called a gravitational manifold. ( ˜U, ξU) and ( ˜U, xK) are called regular coordinate frames on ( ˜ M, ˜f), meanwhile ( ˜U, ˜ξα) and ( ˜U, ˜xµ) are called Minkowski coordinate frames on ( ˜ M, ˜f). Deﬁnition 5.2.1. ˜f is called a classical spacetime reference-system on ˜ M, and ( ˜ M, ˜f) is called a gravitational manifold. ( ˜U, ξU) and ( ˜U, xK) are called regular coordinate frames on ( ˜ M, ˜f), meanwhile ( ˜U, ˜ξα) and ( ˜U, ˜xµ) are called Minkowski coordinate frames on ( ˜ M, ˜f). This is the intrinsic geometrical treatment of the inertial relative motion of physics. Deﬁnition 5.2.1. ˜f is called a classical spacetime reference-system on ˜ M, and ( ˜ M, ˜f) is called a gravitational manifold. ( ˜U, ξU) and ( ˜U, xK) are called regular coordinate frames on ( ˜ M, ˜f), meanwhile ( ˜U, ˜ξα) and ( ˜U, ˜xµ) are called Minkowski coordinate frames on ( ˜ M, ˜f). In regular coordinate frame ( ˜U, xK), ˜UL can be described by equations xi = xi(x0) and xτ = const about parameter x0. We notice that xτ = const, so there does not exist an equation of ˜UL with respect to parameter ˜xτ in Minkowski coordinate frame ( ˜U, ˜xµ). Therefore, we always have d˜xτ = dxτ = 0 on ˜UL. Thus, c = dxi/dx0 = ±dxi/ p (dxi)2 + (dxτ)2 = ±dxi/dxi = 1. According to Deﬁnition 5.2.2 , an orthogonal transformation satisﬁes d˜ζτ = d˜xτ = 0, hence A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 41 c′ = dζs/dζ0 = \|±dζs/dζs\| = 1. It is naturally true for Lorentz transformation as an orthogonal one. ⊓⊔ ⊓⊔ Remark 5.2.2. The above proposition indicates the limitation of Minkowski coordinate, and also turns the principle of constancy of light velocity to a theorem, i.e. conclusion (iii). Its mathematical origin can be attributed to Deﬁnition 3.2.1 . Discussion 5.2.1. Let ˜ρ be a charge of ˜f, and L be a gradient line of ˜ρ on ( ˜ M, ˜g). (1) In the basis coordinate frame {˜ζα} of ˜g, suppose the parameter equation of L satisﬁes ˜ζs = const. Thus on L we have velocity d˜ζs dτ = 0 and proper time d˜ζ0 = dτ. This is the intrinsic geometrical treatment of the inertial relative rest state of physics, which is not a stopped evolution, but just an evolution in a special direction. (1) In the basis coordinate frame {˜ζα} of ˜g, suppose the parameter equation of L satisﬁes ˜ζs = const. Thus on L we have velocity d˜ζs dτ = 0 and proper time d˜ζ0 = dτ. This is the intrinsic geometrical treatment of the inertial relative rest state of physics, which is not a stopped evolution, but just an evolution in a special direction. (2) In the performance coordinate frame {˜xµ} of ˜g, on L we have velocity d˜xi dτ = ˜Ci 0 and coordinate time d˜x0 = ˜C0 τ dτ, where the slack-tights ˜Cµ α are constants. This is the intrinsic geometrical treatment of the inertial relative motion of physics. (2) In the performance coordinate frame {˜xµ} of ˜g, on L we have velocity d˜xi dτ = ˜Ci 0 and coordinate time d˜x0 = ˜C0 τ dτ, where the slack-tights ˜Cµ α are constants. 5.3 Two coordinate representations of intrinsic geometrical property Discussion 5.3.1. According to Deﬁnition 3.3.2.2 , suppose the slack-tights of ( ˜ M, ˜f) about regular coordinate xK are BS I and CI S, such that      dξS = BS I dxI ≃BS 0 dx0 CI 0 ∂ ∂xI ∼= C0 0 d dx0 = d dξ0 ,      dxI = CI SdξS ≃CI 0dξ0 BS 0 ∂ ∂ξS ∼= B0 0 d dξ0 = d dx0 . (19) (19) The slack-tights of ( ˜ M, ˜f) about Minkowski coordinate ˜xµ are ˜Bα µ and ˜Cµ α, such that      d˜ξα = ˜Bα µd˜xµ ≃˜Bα τ d˜xτ ˜Cµ τ ∂ ∂˜xµ ∼= ˜Cτ τ d d˜xτ = d d˜ξτ ,      d˜xµ = ˜Cµ αd˜ξα ≃˜Cµ τ d˜ξτ ˜Bα τ ∂ ∂˜ξα ∼= ˜Bτ τ d d˜ξτ = d d˜xτ . (20) (20) Applying the chain rule of diﬀerentiation, it is not diﬃcult to obtain the following relations between regular slack-tights and Minkowski slack-tights via formula (18). Applying the chain rule of diﬀerentiation, it is not diﬃcult to obtain the following relations between regular slack-tights and Minkowski slack-tights via formula (18). 5.3 Two coordinate representations of intrinsic geometrical property                ˜Bs i = −Bs i ˜B0 i = −Bτ i δτ 0 ˜Ci τ = Ci 0 δτ 0 ,                ˜Bs τ = Bs τ ˜B0 τ = Bτ τ δτ 0 ˜Cτ τ = Cτ 0 δτ 0 ,                ˜Bs 0 = Bs 0 ˜B0 0 = Bτ 0 δτ 0 ˜C0 τ = C0 0 δτ 0 ;                ˜Ci s = −Ci s ˜C0 s = −Cτ s ετ 0 ˜Bs τ = Bs 0 ετ 0 ,                ˜Ci τ = Ci τ ˜C0 τ = Cτ τ ετ 0 ˜Bτ τ = Bτ 0 ετ 0 ,                ˜Ci 0 = Ci 0 ˜C0 0 = Cτ 0 ετ 0 ˜B0 τ = B0 0 ετ 0 ,                  Bs i = −˜Bs i Bτ i = − ˜B0 i ˜δ0τ Ci 0 = − ˜Ci τ ˜δ0τ ,                  Bs 0 = ˜Bs 0 Bτ 0 = ˜B0 0 ˜δ0τ C0 0 = ˜C0 τ ˜δ0τ ,                  Bs τ = ˜Bs τ Bτ τ = ˜B0 τ ˜δ0τ Cτ 0 = ˜Cτ τ ˜δ0τ ;                Ci s = −˜Ci s Cτ s = − ˜C0 s ˜ε0τ Bs 0 = ˜Bs τ ˜ε0τ ,                Ci 0 = ˜Ci 0 Cτ 0 = ˜C0 0 ˜ε0τ B0 0 = ˜B0 τ ˜ε0τ ,                Ci τ = ˜Ci τ Cτ τ = ˜C0 τ ˜ε0τ Bτ 0 = ˜Bτ τ ˜ε0τ ,                ˜Bs i = −Bs i ˜B0 i = −Bτ i δτ 0 ˜Ci τ = Ci 0 δτ 0 ,                ˜Bs τ = Bs τ ˜B0 τ = Bτ τ δτ 0 ˜Cτ τ = Cτ 0 δτ 0 ,                ˜Bs 0 = Bs 0 ˜B0 0 = Bτ 0 δτ 0 ˜C0 τ = C0 0 δτ 0 ;                ˜Ci s = −Ci s ˜C0 s = −Cτ s ετ 0 ˜Bs τ = Bs 0 ετ 0 ,                ˜Ci τ = Ci τ ˜C0 τ = Cτ τ ετ 0 ˜Bτ τ = Bτ 0 ετ 0 ,                ˜Ci 0 = Ci 0 ˜C0 0 = Cτ 0 ετ 0 ˜B0 τ = B0 0 ετ 0 ,                  Bs i = −˜Bs i Bτ i = − ˜B0 i ˜δ0τ Ci 0 = − ˜Ci τ ˜δ0τ ,                  Bs 0 = ˜Bs 0 Bτ 0 = ˜B0 0 ˜δ0τ C0 0 = ˜C0 τ ˜δ0τ ,                  Bs τ = ˜Bs τ Bτ τ = ˜B0 τ ˜δ0τ Cτ 0 = ˜Cτ τ ˜δ0τ ;                Ci s = −˜Ci s Cτ s = − ˜C0 s ˜ε0τ Bs 0 = ˜Bs τ ˜ε0τ ,                Ci 0 = ˜Ci 0 Cτ 0 = ˜C0 0 ˜ε0τ B0 0 = ˜B0 τ ˜ε0τ ,                Ci τ = ˜Ci τ Cτ τ = ˜C0 τ ˜ε0τ Bτ 0 = ˜Bτ τ ˜ε0τ , 42 Zhao-Hui Man where where where    εI J ≜CI SBS J , δS T = BS I CI T , εI 0 ≜B0 0CI 0 = BS 0 CI S, δS 0 ≜C0 0BS 0 = CI 0BS I . 5.3 Two coordinate representations of intrinsic geometrical property ˜εµ ν ≜˜Cµ α ˜Bα ν , ˜δα β = ˜Bα µ ˜Cµ β, ˜εµ τ ≜˜Bτ τ ˜Cµ τ = ˜Bα τ ˜Cµ α, ˜δα τ ≜˜Cτ τ ˜Bα τ = ˜Cµ τ ˜Bα µ. The evolution lemma of Proposition 3.3.2.2 can be expressed in Minkowski coordinate as      wµ ∂ ∂˜xµ ∼= wτ d d˜xτ ⇔wµ = wτ ˜εµ τ wµd˜xµ ≃wτd˜xτ ⇔˜εµ τ wµ = wτ ,      ¯wµ ∂ ∂˜xµ ∼= ¯wτ d d˜xτ ⇔¯wµ = ¯wτ ˜¯ετ µ ¯wµd˜xµ ≃¯wτd˜xτ ⇔˜¯ετ µ ¯wµ = ¯wτ , where ˜¯εµ ν ≜˜¯Bµ α ˜¯Cα ν = εµ ν, ˜¯δα β ≜˜¯Cα µ ˜¯Bµ β = δα β, ˜¯ετ µ ≜˜¯Bτ τ ˜¯Cτ µ = ˜¯Bτ α ˜¯Cα µ, ˜¯δτ α ≜˜¯Cτ τ ˜¯Bτ α = ˜¯Cτ µ ˜¯Bµ α. where ˜¯εµ ν ≜˜¯Bµ α ˜¯Cα ν = εµ ν, ˜¯δα β ≜˜¯Cα µ ˜¯Bµ β = δα β, ˜¯ετ µ ≜˜¯Bτ τ ˜¯Cτ µ = ˜¯Bτ α ˜¯Cα µ, ˜¯δτ α ≜˜¯Cτ τ ˜¯Bτ α = ˜¯Cτ µ ˜¯Bµ α. Discussion 5.3.2. According to Deﬁnition 3.2.1 , on the neighborhood ˜U of p on ( ˜ M, f), time metrics dξ0 and dx0 of ˜f(p) in coordinate frames ( ˜U, ξS) and ( ˜U, xI) satisfy Discussion 5.3.2. According to Deﬁnition 3.2.1 , on the neighborhood ˜U of p on ( ˜ M, f), time metrics dξ0 and dx0 of ˜f(p) in coordinate frames ( ˜U, ξS) and ( ˜U, xI) satisfy            (dξ0)2 ≜ r X s=1 (dξs)2 + (dξτ)2 = δST dξSdξT = δST bS I bT J dxIdxJ = gIJdxIdxJ, (dx0)2 ≜ r X i=1 (dxi)2 + (dxτ)2 = εIJdxIdxJ = εIJcI ScJ T dξSdξT = hST dξSdξT . where (dξτ)2 ≜ D P a=r+1 (dξa)2 and (dxτ)2 ≜ D P m=r+1 (dxm)2. As diﬀerential forms deﬁned on mani- fold, time metrics of ( ˜ M, ˜f) satisfy where (dξτ)2 ≜ D P a=r+1 (dξa)2 and (dxτ)2 ≜ D P m=r+1 (dxm)2. As diﬀerential forms deﬁned on mani- fold, time metrics of ( ˜ M, ˜f) satisfy where (dξτ)2 ≜ D P a=r+1 (dξa)2 and (dxτ)2 ≜ D P m=r+1 (dxm)2. 5.3 Two coordinate representations of intrinsic geometrical property As diﬀerential forms deﬁned on mani- fold, time metrics of ( ˜ M, ˜f) satisfy    (dξ0)2 ≜∆ST dξSdξT = GIJdxIdxJ, (dx0)2 ≜EIJdxIdxJ = HST dξSdξT .    GIJ ≜∆ST BS I BT J , HST ≜EIJCI SCJ T . Denote ˜εµν = ˜εµν ≜          1 µ = ν = 0 −1, µ = ν ̸= 0 0, µ ̸= ν , ˜δαβ = ˜δαβ ≜          1 α = β = 0 −1, α = β ̸= 0 0, α ̸= β . Thus, we can deﬁne proper-times d˜ξτ and d˜xτ in Minkowski coordinate frames ( ˜U, ˜ξα) and ( ˜U, ˜xµ) as below: Thus, we can deﬁne proper-times d˜ξτ and d˜xτ in Minkowski coordinate frames ( ˜U, ˜ξα) and ( ˜U, ˜xµ) as below:            (d˜ξτ)2 = (dξ0)2 − r X s=1 (dξs)2 = ˜δαβd˜ξαd˜ξβ = ˜δαβ˜bα µ˜bβ νd˜xµd˜xν = ˜gµνd˜xµd˜xν, (d˜xτ)2 = (dx0)2 − r X i=1 (dxi)2 = ˜εµνd˜xµd˜xν = ˜εµν˜cµ α˜cν βd˜ξαd˜ξβ = ˜hαβd˜ξαd˜ξβ. And there are diﬀerential forms on ( ˜ M, ˜f) as below: And there are diﬀerential forms on ( ˜ M, ˜f) as below: And there are diﬀerential forms on ( ˜ M, ˜f) as below:    (d˜ξτ)2 ≜˜ ∆αβd˜ξαd˜ξβ = ˜Gµνd˜xµd˜xν, (d˜xτ)2 ≜˜Eµνd˜xµd˜xν = ˜Hαβd˜ξαd˜ξβ.    ˜Gµν ≜˜ ∆αβ ˜Bα µ ˜Bβ ν , ˜Hαβ ≜˜Eµν ˜Cµ α ˜Cν β. 5.3 Two coordinate representations of intrinsic geometrical property A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 43 It is easy to know there are relations between regular metric GIJ and Minkowski metric ˜Gµν as below:                              Gττ = ˜Gττ ˜G00 G00 = ˜δ0 τ ˜δ0 τ ˜ε0τ ˜ε0τ ˜Gττ ˜G00 ˜Gττ Giτ = − ˜Gi0 ˜G00 ˜ε0 τG00 = − ˜δ0 τ ˜δ0 τ ˜ε0τ ˜Gττ ˜G00 ˜Gi0 Gτj = − ˜G0j ˜G00 ˜ε0 τG00 = − ˜δ0 τ ˜δ0 τ ˜ε0τ ˜Gττ ˜G00 ˜G0j Gij = − ˜Gij ˜G00 G00 = − ˜δ0 τ ˜δ0 τ ˜ε0τ ˜ε0τ ˜Gττ ˜G00 ˜Gij ,                            ˜G00 = G00 Gττ ˜Gττ = δτ 0δτ 0 ετ 0ετ 0 G00 Gττ G00 ˜Gi0 = −Giτ Gττ ετ 0 ˜Gττ = −δτ 0δτ 0 ετ 0 G00 Gττ Giτ ˜G0j = −Gτj Gττ ετ 0 ˜Gττ = −δτ 0δτ 0 ετ 0 G00 Gττ Gτj ˜Gij = −Gij Gττ ˜Gττ = −δτ 0δτ 0 ετ 0ετ 0 G00 Gττ Gij . Denote ˜Gττ ≜˜Bτ τ ˜Bτ τ , ˜Gττ ≜˜Cτ τ ˜Cτ τ , then it is easy to obtain relations: ˜Gττ = δτ 0δτ 0 ετ 0ετ 0 G00, G00 = ˜δ0 τ ˜δ0 τ ˜ε0τ ˜ε0τ ˜Gττ. 5.4 Geometrical treatment of classical spacetime evolution As what Remark 5.2.1 says, ( ˜ M, ˜f) cannot totally reﬂect all the intrinsic geometrical properties of internal space Now there is a problem. Several internal dimensions of (M, f) become just one dimension of ( ˜ M, ˜f) via the encapsulation of classical spacetime. (M, f) has several internal charges ρm n0, but ( ˜ M, ˜f) has just one, which is ρτ τ0 in regular form, or ˜ρ0 0τ in Minkowski form. As what Remark 5.2.1 says, ( ˜ M, ˜f) cannot totally reﬂect all the intrinsic geometrical properties of internal space of (M, f). ( ˜ M, ˜f) has just one, which is ρτ τ0 in regular form, or ˜ρ0 0τ in Minkowski form. As what Remark 5.2.1 says, ( ˜ M, ˜f) cannot totally reﬂect all the intrinsic geometrical properties of internal space of (M, f). On the premise of not abandoning the four-dimensional spacetime, if we want to describe gauge ﬁelds, the only way is to put those degrees of freedom of internal space to the phase of complex-valued ﬁeld function. This way is eﬀective, but not natural at all. The logically more natural way is to abandon the framework of four-dimensional spacetime. We should put internal space and external space together to describe their uniﬁed intrinsic geometry, rather than based on the rigid intuition of four-dimensional spacetime, artiﬁcially setting up several abstract degrees of freedom which are irrelevant to the concept of time and space to describe the so called gauge ﬁelds. Both for gauge ﬁelds and for gravitational ﬁelds, their concepts of time and space should be uniﬁed. The gravitational ﬁelds are described by the intrinsic geometry of external space, and the gauge ﬁelds are described by the intrinsic geometry of internal space. They are uniﬁed in intrinsic geometry. Therefore, the complex-valued expression form of traditional gauge ﬁeld theory is a historical necessity, but not a logical necessity. It can be seen later that as long as expanding those encapsulated dimensions, we can clearly illustrate the geometrical properties of internal space. Especially, if understanding in way of intrinsic geometry, some man-made postulates of Standard Model of particle physics will be unnecessary, because they will appear automatically. Deﬁnition 5.4.1. Denote ˜ρµν τ of ˜f concisely by ˜ρ. According to section 3.3.6 , we have the following deﬁnitions in Minkowski coordinate on ( ˜ M, ˜g). 5.4 Geometrical treatment of classical spacetime evolution Discussion 5.4.1. The absolute diﬀerential and absolute gradient of section 3.3.4 can be ex- pressed on ˜ M in Minkowski coordinate as: (1) Let ˜D be aﬃne connection on ˜ M, and denote ˜tL;τ ≜˜t;σ ˜εσ τ , then the absolute diﬀerential of ˜T and ˜TL are (1) Let ˜D be aﬃne connection on ˜ M, and denote ˜tL;τ ≜˜t;σ ˜εσ τ , then the absolute diﬀerential of ˜T and ˜TL are        ˜D ˜T ≜˜D˜t ⊗ ∂ ∂˜x ⊗d˜x ≜˜t;σd˜xσ ⊗ ∂ ∂˜x ⊗d˜x , ˜DL ˜TL ≜˜DL˜tL ⊗ ∂ ∂˜x ⊗d˜x ≜˜tL;τd˜xτ ⊗ ∂ ∂˜x ⊗d˜x , where ˜D˜t ≜˜t;σd˜xσ, ˜DL˜tL ≜˜tL;τd˜xτ. where ˜D˜t ≜˜t;σd˜xσ, ˜DL˜tL ≜˜tL;τd˜xτ. where ˜D˜t ≜˜t;σd˜xσ, ˜DL˜tL ≜˜tL;τd˜xτ. (2) The gradient operator ˜∇is the actual evolution on ˜ M. Thus, the absolute gradient of ˜T and ˜TL are (2) The gradient operator ˜∇is the actual evolution on ˜ M. Thus, the absolute gradient of ˜T and ˜TL are        ˜∇˜T ≜˜∇˜t ⊗ ∂ ∂˜x ⊗d˜x ≜˜t;σ ∂ ∂˜xσ ⊗ ∂ ∂˜x ⊗d˜x , ˜∇L ˜TL ≜˜∇L˜tL ⊗ ∂ ∂˜x ⊗d˜x ≜˜tL;τ d d˜xτ ⊗ ∂ ∂˜x ⊗d˜x , Now Proposition 3.3.4.1 can be expressed as: ˜D ˜T ≃ ˜DL ˜TL if L is an arbitrary path. ˜∇˜T ∼= ˜∇L ˜TL if and only if L is the gradient line of ˜T. The actual evolution equation of ˜T is ˜t;σ = ˜tL;τ ˜¯ετ σ or ˜t;σ = ˜t ;τ L ˜εσ τ . Discussion 5.4.2. Similar to Discussion 3.3.5.1 we have ˜Kµ νρσ :ρ = ˜jµ νσ, where ˜ρµ ντ ≜˜Kµ νρσ :ρ ˜εσ τ , ˜jµ νσ ≜˜ρµ ντ ˜¯ετ σ. Consider the case where external space is ﬂat, then just only the internal component ˜ρ0 0τ does not vanish. Thus we have Minkowski Yang-Mills ﬁeld equation ˜K0 0ρσ :ρ = ˜j0 0σ, where ˜j0 0σ ≜˜ρ0 0τ ˜¯ετ σ. where ˜j0 0σ ≜˜ρ0 0τ ˜¯ετ σ. 44 Zhao-Hui Man Now there is a problem. Several internal dimensions of (M, f) become just one dimension of ( ˜ M, ˜f) via the encapsulation of classical spacetime. (M, f) has several internal charges ρm n0, but ( ˜ M, ˜f) has just one, which is ρτ τ0 in regular form, or ˜ρ0 0τ in Minkowski form. Similar to discussions of section 3.3.7 , denote Similar to discussions of section 3.3.7 , denote        [˜ρ ˜Γω] ≜[˜ρµν ˜Γω] ≜∂˜ρ ∂˜xω −˜ρ;ω ≜∂˜ρµν ∂˜xω −˜ρµν;ω = ˜ρµχ ˜Γ χ νω + ˜ρχν ˜Γ χ µω, [˜ρ ˜Γτ] ≜[˜ρµν ˜Γτ] ≜d˜ρ d˜xτ −˜ρ;τ ≜d˜ρµν d˜xτ −˜ρµν;τ = ˜ρµχ ˜Γ χ ντ + ˜ρχν ˜Γ χ µτ.    [˜ρ ˜Γ ω] ≜gχω[˜ρ ˜Γχ], [˜ρ ˜Γ τ] ≜gττ[˜ρ ˜Γτ].        [˜ρ ˜Bρσ] ≜˜ρµχ ∂˜Γ χ νσ ∂˜xρ −∂˜Γ χ νρ ∂˜xσ ! + ˜ρχν ∂˜Γ χ µσ ∂˜xρ −∂˜Γ χ µρ ∂˜xσ ! , [˜ρ ˜Rρσ] ≜˜ρµχ ˜Rχ νρσ + ˜ρχν ˜Rχ µρσ,      [˜ρ ˜Fρσ] ≜∂[˜ρ ˜Γσ] ∂˜xρ −∂[˜ρ ˜Γρ] ∂˜xσ , [˜ρ ˜Eρσ] ≜[˜ρ ˜Γσ];ρ −[˜ρ ˜Γρ];σ. Then for the same reason as Proposition 3.3.7.2 , we can strictly prove the Lorentz force equation of ˜ρ, which is ˜Fρ ≜d˜pρ d˜xτ = ∂˜mτ ∂˜xρ −˜pσ ∂˜εσ τ ∂˜xρ +[˜ρ ˜Fρσ]˜εσ τ . And for the same reason as Proposition 3.3.7.5 , we have conservation of energy-momentum ˜Tµν ;µ = 0. Then for the same reason as Proposition 3.3.7.2 , we can strictly prove the Lorentz force equation of ˜ρ, which is ˜Fρ ≜d˜pρ d˜xτ = ∂˜mτ ∂˜xρ −˜pσ ∂˜εσ τ ∂˜xρ +[˜ρ ˜Fρσ]˜εσ τ . And for the same reason as Proposition 3.3.7.5 , we have conservation of energy-momentum ˜Tµν ;µ = 0. Deﬁnition 5.4.2. The following three conditions are uniformly called traditional standard conditions: ) Rest mass condition: ∂µ ˜mτ = 0. (1) Rest mass condition: ∂µ ˜mτ = 0. (1) Rest mass condition: ∂µ ˜mτ = 0. (1) Rest mass condition: ∂µ ˜mτ = 0. (2) Canonical mass condition: ˜Γ µ ντ ≜˜Γ µ νρ˜ερ τ = 0 and ˜Γ µ νργρ = 0, where γρ are generators of Dirac algebra. (3) Simple perspective condition: ∂ν ˜εµ τ = 0. (2) Canonical mass condition: ˜Γ µ ντ ≜˜Γ µ νρ˜ερ τ = 0 and ˜Γ µ νργρ = 0, where γρ are generators of Dirac algebra. (2) Canonical mass condition: ˜Γ µ ντ ≜˜Γ µ νρ˜ερ τ = 0 and ˜Γ µ νργρ = 0, where γρ are generators of Dirac algebra. (3) Simple perspective condition: ∂ν ˜εµ τ = 0. (3) Simple perspective condition: ∂ν ˜εµ τ = 0. (3) Simple perspective condition: ∂ν ˜εµ τ = 0. Remark 5.4.1. 5.4 Geometrical treatment of classical spacetime evolution Call ˜mτ ≜˜ρ;τ and ˜mτ ≜˜ρ;τ the rest mass of ˜ρ. (1) Call ˜mτ ≜˜ρ;τ and ˜mτ ≜˜ρ;τ the rest mass of ˜ρ. (2) Call ˜pµ ≜˜ρ;µ and ˜pµ ≜˜ρ;µ the energy-momentum of ˜ρ, and ˜E0 ≜˜p0, ˜E0 ≜˜p0 the energy of ˜ρ. (3) Call ˜ Mτ ≜ d˜ρ d˜xτ and ˜ Mτ ≜ d˜ρ d˜xτ the canonical rest mass of ˜ρ. (3) Call ˜ Mτ ≜ d˜ρ d˜xτ and ˜ Mτ ≜ d˜ρ d˜xτ the canonical rest mass of ˜ρ. (4) Call ˜P µ ≜ ∂˜ρ ∂˜xµ and ˜Pµ ≜ ∂˜ρ ∂˜xµ the canonical energy-momentum of ˜ρ, and ˜H0 ≜−˜P 0, ˜H0 ≜−˜P0 the canonical energy of ˜ρ. (4) Call ˜P µ ≜ ∂˜ρ ∂˜xµ and ˜Pµ ≜ ∂˜ρ ∂˜xµ the canonical energy-momentum of ˜ρ, and ˜H0 ≜−˜P 0, ˜H0 ≜−˜P0 the canonical energy of ˜ρ. Discussion 5.4.3. Similar to Proposition 3.3.6.2 , if and only if the evolution direction of ˜ρ is the gradient direction ˜∇˜ρ on ( ˜ M, ˜g), the directional derivative is D ˜mτ d d˜xτ , ˜mτd˜xτE = D ˜pµ ∂ ∂˜xµ , ˜pµd˜xµE , that is ˜Gττ ˜mτ ˜mτ = ˜Gµν ˜pµ˜pν, or ˜mτ ˜mτ = ˜pµ˜pµ, which is the mathematical origin of energy- momentum equation of physics. In addition, as what Proposition 3.3.6.3 says, according to evolution lemma, if and only if we take the gradient direction ˜∇˜ρ, we have ˜pµ = ˜mτ d˜xµ d˜xτ and ˜pµ = ˜mτ d˜xµ d˜xτ . This is the mathematical origin of traditional deﬁnition of momentum. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 45 Similar to discussions of section 3.3.7 , denote Similar to discussions of section 3.3.7 , denote Similar to discussions of section 3.3.7 , denote Conditions (1) and (3) make the above Lorentz force simplify to ˜Fρ = [˜ρ ˜Fρσ]˜εσ τ , which is the general essence of interaction force of physics, and also is the mathematical origin of Lorentz force FFF = q (EEE + vvv × BBB) or Fρ = jσFρσ of electrodynamics. In this sense, we can argue that Lorentz force equation has become a theorem, and no longer as a principle. Condition (2) is the general mathematical expression of the following example. Take electro- dynamics with natural units for example. The canonical energy-momentum of electric charged particle is H = E + qφ, PPP = ppp + qAAA. We notice that there is no concept of canonical mass ˜ Mτ in physics. It is because that there is H = qφ + p (PPP −qAAA)2 + m2 in electrodynamics, which indicates that electromagnetic potential ﬁeld (φ,AAA) contributes qφ to energy and qAAA to momentum, but it contributes nothing to rest mass of q. This implies that if we deﬁne ˜ Mτ ≜˜mτ + q ˜Aτ, ˜Aτ ≜φγ + AAA · uuu = Aρ dxρ dτ , then electrodynamics actually requires ˜ Mτ = ˜mτ, ˜Aτ = Aρ dxρ dτ = 0 by default, the general mathematical expression of which is ˜Γ µ ντ ≜˜Γ µ νρ˜ερ τ = 0. In a word, the above three conditions are necessary conditions for transitioning in pure mathematical sense to traditional theory of physics. Discussion 5.4.4. Suppose ˜C(˜x)µν is a zero-order or two-order tensor, such as ˜Rµν −1 2 ˜Gµν ˜R, which just only depends on intrinsic geometrical properties of ( ˜ M, ˜g), such that ˜C(˜x)µν ;µ = 0. 46 Zhao-Hui Man We distinguish them with index (˜x). And suppose T(˜ρ)µν ;µ = 0 is the conservation of energy- momentum of ˜ρ. We distinguish various T(˜ρ)µν with index (˜ρ). Hence, ∀c(˜x), c(˜ρ) ∈R, we have  X ˜x c(˜x)C(˜x)µν + X ˜ρ c(˜ρ) ˜T(˜ρ)µν   ;µ = 0. If the ergodic ranges of the summations are suﬃciently large, we immediately obtain X ˜x c(˜x)C(˜x)µν + X ˜ρ c(˜ρ) ˜T(˜ρ)µν = 0, which is the general gravitational ﬁeld equation, where the dimensions among various terms are harmonized by constants c(˜x), c(˜ρ). It is the mathematical origin of Einstein’s gravitational ﬁeld equation. which is the general gravitational ﬁeld equation, where the dimensions among various terms are harmonized by constants c(˜x), c(˜ρ). Similar to discussions of section 3.3.7 , denote It is the mathematical origin of Einstein’s gravitational ﬁeld equation. Deﬁnition 5.4.3. It is similar to Deﬁnition 3.3.6 . Let ˜L be the totality of paths on ˜ M from point a to point b. And let L˜ρ ∈˜L, and parameter ˜xτ satisfy τa ≜˜xτ(a) < ˜xτ(b) ≜τb. We say the functional ˜s˜ρ ˜ W (L˜ρ) ≜ Z L˜ρ ˜D˜ρ = Z τb τa ˜mτd˜xτ = Z τb τa ˜pµd˜xµ = Z τb τa d˜xτ d˜x0 ˜ Mτ −[˜ρ ˜Γσ]˜εσ τ d˜x0 is the action of ˜ρ. For the same reason as the proof of Proposition 3.3.6.4 , we have the following theorem, which is the mathematical origin of the principle of least action of physics. Proposition 5.4.1. (Theorem of least action) L˜ρ is the gradient line of ˜ρ if and only if δ˜s˜ρ ˜ W L˜ρ = 0. Proposition 5.4.1. (Theorem of least action) L˜ρ is the gradient line of ˜ρ if and only if δ˜s˜ρ ˜ W L˜ρ = 0. 5.5 Intrinsic geometrical treatment of Legendre transformation and equation of motion It should be clariﬁed that the above Euler-Lagrange equation holds in arbitrary directions, while the Euler-Lagrange equation of traditional theory holds just only in gradient direction. Remark 5.5.1. The above proposition is the mathematical origin of Euler-Lagrange equation of motion of physics. It should be clariﬁed that the above Euler-Lagrange equation holds in arbitrary directions, while the Euler-Lagrange equation of traditional theory holds just only in gradient direction. The reason why such a situation happens is that their deﬁnitions of momentum are diﬀerent. Deﬁnition 3.3.6.1 deﬁnes the momentum in arbitrary directions. Due to Proposition 3.3.6.3 and Remark 3.3.6.1 , pR = E0 dxR dx0 and ˜pµ = ˜mτ d˜xµ d˜xτ just hold in gradient direction. But traditional theory denotes p ≜mv in arbitrary directions. Such two diﬀerent ways of deﬁning momentum make the conditions of the holding of Euler-Lagrange equation diﬀerent. (1) When ˜pµ = ˜mτ d˜xµ d˜xτ holds just only in gradient direction, Euler-Lagrange equation holds in arbitrary directions. At this time, what we can obtain from Proposition 5.4.1 is just only the former. (1) When ˜pµ = ˜mτ d˜xµ d˜xτ holds just only in gradient direction, Euler-Lagrange equation holds in arbitrary directions. At this time, what we can obtain from Proposition 5.4.1 is just only the former. (2) When we denote p ≜mv in arbitrary directions, Euler-Lagrange equation holds just only in gradient direction. At this time, what we can obtain from Proposition 5.4.1 is just only the latter. (2) When we denote p ≜mv in arbitrary directions, Euler-Lagrange equation holds just only in gradient direction. At this time, what we can obtain from Proposition 5.4.1 is just only the latter. No matter we use which way of deﬁnition, there is always a formula that can describe gradient direction, which is either ˜pµ = ˜mτ d˜xµ d˜xτ or Euler-Lagrange equation. 5.5 Intrinsic geometrical treatment of Legendre transformation and equation of motion This section does not discuss the general abstract theory of Legendre transformation, but discusses the relationship between energy-momentum equation and the concrete construction of Legendre transformation. Deﬁnition 5.5.1. Denote        ˜L0 ≜˜mτ d˜xτ d˜x0 = d˜xτ d˜x0 ˜ Mτ −[˜ρ ˜Γσ]˜εσ τ , ˜L0 ≜˜ Mτ d˜xτ d˜x0 = d˜xτ d˜x0 ˜mτ + [˜ρ ˜Γσ]˜εσ τ . Evidently we have ˜L0 = ˜L0 on canonical mass condition. We say ˜L0 is Lagrangian densigy of ˜ρ. According to Deﬁnition 5.4.1 , we have ˜ Mτd˜xτ = ˜Pkd˜xk −˜H0d˜x0, therefore ˜H0 = ˜Pk d˜xk d˜x0 − ˜ Mτ d˜xτ d˜x0 , that is ˜H0 = ˜Pk d˜xk d˜x0 −˜L0. We say ˜H0 is Hamiltonian density of ˜ρ. This is the intrinsic geometrical origin of Legendre transformation of physics. Proposition 5.5.1. Denote ˜vk ≜d˜xk d˜x0 , and regard ˜L0 = ˜Pk˜vk −˜H0 as function ˜L0(˜xk, ˜vk). Thus, on traditional standard conditions we have d d˜x0 ∂˜L0 ∂˜vk ! −∂˜L0 ∂˜xk = 0. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 47 Proof. On traditional standard conditions, we can obtain Euler-Lagrange equation from the deﬁnition of Lagrangian density. Concretely: Proof. On traditional standard conditions, we can obtain Euler-Lagrange equation from the deﬁnition of Lagrangian density. Concretely: ˜L0 ≜˜ Mτ d˜xτ d˜x0 = d˜ρ d˜xτ d˜xτ d˜x0 = ∂˜ρ ∂˜xµ d˜xµ d˜xτ d˜xτ d˜x0 . Accordingly, ∂˜L0 ∂˜xσ = ∂ ∂˜xσ ∂˜ρ ∂˜xµ d˜xµ d˜xτ d˜xτ d˜x0 . ∂˜L0 ∂˜xσ = ∂ ∂˜xσ ∂˜ρ ∂˜xµ d˜xµ d˜xτ d˜xτ d˜x0 . On traditional standard conditions, On traditional standard conditions, ∂˜L0 ∂˜xσ = d˜xµ d˜xτ d˜xτ d˜x0 ∂ ∂˜xσ ∂˜ρ ∂˜xµ = d˜xµ d˜xτ d˜xτ d˜x0 ∂ ∂˜xµ ∂˜ρ ∂˜xσ = d˜xµ d˜xτ d˜xτ d˜x0 ∂˜Pσ ∂˜xµ = d ˜Pσ d˜x0 . Thus we obtain Euler-Lagrange equation d ˜Pk d˜x0 −∂˜L0 ∂˜xk = 0. In consideration of ˜Pk = ∂˜L0 ∂˜vk , then ˜ ! ˜ Thus we obtain Euler-Lagrange equation d ˜Pk d˜x0 −∂˜L0 ∂˜xk = 0. In consideration of ˜Pk = ∂˜L0 ∂˜vk , then ! d d˜x0 ∂˜L0 ∂˜vk ! −∂˜L0 ∂˜xk = 0. ˜L0 ˜vk ! −∂˜L0 ∂˜xk = 0. ⊓⊔ d d˜x0 ∂L0 ∂˜vk ! −∂L0 ∂˜xk = 0. ⊓⊔ ⊓⊔ Remark 5.5.1. The above proposition is the mathematical origin of Euler-Lagrange equation of motion of physics. 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation This section proves two propositions. The ﬁrst one illustrates the intrinsic geometrical origin of Dirac equation, and makes it no longer a principle but a theorem. The second one clariﬁes the intrinsic geometrical origin of gauge transformation and gauge invariance, and shows how the transformation of reference-systems characterizes the general gauge transformation. Proposition 5.6.1. On traditional standard conditions, suppose there is a smooth real function f(˜xµ) on an isotropic ( ˜ M, ˜g), and deﬁne Dirac algebras γµ and γα such that γµ = ˜Cµ αγα, Zhao-Hui Man 48 γβγα = 2˜δαβ, γµγν + γνγµ = 2 ˜Gµν, ˜Γ µ νσγσ = 0, γµ ∂f ∂˜xµ = 0, Z f2dV = 1. Let ˜ρ ≜˜ρων be γαγβ + γβγα = 2˜δαβ, γµγν + γνγµ = 2 ˜Gµν, ˜Γ µ νσγσ = 0, γµ ∂f ∂˜xµ = 0, Z f2dV = 1. Let ˜ρ ≜˜ρων be ∂x Z a charge of reference-system ˜f, and denote a charge of reference-system ˜f, and denote a charge of reference-system ˜f, and denote        ˜S ≜ Z ˜sdV , ˜s = Z ˜L0d˜x0 = f2 ˜S,      ˜Mτ ≜ Z ˜mτdV , ˜mτ = f2 ˜Mτ,      ˜P ≜ Z ˜ρdV , ˜ρ = f2˜P. Then denote ψ(˜xµ) ≜f(˜xµ)ei ˜S, ˜Dµ ≜ ∂ ∂˜xµ −i[˜P ˜Γµ]. If and only if we take the gradient direction ˜∇˜ρ, equation iγµ ˜Dµψ = ˜Mτψ holds. Proof. Due to Discussion 5.4.3 , if and only if we take the gradient direction of ˜ρ there exists energy-momentum equation ˜ρ;µ ˜ρ;µ = ˜ρ;τ ˜ρ;τ, that is ˜Gµν ˜ρ;µ ˜ρ;ν = ˜m2 τ due to isotropy. Then, (γµ ˜ρ;µ)(γν ˜ρ;ν)+(γν ˜ρ;ν)(γµ ˜ρ;µ) = 2 ˜m2 τ, hence (γµ ˜ρ;µ)(γν ˜ρ;ν) = ˜m2 τ, that is (γµ ˜ρ;µ)2 = ˜m2 τ. Without loss of generality, we take γµ ˜ρ;µ = −˜mτ. Due to canonical mass condition ˜Γ µ νσγσ = 0 we have γµ[˜P ˜Γµ] = 0. And with condition γµ ∂f ∂˜xµ = 0 it can be obtained that γµ ˜ρ;µ = −˜mτ ⇔γµ ∂˜s ∂xµ = −˜mτ ⇔γµ ∂˜S ∂xµ = −˜Mτ ⇔γµ ∂˜S ∂xµ −[˜P ˜Γµ] ! = −˜Mτ ⇔iγµ i ∂˜S ∂xµ fei ˜S −i[˜P ˜Γµ]fei ˜S ! = ˜Mτfei ˜S ⇔iγµ ∂f ∂˜xµ ei ˜S + i ∂˜S ∂xµ fei ˜S −i[˜P ˜Γµ]fei ˜S ! 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation = ˜Mτfei ˜S ⇔iγµ  ∂ fei ˜S ∂˜xµ −i[˜P ˜Γµ]fei ˜S  = ˜Mτfei ˜S ⇔iγµ ∂ψ ∂˜xµ −i[˜P ˜Γµ]ψ = ˜Mτψ ∂ ⇔iγµ ∂ ∂˜xµ −i[˜P ˜Γµ] ψ = ˜Mτψ ⇔iγµ ˜Dµψ = ˜Mτψ. ⊓⊔ ⊓⊔ Remark 5.6.1. Until now, Dirac equation has become a theorem. According to this theorem, Dirac equation also reﬂects the notion of gradient direction, it thereby describes the eﬀects of intrinsic geometry of manifold on gradient direction. This is the mathematical origin of the eﬀectiveness of Dirac equation of physics. Proposition 5.6.2. On ˜ M let the slack-tights of ˜g be ˜Bα µ and ˜Cµ α, and the slack-tights of ˜k be ˜Bµ µ′ and ˜Cµ′ µ . Let ˜g′ ≜L[˜k](˜g), then the slack-tights of ˜g′ are ˜Bα µ′ = Bα µ ˜Bµ µ′ and ˜Cµ′ α = ˜Cµ α ˜Cµ′ µ . Proposition 5.6.2. On ˜ M let the slack-tights of ˜g be ˜Bα µ and ˜Cµ α, and the slack-tights of ˜k be ˜Bµ µ′ and ˜Cµ′ µ . Let ˜g′ ≜L[˜k](˜g), then the slack-tights of ˜g′ are ˜Bα µ′ = Bα µ ˜Bµ µ′ and ˜Cµ′ α = ˜Cµ α ˜Cµ′ µ . Deﬁne Dirac algebras γα, γµ = ˜Cµ αγα and γµ′ = ˜Cµ′ α γα, such that γαγβ + γβγα = 2˜δαβ, γµγν + γνγµ = 2 ˜Gµν, γµ′γν′ + γν′γµ′ = 2 ˜Gµ′ν′, where ˜Gµν = ˜δαβ ˜Cµ α ˜Cν β is the metric tensor of ˜g, and ˜Gµ′ν′ = ˜δαβ ˜Cµ′ α ˜Cν′ β is the metric tensor of ˜g′. Proposition 5.6.2. On ˜ M let the slack-tights of ˜g be ˜Bα µ and ˜Cµ α, and the slack-tights of ˜k be ˜Bµ µ′ and ˜Cµ′ µ . Let ˜g′ ≜L[˜k](˜g), then the slack-tights of ˜g′ are ˜Bα µ′ = Bα µ ˜Bµ µ′ and ˜Cµ′ α = ˜Cµ α ˜Cµ′ µ . Deﬁne Dirac algebras γα, γµ = ˜Cµ αγα and γµ′ = ˜Cµ′ α γα, such that γαγβ + γβγα = 2˜δαβ, γµγν + γνγµ = 2 ˜Gµν, γµ′γν′ + γν′γµ′ = 2 ˜Gµ′ν′, where ˜Gµν = ˜δαβ ˜Cµ α ˜Cν β is the metric tensor of ˜g, and ˜Gµ′ν′ = ˜δαβ ˜Cµ′ α ˜Cν′ β is the metric tensor of ˜g′. Let ˜ρ be a charge of ˜f, and ˜D be the simple connection on ( ˜ M, ˜g) and ( ˜ M, ˜g′). [ ] or the deﬁnition of general linear group GL(M). a selected ˜ρ, the smooth real function θ is deternimed by L[k] ∈GL(M). See Discussion he deﬁnition of general linear group GL(M). 2.3.1 for the deﬁnition of general linear group GL(M). 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation L[˜k] : ˜Bα µ 7→˜Bα µ′ = Bα µ ˜Bµ µ′, ˜Cµ α 7→˜Cµ′ α = ˜Cµ α ˜Cµ′ µ , ˜Bτ τ 7→˜Bτ τ ′ = B L[˜k] : ∂ ∂˜xµ 7→ ∂ ∂˜xµ′ = ˜Bµ µ′ ∂ ∂˜xµ , d d˜xτ 7→ d d˜xτ ′ = ˜Bτ τ ′ d d˜xτ . L[˜k] : ρ;µ 7→ρ;µ′ = ρ;µ ˜Bµ µ′, ˜Mτ 7→˜Mτ ′ = ˜Mτ ˜Bτ τ ′. L[˜k] : γµ 7→γµ′ = ˜Cµ′ µ γµ. According to equation(21) below, it is obtained that L[˜k] : [˜P ˜Γµ] 7→[˜P ˜Γµ′] = ˜Bµ µ′([˜P ˜Γµ] + rµ). Correspondingly, the transformation of ˜Dµ is Correspondingly, the transformation of ˜Dµ is ˜Dµ ≜ ∂ ∂˜xµ −i[˜P ˜Γµ] 7→˜Dµ′ ≜ ∂ ∂˜xµ′ −i[˜P ˜Γµ′] = ˜Bµ µ′ ∂ ∂˜xµ −i ˜Bµ µ′([˜P ˜Γµ] + rµ) = ˜Bµ µ′( ˜Dµ −irµ). The transformation of ψ is The transformation of ψ is The transformation of ψ is ψ = fe i Z ˜P;µ + [˜P ˜Γµ] d˜xµ 7→ψ′ = fe i Z ˜P;µ′ + [˜P ˜Γµ′] d˜xµ′ = fe i Z ˜P;µ′ + ˜Bµ µ′([˜P ˜Γµ] + rµ) d˜xµ , xµ . Denote θ ≜ Z rµd˜xµ, rµ = ∂µθ, thus we obtain ψ 7→ψ′ = ψeiθ. that is ψ 7→ψ′ = ψe i Z rµdxµ . Denote θ ≜ Z rµd˜xµ, rµ = ∂µθ, thus we obtain ψ 7→ψ′ = ψeiθ. Z We have proved (1) and (2) as the above. Now in order to prove (4), we can substitute the above transformations into iγµ ˜Dµψ = ˜Mτψ of ˜g. iγµ ˜Dµψ = ˜Mτψ ⇔i ˜Bµ µ′γµ′ ˜Cν′ µ ( ˜Dν′ + i∂ν′θ) ψ′e−iθ = ˜Mτ ψ′e−iθ ⇔iγµ′( ˜Dµ′ + i∂µ′θ) ψ′e−iθ = ˜Mτψ′e−iθ ⇔iγµ′ ∂µ′ −i[˜P ˜Γµ′] + i∂µ′θ ψ′e−iθ = ˜Mτψ′e−iθ ⇔iγµ′∂µ′ψ′e−iθ + iγµ′ψ′∂µe−iθ + γµ′[˜P ˜Γµ′]ψ′e−iθ −γµ′ψ′e−iθ∂µ′θ = ˜Mτψ′e−iθ ⇔iγµ′∂µ′ψ′ + γµ′ψ′∂µ′θ + γµ′[˜P ˜Γµ′]ψ′ −γµ′ψ′∂µ′θ = ˜Mτψ′ ⇔iγµ′∂µ′ψ′ + γµ′[˜P ˜Γµ′]ψ′ = ˜Mτψ′ ⇔iγµ′ ˜Dµ′ψ′ = ˜Mτψ′. ⇔iγµ′ ˜Dµ′ψ′ = ˜Mτψ′. Consequently, iγµ′ ˜Dµ′ψ′ = ˜Mτψ′ = ˜Mτψeiθ = iγµ ˜Dµψ eiθ, hence iγµ′ ˜Dµ′ψ′ = iγµ ˜Dµψ . In addition, it is evident that \|ψ′\| = \|ψ\|. It indicates that iγµ ˜Dµψ and \|ψ\| remain unchanged under transformation L[˜k]. Due to the arbitrariness of [˜k], according to section 2.6 , iγµ ˜Dµψ is a universal geometrical property of ˜ M. 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation According to Proposition 5.6.1 , suppose ( ˜Γ˜g) µ νσγσ = 0, the Dirac equation of ˜ρ on ( ˜ M, ˜g) is iγµ ˜Dµψ = ˜Mτψ, and suppose ( ˜Γ˜g′) µ νσγσ = 0, the Dirac equation of ˜ρ on ( ˜ M, ˜g′) is iγµ′ ˜Dµ′ψ′ = ˜Mτ ′ψ′. Then we have the following conclusions under transformation L[˜k] : ˜g 7→˜g′. (1) L[˜k] : ˜Dµ 7→˜Dµ′ = ˜Bµ µ′( ˜Dµ −i∂µθ). [ ] µ (2) L[˜k] : ψ 7→ψ′ = ψeiθ. [ ] or a selected ˜ρ, the smooth real function θ is deternimed by L[k] ∈GL(M). See Discussion [ ] (3) For a selected ˜ρ, the smooth real function θ is deternimed by L[k] ∈GL(M). See Discussion 2.3.1 for the deﬁnition of general linear group GL(M). ( ) ρ, y [k] ∈ ( ) 2.3.1 for the deﬁnition of general linear group GL(M). 2.3.1 for the deﬁnition of general linear group GL(M). 2.3.1 for the deﬁnition of general linear group GL(M). A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 49 (4) Suppose ˜g satisﬁes the condition of Proposition 5.6.1 , then \|ψ\| and iγµ ˜Dµψ are both universal geometrical properties of ˜ M. (4) Suppose ˜g satisﬁes the condition of Proposition 5.6.1 , then \|ψ\| and iγµ ˜Dµψ are both universal geometrical properties of ˜ M. (5) The necessary condition of simultaneous ( ˜Γ˜g) µ νσγσ = 0 and ( ˜Γ˜g′) µ νσγσ = 0 is that L[˜k] is an orthogonal transformation. (5) The necessary condition of simultaneous ( ˜Γ˜g) µ νσγσ = 0 and ( ˜Γ˜g′) µ νσγσ = 0 is that L[˜k] is an orthogonal transformation. Proof. Under the transformation L[˜k] : ˜g 7→˜g′ we have Proof. Under the transformation L[˜k] : ˜g 7→˜g′ we have Proof. Under the transformation L[˜k] : ˜g 7→˜g′ we have L[˜k] : ˜Bα µ 7→˜Bα µ′ = Bα µ ˜Bµ µ′, ˜Cµ α 7→˜Cµ′ α = ˜Cµ α ˜Cµ′ µ , ˜Bτ τ 7→˜Bτ τ ′ = Bτ τ ˜Bτ τ ′, ˜Cτ τ 7→˜Cτ ′ τ = ˜Cτ τ ˜Cτ ′ τ . L[˜k] : ∂ ∂˜xµ 7→ ∂ ∂˜xµ′ = ˜Bµ µ′ ∂ ∂˜xµ , d d˜xτ 7→ d d˜xτ ′ = ˜Bτ τ ′ d d˜xτ . L[˜k] : ρ;µ 7→ρ;µ′ = ρ;µ ˜Bµ µ′, ˜Mτ 7→˜Mτ ′ = ˜Mτ ˜Bτ τ ′. L[˜k] : γµ 7→γµ′ = ˜Cµ′ µ γµ. 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation Next we consider (5). The above iγµ′ ˜Dµ′ψ′ = ˜Mτψ′ is obtained in the case where ˜g satisﬁes the condition ( ˜Γ˜g) µ νσγσ = 0 of Proposition 5.6.1 . When ˜g′ satisﬁes condition ( ˜Γ˜g′) µ′ ν′σ′γσ′ = 0, 50 Zhao-Hui Man di t P iti 5 6 1 h i µ′ ˜D ψ′ ˜M ψ′ C h t ti 50 Zhao-Hui Man according to Proposition 5.6.1 we have iγµ′ ˜Dµ′ψ′ = ˜Mτ ′ψ′. Compare such two equations we obtain ˜Mτ = ˜Mτ ′, that is ˜Mτ = ˜Bτ τ ′ ˜Mτ, or ˜Bτ τ ′ = 1. According to Deﬁnition 5.2.2 , L[˜k] is an orthogonal transformation 50 Zhao-Hui Man according to Proposition 5.6.1 we have iγµ′ ˜Dµ′ψ′ = ˜Mτ ′ψ′. Compare such two equations we obtain ˜Mτ = ˜Mτ ′, that is ˜Mτ = ˜Bτ τ ′ ˜Mτ, or ˜Bτ τ ′ = 1. According to Deﬁnition 5.2.2 , L[˜k] is an orthogonal transformation. according to Proposition 5.6.1 we have iγµ′ ˜Dµ′ψ′ = ˜Mτ ′ψ′. Compare such two equations we obtain ˜Mτ = ˜Mτ ′, that is ˜Mτ = ˜Bτ τ ′ ˜Mτ, or ˜Bτ τ ′ = 1. According to Deﬁnition 5.2.2 , L[˜k] is an orthogonal transformation. In order to prove (3), we need to calculate rµ. According to Proposition 2.7.2 , we have ( ˜Γ˜g′) µ′ ν′σ′ = ( ˜Γ˜g) µ νσ ˜Cµ′ µ ˜Bν ν′ ˜Bσ σ′ + ( ˜Γ˜k) µ′ ν′σ′. Due to Discussion 5.4.3 we know [˜ρ ˜Γµ] ≜[˜ρων ˜Γµ] ≜ ˜ρωχ( ˜Γ˜g) χ νµ + ˜ρχν( ˜Γ˜g) χ ωµ and [˜ρ′ ˜Γµ′] ≜[˜ρω′ν′ ˜Γµ′] = ˜ρω′χ′( ˜Γ˜g′) χ′ ν′µ′ + ˜ρχ′ν′( ˜Γ˜g′) χ′ ω′µ′. 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation [˜ρ ˜Γµ′] ≜[˜ρων ˜Γµ′] ≜˜Cω′ ω ˜Cν′ ν [˜ρω′ν′ ˜Γµ′] = ˜Cω′ ω ˜Cν′ ν ˜ρω′χ′( ˜Γ˜g′) χ′ ν′µ′ + ˜ρχ′ν′( ˜Γ˜g′) χ′ ω′µ′ = ˜Cω′ ω ˜Cν′ ν ˜ρω′χ′ ( ˜Γ˜g) χ ρµ ˜Cχ′ χ ˜Bρ ν′ ˜Bµ µ′ + ( ˜Γ˜k) χ′ ν′µ′ + ˜ρχ′ν′ ( ˜Γ˜g) χ σµ ˜Cχ′ χ ˜Bσ ω′ ˜Bµ µ′ + ( ˜Γ˜k) χ′ ω′µ′ = ˜ρωχ( ˜Γ˜g) χ νµ ˜Bµ µ′ + ˜ρχν( ˜Γ˜g) χ ωµ ˜Bµ µ′ + ˜ρωχ′( ˜Γ˜k) χ′ ν′µ′ ˜Cν′ ν + ˜ρχ′ν′( ˜Γ˜k) χ′ ω′µ′ ˜Cω′ ω = ˜ρωχ( ˜Γ˜g) χ νµ ˜Bµ µ′ + ˜ρχν( ˜Γ˜g) χ ωµ ˜Bµ µ′ + ˜ρωχ( ˜Γ˜k) χ′ ν′σ′ ˜Cσ′ µ ˜Bχ χ′ ˜Cν′ ν ˜Bµ µ′ + ˜ρχν( ˜Γ˜k) χ′ ω′σ′ ˜Cσ′ µ ˜Bχ χ′ ˜Cω′ ω ˜Bµ µ′ = [˜ρ ˜Γµ] + ˜ρωχ( ˜Γ˜k) χ′ ν′σ′ ˜Bχ χ′ ˜Cν′ ν ˜Cσ′ µ + ˜ρχν( ˜Γ˜k) χ′ ω′σ′ ˜Bχ χ′ ˜Cω′ ω ˜Cσ′ µ ˜Bµ µ′, hence [˜P ˜Γµ′] = [˜P ˜Γµ] + ˜Pωχ( ˜Γ˜k) χ′ ν′σ′ ˜Bχ χ′ ˜Cν′ ν ˜Cσ′ µ + ˜Pχν( ˜Γ˜k) χ′ ω′σ′ ˜Bχ χ′ ˜Cω′ ω ˜Cσ′ µ ˜Bµ µ′ = [˜P ˜Γµ] + rωνµ ˜Bµ µ′ = [˜P ˜Γµ] + rµ ˜Bµ µ′, (21) where rµ ≜rωνµ ≜˜Pωχ( ˜Γ˜k) χ′ ν′σ′ ˜Bχ χ′ ˜Cν′ ν ˜Cσ′ µ + ˜Pχν( ˜Γ˜k) χ′ ω′σ′ ˜Bχ χ′ ˜Cω′ ω ˜Cσ′ µ . where rµ ≜rωνµ ≜˜Pωχ( ˜Γ˜k) χ′ ν′σ′ ˜Bχ χ′ ˜Cν′ ν ˜Cσ′ µ + ˜Pχν( ˜Γ˜k) χ′ ω′σ′ ˜Bχ χ′ ˜Cω′ ω ˜Cσ′ µ . Wenotice that ˜C and ˜B intheabove rµ areslack-tightsof [˜k],and ( ˜Γ˜k) µ′ ν′σ′ = 1 2 ˜Cµ′ µ ∂Bµ ν′ ∂˜xσ′ + ∂Bµ σ′ ∂xν′ ! is simple connection of [k], therefore for a selected ˜ρ we know rµ is uniquely determined by L[˜k], and furthermore θ ≜ Z rµd˜xµ is uniquely determined by L[˜k]. In consideration of that L[˜k] of ˜ M is determined by L[k] of M, so rµ and θ are ﬁnally determined by L[k]. ⊓⊔ Remark 5.6.2. Conclusions (1)(2)(3) are the reasons why Deﬁnition 2.3.3 calls L[k] a general gauge transformation. Conclusion (4) can also be called the gauge invariance, which utill now has become a theorem, no longer been as a principle. 5.6 Intrinsic geometrical treatment of Dirac equation and gauge transformation This proposition indicates that ψ 7→ψ′ and ˜Dµ 7→˜Dµ′ and gauge invariance such three things have the same mathematical origin, which is the intrinsic transformation L[k], and their physical connotations just only come from the axiom and corollary of section 3.1 . L[k] is an element of general linear group, therefore gauge ﬁelds and gauge transformations deﬁned by whatever subgroup of general linear group can always be characterized by [k] and L[k]. This is the intrinsic geometrical origin of gauge ﬁeld and gauge transformation. In summary, without the viewpoints of intrinsic geometry, it is impossible to clarify that there exists a more fundamental mathematical essence than gauge transformations ψ 7→ψ′ and ˜Dµ 7→˜Dµ′. In consideration of Discussion 5.4.2 , total superiority of viewpoints of intrinsic geometry can be brought into full play just only on manifold M rather than ˜ M, complete details of various intrinsic geometrical properties of gauge ﬁeld can thereby be presented on M. Hence, next we are going to stop the discussions about intrinsic geometry of ˜ M, but to focus on intrinsic A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 51 geometry of M. The intrinsic geometrical properties of the following sections still can only be described by simple connection, but not Levi-Civita connection. Deﬁnition 5.6.1. Let there be a geometrical manifold (M, k), such that M = P ×N, r ≜dimP = 3 and D ≜dimM = 5 or 6 or 8. Deﬁnition 5.6.1. Let there be a geometrical manifold (M, k), such that M = P ×N, r ≜dimP = 3 and D ≜dimM = 5 or 6 or 8. (1) Suppose the local coordinate representation of k is xm′ = xm′(xm) and xi′ = δi′ i xi, which satisﬁes internal standard conditions: (i)Gmn = const, (ii)Gmn = 0 when m ̸= n. We say k is a typical gauge ﬁeld, and L[k] is a typical gauge transformation. [ ] (2) Suppose the local coordinate representation of k is xm′ = xm′(xM) and xi′ = xi′(xi), we say k is a typical gauge ﬁeld with gravitation, and L[k] is a typical gravitational gauge transformation. (2) Suppose the local coordinate representation of k is xm′ = xm′(xM) and xi′ = xi′(xi), we say k is a typical gauge ﬁeld with gravitation, and L[k] is a typical gravitational gauge transformation. 6 Several intrinsic geometrical properties in 5-dimensional case Deﬁnition 6.1. Suppose f satisﬁes the (1) of Deﬁnition 5.6.1 , and geometrical manifold (M, f) satisﬁes D = r + 2 = 5. On a neighborhood U of any point p the coordinate representation of f(p) is ξa = ξa(xm) and ξs = δs i xi, such that G(D−1)(D−1) = GDD. We say f is a weak and electromagnetic uniﬁed ﬁeld. The reason for such naming lies in the following proposition. Proposition 6.1. Let the simple connection of the above (M, f) be ΛM NP and ΛMNP. And let the coeﬃcients of curvature of (M, f) be KM NPQ and KMNPQ. Denote        BP ≜ 1 √ 2 ΛDDP + Λ(D−1)(D−1)P A3 P ≜ 1 √ 2 ΛDDP −Λ(D−1)(D−1)P ,        A1 P ≜ 1 √ 2 Λ(D−1)DP + ΛD(D−1)P A2 P ≜ 1 √ 2 Λ(D−1)DP −ΛD(D−1)P .        BPQ ≜ 1 √ 2 KDDPQ + K(D−1)(D−1)PQ F 3 PQ ≜ 1 √ 2 KDDPQ −K(D−1)(D−1)PQ ,        F 1 PQ ≜ 1 √ 2 K(D−1)DPQ + KD(D−1)PQ F 2 PQ ≜ 1 √ 2 K(D−1)DPQ −KD(D−1)PQ . And denote g ≜ qG(D−1)(D−1)2 + GDD2. Thus the following equations hold, and they are all intrinsic geometrical properties of (M, f). note g ≜ qG(D−1)(D−1)2 + GDD2. Thus the following equations hold, and they are all And denote g ≜ qG(D−1)(D−1)2 + GDD2. Thus the following equations hold, and they And denote g ≜ qG(D−1)(D−1)2 + GDD2. Thus the following equations hold, and they are all intrinsic geometrical properties of (M, f). q intrinsic geometrical properties of (M, f).                          BPQ = ∂BQ ∂xP −∂BP ∂xQ , F 3 PQ = ∂A3 Q ∂xP −∂A3 P ∂xQ + g A1 P A2 Q −A2 P A1 Q , F 1 PQ = ∂A1 Q ∂xP −∂A1 P ∂xQ + g A2 P A3 Q −A3 P A2 Q , F 2 PQ = ∂A2 Q ∂xP −∂A2 P ∂xQ −g A3 P A1 Q −A1 P A3 Q . Proof. 6 Several intrinsic geometrical properties in 5-dimensional case According to the deﬁnition, the slack-tights of f satisfy that Bs m = 0, Ci a = 0. Bs i = δs i , Ba i = 0, Ci s = δi s, Cm s = 0. The metric of f satisﬁes that Gmn = 0(m ̸= n), Gmn = const, GMN ≜δABBA MBB N and GMN = δABCM A CN B . Concretely: Proof. According to the deﬁnition, the slack-tights of f satisfy that Bs m = 0, Ci a = 0. Bs i = δs i , Ba i = 0, Ci s = δi s, Cm s = 0. The metric of f satisﬁes that Gmn = 0(m ̸= n), Gmn = const, GMN ≜δABBA MBB N and GMN = δABCM A CN B . Concretely: 52 Zhao-Hui Man                Gij = δstBs i Bt j + δabBa i Bb j = δstδs i δt j = δij Gin = δstBs i Bt n + δabBa i Bb n = 0 Gmj = δstBs mBt j + δabBa mBb j = 0 Gmn = BD−1 m BD−1 n + BD mBD n ,                Gij = δstCi sCj t = δstδi sδj t = δij Gin = δstCi sCn t = 0 Gmj = δstCm s Cj t = 0 Gmn = Cm D−1Cn D−1 + Cm D Cn D . 6 Several intrinsic geometrical properties in 5-dimensional case Calculate the simeple connection of f, that is ΛM NP ≜ 1 2CM A ∂BA N ∂xP + ∂BA P ∂xN and ΛMNP ≜ GMM′ΛM′ NP, then we obtain                      Λi NP = 0 Λm jk = 0 Λm nP = 1 2Cm a ∂Ba n ∂xP + ∂Ba P ∂xn Λm Np = 1 2Cm a ∂Ba N ∂xp + ∂Ba p ∂xN ,                      ΛiNP = GiM′ΛM′ NP = Gii′Λi′ NP = 0 Λmjk = GmM′ΛM′ jk = Gmm′Λm′ jk = 0 ΛmnP = 1 2δabBb m ∂Ba n ∂xP + ∂Ba P ∂xn ΛmNp = 1 2δabBb m ∂Ba N ∂xp + ∂Ba p ∂xN . Calculate the coeﬃcients of curvature of f, that is Calculate the coeﬃcients of curvature of f, that is Km nPQ ≜ ∂Λm nQ ∂xP −∂Λm nP ∂xQ + Λm HP ΛH nQ −ΛH nP Λm HQ Km nPQ ≜ Q ∂xP − nP ∂xQ + Λm HP ΛH nQ −ΛH nP Λm HQ and KmnPQ ≜GmM′KM′ nPQ = Gmm′Km′ nPQ, then we obtain                                    KD−1 (D−1)PQ = ∂ΛD−1 (D−1)Q ∂xP − ∂ΛD−1 (D−1)P ∂xQ + ΛD−1 DP ΛD (D−1)Q −ΛD (D−1)P ΛD−1 DQ KD−1 DPQ = ∂ΛD−1 DQ ∂xP −∂ΛD−1 DP ∂xQ + ΛD−1 (D−1)P ΛD−1 DQ + ΛD−1 DP ΛD DQ −ΛD−1 DP ΛD−1 (D−1)Q −ΛD DP ΛD−1 DQ KD (D−1)PQ = ∂ΛD (D−1)Q ∂xP − ∂ΛD (D−1)P ∂xQ + ΛD (D−1)P ΛD−1 (D−1)Q + ΛD DP ΛD (D−1)Q −ΛD−1 (D−1)P ΛD (D−1)Q −ΛD (D−1)P ΛD DQ KD DPQ = ∂ΛD DQ ∂xP −∂ΛD DP ∂xQ + ΛD (D−1)P ΛD−1 DQ −ΛD−1 DP ΛD (D−1)Q . 6 Several intrinsic geometrical properties in 5-dimensional case Hence, A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 53 BPQ ≜ 1 √ 2 KDDPQ + K(D−1)(D−1)PQ = 1 √ 2 ∂ ΛDDQ + Λ(D−1)(D−1)Q ∂xP −1 √ 2 ∂ ΛDDP + Λ(D−1)(D−1)P ∂xQ = ∂BQ ∂xP −∂BP ∂xQ . F 3 PQ ≜ 1 √ 2 KDDPQ −K(D−1)(D−1)PQ √ 2 = 1 √ 2 ∂ΛDDQ ∂xP −∂ΛDDP ∂xQ + G(D−1)(D−1) ΛD(D−1)P Λ(D−1)DQ −Λ(D−1)DP ΛD(D−1)Q −1 √ 2 ∂Λ(D−1)(D−1)Q ∂xP −∂Λ(D−1)(D−1)P ∂xQ + GDD Λ(D−1)DP ΛD(D−1)Q −ΛD(D−1)P Λ(D−1)DQ = ∂A3 Q ∂xP −∂A3 P ∂xQ + g ΛD(D−1)P Λ(D−1)DQ −Λ(D−1)DP ΛD(D−1)Q = ∂A3 Q ∂xP −∂A3 P ∂xQ + g A1 P A2 Q −A2 P A1 Q . √ 2 = 1 √ 2 ∂ΛDDQ ∂xP −∂ΛDDP ∂xQ + G(D−1)(D−1) ΛD(D−1)P Λ(D−1)DQ −Λ(D−1)DP ΛD(D−1)Q −1 √ 2 ∂Λ(D−1)(D−1)Q ∂xP −∂Λ(D−1)(D−1)P ∂xQ + GDD Λ(D−1)DP ΛD(D−1)Q −ΛD(D−1)P Λ(D−1)DQ Similarly we also obtain F 1 PQ = ∂A1 Q ∂xP −∂A1 P ∂xQ + g A2 P A3 Q −A3 P A2 Q and F 2 PQ = ∂A2 Q ∂xP −∂A2 P ∂xQ − g A3 P A1 Q −A1 P A3 Q . ⊓⊔ Similarly we also obtain F 1 PQ = ∂A1 Q ∂xP −∂A1 P ∂xQ + g A2 P A3 Q −A3 P A2 Q and F 2 PQ = ∂A2 Q ∂xP −∂A2 P ∂xQ − g A3 P A1 Q −A1 P A3 Q . ⊓⊔ ⊓⊔ Remark 6.1. Comparing the conclusions of the above proposition and the Glashow-Weinberg- Salam theory of physics, we know that this proposition gives another mathematical treatment of weak and electromagnetic ﬁeld of physics. It does not abstractly deﬁne gauge potentials as the adjoint representation of group U(1) × SU(2), but gives concrete intrinsic geometrical constructions. In addition, the following proposition will prove the characteristics of chirality of leptons from the perspective of intrinsic geometry. This is beyond the reach of the traditional method that abstractly regards leptons as the fundamental representation of group U(1) × SU(2). Deﬁnition 6.2. Suppose f and g both satisfy Deﬁnition 6.1 . According to Deﬁnition 3.3.1.1 and Deﬁnition 3.3.5.2 , let ρmn of f evolve on (M, g). Then l ≜(ρ(D−1)(D−1), ρDD) is called electric charged lepton, and ν ≜(ρD(D−1), ρ(D−1)D) is called neutrino. 6 Several intrinsic geometrical properties in 5-dimensional case                                            K(D−1)(D−1)PQ = ∂Λ(D−1)(D−1)Q ∂xP −∂Λ(D−1)(D−1)P ∂xQ +GDD Λ(D−1)DP ΛD(D−1)Q −ΛD(D−1)P Λ(D−1)DQ KD(D−1)PQ = ∂ΛD(D−1)Q ∂xP −∂ΛD(D−1)P ∂xQ + GDD ΛDDP ΛD(D−1)Q −ΛD(D−1)P ΛDDQ +G(D−1)(D−1) ΛD(D−1)P Λ(D−1)(D−1)Q −Λ(D−1)(D−1)P ΛD(D−1)Q K(D−1)DPQ = ∂Λ(D−1)DQ ∂xP −∂Λ(D−1)DP ∂xQ + GDD Λ(D−1)DP ΛDDQ −ΛDDP Λ(D−1)DQ +G(D−1)(D−1) Λ(D−1)(D−1)P Λ(D−1)DQ −Λ(D−1)DP Λ(D−1)(D−1)Q KDDPQ = ∂ΛDDQ ∂xP −∂ΛDDP ∂xQ + G(D−1)(D−1) ΛD(D−1)P Λ(D−1)DQ −Λ(D−1)DP ΛD(D−1)Q . Hence, and KmnPQ ≜GmM′KM′ nPQ = Gmm′Km′ nPQ, KmnPQ ≜GmM′KM′ nPQ = Gmm′Km′ nPQ then we obtain                                  KD−1 (D−1)PQ = ∂ΛD−1 (D−1)Q ∂xP − ∂ΛD−1 (D−1)P ∂xQ + ΛD−1 DP ΛD (D−1)Q −ΛD (D−1)P ΛD−1 DQ KD−1 DPQ = ∂ΛD−1 DQ ∂xP −∂ΛD−1 DP ∂xQ + ΛD−1 (D−1)P ΛD−1 DQ + ΛD−1 DP ΛD DQ −ΛD−1 DP ΛD−1 (D−1)Q −ΛD DP ΛD−1 DQ KD (D−1)PQ = ∂ΛD (D−1)Q ∂xP − ∂ΛD (D−1)P ∂xQ + ΛD (D−1)P ΛD−1 (D−1)Q + ΛD DP ΛD (D−1)Q −ΛD−1 (D−1)P ΛD (D−1)Q −ΛD (D−1)P ΛD DQ D ∂ΛD DQ ∂ΛD DP D D 1 D 1 D .    KD DPQ = ∂ΛDQ ∂xP −∂ΛDP ∂xQ + ΛD (D−1)P ΛD−1 DQ −ΛD−1 DP ΛD (D−1)Q                                            K(D−1)(D−1)PQ = ∂Λ(D−1)(D−1)Q ∂xP −∂Λ(D−1)(D−1)P ∂xQ +GDD Λ(D−1)DP ΛD(D−1)Q −ΛD(D−1)P Λ(D−1)DQ KD(D−1)PQ = ∂ΛD(D−1)Q ∂xP −∂ΛD(D−1)P ∂xQ + GDD ΛDDP ΛD(D−1)Q −ΛD(D−1)P ΛDDQ +G(D−1)(D−1) ΛD(D−1)P Λ(D−1)(D−1)Q −Λ(D−1)(D−1)P ΛD(D−1)Q K(D−1)DPQ = ∂Λ(D−1)DQ ∂xP −∂Λ(D−1)DP ∂xQ + GDD Λ(D−1)DP ΛDDQ −ΛDDP Λ(D−1)DQ +G(D−1)(D−1) Λ(D−1)(D−1)P Λ(D−1)DQ −Λ(D−1)DP Λ(D−1)(D−1)Q KDDPQ = ∂ΛDDQ ∂xP −∂ΛDDP ∂xQ + G(D−1)(D−1) ΛD(D−1)P Λ(D−1)DQ −Λ(D−1)DP ΛD(D−1)Q . 6 Several intrinsic geometrical properties in 5-dimensional case l and ν are uniformly called leptons, denoted by L. And L 1 √ 2 1 1 is called left-handed lepton, L 1 √ 2 1 −1 is called right-handed lepton, denoted by        lL ≜ 1 √ 2 ρ(D−1)(D−1) + ρDD , lR ≜ 1 √ 2 ρ(D−1)(D−1) −ρDD ,        νL ≜ 1 √ 2 ρD(D−1) + ρ(D−1)D , νR ≜ 1 √ 2 ρD(D−1) −ρ(D−1)D . On (M, g) we deﬁne On (M, g) we deﬁne On (M, g) we deﬁne        W 1 P ≜ 1 √ 2 Γ(D−1)DP + ΓD(D−1)P , W 2 P ≜ 1 √ 2 Γ(D−1)DP −ΓD(D−1)P ,        ZP ≜ 1 √ 2 Γ(D−1)(D−1)P + ΓDDP , AP ≜ 1 √ 2 Γ(D−1)(D−1)P −ΓDDP , and say the intrinsic geometrical property Ap is electromagnetic potential, Zp is Z potential, W 1 P and W 2 P are W potential. Then denote and say the intrinsic geometrical property Ap is electromagnetic potential, Zp is Z potential, W 1 P and W 2 P are W potential. Then denote 54 Zhao-Hui Man        W + P ≜ 1 √ 2 W 1 P −iW 2 P W − P ≜ 1 √ 2 W 1 P + iW 2 P ,        l+ L = 1 √ 2 (lL −ilR) l− L = 1 √ 2 (lL + ilR) ,        l+ R ≜ 1 √ 2 (lR −ilL) l− R ≜ 1 √ 2 (lR + ilL) . Proposition 6.2. If (M, g) satisﬁes symmetry condition Γ(D−1)DP = ΓD(D−1)P, then intrinsic geometrical properties l and ν of f satisfy the following conclusions on (M, g). 6 Several intrinsic geometrical properties in 5-dimensional case ⇒               lL;P = ∂P lL − √ 2ρ(D−1)(D−1)Γ D−1 (D−1)P − √ 2ρDDΓ D DP −gνLW 1 P , lR;P = ∂P lR − √ 2ρ(D−1)(D−1)Γ D−1 (D−1)P + √ 2ρDDΓ D DP , νL;P = ∂P νL −glLW 1 P −νL Γ D−1 (D−1)P + Γ D DP ,           ρDD;P = ∂P ρDD −2ρDDΓDP − ρD(D−1) + ρ(D−1)D ΓDP , ρD(D−1);P = ∂P ρD(D−1) − ρ(D−1)(D−1)Γ D−1 DP + ρDDΓ D (D−1)P −ρD(D−1) Γ D−1 (D−1)P + Γ D DP , ρ(D−1)D;P = ∂P ρ(D−1)D − ρ(D−1)(D−1)Γ D−1 DP + ρDDΓ D (D−1)P −ρ(D−1)D Γ D−1 (D−1)P + Γ D DP . ⇒                  lL;P = ∂P lL − √ 2ρ(D−1)(D−1)Γ D−1 (D−1)P − √ 2ρDDΓ D DP −gνLW 1 P , lR;P = ∂P lR − √ 2ρ(D−1)(D−1)Γ D−1 (D−1)P + √ 2ρDDΓ D DP , νL;P = ∂P νL −glLW 1 P −νL Γ D−1 (D−1)P + Γ D DP , νR;P = ∂P νR −νR Γ D−1 (D−1)P + Γ D DP . ⇒                lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P , lR;P = ∂P lR −glRZP −glLAP , νL;P = ∂P νL −gνLZP −glLW 1 P , νR;P = ∂P νR −gνRZP . ⇔                                 l− L;P = ∂P l− L −gl− LZP −gl− RAP −gνLW − P , l+ L;P = ∂P l+ L −gl+ LZP −gl+ RAP −gνLW + P ,    l− R;P = ∂P l− R −gl− RZP −gl− LAP −igνLW − P , l+ R;P = ∂P l+ R −gl+ RZP −gl+ LAP + igνLW + P ,    νL;P = ∂P νL −gνLZP −gl+ LW − P −gl− LW + P , νR;P = ∂P νR −gνRZP . 6 Several intrinsic geometrical properties in 5-dimensional case                            l− L;P = ∂P l− L −gl− LZP −gl− RAP −gνLW − P , l+ L;P = ∂P l+ L −gl+ LZP −gl+ RAP −gνLW + P , l− R;P = ∂P l− R −gl− RZP −gl− LAP −igνLW − P , l+ R;P = ∂P l+ R −gl+ RZP −gl+ LAP + igνLW + P , νL;P = ∂P νL −gνLZP −gl+ LW − P −gl− LW + P , νR;P = ∂P νR −gνRZP .                lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P , lR;P = ∂P lR −glRZP −glLAP , νL;P = ∂P νL −gνLZP −glLW 1 P , νR;P = ∂P νR −gνRZP .                            l− L;P = ∂P l− L −gl− LZP −gl− RAP −gνLW − P , l+ L;P = ∂P l+ L −gl+ LZP −gl+ RAP −gνLW + P , l− R;P = ∂P l− R −gl− RZP −gl− LAP −igνLW − P , l+ R;P = ∂P l+ R −gl+ RZP −gl+ LAP + igνLW + P , νL;P = ∂P νL −gνLZP −gl+ LW − P −gl− LW + P , νR;P = ∂P νR −gνRZP .                lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P , lR;P = ∂P lR −glRZP −glLAP , νL;P = ∂P νL −gνLZP −glLW 1 P , νR;P = ∂P νR −gνRZP . Proof. 6 Several intrinsic geometrical properties in 5-dimensional case Due to ρmn;P = ∂P ρmn −ρHnΓ H mP −ρmHΓ H nP = ∂P ρmn −ρhnΓ h mP −ρmhΓ h nP, we have                  ρ(D−1)(D−1);P = ∂P ρ(D−1)(D−1) −2ρ(D−1)(D−1)Γ D−1 (D−1)P − ρD(D−1) + ρ(D−1)D Γ D (D−1)P , ρDD;P = ∂P ρDD −2ρDDΓ D DP − ρD(D−1) + ρ(D−1)D Γ D−1 DP , ρD(D−1);P = ∂P ρD(D−1) − ρ(D−1)(D−1)Γ D−1 DP + ρDDΓ D (D−1)P −ρD(D−1) Γ D−1 (D−1)P + Γ D DP , ρ(D−1)D;P = ∂P ρ(D−1)D − ρ(D−1)(D−1)Γ D−1 DP + ρDDΓ D (D−1)P −ρ(D−1)D Γ D−1 (D−1)P + Γ D DP . Proof. Due to ρmn;P = ∂P ρmn −ρHnΓ H mP −ρmHΓ H nP = ∂P ρmn −ρhnΓ h mP −ρmhΓ h nP, we have     ρ(D−1)(D−1);P = ∂P ρ(D−1)(D−1) −2ρ(D−1)(D−1)Γ D−1 (D−1)P − ρD(D−1) + ρ(D−1)D Γ D (D−1)P , Proof. Due to ρmn;P = ∂P ρmn −ρHnΓ H mP −ρmHΓ H nP = ∂P ρmn −ρhnΓ h mP −ρmhΓ h nP, we have  ρ(D )(D ) P = ∂P ρ(D )(D ) 2ρ(D )(D )Γ D−1 ρD(D ) + ρ(D )D Γ D Proof. Due to ρmn;P = ∂P ρmn −ρHnΓ H mP −ρmHΓ H nP = ∂P ρmn −ρhnΓ h mP −ρmhΓ h nP, we have                  ρ(D−1)(D−1);P = ∂P ρ(D−1)(D−1) −2ρ(D−1)(D−1)Γ D−1 (D−1)P − ρD(D−1) + ρ(D−1)D Γ D (D−1)P , ρDD;P = ∂P ρDD −2ρDDΓ D DP − ρD(D−1) + ρ(D−1)D Γ D−1 DP , ρD(D−1);P = ∂P ρD(D−1) − ρ(D−1)(D−1)Γ D−1 DP + ρDDΓ D (D−1)P −ρD(D−1) Γ D−1 (D−1)P + Γ D DP , ρ(D−1)D;P = ∂P ρ(D−1)D − ρ(D−1)(D−1)Γ D−1 DP + ρDDΓ D (D−1)P −ρ(D−1)D Γ D−1 (D−1)P + Γ D DP . 6 Several intrinsic geometrical properties in 5-dimensional case 55 A generalization of intrinsic geometry and its application to Hilbert’s 6th problem We can obtain whatever subgeometry of intrinsic geomtry by way of restricting slack-tights via some symmetry conditions, like what section 2.4 and section 2.5 do. Γ(D−1)DP = ΓD(D−1)P in Proposition 6.2 is just exactly such a symmetry condition, which can be regarded as an equivalent condition to deﬁne a kind of subgeometry of intrinsic geometry to describe the weak d l t ti iﬁd ﬁld I We can obtain whatever subgeometry of intrinsic geomtry by way of restricting slack-tights via some symmetry conditions, like what section 2.4 and section 2.5 do. Γ(D−1)DP = ΓD(D−1)P in Proposition 6 2 is just exactly such a symmetry condition which can be regarded as an in Proposition 6.2 is just exactly such a symmetry condition, which can be regarded as an equivalent condition to deﬁne a kind of subgeometry of intrinsic geometry to describe the weak and eletromagnetic uniﬁed ﬁeld. In summary: (1) The traditional physics starts from a very large symmetry group, and reduces symmetries in way of symmetry breaking to approach the target geometry. (2) The viewpoint of intrinsic geometry starts from the smallest symmetry group {e}, and adds symmetries in way of symmetry condition to approach the target geometry. Such two ways must lead to the same destination. They both go towards the same speciﬁc geometry. Such two ways must lead to the same destination. They both go towards the same speciﬁc geometry. From the perspective of uniﬁcation of time and space, it is better to focus on concrete geometrical construction than to focus on abstract algebraic structure, and it is better to study how to add symmetry conditions than to introduce symmetry breaking. From the perspective of uniﬁcation of time and space, it is better to focus on concrete geometrical construction than to focus on abstract algebraic structure, and it is better to study how to add symmetry conditions than to introduce symmetry breaking. Remark 6.2. If we do not consider from the perspective of intrinsic geometry, the Hilbert’s 6th problem can never be solved at the most basic level. In addition: Remark 6.2. If we do not consider from the perspective of intrinsic geometry, the Hilbert’s 6th problem can never be solved at the most basic level. 6 Several intrinsic geometrical properties in 5-dimensional case In addition: (1) Proposition 6.2 gives the mathematical essence of Glashow-Weinberg-Salam theory from the perspective of intrinsic geometry, even the coupling constant g becomes an intrinsic geometrical property. Furthermore, we are able to study Dirac equations of lL, lR, νL, νR in way of section 5.6 , and moreover to construct complex-valued Lagrangian density, and to treat QFT from the perspective of intrinsic geometry in sense of section 3.4 . However, such topics are beyond the subject of this paper, we will not discuss them. (2) We notice that the coupling constants of ZP and AP in Proposition 6.2 satisfy gZ = gA = g, which is comprehensible, because it is just a conclusion at the most basic level. Only when we consider a kind of medium that is called Higgs ﬁeld, there appears a Weinberg angle and thereby we have gZ ̸= gA. When we consider Higgs boson as a zero-spin pair of neutrinos, the Higgs boson will lose its fundamentality and it thereby does not have enough importance in theory of the most basic level. This paper does not concern such non-fundamental objects and properties. (3) The mixing of leptons of three generations will automatically appear as an intrinsic geometrical property on the geometrical manifold of Deﬁnition 8.1 . (3) The mixing of leptons of three generations will automatically appear as an intrinsic geometrical property on the geometrical manifold of Deﬁnition 8.1 . 6 Several intrinsic geometrical properties in 5-dimensional case ⊓⊔ ⇔                                 l− L;P = ∂P l− L −gl− LZP −gl− RAP −gνLW − P , l+ L;P = ∂P l+ L −gl+ LZP −gl+ RAP −gνLW + P ,    l− R;P = ∂P l− R −gl− RZP −gl− LAP −igνLW − P , l+ R;P = ∂P l+ R −gl+ RZP −gl+ LAP + igνLW + P ,    νL;P = ∂P νL −gνLZP −gl+ LW − P −gl− LW + P , νR;P = ∂P νR −gνRZP . ⇒                lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P , lR;P = ∂P lR −glRZP −glLAP , νL;P = ∂P νL −gνLZP −glLW 1 P , νR;P = ∂P νR −gνRZP . ⇔                                 l− L;P = ∂P l− L −gl− LZP −gl− RAP −gνLW − P , l+ L;P = ∂P l+ L −gl+ LZP −gl+ RAP −gνLW + P ,    l− R;P = ∂P l− R −gl− RZP −gl− LAP −igνLW − P , l+ R;P = ∂P l+ R −gl+ RZP −gl+ LAP + igνLW + P ,    νL;P = ∂P νL −gνLZP −gl+ LW − P −gl− LW + P , νR;P = ∂P νR −gνRZP . ⊓⊔ ⊓⊔ ⊓⊔ Discussion 6.1. According to Discussion 2.6.2 , the transformation group of intrinsic geometry is the subgroup {e} which is uniquely made up of the unit element e of general linear group. Its symmetry is the smallest and never breaks, because it is too small to break. In other words, intrinsic geometry is the largest geometry of geometrical manifold, and its geometrical properties are the richest, so that any irregular smooth shape is an intrinsic geometrical property, it thereby can anyway be precisely characterized by slack-tights. Deﬁnition 7.2. Suppose f and g both satisfy Deﬁnition 7.1 . According to Deﬁnition 3.3.1.1 and Deﬁnition 3.3.5.2 , let ρmn of f evolve on (M, g). Deﬁne 7 Several intrinsic geometrical properties in 6-dimensional case Deﬁnition 7.1. Suppose f satisﬁes the (1) of Deﬁnition 5.6.1 , and geometrical manifold (M, f) satisﬁes D = r + 3 = 6. On a neighborhood U of any point p the coordinate representation of f(p) is ξa = ξa(xm) and ξs = δs i xi, such that G(D−2)(D−2) = G(D−1)(D−1) = GDD. We say f is a strong interaction ﬁeld. Deﬁnition 7.2. Suppose f and g both satisfy Deﬁnition 7.1 . According to Deﬁnition 3.3.1.1 and Deﬁnition 3.3.5.2 , let ρmn of f evolve on (M, g). Deﬁne Zhao-Hui Man 56          d1 ≜(ρ(D−2)(D−2), ρ(D−1)(D−1)) d2 ≜(ρ(D−1)(D−1), ρDD) d3 ≜(ρDD, ρ(D−2)(D−2)) ,          u1 ≜(ρ(D−2)(D−1), ρ(D−1)(D−2)) u2 ≜(ρ(D−1)D, ρD(D−1)) u3 ≜(ρD(D−2), ρ(D−2)D) . We say d1 and u1 are red color charge, d2 and u2 are blue color charge, d3 and u3 are green color charge. Then d1, d2, d3 are called down-type color charge, uniformly denoted by d, and u1, u2, u3 are called up-type color charge, uniformly denoted by u. d and u are uniformly called color charge, denoted by q. We say q 1 √ 2 1 1 is left-handed color charge, and q 1 √ 2 1 −1 are called right-handed color charge. They are                  d1L ≜ 1 √ 2 ρ(D−2)(D−2) + ρ(D−1)(D−1) d2L ≜ 1 √ 2 ρ(D−1)(D−1) + ρDD d3L ≜ 1 √ 2 ρDD + ρ(D−2)(D−2) ,                  d1R ≜ 1 √ 2 ρ(D−2)(D−2) −ρ(D−1)(D−1) d2R ≜ 1 √ 2 ρ(D−1)(D−1) −ρDD d3R ≜ 1 √ 2 ρDD −ρ(D−2)(D−2) .                  u1L ≜ 1 √ 2 ρ(D−2)(D−1) + ρ(D−1)(D−2) u2L ≜ 1 √ 2 ρ(D−1)D + ρD(D−1) u3L ≜ 1 √ 2 ρD(D−2) + ρ(D−2)D ,                  u1R ≜ 1 √ 2 ρ(D−2)(D−1) −ρ(D−1)(D−2) u2R ≜ 1 √ 2 ρ(D−1)D −ρD(D−1) u3R ≜ 1 √ 2 ρD(D−2) −ρ(D−2)D . 7 Several intrinsic geometrical properties in 6-dimensional case On (M g) we denote  √ On (M, g) we denote gs ≜ qG(D−1)(D−1)2 + GDD2 = qG(D−1)(D−1)2 + G(D−2)(D−2)2 = qG(D−2)(D−2)2 + GDD2.        U 1 P ≜ 1 √ 2 Γ(D−2)(D−2)P + Γ(D−1)(D−1)P V 1 P ≜ 1 √ 2 Γ(D−2)(D−2)P −Γ(D−1)(D−1)P ,        X23 P ≜ 1 √ 2 Γ(D−2)(D−1)P + Γ(D−1)(D−2)P Y 23 P ≜ 1 √ 2 Γ(D−2)(D−1)P −Γ(D−1)(D−2)P ,        U 2 P ≜ 1 √ 2 Γ(D−1)(D−1)P + ΓDDP V 2 P ≜ 1 √ 2 Γ(D−1)(D−1)P −ΓDDP ,        X31 P ≜ 1 √ 2 Γ(D−1)DP + ΓD(D−1)P Y 31 P ≜ 1 √ 2 Γ(D−1)DP −ΓD(D−1)P ,        U 3 P ≜ 1 √ 2 ΓDDP + Γ(D−2)(D−2)P V 3 P ≜ 1 √ 2 ΓDDP −Γ(D−2)(D−2)P ,        X12 P ≜ 1 √ 2 ΓD(D−2)P + Γ(D−2)DP Y 12 P ≜ 1 √ 2 ΓD(D−2)P −Γ(D−2)DP . gs ≜ qG(D−1)(D−1)2 + GDD2 = qG(D−1)(D−1)2 + G(D−2)(D−2)2 = qG(D−2)(D−2)2 + GDD2        U 1 P ≜ 1 √ 2 Γ(D−2)(D−2)P + Γ(D−1)(D−1)P V 1 P ≜ 1 √ 2 Γ(D−2)(D−2)P −Γ(D−1)(D−1)P ,        X23 P ≜ 1 √ 2 Γ(D−2)(D−1)P + Γ(D−1)(D−2)P Y 23 P ≜ 1 √ 2 Γ(D−2)(D−1)P −Γ(D−1)(D−2)P ,        U 2 P ≜ 1 √ 2 Γ(D−1)(D−1)P + ΓDDP V 2 P ≜ 1 √ 2 Γ(D−1)(D−1)P −ΓDDP ,        X31 P ≜ 1 √ 2 Γ(D−1)DP + ΓD(D−1)P Y 31 P ≜ 1 √ 2 Γ(D−1)DP −ΓD(D−1)P ,        U 3 P ≜ 1 √ 2 ΓDDP + Γ(D−2)(D−2)P V 3 P ≜ 1 √ 2 ΓDDP −Γ(D−2)(D−2)P ,        X12 P ≜ 1 √ 2 ΓD(D−2)P + Γ(D−2)DP Y 12 P ≜ 1 √ 2 ΓD(D−2)P −Γ(D−2)DP . 7 Several intrinsic geometrical properties in 6-dimensional case We just need to substitute the Gell-Mann matrices λ1 ≜     0 1 0 1 0 0 0 0 0    , λ2 ≜     0 −i 0 i 0 0 0 0 0    , λ3 ≜     1 0 0 0 −1 0 0 0 0    , λ4 ≜     0 0 1 0 0 0 1 0 0    , λ5 ≜     0 0 −i 0 0 0 i 0 0    , λ6 ≜     0 0 0 0 0 1 0 1 0    , λ7 ≜     0 0 0 0 0 −i 0 i 0    , λ8 ≜ 1 √ 6     1 0 0 0 1 0 0 0 −2    . to A T Aa and directly verify them ⊓⊔ = TaAa P and directly verify them. ⊓⊔ into AP = TaAa P and directly verify them. into AP = TaAa P and directly verify them. into AP = TaAa P and directly verify them. ⊓⊔ Remark 7.1. On one hand, the above proposition indicates that Deﬁnition 7.1 is another math- ematical treatment of strong interaction ﬁeld of physics. It does not anymore deﬁne the gauge potentials abstractly like QCD based on SU(3) theory, but gives concrete intrinsic geometrical constructions. On the other hand, the above proposition implies that if we take appropriate symmetry conditions, the algebraic properties of SU(3) group can be described by the transfor- mation group GL(3, R) of internal space of g. In other words, there exists a homomorphism from GL(3, R) to SU(3). 7 Several intrinsic geometrical properties in 6-dimensional case We notice that there are just only three independent ones in U 1 P, U 2 P, U 3 P, V 1 P, V 2 P and V 3 P. Without loss of generality, let We notice that there are just only three independent ones in U 1 P, U 2 P, U 3 P, V 1 P, V 2 P and V 3 P. Without loss of generality, let          RP ≜aRU 1 P + bRU 2 P + cRU 3 P SP ≜aSU 1 P + bSU 2 P + cSU 3 P TP ≜aT U 1 P + bT U 2 P + cT U 3 P ,          U 1 P ≜αRRP + αSSP + αT TP U 2 P ≜βRRP + βSSP + βT TP U 3 P ≜γRRP + γSSP + γT TP , where the coeﬃcients matrix are non-singular. here the coeﬃcients matrix are non-singular. where the coeﬃcients matrix are non-singular. Proposition 7.1. Let λa (a = 1, 2, · · · , 8) be the Gell-Mann matrices, and Ta ≜1 2λa be the gener- ators of SU(3) group. When (M, g) satisﬁes symmetry condition Γ(D−2)(D−2)P + Γ(D−1)(D−1)P + ΓDDP = 0, denote Proposition 7.1. Let λa (a = 1, 2, · · · , 8) be the Gell-Mann matrices, and Ta ≜1 2λa be the gener- ators of SU(3) group. When (M, g) satisﬁes symmetry condition Γ(D−2)(D−2)P + Γ(D−1)(D−1)P + ΓDDP = 0, denote Proposition 7.1. Let λa (a = 1, 2, · · · , 8) be the Gell-Mann matrices, and Ta ≜1 2λa be the gener- ators of SU(3) group. 7 Several intrinsic geometrical properties in 6-dimensional case When (M, g) satisﬁes symmetry condition Γ(D−2)(D−2)P + Γ(D−1)(D−1)P + ΓDDP = 0, denote A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 57 AP ≜1 2     A11 P A12 P A13 P A21 P A22 P A23 P A31 P A32 P A33 P    ,            A32 P ≜X23 P + iY 23 P A23 P ≜X23 P −iY 23 P A11 P ≜SP + 1 √ 6TP ,            A31 P ≜X31 P + iY 31 P A13 P ≜X31 P −iY 31 P A22 P ≜−SP + 1 √ 6TP ,            A21 P ≜X12 P + iY 12 P A12 P ≜X12 P −iY 12 P A33 P ≜−2 √ 6TP . Thus, AP = TaAa P if and only if A1 P ≜X12 P , A2 P ≜Y 12 P , A3 P ≜SP, A4 P ≜X31 P , A5 P ≜Y 31 P , A6 P ≜X23 P , A7 P ≜Y 23 P and A8 P ≜TP. P f W d b h G ll M AP ≜1 2     A11 P A12 P A13 P A21 P A22 P A23 P A31 P A32 P A33 P    ,            A32 P ≜X23 P + iY 23 P A23 P ≜X23 P −iY 23 P A11 P ≜SP + 1 √ 6TP ,            A31 P ≜X31 P + iY 31 P A13 P ≜X31 P −iY 31 P A22 P ≜−SP + 1 √ 6TP ,            A21 P ≜X12 P + iY 12 P A12 P ≜X12 P −iY 12 P A33 P ≜−2 √ 6TP . Thus, AP = TaAa P if and only if A1 P ≜X12 P , A2 P ≜Y 12 P , A3 P ≜SP, A4 P ≜X31 P , A5 P ≜Y 31 P , A6 P ≜X23 P , A7 P ≜Y 23 P and A8 P ≜TP. Proof. We just need to substitute the Gell-Mann matrices Proof. 8 Several intrinsic geometrical properties in 8-dimensional case On (M g) we denote On (M, g) we denote      g ≜ qG(D−4)(D−4)2 + G(D−3)(D−3)2, gs ≜ qG(D−1)(D−1)2 + GDD2 = qG(D−1)(D−1)2 + G(D−2)(D−2)2 = qG(D−2)(D−2)2 + GDD2,        ZP ≜ 1 √ 2 (Γ(D−4)(D−4)P + Γ(D−3)(D−3)P ) AP ≜ 1 √ 2 (Γ(D−4)(D−4)P −Γ(D−3)(D−3)P ) ,        W 1 P ≜ 1 √ 2 (Γ(D−4)(D−3)P + Γ(D−3)(D−4)P ) W 2 P ≜ 1 √ 2 (Γ(D−4)(D−3)P −Γ(D−3)(D−4)P ) ,        U 1 P ≜ 1 √ 2 Γ(D−2)(D−2)P + Γ(D−1)(D−1)P V 1 P ≜ 1 √ 2 Γ(D−2)(D−2)P −Γ(D−1)(D−1)P ,        X23 P ≜ 1 √ 2 Γ(D−2)(D−1)P + Γ(D−1)(D−2)P Y 23 P ≜ 1 √ 2 Γ(D−2)(D−1)P −Γ(D−1)(D−2)P ,        U 2 P ≜ 1 √ 2 Γ(D−1)(D−1)P + ΓDDP V 2 P ≜ 1 √ 2 Γ(D−1)(D−1)P −ΓDDP ,        X31 P ≜ 1 √ 2 Γ(D−1)DP + ΓD(D−1)P Y 31 P ≜ 1 √ 2 Γ(D−1)DP −ΓD(D−1)P ,        U 3 P ≜ 1 √ 2 ΓDDP + Γ(D−2)(D−2)P V 3 P ≜ 1 √ 2 ΓDDP −Γ(D−2)(D−2)P ,        X12 P ≜ 1 √ 2 ΓD(D−2)P + Γ(D−2)DP Y 12 P ≜ 1 √ 2 ΓD(D−2)P −Γ(D−2)DP . Deﬁnition 8.3. Deﬁne the symmetry condition of uniﬁcation: (1) Basic conditions, No.1: Deﬁnition 8.3. 8 Several intrinsic geometrical properties in 8-dimensional case O (M ) d t                  d1L ≜ 1 √ 2 ρ(D−2)(D−2) + ρ(D−1)(D−1) d2L ≜ 1 √ 2 ρ(D−1)(D−1) + ρDD d3L ≜ 1 √ 2 ρDD + ρ(D−2)(D−2) ,                  d1R ≜ 1 √ 2 ρ(D−2)(D−2) −ρ(D−1)(D−1) d2R ≜ 1 √ 2 ρ(D−1)(D−1) −ρDD d3R ≜ 1 √ 2 ρDD −ρ(D−2)(D−2) ,                  u1L ≜ 1 √ 2 ρ(D−2)(D−1) + ρ(D−1)(D−2) u2L ≜ 1 √ 2 ρ(D−1)D + ρD(D−1) u3L ≜ 1 √ 2 ρD(D−2) + ρ(D−2)D ,                  u1R ≜ 1 √ 2 ρ(D−2)(D−1) −ρ(D−1)(D−2) u2R ≜ 1 √ 2 ρ(D−1)D −ρD(D−1) u3R ≜ 1 √ 2 ρD(D−2) −ρ(D−2)D . 8 Several intrinsic geometrical properties in 8-dimensional case                l ≜ ρ(D−4)(D−4), ρ(D−3)(D−3) d1 ≜(ρ(D−2)(D−2), ρ(D−1)(D−1)) d2 ≜(ρ(D−1)(D−1), ρDD) d3 ≜(ρDD, ρ(D−2)(D−2)) And Denote And Denote e ote        lL ≜ 1 √ 2 ρ(D−4)(D−4) + ρ(D−3)(D−3) lR ≜ 1 √ 2 ρ(D−4)(D−4) −ρ(D−3)(D−3) ,        νL ≜ 1 √ 2 ρ(D−3)(D−4) + ρ(D−4)(D−3) νR ≜ 1 √ 2 ρ(D−3)(D−4) −ρ(D−4)(D−3) , 58 Zhao-Hui Man                  d1L ≜ 1 √ 2 ρ(D−2)(D−2) + ρ(D−1)(D−1) d2L ≜ 1 √ 2 ρ(D−1)(D−1) + ρDD d3L ≜ 1 √ 2 ρDD + ρ(D−2)(D−2) ,                  d1R ≜ 1 √ 2 ρ(D−2)(D−2) −ρ(D−1)(D−1) d2R ≜ 1 √ 2 ρ(D−1)(D−1) −ρDD d3R ≜ 1 √ 2 ρDD −ρ(D−2)(D−2) ,                  u1L ≜ 1 √ 2 ρ(D−2)(D−1) + ρ(D−1)(D−2) u2L ≜ 1 √ 2 ρ(D−1)D + ρD(D−1) u3L ≜ 1 √ 2 ρD(D−2) + ρ(D−2)D ,                  u1R ≜ 1 √ 2 ρ(D−2)(D−1) −ρ(D−1)(D−2) u2R ≜ 1 √ 2 ρ(D−1)D −ρD(D−1) u3R ≜ 1 √ 2 ρD(D−2) −ρ(D−2)D . 8 Several intrinsic geometrical properties in 8-dimensional case Deﬁnition 8.1. Suppose f satisﬁes the (1) of Deﬁnition 5.6.1 , and geometrical manifold (M, f) satisﬁes D = r + 5 = 8. On a neighborhood U of any point p the coordinate representation of f(p) is ξa = ξa(xm) and ξs = δs i xi, such that G(D−4)(D−4) = G(D−3)(D−3) and G(D−2)(D−2) = G(D−1)(D−1) = GDD. We say f is a typical uniﬁed gauge ﬁeld. Deﬁnition 8.2. Suppose f and g both satisfy Deﬁnition 8.1 . According to Deﬁnition 3.3.1.1 and Deﬁnition 3.3.5.2 , let ρmn of f evolve on (M, g). Deﬁne                l ≜ ρ(D−4)(D−4), ρ(D−3)(D−3) d1 ≜(ρ(D−2)(D−2), ρ(D−1)(D−1)) d2 ≜(ρ(D−1)(D−1), ρDD) d3 ≜(ρDD, ρ(D−2)(D−2)) ,                ν ≜ ρ(D−3)(D−4), ρ(D−4)(D−3) u1 ≜(ρ(D−2)(D−1), ρ(D−1)(D−2)) u2 ≜(ρ(D−1)D, ρD(D−1)) u3 ≜(ρD(D−2), ρ(D−2)D) . (5) CKM mixing conditions of strong interaction, No.1: (5) CKM mixing conditions of strong interaction, No.1: (5) CKM mixing conditions of strong interaction, No.1: 5) CKM mixing conditions of strong interaction, No.1:          Γ D−3 (D−2)P = cD−4 D−2Γ D−3 (D−4)P , Γ D−3 (D−1)P = cD−4 D−1Γ D−3 (D−4)P , Γ D−3 DP = cD−4 D Γ D−3 (D−4)P ,          Γ D−4 (D−2)P = cD−3 D−2Γ D−4 (D−3)P , Γ D−4 (D−1)P = cD−3 D−1Γ D−4 (D−3)P , Γ D−4 DP = cD−3 D Γ D−4 (D−3)P ,    cD−4 D−2 = cD−4 D−1 = cD−4 D , cD−3 D−2 = cD−3 D−1 = cD−3 D , (6) CKM mixing conditions of strong interaction, No.2:    ρ(D−2)(D−3) = ρ(D−1)(D−3) = ρD(D−3), ρ(D−2)(D−4) = ρ(D−1)(D−4) = ρD(D−4),    ρ(D−3)(D−2) = ρ(D−3)(D−1) = ρ(D−3)D, ρ(D−4)(D−2) = ρ(D−4)(D−1) = ρ(D−4)D, where cm n are constants. where cm n are constants. Proposition 8.1. When (M, g) satisﬁes the symmetry conditions of Deﬁnition 8.3 , denote p ( , g) y y , l′ ≜ ρ(D−4)(D−4) + cD−2 D−4 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−4 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−4 2 ρD(D−4) + ρ(D−4)D , ρ(D−3)(D−3) + cD−2 D−3 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) +cD−1 D−3 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−3 2 ρD(D−3) + ρ(D−3)D ! . ν′ ≜ ρ(D−3)(D−4) + cD−2 D−3 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−3 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−3 2 ρD(D−4) + ρ(D−4)D , ρ(D−4)(D−3) + cD−2 D−4 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) +cD−1 D−4 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−4 2 ρD(D−3) + ρ(D−3)D ! . l′ ≜ ρ(D−4)(D−4) + cD−2 D−4 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−4 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−4 2 ρD(D−4) + ρ(D−4)D , ρ(D−3)(D−3) + cD−2 D−3 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) +cD−1 D−3 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−3 2 ρD(D−3) + ρ(D−3)D ! . 8 Several intrinsic geometrical properties in 8-dimensional case Deﬁne the symmetry condition of uniﬁcation: (1) Basic conditions, No.1:    G(D−4)(D−4) = G(D−3)(D−3), G(D−2)(D−2) = G(D−1)(D−1) = GDD, (2) Basic conditions, No.2: (2) Basic conditions, No.2: (2) Basic conditions, No.2:    Γ(D−3)(D−4)P = Γ(D−4)(D−3)P , Γ(D−2)(D−2)P + Γ(D−1)(D−1)P + ΓDDP = 0, (3) MNS mixing conditions of weak interaction, No.1: (3) MNS mixing conditions of weak interaction, No.1:          Γ D−2 (D−4)P = cD−2 D−3Γ D−3 (D−4)P , Γ D−1 (D−4)P = cD−1 D−3Γ D−3 (D−4)P , Γ D (D−4)P = cD D−3Γ D−3 (D−4)P ,          Γ D−2 (D−3)P = cD−2 D−4Γ D−4 (D−3)P , Γ D−1 (D−3)P = cD−1 D−4Γ D−4 (D−3)P , Γ D (D−3)P = cD D−4Γ D−4 (D−3)P ,          cD−2 D−3 = cD−2 D−4, cD−1 D−3 = cD−1 D−4, cD D−3 = cD D−4,          Γ D−2 (D−3)P = cD−2 D−4Γ D−4 (D−3)P , Γ D−1 (D−3)P = cD−1 D−4Γ D−4 (D−3)P , Γ D (D−3)P = cD D−4Γ D−4 (D−3)P ,          cD−2 D−3 = cD−2 D−4, cD−1 D−3 = cD−1 D−4, cD D−3 = cD D−4, A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 59 (4) MNS mixing conditions of weak interaction, No.2: (4) MNS mixing conditions of weak interaction, No.2: (4) MNS mixing conditions of weak interaction, No.2:          ρ(D−2)(D−3) = ρ(D−2)(D−4), ρ(D−1)(D−3) = ρ(D−1)(D−4), ρD(D−3) = ρD(D−4),          ρ(D−3)(D−2) = ρ(D−4)(D−2), ρ(D−3)(D−1) = ρ(D−4)(D−1), ρ(D−3)D = ρ(D−4)D, (5) CKM mixing conditions of strong interaction, No.1: ρmn;P = ∂P ρmn −ρHnΓ H mP −ρmHΓ H nP According to Deﬁnition 8.2 and Deﬁnition 8.3 , by calculation we obtain that lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P −1 2 h cD−2 D−4 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−2 D−3 ρ(D−2)(D−4) + ρ(D−4)(D−2) i g √ 2W 1 P −1 2 h cD−1 D−4 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD−1 D−3 ρ(D−1)(D−4) + ρ(D−4)(D−1) i g √ 2W 1 P lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P −1 2 h cD−2 D−4 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−2 D−3 ρ(D−2)(D−4) + ρ(D−4)(D−2) i g √ 2W 1 P lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P lL;P = ∂P lL −glLZP −glRAP −gνLW 1 P −1 2 h cD−2 D−4 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−2 D−3 ρ(D−2)(D−4) + ρ(D−4)(D−2) i g √ 2W 1 P −1 2 h cD−1 D−4 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD−1 D−3 ρ(D−1)(D−4) + ρ(D−4)(D−1) i g √ 2W 1 P −1 2 h cD D−4 ρD(D−3) + ρ(D−3)D + cD D−3 ρD(D−4) + ρ(D−4)D i g √ 2W 1 P , lR;P = ∂P lR −glRZP −glLAP , −1 2 h cD−2 D−4 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−2 D−3 ρ(D−2)(D−4) + ρ(D−4)(D−2) i g √ 2W 1 P −1 2 h cD−1 D−4 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD−1 D−3 ρ(D−1)(D−4) + ρ(D−4)(D−1) i g √ 2W 1 P 1 h i g −2 h cD 2 D−4 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD 2 D−3 ρ(D−2)(D−4) + ρ(D−4)(D−2) i g √ 2W 1 P −1 2 h cD−1 D−4 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD−1 D−3 ρ(D−1)(D−4) + ρ(D−4)(D−1) i g √ 2W 1 P −1 2 h cD D−4 ρD(D−3) + ρ(D−3)D + cD D−3 ρD(D−4) + ρ(D−4)D i g √ 2W 1 P , √ −1 2 h cD−1 D−4 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD−1 D−3 ρ(D−1)(D−4) + ρ(D−4)(D−1) i g √ 2W 1 P −1 2 h cD D−4 ρD(D−3) + ρ(D−3)D + cD D−3 ρD(D−4) + ρ(D−4)D i g √ 2W 1 P , lR;P = ∂P lR −glRZP −glLAP , lR;P = ∂P lR −glRZP −glLAP , νL;P = ∂P νL −gνLZP −glLW 1 P lR;P = ∂P lR −glRZP −glLAP , ; g g P −1 2 h cD−2 D−4 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−2 D−3 ρ(D−3)(D−2) + ρ(D−2)(D−3) i g √ 2W 1 P −1 2 h cD−1 D−4 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD−1 D−3 ρ(D−3)(D−1) + ρ(D−1)(D−3) i g √ 2W 1 P −1 2 h cD D−4 ρD(D−4) + ρ(D−4)D + cD D−3 ρ(D−3)D + ρD(D−3) i g √ 2W 1 P , νR;P = ∂P νR −gνRZP . (5) CKM mixing conditions of strong interaction, No.1: Then, according to deﬁnitions of l′ and ν′, we obtain that l′ L = lL l′ L = lL + cD−2 D−4 2 √ 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−4 2 √ 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−4 2 √ 2 ρD(D−4) + ρ(D−4)D + cD−2 D−3 2 √ 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−1 D−3 2 √ 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−3 2 √ 2 ρD(D−3) + ρ(D−3)D ν′ L = νL + cD−2 D−3 2 √ 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−3 2 √ 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−3 2 √ 2 ρD(D−4) + ρ(D−4)D + cD−2 D−4 2 √ 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−1 D−4 2 √ 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−4 2 √ 2 ρD(D−3) + ρ(D−3)D . D−2 D−4 2 √ 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−4 2 √ 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−4 2 √ 2 ρD(D−4) + ρ(D−4)D D−2 D−3 2 √ 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−1 D−3 2 √ 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−3 2 √ 2 ρD(D−3) + ρ(D−3)D + cD−2 D−3 2 √ 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−3 2 √ 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−3 2 √ 2 ρD(D−4) + ρ(D−4)D + cD−2 D−4 2 √ 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) + cD−1 D−4 2 √ 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−4 2 √ 2 ρD(D−3) + ρ(D−3)D . Substitute them into the previous equations, and we obtain that Substitute them into the previous equations, and we obtain that ubstitute them into the previous equations, and we obtain that    lL;P = ∂P lL −glLZP −glRAP −gν′ LW 1 P , lR;P = ∂P lR −glRZP −glLAP , ,    νL;P = ∂P νL −gνLZP −gl′ LW 1 P , νR;P = ∂P νR −gνRZP . ⊓⊔ Remark 8.1. Reviewing Discussion 6.1 , we know the above proposition gives the mathematical essence of MNS mixing of weak interaction from the perspective of intrinsic geometry. In math- ematics, the MNS mixing automatically appears as an intrinsic geometrical property, therefore it is not necessary to postulate artiﬁcially like that in physics. Remark 8.1. (5) CKM mixing conditions of strong interaction, No.1: ν′ ≜ ρ(D−3)(D−4) + cD−2 D−3 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) ν′ ≜ ρ(D−3)(D−4) + D−3 2 ρ(D−2)(D−4) + ρ(D−4)(D−2) + cD−1 D−3 2 ρ(D−1)(D−4) + ρ(D−4)(D−1) + cD D−3 2 ρD(D−4) + ρ(D−4)D , ρ(D−4)(D−3) + cD−2 D−4 2 ρ(D−2)(D−3) + ρ(D−3)(D−2) +cD−1 D−4 2 ρ(D−1)(D−3) + ρ(D−3)(D−1) + cD D−4 2 ρD(D−3) + ρ(D−3)D ! . Thus, the intrinsic geometrical properties l and ν of f satisfy the following conclusions on (M, g). Thus, the intrinsic geometrical properties l and ν of f satisfy the following conclusions on (M, g).                lL;P = ∂P lL −glLZP −glRAP −gν′ LW 1 P , lR;P = ∂P lR −glRZP −glLAP , νL;P = ∂P νL −gνLZP −gl′ LW 1 P , νR;P = ∂P νR −gνRZP . (22) (22) Zhao-Hui Man 60 Proof. First, ρmn of f can be calculated as below: f. First, ρmn of f can be calculated as below: Proof. First, ρmn of f can be calculated as below: ρmn;P = ∂P ρmn −ρHnΓ H mP −ρmHΓ H nP = ∂P ρmn −ρ(D−4)nΓ D−4 mP −ρ(D−3)nΓ D−3 mP −ρ(D−2)nΓ D−2 mP −ρ(D−1)nΓ D−1 mP −ρDnΓ D mP −ρm(D−4)Γ D−4 nP −ρm(D−3)Γ D−3 nP −ρm(D−2)Γ D−2 nP −ρm(D−1)Γ D−1 nP −ρmDΓ D nP . (5) CKM mixing conditions of strong interaction, No.1: When (M, g) satisﬁes the symmetry conditions of Deﬁnition 8.3 , denote d′ 1L ≜ 1 2 √ 2cD−3 D−1(ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−3 D−2(ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−4 D−1(ρ(D−3)(D−2) + ρ(D−2)(D−3)) + 1 2 √ 2cD−4 D−2(ρ(D−3)(D−1) + ρ(D−1)(D−3)) d′ 2L ≜ 1 2 √ 2cD−3 D (ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−3 D−1(ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−4 D (ρ(D−3)(D−1) + ρ(D−1)(D−3)) + 1 2 √ 2cD−4 D−1(ρ(D−3)D + ρD(D−3)) d′ 3L ≜ 1 2 √ 2cD−3 D−2(ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−3 D (ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−4 D−2(ρ(D−3)D + ρD(D−3)) + 1 2 √ 2cD−4 D (ρ(D−3)(D−2) + ρ(D−2)(D−3)) u′ 1L ≜ 1 2 √ 2cD−3 D−2(ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−4 D−2(ρ(D−3)(D−2) + ρ(D−2)(D−3)) + 1 2 √ 2cD−3 D−1(ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−4 D−1(ρ(D−3)(D−1) + ρ(D−1)(D−3)) u′ 2L ≜ 1 2 √ 2cD−3 D−1(ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−4 D−1(ρ(D−3)(D−1) + ρ(D−1)(D−3)) + 1 2 √ 2cD−3 D (ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−4 D (ρ(D−3)D + ρD(D−3)) u′ 3L ≜ 1 2 √ 2cD−3 D (ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−4 D (ρ(D−3)D + ρD(D−3)) + 1 2 √ 2cD−3 D−2(ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−4 D−2(ρ(D−3)(D−2) + ρ(D−2)(D−3)). (5) CKM mixing conditions of strong interaction, No.1: Reviewing Discussion 6.1 , we know the above proposition gives the mathematical essence of MNS mixing of weak interaction from the perspective of intrinsic geometry. In math- ematics, the MNS mixing automatically appears as an intrinsic geometrical property, therefore it is not necessary to postulate artiﬁcially like that in physics. A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 61 In physics, e, µ and τ have just only ontological diﬀerences, but they have no diﬀerence in mathematical connotation. By contrast, Proposition 8.1 tells us that leptons of three generations should be constructed by diﬀerent linear combinations of ρ(D−2)(D−3), ρ(D−3)(D−2), ρ(D−1)(D−3), ρ(D−3)(D−1), ρD(D−3), ρ(D−3)D, ρ(D−2)(D−4), ρ(D−4)(D−2), ρ(D−1)(D−4), ρ(D−4)(D−1), ρD(D−4) and ρ(D−4)D. Thus, e, µ and τ may have concrete and distinguishable mathematical connotations. For example, suppose aτ, bτ, aτ m n , bτ m n are constants, then we can image that    e ≜l = (ρ(D−4)(D−4), ρ(D−3)(D−3)), νe ≜ν = (ρ(D−3)(D−4), ρ(D−4)(D−3)).                    µ ≜aµe + 1 2 aµD−2 D−4ρ(D−2)(D−4) + aµD−1 D−4ρ(D−1)(D−4) + aµD D−4ρD(D−4), aµD−2 D−3ρ(D−2)(D−3) + aµD−1 D−3ρ(D−1)(D−3) + aµD D−3ρD(D−3) . νµ ≜bµνe + 1 2 bµD−2 D−3ρ(D−2)(D−4) + bµD−1 D−3ρ(D−1)(D−4) + bµD D−3ρD(D−4), bµD−2 D−4ρ(D−2)(D−3) + bµD−1 D−4ρ(D−1)(D−3) + bµD D−4ρD(D−3) .                    τ ≜aτµ + 1 2 aτ D−2 D−4ρ(D−4)(D−2) + aτ D−1 D−4ρ(D−4)(D−1) + aτ D D−4ρ(D−4)D, aτ D−2 D−3ρ(D−3)(D−2) + aτ D−1 D−3ρ(D−3)(D−1) + aτ D D−3ρ(D−3)D . ντ ≜bτνµ + 1 2 bτ D−2 D−3ρ(D−4)(D−2) + bτ D−1 D−3ρ(D−4)(D−1) + bτ D D−3ρ(D−4)D, bτ D−2 D−4ρ(D−3)(D−2) + bτ D−1 D−4ρ(D−3)(D−1) + bτ D D−4ρ(D−3)D . Proposition 8.2. (5) CKM mixing conditions of strong interaction, No.1: d1L;P = ∂P d1L −gsd1LU 1 P + gsd2LV 1 P −gsd3LV 1 P −gsu1LX23 P −gs 2 u2LX31 P + gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P −gu′ 1LW 1 P d2L;P = ∂P d2L −gsd2LU 2 P + gsd3LV 2 P −gsd1LV 2 P −gsu2LX31 P −gs 2 u3LX12 P + gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P −gu′ 2LW 1 P d3L;P = ∂P d3L −gsd3LU 3 P + gsd1LV 3 P −gsd2LV 3 P −gsu3LX12 P −gs 2 u1LX23 P + gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P −gu′ 3LW 1 P d1R;P = ∂P d1R −gsd1LV 1 P + gsd2LU 1 P −gsd3LU 1 P + gsu1LY 23 P + gs 2 u2LX31 P −gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P d2R;P = ∂P d2R −gsd2LV 2 P + gsd3LU 2 P −gsd1LU 2 P + gsu2LY 31 P + gs 2 u3LX12 P −gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P d3R;P = ∂P d3R −gsd3LV 3 P + gsd1LU 3 P −gsd2LU 3 P + gsu3LY 12 P + gs 2 u1LX23 P −gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P u1L;P = ∂P u1L −gsu1LU 1 P −gs 2 u2LX12 P −gs 2 u2LY 12 P −gs 2 u3LX31 P + gs 2 u3LY 31 P −gsd1LX23 P + gsd2LY 23 P −gsd3LY 23 P −gd′ 1LW 1 P u2L;P = ∂P u2L −gsu2LU 2 P −gs 2 u3LX23 P −gs 2 u3LY 23 P −gs 2 u1LX12 P + gs 2 u1LY 12 P −gsd2LX31 P + gsd3LY 31 P −gsd1LY 31 P −gd′ 2LW 1 P u3L;P = ∂P u3L −gsu3LU 3 P −gs 2 u1LX31 P −gs 2 u1LY 31 P −gs 2 u2LX23 P + gs 2 u2LY 23 P −gsd3LX12 P + gsd1LY 12 P −gsd2LY 12 P −gd′ 3LW 1 P u1R;P = ∂P u1R −gsu1RU 1 P + gs 2 u2RX12 P + gs 2 u2RY 12 P + gs 2 u3RX31 P −gs 2 u3RY 31 P u2R;P = ∂P u2R −gsu2RU 2 P + gs 2 u3RX23 P + gs 2 u3RY 23 P + gs 2 u1RX12 P −gs 2 u1RY 12 P u3R;P = ∂P u3R −gsu3RU 3 P + gs 2 u1RX31 P + gs 2 u1RY 31 P + gs 2 u2RX23 P −gs 2 u2RY 23 P . (5) CKM mixing conditions of strong interaction, No.1: d′ 1L ≜ 1 2 √ 2cD−3 D−1(ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−3 D−2(ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−4 D−1(ρ(D−3)(D−2) + ρ(D−2)(D−3)) + 1 2 √ 2cD−4 D−2(ρ(D−3)(D−1) + ρ(D−1)(D−3)) d′ 2L ≜ 1 2 √ 2cD−3 D (ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−3 D−1(ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−4 D (ρ(D−3)(D−1) + ρ(D−1)(D−3)) + 1 2 √ 2cD−4 D−1(ρ(D−3)D + ρD(D−3)) d′ 3L ≜ 1 2 √ 2cD−3 D−2(ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−3 D (ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−4 D−2(ρ(D−3)D + ρD(D−3)) + 1 2 √ 2cD−4 D (ρ(D−3)(D−2) + ρ(D−2)(D−3)) u′ 1L ≜ 1 2 √ 2cD−3 D−2(ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−4 D−2(ρ(D−3)(D−2) + ρ(D−2)(D−3)) + 1 2 √ 2cD−3 D−1(ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−4 D−1(ρ(D−3)(D−1) + ρ(D−1)(D−3)) u′ 2L ≜ 1 2 √ 2cD−3 D−1(ρ(D−4)(D−1) + ρ(D−1)(D−4)) + 1 2 √ 2cD−4 D−1(ρ(D−3)(D−1) + ρ(D−1)(D−3)) + 1 2 √ 2cD−3 D (ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−4 D (ρ(D−3)D + ρD(D−3)) u′ 3L ≜ 1 2 √ 2cD−3 D (ρ(D−4)D + ρD(D−4)) + 1 2 √ 2cD−4 D (ρ(D−3)D + ρD(D−3)) + 1 2 √ 2cD−3 D−2(ρ(D−4)(D−2) + ρ(D−2)(D−4)) + 1 2 √ 2cD−4 D−2(ρ(D−3)(D−2) + ρ(D−2)(D−3)). 62 Zhao-Hui Man Then the intrinsic geometrical properties d1, d2, d3, u1, u2 and u3 of f satisfy the following conclusions on (M, g). (5) CKM mixing conditions of strong interaction, No.1: d1L;P = ∂P d1L −gsd1LU 1 P + gsd2LV 1 P −gsd3LV 1 P −gsu1LX23 P −gs 2 u2LX31 P + gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P −gu′ 1LW 1 P d2L;P = ∂P d2L −gsd2LU 2 P + gsd3LV 2 P −gsd1LV 2 P −gsu2LX31 P −gs 2 u3LX12 P + gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P −gu′ 2LW 1 P d3L;P = ∂P d3L −gsd3LU 3 P + gsd1LV 3 P −gsd2LV 3 P −gsu3LX12 P −gs 2 u1LX23 P + gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P −gu′ 3LW 1 P d1R;P = ∂P d1R −gsd1LV 1 P + gsd2LU 1 P −gsd3LU 1 P + gsu1LY 23 P + gs 2 u2LX31 P −gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P d2R;P = ∂P d2R −gsd2LV 2 P + gsd3LU 2 P −gsd1LU 2 P + gsu2LY 31 P + gs 2 u3LX12 P −gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P d3R;P = ∂P d3R −gsd3LV 3 P + gsd1LU 3 P −gsd2LU 3 P + gsu3LY 12 P + gs 2 u1LX23 P −gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P u1L;P = ∂P u1L −gsu1LU 1 P −gs 2 u2LX12 P −gs 2 u2LY 12 P −gs 2 u3LX31 P + gs 2 u3LY 31 P −gsd1LX23 P + gsd2LY 23 P −gsd3LY 23 P −gd′ 1LW 1 P u2L;P = ∂P u2L −gsu2LU 2 P −gs 2 u3LX23 P −gs 2 u3LY 23 P −gs 2 u1LX12 P + gs 2 u1LY 12 P −gsd2LX31 P + gsd3LY 31 P −gsd1LY 31 P −gd′ 2LW 1 P u3L;P = ∂P u3L −gsu3LU 3 P −gs 2 u1LX31 P −gs 2 u1LY 31 P −gs 2 u2LX23 P + gs 2 u2LY 23 P −gsd3LX12 P + gsd1LY 12 P −gsd2LY 12 P −gd′ 3LW 1 P u1R;P = ∂P u1R −gsu1RU 1 P + gs 2 u2RX12 P + gs 2 u2RY 12 P + gs 2 u3RX31 P −gs 2 u3RY 31 P u2R;P = ∂P u2R −gsu2RU 2 P + gs 2 u3RX23 P + gs 2 u3RY 23 P + gs 2 u1RX12 P −gs 2 u1RY 12 P u3R;P = ∂P u3R −gsu3RU 3 P + gs 2 u1RX31 P + gs 2 u1RY 31 P + gs 2 u2RX23 P −gs 2 u2RY 23 P . (5) CKM mixing conditions of strong interaction, No.1: d1L;P = ∂P d1L −gsd1LU 1 P + gsd2LV 1 P −gsd3LV 1 P −gsu1LX23 P −gs 2 u2LX31 P + gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P −gu′ 1LW 1 P d2L;P = ∂P d2L −gsd2LU 2 P + gsd3LV 2 P −gsd1LV 2 P −gsu2LX31 P −gs 2 u3LX12 P + gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P −gu′ 2LW 1 P d3L;P = ∂P d3L −gsd3LU 3 P + gsd1LV 3 P −gsd2LV 3 P −gsu3LX12 P −gs 2 u1LX23 P + gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P −gu′ 3LW 1 P d1R;P = ∂P d1R −gsd1LV 1 P + gsd2LU 1 P −gsd3LU 1 P + gsu1LY 23 P + gs 2 u2LX31 P −gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P d2R;P = ∂P d2R −gsd2LV 2 P + gsd3LU 2 P −gsd1LU 2 P + gsu2LY 31 P + gs 2 u3LX12 P −gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P d3R;P = ∂P d3R −gsd3LV 3 P + gsd1LU 3 P −gsd2LU 3 P + gsu3LY 12 P + gs 2 u1LX23 P −gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P u1L;P = ∂P u1L −gsu1LU 1 P −gs 2 u2LX12 P −gs 2 u2LY 12 P −gs 2 u3LX31 P + gs 2 u3LY 31 P −gsd1LX23 P + gsd2LY 23 P −gsd3LY 23 P −gd′ 1LW 1 P u2L;P = ∂P u2L −gsu2LU 2 P −gs 2 u3LX23 P −gs 2 u3LY 23 P −gs 2 u1LX12 P + gs 2 u1LY 12 P −gsd2LX31 P + gsd3LY 31 P −gsd1LY 31 P −gd′ 2LW 1 P u3L;P = ∂P u3L −gsu3LU 3 P −gs 2 u1LX31 P −gs 2 u1LY 31 P −gs 2 u2LX23 P + gs 2 u2LY 23 P −gsd3LX12 P + gsd1LY 12 P −gsd2LY 12 P −gd′ 3LW 1 P u1R;P = ∂P u1R −gsu1RU 1 P + gs 2 u2RX12 P + gs 2 u2RY 12 P + gs 2 u3RX31 P −gs 2 u3RY 31 P u2R;P = ∂P u2R −gsu2RU 2 P + gs 2 u3RX23 P + gs 2 u3RY 23 P + gs 2 u1RX12 P −gs 2 u1RY 12 P u3R;P = ∂P u3R −gsu3RU 3 P + gs 2 u1RX31 P + gs 2 u1RY 31 P + gs 2 u2RX23 P −gs 2 u2RY 23 P . (5) CKM mixing conditions of strong interaction, No.1: Proof. Substitute Deﬁnition 8.2 into ρmn and consider Deﬁnition 8.3 , then by calculation we ﬁnally obtain that Proof. (5) CKM mixing conditions of strong interaction, No.1: Substitute Deﬁnition 8.2 into ρmn and consider Deﬁnition 8.3 , then by calculation we ﬁnally obtain that d1L;P = ∂P d1L −gsd1LU 1 P + gsd2LV 1 P −gsd3LV 1 P −gsu1LX23 P −gs 2 u2LX31 P + gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P −1 2cD−3 D−2 ρ(D−4)(D−2) + ρ(D−2)(D−4) g √ 2W 1 P −1 2cD−4 D−2 ρ(D−3)(D−2) + ρ(D−2)(D−3) g √ 2W 1 P −1 2cD−3 D−1 ρ(D−4)(D−1) + ρ(D−1)(D−4) g √ 2W 1 P −1 2cD−4 D−1 ρ(D−3)(D−1) + ρ(D−1)(D−3) g √ 2W 1 P d2L;P = ∂P d2L −gsd2LU 2 P + gsd3LV 2 P −gsd1LV 2 P −gsu2LX31 P −gs 2 u3LX12 P + gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P A generalization of intrinsic geometry and its application to Hilbert’s 6th problem A generalization of intrinsic geometry and its application to Hilbert’s 6th problem 63 −1 2cD−3 D−1 ρ(D−4)(D−1) + ρ(D−1)(D−4) g √ 2W 1 P −1 2cD−4 D−1 ρ(D−3)(D−1) + ρ(D−1)(D−3) g √ 2W 1 P −1 2cD−3 D ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−4 D ρ(D−3)D + ρD(D−3) g √ 2W 1 P d3L;P = ∂P d3L −gsd3LU 3 P + gsd1LV 3 P −gsd2LV 3 P −gsu3LX12 P −gs 2 u1LX23 P + gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P −1 2cD−3 D ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−4 D ρ(D−3)D + ρD(D−3) g √ 2W 1 P −1 2cD−3 D−2 ρ(D−4)(D−2) + ρ(D−2)(D−4) g √ 2W 1 P −1 2cD−4 D−2 ρ(D−3)(D−2) + ρ(D−2)(D−3) g √ 2W 1 P d1R;P = ∂P d1R −gsd1LV 1 P + gsd2LU 1 P −gsd3LU 1 P + gsu1LY 23 P + gs 2 u2LX31 P −gs 2 u2LY 31 P −gs 2 u3LX12 P −gs 2 u3LY 12 P d2R;P = ∂P d2R −gsd2LV 2 P + gsd3LU 2 P −gsd1LU 2 P + gsu2LY 31 P + gs 2 u3LX12 P −gs 2 u3LY 12 P −gs 2 u1LX23 P −gs 2 u1LY 23 P d3R;P = ∂P d3R −gsd3LV 3 P + gsd1LU 3 P −gsd2LU 3 P + gsu3LY 12 P + gs 2 u1LX23 P −gs 2 u1LY 23 P −gs 2 u2LX31 P −gs 2 u2LY 31 P u1L;P = ∂P u1L −gsu1LU 1 P −gs 2 u2LX12 P −gs 2 u2LY 12 P −gs 2 u3LX31 P + gs 2 u3LY 31 P −gsd1LX23 P + gsd2LY 23 P −gsd3LY 23 P −1 2cD−3 D−1 ρ(D−4)(D−1) + ρ(D−1)(D−4) g √ 2W 1 P −1 2cD−4 D−1 ρ(D−3)(D−1) + ρ(D−1)(D−3) g √ 2W 1 P −1 2cD−3 D ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−4 D ρ(D−3)D + ρD(D−3) g √ 2W 1 P d3L;P = ∂P d3L −gsd3LU 3 P + gsd1LV 3 P −gsd2LV 3 P g g g g −1 2cD−3 D−1 ρ(D−4)(D−2) + ρ(D−2)(D−4) g √ 2W 1 P −1 2cD−3 D−2 ρ(D−4)(D−1) + ρ(D−1)(D−4) −1 2cD−3 D−1 ρ(D−4)(D−2) + ρ(D−2)(D−4) g √ 2W 1 P −1 2cD−3 D−2 ρ(D−4)(D−1) + ρ(D−1)(D−4) g √ 2W 1 P −1 2cD−4 D−1 ρ(D−3)(D−2) + ρ(D−2)(D−3) g √ 2W 1 P −1 2cD−4 D−2 ρ(D−3)(D−1) + ρ(D−1)(D−3) g √ 2W 1 P u2L;P = ∂P u2L −gsu2LU 2 P −gs 2 u3LX23 P −gs 2 u3LY 23 P −gs 2 u1LX12 P + gs 2 u1LY 12 P −gsd2LX31 P + gsd3LY 31 P −gsd1LY 31 P √ −1 2cD−4 D−1 ρ(D−3)(D−2) + ρ(D−2)(D−3) g √ 2W 1 P −1 2cD−4 D−2 ρ(D−3)(D−1) + ρ(D−1)(D−3) u2L;P = ∂P u2L −gsu2LU 2 P −gs 2 u3LX23 P −gs 2 u3LY 23 P −gs 2 u1LX12 P + gs 2 u1LY 12 P −gsd2LX31 P + gsd3LY 31 P −gsd1LY 31 P −2cD−1 ρ(D−3)(D−2) + ρ(D−2)(D−3) √ 2WP −2cD−2 ρ(D−3)(D−1) + ρ(D−1)(D−3) √ 2 u2L;P = ∂P u2L −gsu2LU 2 P −gs 2 u3LX23 P −gs 2 u3LY 23 P −gs 2 u1LX12 P + gs 2 u1LY 12 P −gsd2LX31 P + gsd3LY 31 P −gsd1LY 31 P −1 2cD−3 D ρ(D−4)(D−1) + ρ(D−1)(D−4) g √ 2W 1 P −1 2cD−3 D−1 ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−4 D ρ(D−3)(D−1) + ρ(D−1)(D−3) g √ 2W 1 P −1 2cD−4 D−1 ρ(D−3)D + ρD(D−3) g √ 2W 1 P u3L;P = ∂P u3L −gsu3LU 3 P −gs 2 u1LX31 P −gs 2 u1LY 31 P −gs 2 u2LX23 P + gs 2 u2LY 23 P −gsd3LX12 P + gsd1LY 12 P −gsd2LY 12 P −1 2cD−3 D−2 ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−3 D ρ(D−4)(D−2) + ρ(D−2)(D−4) g √ 2W 1 P −1 2cD−4 D−2 ρ(D−3)D + ρD(D−3) g √ 2W 1 P −1 2cD−4 D ρ(D−3)(D−2) + ρ(D−2)(D−3) g √ 2W 1 P u1R;P = ∂P u1R −gsu1RU 1 P + gs 2 u2RX12 P + gs 2 u2RY 12 P + gs 2 u3RX31 P −gs 2 u3RY 31 P u2R;P = ∂P u2R −gsu2RU 2 P + gs 2 u3RX23 P + gs 2 u3RY 23 P + gs 2 u1RX12 P −gs 2 u1RY 12 P u3R;P = ∂P u3R −gsu3RU 3 P + gs 2 u1RX31 P + gs 2 u1RY 31 P + gs 2 u2RX23 P −gs 2 u2RY 23 P . (5) CKM mixing conditions of strong interaction, No.1: The above proposition gives the mathematical essence of CKM mixing of strong in- teraction from the perspective of intrinsic geometry. In mathematics, d′ 1L, d′ 2L, d′ 3L, u′ 1L, u′ 2L, u′ 3L automatically appear as intrinsic geometrical properties, which are thereby not necessary to be postulated artiﬁcially like that in physics. Remark 8.2. The above proposition gives the mathematical essence of CKM mixing of strong in- teraction from the perspective of intrinsic geometry. In mathematics, d′ 1L, d′ 2L, d′ 3L, u′ 1L, u′ 2L, u′ 3L automatically appear as intrinsic geometrical properties, which are thereby not necessary to be postulated artiﬁcially like that in physics. Deﬁnition 8.4. If reference-system f satisﬁes ρ(D−2)(D−2) = ρ(D−1)(D−1) = ρDD = ρ(D−2)(D−1) = ρ(D−1)(D−2) = ρ(D−1)D = ρD(D−1) = ρD(D−2) = ρ(D−2)D = 0, we say f is a lepton ﬁeld, otherwise f is a hadron ﬁeld. Suppose f is a hadron ﬁeld. For d1, d2, d3, u1, u2, u3, if f satisﬁes that ﬁve of them are zero and the other one is non-zero, we say f is a single quark. Proposition 8.3. There does not exist a single quark. In other words, if ﬁve of d1, d2, d3, u1, u2, u3 are zero, then d1 = d2 = d3 = u1 = u2 = u3 = 0. Proposition 8.3. There does not exist a single quark. In other words, if ﬁve of d1, d2, d3, u1, u2, u3 are zero, then d1 = d2 = d3 = u1 = u2 = u3 = 0. Remark 8.3. For a single down-type quark, the above proposition is evident. Without loss of generality let u1 = u2 = u3 = 0 and d1 = d2 = 0, thus ρ(D−2)(D−2) = ρ(D−1)(D−1) = ρDD = 0, hence we must have d3 = 0. Remark 8.3. For a single down-type quark, the above proposition is evident. Without loss of generality let u1 = u2 = u3 = 0 and d1 = d2 = 0, thus ρ(D−2)(D−2) = ρ(D−1)(D−1) = ρDD = 0, hence we must have d3 = 0. For a single up-type quark, this paper has not made progress on the proof yet. Anyway, in consideration of that in physics the color conﬁnement has no clear mathematical connotation, by contrast, Proposition 8.3 explicitly gives the mathematical connotation of color conﬁnement from the perspective of intrinsic geometry, which itself is very signiﬁcant. (5) CKM mixing conditions of strong interaction, No.1: u2L;P = ∂P u2L −gsu2LU 2 P −gs 2 u3LX23 P −gs 2 u3LY 23 P −gs 2 u1LX12 P + gs 2 u1LY 12 P −gsd2LX31 P + gsd3LY 31 P −gsd1LY 31 P −1 2cD−3 D ρ(D−4)(D−1) + ρ(D−1)(D−4) g √ 2W 1 P −1 2cD−3 D−1 ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−4 D ρ(D−3)(D−1) + ρ(D−1)(D−3) g √ 2W 1 P −1 2cD−4 D−1 ρ(D−3)D + ρD(D−3) g √ 2W 1 P u3L;P = ∂P u3L −gsu3LU 3 P −gs 2 u1LX31 P −gs 2 u1LY 31 P −gs 2 u2LX23 P + gs 2 u2LY 23 P −gsd3LX12 P + gsd1LY 12 P −gsd2LY 12 P −1cD−3 D 2 ρ(D 4)D + ρD(D 4) g √W 1 P −1cD−3 D ρ(D 4)(D 2) + ρ(D 2)(D 4) g √W 1 P −1 2cD−3 D ρ(D−4)(D−1) + ρ(D−1)(D−4) g √ 2W 1 P −1 2cD−3 D−1 ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−4 D ρ(D−3)(D−1) + ρ(D−1)(D−3) g √ 2W 1 P −1 2cD−4 D−1 ρ(D−3)D + ρD(D−3) g √ 2W 1 P u3L;P = ∂P u3L −gsu3LU 3 P −gs 2 u1LX31 P −gs 2 u1LY 31 P −gs 2 u2LX23 P + gs 2 u2LY 23 P −gsd3LX12 P + gsd1LY 12 P −gsd2LY 12 P −1 2cD−3 D−2 ρ(D−4)D + ρD(D−4) g √ 2W 1 P −1 2cD−3 D ρ(D−4)(D−2) + ρ(D−2)(D−4) g √ 2W 1 P −1 2cD−4 D−2 ρ(D−3)D + ρD(D−3) g √ 2W 1 P −1 2cD−4 D ρ(D−3)(D−2) + ρ(D−2)(D−3) g √ 2W 1 P u1R;P = ∂P u1R −gsu1RU 1 P + gs 2 u2RX12 P + gs 2 u2RY 12 P + gs 2 u3RX31 P −gs 2 u3RY 31 P u2R;P = ∂P u2R −gsu2RU 2 P + gs 2 u3RX23 P + gs 2 u3RY 23 P + gs 2 u1RX12 P −gs 2 u1RY 12 P u3R;P = ∂P u3R −gsu3RU 3 P + gs 2 u1RX31 P + gs 2 u1RY 31 P + gs 2 u2RX23 P −gs 2 u2RY 23 P . 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https://openalex.org/W1591551585	https://europepmc.org/articles/pmc5312909?pdf=render	English	null	Nanostructured Electrode Materials for Electrochemical Capacitor Applications	Nanomaterials	2,015	cc-by	14,552	Hojin Choi 1 and Hyeonseok Yoon 1,2,* 1 Department of Polymer Engineering, Graduate School, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Korea; E-Mail: iyzvnzzang@gmail.com 2 School of Polymer Science and Engineering, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Korea * Author to whom correspondence should be addressed; E-Mail: hyoon@chonnam.ac.kr; Tel.: +82-62-530-1778; Fax: +82-62-530-1779. * Author to whom correspondence should be addressed; E-Mail: hyoon@chonnam.ac.kr; Tel.: +82-62-530-1778; Fax: +82-62-530-1779. Academic Editor: Jiye Fang Academic Editor: Jiye Fang Received: 1 April 2015 / Accepted: 27 May 2015 / Published: 2 June 2015 Abstract: The advent of novel organic and inorganic nanomaterials in recent years, particularly nanostructured carbons, conducting polymers, and metal oxides, has enabled the fabrication of various energy devices with enhanced performance. In this paper, we review in detail different nanomaterials used in the fabrication of electrochemical capacitor electrodes and also give a brief overview of electric double-layer capacitors, pseudocapacitors, and hybrid capacitors. From a materials point of view, the latest trends in electrochemical capacitor research are also discussed through extensive analysis of the literature and by highlighting notable research examples (published mostly since 2013). Finally, a perspective on next-generation capacitor technology is also given, including the challenges that lie ahead. Keywords: nanomaterials; electrochemical capacitors; electric double-layer capacitors (EDLCs); pseudocapacitors; hybrid capacitors Nanomaterials 2015, 5, 906-936; doi:10.3390/nano5020906 Nanomaterials 2015, 5, 906-936; doi:10.3390/nano5020906 nanomaterials ISSN 2079-4991 www.mdpi.com/journal/nanomaterials OPEN ACCESS 1. Introduction Electrochemical capacitors are a special class of electric energy storage devices that are based on nonfaradaic and/or faradaic charging/discharging at the interface between an electrode and an electrolyte [1,2]. In other words, they take advantage of ion adsorption and redox reactions to store 907 Nanomaterials 2015, 5 electric energy in the electrode. They can complement or replace batteries used in electric energy storage devices and harvesting applications when high power delivery or uptake is required. The different properties of each type of electrochemical capacitor impart unique characteristics to such devices and enable their unique applications [3]. Two primary attributes of an electrochemical capacitor are its energy and power density, both of which are mostly expressed as a quantity per unit weight. Since the energy stored in electrochemical capacitors is related to the charge arising from the potential difference at each interface, especially in the case of nonfaradaic charge storage, they can offer more rapid charge/discharge rates as compared with batteries, whereas their energy density is lower than that of batteries [4]. Many researchers have made an effort to improve the energy density while maintaining high power density. Current research on electrochemical capacitors can be categorized into three device types: electric double-layer capacitors (EDLCs), pseudocapacitors, and hybrid capacitors [5]. In most cases, EDLCs are based on porous carbon electrode materials. EDLCs are often referred to as supercapacitors or ultracapacitors that electrostatically store the charge by using reversible adsorption of the electrolyte ions onto electrochemically stable, high-surface-area carbonaceous electrodes. The specific surface area of EDLCs is enlarged by making the bulk of the carbon material porous [6]. In principle, the main requirements for EDLC electrodes include: (i) fast charge/discharge rate; (ii) large potential window; (iii) high conductivity; and (iv) large effective surface area [7,8]. Pseudocapacitive materials show fast redox reactions during the charge/discharge process at their surface. The fact that charge storage is based on a redox process means that pseudocapacitors have some battery-like behavior (faradaic reaction) in their charge/discharge process [9,10]. Therefore, pseudocapacitors show higher capacitances than EDLCs, although they have somewhat slower charge/discharge rates than EDLCs. Pseudocapacitance is typically shown by materials such as conducting polymers and transition metal oxides. The interest is increasing in the development of improved materials for pseudocapacitors. 1. Introduction To improve the capacitance of pseudocapacitors, four key factors are required: (i) doping of the conducting polymer to increase the redox state and conductivity; (ii) high charge/discharge rate; (iii) high surface area for the redox reaction; and (iv) a wide potential window [11,12]. Hybrid capacitors comprising EDLCs and pseudocapacitors combine their advantages, namely high energy and power densities [13,14]. The charge storage mechanisms in such devices are a combination of purely electrostatic adsorption–desorption phenomenon at the nonfaradaic electrode and a reversible faradaic reaction at the electrode. To achieve high energy density, hybrid capacitor systems comprising redox materials have been actively researched and developed in recent years [15]. Both EDLCs and pseudocapacitors are essential for fabricating high-performance hybrid capacitors. Electrochemical capacitors consist of electrolytes, separators, binders, and electrode materials. Here, we focus on the nanostructured electrode materials for use in the three different types of electrochemical capacitors, i.e., EDLCs, pseudocapacitors, and hybrid capacitors. The latest important works and achievements in electrochemical capacitor research are highlighted to provide information on what material factors are critical in determining the performance of electrochemical capacitors. We will mainly cover studies that have been conducted over the last few years, with major emphasis placed on the literature from 2013 to the present day. Lastly, future trends in the research field will be discussed, along with remaining technical challenges. Nanomaterials 2015, 5 908 2. EDLC Materials Carbon materials are considered prospective electrode materials for industrialization. The advantages of carbon materials include large specific surface area, good electronic conductivity, and high chemical stability [16]. The charge storage mechanism of carbon-based electrode materials mostly conforms to that of EDLCs. The important factors influencing their electrochemical performance are specific surface area, pore-size distribution, pore shape and structure, electrical conductivity, and surface functionality. Among these, specific surface area and pore-size distribution are the two most important factors affecting the performance of EDLCs [17]. Nanomaterials 2015, 5 Nanomaterials 2015, 5 a high-surface-area microporous carbon shell. Therefore, these CNT/microporous carbon core-shell nanocomposites are promising electrode materials for EDLCs. In another case, porous carbon–CNT–graphene ternary all-carbon foams were obtained through multicomponent surface self-assembly of graphene oxide (GO)-dispersed pristine CNTs (GOCs) supported on a commercial sponge [29]. The GO acted as a “surfactant” that dispersed the CNTs, thereby preserving their excellent electronic structure and preventing the aggregation of graphene, which resulted in an overall improvement of the conductivity. The fabrication of 3D hierarchically porous carbon–CNT–graphene ternary all-carbon foams (3D-HPCFs) is illustrated in Figure 2. The large number of high-surface-area mesopores promotes ion transport/charge storage. Figure 2. Fabrication of 3D hierarchically porous carbon–CNT–graphene ternary all-carbon foams (3D-HPCFs): (a) Impregnating sponge into the GOC solution; (b) Impregnating the resulting GOC/commercial sponge into a multicomponent ethanol solution of F127, phenol–formaldehyde resol, and tetraethoxysilane (TEOS) for self-assembly; (c) Carbonization and subsequent etching removal of SiO2 from TEOS. With permission from [29]; Copyright 2014, American Chemical Society. GO Graphene CNTs a) c) b) PF F127 TEOS Mesoporous carbons GO Graphene CNTs a) c) b) PF F127 TEOS Mesoporous carbons c) b) Mesoporous carbons Mesoporous carbons ure 2. Fabrication of 3D hierarchically porous carbon–CNT–graphene ternary all-carbon Figure 2. Fabrication of 3D hierarchically porous carbon–CNT–graphene ternary all-carbon foams (3D-HPCFs): (a) Impregnating sponge into the GOC solution; (b) Impregnating the resulting GOC/commercial sponge into a multicomponent ethanol solution of F127, phenol–formaldehyde resol, and tetraethoxysilane (TEOS) for self-assembly; (c) Carbonization and subsequent etching removal of SiO2 from TEOS. With permission from [29]; Copyright 2014, American Chemical Society. Figure 2. Fabrication of 3D hierarchically porous carbon–CNT–graphene ternary all-carbon foams (3D-HPCFs): (a) Impregnating sponge into the GOC solution; (b) Impregnating the resulting GOC/commercial sponge into a multicomponent ethanol solution of F127, phenol–formaldehyde resol, and tetraethoxysilane (TEOS) for self-assembly; (c) Carbonization and subsequent etching removal of SiO2 from TEOS. With permission from [29]; Copyright 2014, American Chemical Society. 2.1. Porous Carbon To realize the enhanced performance that stems from increasing the surface area of electrode materials, the pore size of the materials should be limited to the range of subnanometers to a few nanometers [18]. Extensive research has been conducted on developing porous structures [19]. The small pore sizes of carbon materials result in large specific surface areas. However, at a certain, optimal pore size, the effective surface area of the double layer is maximized while maintaining the optimal ion exchange with the electrolyte [20]. For example, a colloidal crystal templating method was optimized for the synthesis of three-dimensionally ordered mesoporous (3DOm) carbon with a well-defined geometry, a three-dimensional (3D) interconnected pore structure, and tunable pore sizes of 8–40 nm (Figure 1) [21]. To achieve precise control over the pore sizes in the carbon products, parameters were established for direct syntheses or seed growth of monodispersed silica nanospheres of specific sizes [22]. The 3DOms exhibited higher capacitance than conventional porous carbon electrodes. Moreover, using an ionic liquid as an electrolyte resulted in an increased cell voltage, which in turn enhanced the power density of the electrode [23]. Figure 1. Schematic of fabrication of three-dimensionally ordered mesoporous (3DOm) carbon. With permission from [21]; Copyright 2013, American Chemical Society. Silica template Silica-polymer composite 3DOm carbon Infiltration of carbon precursor 1) Pyrolysis 900 °C, 6 h 2) Silica etching Silica-polymer composite Infiltration of carbon precursor Silica-polymer composite Silica-polymer composite 3DOm carbon 3DOm carbon Silica template Silica template Figure 1. Schematic of fabrication of three-dimensionally ordered mesoporous (3DOm) carbon. With permission from [21]; Copyright 2013, American Chemical Society. These porous carbon materials have a synergistic effect when used in conjunction with other carbon materials [24]. A judicious combination of porous carbon and so-called nanocarbons, such as graphene [25], carbon nanotubes (CNTs) [26], and carbon nanofibers (CNFs) [27], could improve the capacitive performance based purely on the principle of an electrochemical double layer. High-surface-area carbon nanotube (CNT)/microporous carbon composite materials were prepared for EDLC electrodes [28]. All-carbon-based CNT/microporous carbon core-shell nanocomposites have 909 2.2. CNFs, CNTs, and Graphene Graphene has a maximum theoretical specific surface area of ca. 2600 m2·g−1, which is twice that of single-walled CNTs and substantially higher than those of most carbon black and activated carbons [30]. Graphene is a unique and attractive electrode material owing to its atom-thick two-dimensional (2D) structure and excellent properties. However, graphene sheets easily form irreversible agglomerates and restack to the graphitic structure. To overcome this problem, graphene can be hybridized with CNTs, CNFs, and porous carbon. Various carbon hybrids have been fabricated by electrospinning followed by heat treatment [31], since electrospinning can readily create multicomponent nanofibers as the carbon precursor [32]. Graphene nanoribbon (GNR)/carbon composite nanofibers have been prepared by 910 Nanomaterials 2015, 5 electrospinning from polyacrylonitrile (PAN)-containing GO nanoribbons (GONRs) and successive twisting and carbonization (Figure 3) [33]. Figure 3. Schematic of the fabrication process of graphene nanoribbon (GNR)/carbon nanofiber (CNF) via carbonization. With permission from [33]; Copyright 2013, American Chemical Society. GONR GNR (thermally annealed) Graphitic structure Stabilization Carbonization GONR/PAN composite nanofiber GNR/carbon composite nanofiber GONR/PAN composite nanofiber GNR/carbon composite nanofiber Graphitic structure GONR GNR (thermally annealed) GNR (thermally annealed) Figure 3. Schematic of the fabrication process of graphene nanoribbon (GNR)/carbon nanofiber (CNF) via carbonization. With permission from [33]; Copyright 2013, American Chemical Society. Many studies have shown that highly oriented CNFs have a large surface area and excellent electrical properties [34]. PAN-containing GONR nanocomposites also become highly oriented during electrospinning. In addition, GONRs have been converted to an all-carbon material, GNR/CNF, by carbonization [35]. Both the graphene ribbons and CNFs exhibit high performance as electrochemical capacitors; it is expected that the structural synergistic effect would further improve this performance. Various methods of fabricating these graphene fiber materials have been investigated. Graphene-coated nanotube aerogels, a type of graphene fiber material, have been developed by coating the nodes of an isotropic single-wall carbon nanotube network within an aerogel with a few layers of graphene. These graphene-based CNT and CNF materials are especially important for textile-enabled materials and devices. They can also act as the building blocks for forming 2D and 3D macroscopic structures for EDLCs [36,37]. The use of CNFs and CNTs has further enhanced the properties of microelectrochemical capacitors, thereby enabling the fabrication of flexible and adaptable devices [38]. Many researchers have reported on combinations of CNTs, CNFs, metals, and graphene. 2.2. CNFs, CNTs, and Graphene CNTs and CNFs are connected to the graphene layer through covalent bonds, leading to seamless, high-quality carbon material–graphene–metal interfaces [39]. Ternary hybrid nanostructures consisting of CNFs, manganese oxide (MnO), and graphene were recently fabricated, as shown in Figure 4. The metal oxide MnO is a pseudocapacitive element, which will be discussed in a subsequent section. MnO-decorated CNFs (MCNFs) were dispersed in an aqueous solution containing isolated GO sheets exfoliated from oxidized graphite. GO sheets are highly dispersible in aqueous solution owing to the oxygen-containing functional groups, and they have high affinity with CNFs because of their similar chemical structures. Thus, MCNFs were readily wrapped with GO sheets to yield a 3D nanohybrid architecture. Afterwards, GO was converted to reduced graphene oxide (RGO) by using hydrazine. Intercalated MCNFs improved the conductivity of the MCNF/RGO nanocomposite and facilitated ion diffusion by increasing the spacing between the graphene sheets. The RGO sheet is considered to act as a conductive channel in the nanohybrid. Additionally, the intercalation of CNFs between the RGO sheets induced a 3D opened 911 Nanomaterials 2015, 5 geometry in the electrode, which allowed facile ion and charge transfer. Consequently, high-performance capacitive properties were observed for these new 3D structures [40–43]. Figure 4. (a) Schematic of the fabrication of ternary hybrid nanostructures: MnO-decorated CNFs (MCNFs)/reduced graphene oxide (RGO) nanohybrids; (b) Typical transmission electron microscopy (TEM) image of MCNF; (c) High-resolution TEM image of an MCNF (lattice planes of γ-MnO2, such as (131) and (300), are marked); and (d) Cross-sectional scanning electron microscopy (SEM) image of MCNF/RGO nanohybrids (RGO 50 wt.%) deposited on a silicon wafer. With permission from [39]; Copyright 2013, Wiley-VCH Verlag GmbH & Co. KGaA. PVP(Mn(Ac)2)/PAN nanofiber Polymer mixture MnO2 nanonodules MnO2 decorated CNF 1. Calcination 2. Carbonization Reduction (Hydrazine) ) b) c) d) a) b) c) d) PVP(Mn(Ac)2)/PAN nanofiber Polymer mixture MnO2 nanonodules MnO2 decorated CNF 1. Calcination 2. Carbonization Reduction (Hydrazine) a) c) b) MnO2 nanonodules c) Polymer mixture PVP(Mn(Ac)2)/PAN nanofiber MnO2 decorated CNF d) Figure 4. (a) Schematic of the fabrication of ternary hybrid nanostructures: MnO-decorated CNFs (MCNFs)/reduced graphene oxide (RGO) nanohybrids; (b) Typical transmission electron microscopy (TEM) image of MCNF; (c) High-resolution TEM image of an MCNF (lattice planes of γ-MnO2, such as (131) and (300), are marked); and (d) Cross-sectional scanning electron microscopy (SEM) image of MCNF/RGO nanohybrids (RGO 50 wt.%) deposited on a silicon wafer. 2.2. CNFs, CNTs, and Graphene With permission from [39]; Copyright 2013, Wiley-VCH Verlag GmbH & Co. KGaA. CNT-bridged graphene 3D building blocks were synthesized via the coulombic interaction between positively charged CNTs grafted by cationic surfactants and negatively charged GO sheets (Figure 5) [44]. The CNTs were intercalated into the nanoporous graphene layers to build pillared 3D structures, which increased the accessible surface area and allowed fast ion diffusion. Because of this unique 3D porous structure, the electrodes showed remarkable electrochemical performance in ionic liquid electrolytes [45]. However, it is difficult to prepare electrodes by using uniform nanostructured CNTs on special substrates, especially those with porous surface structures. Cellulose is the most abundant and sustainable natural polymer. Cellulose nanofibrils (CeNFs) derived from cellulose have high aspect ratios, excellent mechanical properties, excellent flexibility, and superior hydrophilicity [46]. A recent work reported the fabrication of a unique CeNF/carbon nanohybrid aerogel electrode material, shown in Figure 6. The CeNF-based aerogel possessed a porous structure and an extremely high porosity (resulting in ultralow density and a high specific surface area), as well as excellent electrolyte-absorption properties. Furthermore, the hydrophilicity of the CeNFs in the aerogel improved the contact between the electrodes and electrolytes, and it provided diffusion channels for the electrolyte ions, thus enhancing the performance of the EDLCs. Various polymer precursors have been used to make new types of carbon nanomaterials. The main characteristics of the resultant carbon nanomaterials depend on the polymer precursors. It is relatively easy to control the morphology and composition of polymers, which offers opportunities for producing carbon nanomaterials with controlled structures and properties [47,48]. Several researchers have fabricated electrode materials via heat treatment of GO/polymer hybrid precursors, such as 912 Nanomaterials 2015, 5 GO/polypyrrole (PPy) nanowires, GO/PPy nanotubes, and GO/polyaniline (PANI). Heat treatment of the GO/polymer hybrids resulted in the formation of graphene-embedded all-carbon nanostructures. These carbonized nanohybrids exhibited good performance as EDLC electrode materials. Figure 5 Schematic for fabricating the graphene/single walled CNT hybrid nanostructure CTAB (Positive head group) Negative head group CTAB -CNT GO COOH – COC – OH – Electrostatic Self -Assembly KOH Activation a) b) Figure 5. Schematic for fabricating the graphene/single-walled CNT hybrid nanostructure. (a) Cetyltrimethylammonium bromide (CTAB)-grafted CNTs are positively charged and the GO layers are negatively charged owing to their respective functional groups; (b) Schematic of the 3D CNT-bridged graphene block. 2.2. CNFs, CNTs, and Graphene KOH activation generates nanoscale pores in the graphene layers, which are expected to provide a simple means of ion diffusion. With permission from [44]; Copyright 2015, American Chemical Society. CTAB (Positive head group) Negative head group CTAB -CNT GO COOH – COC – OH – Electrostatic Self -Assembly KOH Activation a) b) CTAB (Positive head group) Negative head group CTAB -CNT GO COOH – COC – OH – Electrostatic Self -Assembly KOH Activation a) GO a) CTAB -C Electrostatic Self -Assembly KOH Activation b) Figure 5. Schematic for fabricating the graphene/single-walled CNT hybrid nanostructure. (a) Cetyltrimethylammonium bromide (CTAB)-grafted CNTs are positively charged and the GO layers are negatively charged owing to their respective functional groups; (b) Schematic of the 3D CNT-bridged graphene block. KOH activation generates nanoscale pores in the graphene layers, which are expected to provide a simple means of ion diffusion. With permission from [44]; Copyright 2015, American Chemical Society. 913 Nanomaterials 2015, 5 Figure 6. (a) Schematic of the fabrication process of a CeNF/RGO/CNT aerogel electrode. SEM images of compressed CeNF/RGO/CNT hybrid aerogel films, (b and c) bottom surface of the aerogel film, and (d and e) cross section of the aerogel film. With permission from [46]; Copyright 2015, American Chemical Society. 2 µm 2 µm 200 nm 200 nm b) c) d) e) CeNF/RGO/CNT aerogel CeNF/RGO/CNT electrode CeNF/GO/CNT aerogel GO/CNT dispersion CeNF/GO/CNT dispersion 1 MPa 150 °C in air Freeze-drying Adding CeNF solution –78 °C Frozen/CeNF/GO/CNT dispersion a) 2 µm 2 µm 200 nm 200 nm b) c) d) e) GO/CNT dispersion Add s a) Adding CeNF solution b) –78 °C e) 1 MPa CeNF/RGO/CNT aerogel CeNF/GO/CNT aerogel CeNF/RGO/CNT electrode Figure 6. (a) Schematic of the fabrication process of a CeNF/RGO/CNT aerogel electrode. SEM images of compressed CeNF/RGO/CNT hybrid aerogel films, (b and c) bottom surface of the aerogel film, and (d and e) cross section of the aerogel film. With permission from [46]; Copyright 2015, American Chemical Society. 2.3. Summary EDLCs can be applied in the case of stationary and mobile systems requiring high power pulses. Moreover, owing to their low time constant, they can quickly harvest energy, such as during deceleration or braking of vehicles. Although EDLCs are able to provide higher power with a longer cycle life, they suffer from a relatively low energy density. Therefore, the current research is mainly concerned with the optimization of the existing electrode materials and the development of new materials to improve energy density. Many different carbon forms, such as CNTs, CNFs, activated carbon powders, and carbonized materials, can be used as active materials in EDLC electrodes [49,50]. Table 1 summarizes the EDLC electrode materials developed recently and their electrochemical performance. Table 1. EDLC materials and electrochemical performance. Materials Electrode System (1,2) Electrolyte Current Density or Scan Rate Potential Range (V) Specific Capacitance (F·g−1) Ref. Carbon nanosheets Two (3) EMIMBF4 1–10 A·g−1 3.5 155–242 6 Carbon nanosheets Two PVA–H3PO4 gel 0.25–1.3 A·g−1 0.8 4.9–29.2 7 N-doped carbon nanosheets Two 1 M NaOH 0.75–7 A·g−1 1.0 150–180 8 Porous carbon nanowhiskers Two 6 M KOH 0.5–30 A·g−1 1.0 125–210 16 N-doped nanoporous carbon Three 6 M KOH 0.5–40 A·g−1 1.0 90–240 17 Nanofibers/mesoporous carbon Three 0.5 M K2SO4 0.5–6 A·g−1 0.8 72–99 18 N-doped carbon nanospheres Three 6 M KOH 0.5–40 A·g−1 1.0 62–194 19 Nanoporous carbon Two (4) EMI–TFSI 0.5–25 A·g−1 3.5 80–178 21 N-doped nanoporous carbon Three 6 M KOH 1–10 A·g−1 1.0 175–250 22 Mesoporous carbon nanosheets Two (5) 1 M TEABF4/AN 0.1–120 A·g−1 2.7 100–130 24 GO-activate carbon Two EMIMBF4 1–10 A·g−1 3.5 110–135 25 Table 1. EDLC materials and electrochemical performance. Nanomaterials 2015, 5 914 Table 1. Cont. Materials Electrode System (1,2) Electrolyte Current Density or Scan Rate Potential Range (V) Specific Capacitance (F·g−1) Ref. 3. Pseudocapacitive Materials Various methods of preparing pseudocapacitive materials have been recently described [51,52]. Pseudocapacitive materials show redox reactions during the charge/discharge process, thereby resulting in pseudocapacitors whose capacitance is better than that of EDLCs [53–55]. In most cases, however, faradaic redox reactions for pseudocapacitance are confined to the surfaces of the electrode materials. Thus, rational nanostructuring of pseudocapacitive materials for enlarging the effective surface area is essential to obtaining enhanced pseudocapacitance [56]. 2.3. Summary Porous carbon/CNTs Two 3 M H2SO4 0.1–50 A·g−1 0.9 125–237 26 Porous CNFs Two EMI-TFSI 5–100 mV·s−1 2.0 65–150 27 Porous CNTs Three 1 M H2SO4 1–10 A·g−1 0.7 454–710 28 Nanoporous carbon foams Two 6 M KOH 0.2–20 A·g−1 1.1 125–379 29 Carbonization of carbon hydrates Two 5 M KOH 1 A·g−1 0.8 140 32 Carbonized hollow nanocarbon Two 1 M KOH 1–20 A·g−1 0.9 160–183 35 Graphene-coated CNTs Two EMI-TFSI 0.01–10 A·g−1 3.0 60–130 36 Graphene/CNF Three 1 M H2SO4 0.2 A·g−1 1.5 174 39 3D porous graphene-like carbon Two (6) TEMABF4/PC 1–32 A·g−1 2.5 156–178 40 Mesoporous graphene nanoballs Three 1 M H2SO4 5–100 mV·s−1 0.8 206 41 Graphene/CNT composite fibers Two 0.5 M H2SO4 0.2–2 A·g−1 0.8 6–35 42 N-doped 3D nanoporous carbon Two 0.5 M Na2SO4 0.5–20 A·g−1 1.0 226–304 43 Graphene/CNT Two EMIMBF4 10 A·g−11 4.0 199 44 Graphene/carbon Two EMIMBF4 1–10 A·g−1 3.5 160–190 45 RGO/CNT Two PVA–H2SO4 gel 0.5–4 A·g−1 1.0 180–252 46 Carbonized PPy nanostructures Three 1 M H2SO4 5 mV·s−1 0.9 264 47 Carbonized PPy-CNTs Three 1 M KCL 5–100 mV·s−1 1.5 40–140 48 Halogen-containing nanoporous carbon Three 6 M KOH 0.5–40 A·g−1 1.0 110–313 49 Oxygen-rich nanoporous carbon Two 1 M H2SO4 0.5–10 A·g−1 1.0 210–297 50 (1) The “Two” indicates capacitor cells composed of two electrodes (anode/cathode), separator, and electrolyte, containing symmetric or asymmetric electrode configurations; (2) The “Three” indicates electrode measuring systems composed of working electrode, counter electrode, and reference electrode in an electrolyte; (3) EMIMBF4: 1-ethyl-3-methylimidazolium tetrafluoroborate; (4) EMI-TFSI: 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide; (5) TEABF4/AN: tetraethylammonium tetrafluoroborate/acetonitrile; (6) TEMABF4/PC: triethylmethylammonium tetrafluoroborate/propylene carbonate. (1) The “Two” indicates capacitor cells composed of two electrodes (anode/cathode), separator, and electrolyte, containing symmetric or asymmetric electrode configurations; (2) The “Three” indicates electrode measuring systems composed of working electrode, counter electrode, and reference electrode in an electrolyte; (3) EMIMBF4: 1-ethyl-3-methylimidazolium tetrafluoroborate; (4) EMI-TFSI: 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide; (5) TEABF4/AN: tetraethylammonium tetrafluoroborate/acetonitrile; (6) TEMABF4/PC: triethylmethylammonium tetrafluoroborate/propylene carbonate. Nanomaterials 2015, 5 Owing to their unique properties and small dimensions, conducting polymer nanostructures have wide technical applications [59,60]. For example, Figure 7 shows the SEM images of three PANI nanostructures synthesized with different shapes. PANI has been extensively used as an electrode-active material in electrochemical capacitors. The capacitances of these PANI nanostructures were determined via charge/discharge cycling. For the same range of potentials, the discharging time increased in the order of nanospheres < nanorods < nanofibers, indicating that the nanofiber electrode has the highest specific discharge capacitance [61]. In other words, the capacitance of PANI nanostructures depends on their aspect ratio. The nanofibers also had faster electrode kinetics by virtue of the higher oxidation level and crystallinity. Lastly, interparticle resistance varied as a function of the aspect ratio of the nanostructures, which has been identified as one of the significant factors affecting the electrode performance. Several papers have similarly described the morphology effect of nanoparticles on the performance of pseudocapacitors [10,58,61]. As a result, judicious control of the morphology of nanostructures for electrode materials offers many possibilities of tailoring the performance of electrochemical capacitors. 20 30 40 50 60 70 0 15 30 45 60 75 0 150 300 450 600 D Frequency (%) D L Size (nm) c) F (%) 30 40 50 60 0 15 30 45 60 75 90 120 150 180 D Frequency (%) D L Size (nm) b) c Figure 7. SEM images of polyaniline (PANI) nanostructures with different aspect ratios synthesized under the same stirring condition (200 rpm) and histograms showing their size distribution (D, diameter; L, length): (a) Nanospheres; (b) Nanorods; and (c) Nanofibers. With permission from [61]; Copyright 2012, American Chemical Society. 15 30 45 60 75 90 0 15 30 45 60 75 Frequency (%) Size (nm) D 20 30 40 50 60 70 0 15 30 45 60 75 0 150 300 450 600 D Frequency (%) D L Size (nm) 30 40 50 60 0 15 30 45 60 75 90 120 150 180 D Frequency (%) D L Size (nm) b) a) c) a) b) c) 15 30 45 60 75 90 0 15 30 45 60 75 Frequency (%) Size (nm) D L D D D L Figure 7. 3.1. Conducting Polymers Precise control over the size and morphology of conducting polymers on the nanoscale is essential to improving the performance of pseudocapacitors. However, the highly unstable nature of polymers on this scale has hindered the development of polymer nanoarchitectures [57]. Nevertheless, numerous efforts have been made to fabricate polymer nanomaterials with well-defined sizes and morphologies, and various types of conducting polymer nanostructures have been fabricated in a controlled manner [10,58]. 915 Nanomaterials 2015, 5 Nanomaterials 2015, 5 advantages are expected to induce a synergetic effect that enhances device performance, particularly in pseudocapacitors. Electrospinning is a simple and scalable technique that can create continuous and aligned polymer nanofibers and microfibers under a large electric field. Electrospun nanofibers are composed of various polymer melts and solutions, which can serve as templates that direct the formation of conducting polymer nanofibers and nanotubes. In a recent study, ultrathin poly(methyl methacrylate) (PMMA) nanofibers with an average diameter of 60 nm were obtained through electrospinning for use as a template (Figure 8b); these nanofibers were then immersed in a ferric chloride solution in order to adsorb ferric ions onto the PMMA nanofibers (Figure 8c) [64]. Subsequent coating of PEDOT onto the PMMA nanofibers via VDP at controlled temperatures and pressures yielded core–shell-structured nanofibers (Figure 8d,f,h). Unique surface substructures such as nanonodules and nanorods were grown on the nanofibers by adjusting the major kinetic factors. Moreover, the PMMA was removed by selective solvent etching, which resulted in nanotubular structures (Figure 8e,g,i). These multidimensional nanofibers and nanotubes with surface substructures offer significant advantages when used as pseudocapacitors. VDP has attracted significant attention owing to its simplicity and controllability as compared to other techniques used to obtain nanostructured conducting polymers [65]. As a conjugated polymer, PPy shows practical potential for a diverse and promising range of future technologies. Free-standing, flexible, and large-area PPy/cellulose (PPCL) papers were readily prepared using VDP (Figure 9), and PPy coatings on cellulose microfibers were influenced by the properties of co-vapors introduced together with pyrrole during polymerization [66]. It is known that PPy stores electrical charges through a pseudocapacitive charge storage mechanism mediated by a redox reaction. Pseudocapacitance stems from reversible surface or near-surface reactions for charge storage. As a result, use of co-vapors in VDP has offered the possibility of tuning the physical properties of the deposited polymers, as well as the performance of the polymer-based electrochemical capacitors. Conducting polymers can also be coated on metal nanoparticles to improve the electrochemical performance. Uniform coating of the metal nanoparticles with conducting polymer layers prevents interparticle aggregation and imparts unprecedented electrical, optical, and chemical properties to the nanoparticles. Ag–PPy core–shell nanoparticles were obtained in the form of a stable colloidal suspension through a simple one-pot synthesis [67]. Nanomaterials 2015, 5 SEM images of polyaniline (PANI) nanostructures with different aspect ratios synthesized under the same stirring condition (200 rpm) and histograms showing their size distribution (D, diameter; L, length): (a) Nanospheres; (b) Nanorods; and (c) Nanofibers. With permission from [61]; Copyright 2012, American Chemical Society. Precise control of the nanostructure morphology is crucial to realizing a variety of next-generation technologies. Poly(3,4-ethylenedioxythiophene) (PEDOT) and multidimensional PPy nanotubes are only two examples of these promising nanostructures [62]. The surface of the former was decorated with substructures such as nanonodules and nanorods and the latter were fabricated as chemiresistors on a sacrificial nanofiber template by vapor deposition polymerization (VDP) [63]. These unique multidimensional nanostructures have enhanced surface-to-volume ratios, and their unique morphology and anisotropic geometry results in a high surface area and excellent charge-transport properties. These 916 Nanomaterials 2015, 5 Briefly, the silver nanoseed surfaces became the active sites for oxidation of the surrounding pyrrole monomers and PPy short chains, thereby resulting in the formation of a PPy shell (Figure 10). These bottom-up approaches offer an efficient and simple route for the fabrication of nanostructured metal/conducting polymer complexes. 917 Nanomaterials 2015, 5 Figure 8. Multidimensional poly(3,4-ethylenedioxythiophene) (PEDOT) nanostructure with unique surface substructures (a) Schematic of the synthetic routes for fabricatin Conductive collector Polymer Solution 1 torr VDP @ 90 °C Before etching After etching 760 torr PMMA nanofibers Oxidant coated PMMA nanofibers VDP @ 90 °C VDP @ 60 °C Before etching After etching Before etching After etching b) h) g) c) d) e) f) i) a) Figure 8. Multidimensional poly(3,4-ethylenedioxythiophene) (PEDOT) nanostructures with unique surface substructures. (a) Schematic of the synthetic routes for fabricating multidimensional PEDOT nanostructures; (b–i) The poly(methyl methacrylate) (PMMA) nanofibers serve as a template and substrate for the growth of PEDOT under different synthetic conditions (temperature and pressure). The morphologies of the resulting nanomaterials were characterized by SEM and TEM (right top inset images): PMMA nanofibers (b) before and (c) after ferric ion adsorption; PMMA/PEDOT nanofibers with a smooth layer surface (d) before and (e) after core etching; PMMA/PEDOT nanofibers with nanorod surface (f) before and (g) after core etching; PMMA/ PEDOT nanofibers with NN surface (h) before and (i) after core etching. With permission from [64]; Copyright 2012, American Chemical Society. Conductive collector Polymer Solution 1 torr VDP @ 90 °C Before etching After etching 760 torr PMMA nanofibers Oxidant coated PMMA nanofibers VDP @ 90 °C VDP @ 60 °C Before etching After etching Before etching After etching b) h) g) c) d) e) f) i) a) Polymer Solution Conductive collector After etching After etching Before etching Before etching Figure 8. Multidimensional poly(3,4-ethylenedioxythiophene) (PEDOT) nanostructu Figure 8. Multidimensional poly(3,4-ethylenedioxythiophene) (PEDOT) nanostructures with unique surface substructures. (a) Schematic of the synthetic routes for fabricating multidimensional PEDOT nanostructures; (b–i) The poly(methyl methacrylate) (PMMA) nanofibers serve as a template and substrate for the growth of PEDOT under different synthetic conditions (temperature and pressure). Nanomaterials 2015, 5 The morphologies of the resulting nanomaterials were characterized by SEM and TEM (right top inset images): PMMA nanofibers (b) before and (c) after ferric ion adsorption; PMMA/PEDOT nanofibers with a smooth layer surface (d) before and (e) after core etching; PMMA/PEDOT nanofibers with nanorod surface (f) before and (g) after core etching; PMMA/ PEDOT nanofibers with NN surface (h) before and (i) after core etching. With permission from [64]; Copyright 2012, American Chemical Society. 918 Nanomaterials 2015, 5 Figure 9. (a) Photograph showing the flexibility of a large-area, free-standing PPy/cellulose (PPCL) paper; (b) SEM images of a PPCL paper (inset: high-magnification image). With permission from [65]; Copyright 2014, The Royal Society of Chemistry. b) a) a) b) a) a) b) b) a) a) b) b) Figure 9. (a) Photograph showing the flexibility of a large-area, free-standing PPy/cellulose (PPCL) paper; (b) SEM images of a PPCL paper (inset: high-magnification image). With permission from [65]; Copyright 2014, The Royal Society of Chemistry. Figure 10. Schematic of the formation mechanism of Ag–PPy nanoparticles: the reaction process consisted of three stages (I, II, and III). With permission from [67]; Copyright 2012, American Chemical Society. OH OH OH Starch chain Ag+ Ag+ Ag+ Ag Ag+ Ag+ PPy short chains Py Py Py Py Py Ag (II) (I) (III) (I) Starch chain OH PPy short chains Figure 10. Schematic of the formation mechanism of Ag–PPy nanoparticles: the reaction process consisted of three stages (I, II, and III). With permission from [67]; Copyright 2012, American Chemical Society. Nanomaterials 2015, 5 Nanomaterials 2015, 5 can be designed in order to overcome this drawback [66,76,77]. For example, it was found that CoO-based PPy nanowire electrodes had very high capacitance and good rate capability [78]. Using nanoporous silica as a template enables the fabrication of pseudocapacitor electrodes, which have narrow particle size and pore-size distributions, large surface area, and large pore volume. As compared to normal PPy, enhanced conductivity and surface area were observed due to the synergistic effect of the nanohybrid structures. As a result, the CoO/PPy nanowires showed excellent pseudocapacitance behavior [79]. Extensive research has also focused on maximizing the specific surface area of metal oxides through hierarchical structuring of the composite [80–83]. As an example, Ding and Yang reported the preparation of a 1D hierarchical nanostructure of NiCo2O4 nanosheets@halloysite nanotubes through simple co-precipitation followed by thermal annealing (Figure 11) [84]. The hierarchical nanostructure had an excellent specific capacitance of 1728 F·g−1 even after 8600 cycles at a current density of 10 A·g−1. Figure 11. Schematic of the synthetic procedure of NiCo2O4 nanosheets@halloysite nanotube hybrid nanostructures. With permission from [84]; Copyright 2012, American Chemical Society. Figure 11. Schematic of the synthetic procedure of NiCo2O4 nanosheets@halloysite nanotube hybrid nanostructures. With permission from [84]; Copyright 2012, American Chemical Society. 3.2. Metal Oxides Metal oxides are attractive pseudocapacitive electrode materials because of their easy processing [5]. Ruthenium oxides and manganese oxides have been widely studied for electrochemical capacitor applications [68,69]. However, the high cost of ruthenium oxides excludes them from wide practical application. Additionally, manganese oxides have several different crystalline structures, such as α-, β-, γ-, δ-, and ξ-MnO2, and unfortunately their capacitances highly depend on the crystalline structure [70–74]. Therefore, recent research trends have focused on exploring other transition metal oxides. For example, a binary transition metal sulfide, NiCo2S4, with a hollow hexagonal nanoplate structure was successfully prepared through a hydrothermal route with the aid of a sacrificial template. The NiCo2S4 gave a maximum specific capacitance of 437 F·g−1 in a KOH electrolyte at a current density of 1 A·g−1 [75]. However, poor conductivity of metal oxides poses a serious problem to the efficient use of these oxides in electrochemical capacitors. A nanocomposite composed of a metal oxide and a conductive material 919 3.3. Summary In recent years, various methods for fabricating pseudocapacitive materials have been reported. Table 2 summarizes the pseudocapacitive electrode materials and their electrochemical performance. Owing to the redox reaction-mediated charge storage, pseudocapacitive materials have higher energy density than their EDLC counterparts. The enhanced surface area and redox activity was achieved primarily by controlling the morphology and by combining materials. However, pseudocapacitive materials, especially conducting polymers, undergo severe volumetric changes during charge/discharge processes, often leading to structural deterioration and rapid decay of the capacitance. Long-term cycling stability is, therefore, a barrier to the wide use of pseudocapacitors in practical applications. 920 Nanomaterials 2015, 5 Nanomaterials 2015, 5 Table 2. Pseudocapacitor materials and electrochemical performance. Table 2. Pseudocapacitor materials and electrochemical performance. Materials Electrode System (1,2) Electrolyte Current Density or Scan Rate Potential Range (V) Specific Capacitance (F·g−1) Ref. 3.3. Summary CoCl2 nanostructures Three 2 M KOH 1 A·g−1 0.45 1962 9 PANI nanotubes Two 1 M H2SO4 1–30 A·g−1 0.5 477–896 10 PPy-clay core–shell nanoarrays Three 1 M KOH 1–20 A·g−1 1.2 1750–2342 11 Polythiophene nanostructures Two 0.5 M TEABF4 40–100 mV·s−1 4.0 75–250 12 NiO nanoblocks Three 1 M KOH 1.11–111 A·g−1 0.6 680–1336 51 PPy–sepiolite nanocomposites Three 1 M KCl 3 mA·cm−2 1.0 165 52 ZnCo2O4 nanorods/Ni foams Two PVA-KOH gel 1–20 A·g−1 1.0 1015–1400 53 NiCo2(OH)6 nanotubes Two 1.9 M KCL 0.1M KOH 10–100 A·g−1 0.5 169–200 54 3D Co3O4 nanonetworks Three 6 M KOH 2–100 mV·s−1 1.5 546–1049 55 Ni2(CO3)(OH)2 nanosheets Three 3 M KOH 0.5–10 A·g−1 0.4 612–1178 56 PPy–PANI double-wall nanotubes Three 1 M H2SO4 5–250 mV·s−1 0.6 366–693 58 PANI nanofibers Three 1 M H2SO4 0.1–10 A·g−1 0.8 20–192 61 PPy nanofibrils Three 1 M H2SO4 0.1 A·g−1 0.7 280 62 Hollow NiCo2S4 nanoplates Three 3 M KOH 1–20 A·g−1 0.5 231–437 63 α-Fe2O3/MnO2 nanowires Three 0.7 M H3BO3 1–50 A·g−1 0.6 480–838 70 2D TiS2 nanocrystals Three 1 M LiClO4 0.5–10 A·g−1 1.2 320–470 71 CoAl/PEDOT nanoarrays Three 6 M KOH 1–40 A·g−1 0.6 424–672 76 Au-MnO2 core–shell nanomesh Two PVA-LiClO4 gel 0.56 A·g−1 2.0 524 77 CoO/PPy nanowires Two 3 M NaOH 1–50 mA·cm−2 1.6 647–2223 78 V2O5-PPy nanofibers Two PVA-LiCl gel 4.5 mA·cm−2 2.0 412 79 CuO nanowires Three 2 M KOH 1–5 A·g−1 0.45 102–118 80 Nanoporous Ni(OH)2 films Two 6 M KOH 0.9–50 A·g−1 1.6 20–192 81 β-Co(OH)2 nanosheets Two 2 M KOH 1–25 A·g−1 0.5 1530–2080 82 Co3O4 nanostructures Two 2 M KOH 0.5–2.5 A·g−1 0.8 150–476 83 NiCo2O4 nanosheets Three 2 M KOH 6–30 A·g−1 0.5 1500–1886 84 (1) The “Two” indicates capacitor cells composed of two electrodes (anode/cathode), separator, and electrolyte, containing symmetric or asymmetric electrode configurations. (2) The “Three” indicates electrode measuring systems composed of working electrode, counter electrode, and reference electrode in an electrolyte. Nanomaterials 2015, 5 materials [88–90]. These properties could be combined with those of the transition metal oxides and polymers, leading to the development of a new brand of electrochemical capacitors. 4. Hybrid Capacitive Materials Carbon materials have excellent electrical properties such as conductivity, power density, and long-term cycling stability; however, overcoming their low energy density has been the subject of intensive research [85,86]. The specific capacitance of carbon materials can be improved by mixing them with pseudocapacitive active materials [87]. The pseudocapacitance of electronically conducting polymers stems from rapid and reversible oxidation and reduction processes. EDLCs composed of pseudocapacitive-based nanostructures can store charge on the electrode surface both through a double layer and via a redox reaction. The low cycling stability of pseudocapacitive materials can be overcome through the use of carbon materials. Several active materials, including conducting polymers PPy, PANI, or metal oxides, have been directly grown on carbon owing to the properties imparted by these carbon-based 921 4.1. Coupling EDLC and Pseudocapacitive Materials 4.1. Coupling EDLC and Pseudocapacitive Materials Carbon-coated conducting polymers have, in general, excellent cycling stability, as well as high energy and power density [91]. More importantly, carbon materials enhance the corresponding cycling stability and prevent the structural breakdown of pseudocapacitive conducting polymers [92]. The energy density is also improved with the aid of the pseudocapacitance stemming from the conducting polymers. For example, exceptional capacitance retention of 95% and 85% after 10,000 cycles were recently reported for carbon-coated PANI and PPy [93]. These retention rates are the best values ever obtained for polymer-based pseudocapacitive electrodes in an aqueous electrolyte. These results show that the carbon material was very effective in maintaining the structures of the PANI and PPy nanowire electrodes during charge/discharge cycling, thereby resulting in excellent capacitance retention rates. The carbon-coated PANI and PPy electrodes exhibited similar pseudocapacitive behavior and specific capacitance as compared to bare polymer samples. Furthermore, the excellent electrochemical performance of the polymer electrodes was maintained, even with improvement in cycling stability [94]. a) Hydrothermal decomposition of glucose PPy or PANI Carbon-coated PPy or PANI Figure 12. (a) Schematic of the carbonaceous coating procedure using glucose as the carbon precursor. (b–d) TEM images of carbon-coated PPy samples with different carbonaceous shell thicknesses, which was controlled via the reaction time for the hydrothermal deposition of glucose: (b) 1, (c) 2, and (d) 3 h. With permission from [93]; Copyright 2014, American Chemical Society. 5 nm 5 nm 5 nm b) c) d) 5 nm 5 nm 5 nm a) Hydrothermal decomposition of glucose PPy or PANI Carbon-coated PPy or PANI Hydrothermal decomposition of glucose Hydrothermal decomposition of glucose Carbon-coated PPy or PANI 5 nm d) 5 nm d) 5 nm c) 5 nm 5 nm b) 5 nm b) c) 5 nm 5 nm 5 nm 5 nm Figure 12. (a) Schematic of the carbonaceous coating procedure using glucose as the carbon precursor. (b–d) TEM images of carbon-coated PPy samples with different carbonaceous shell thicknesses, which was controlled via the reaction time for the hydrothermal deposition of glucose: (b) 1, (c) 2, and (d) 3 h. With permission from [93]; Copyright 2014, American Chemical Society. Nickel- and manganese-based oxides/hydroxides have high theoretical specific capacitance. They are also inexpensive, naturally abundant, and environmentally friendly. Metal–oxide and graphene–oxide hybrid capacitors have many outstanding properties. Nickel hydroxide-manganese dioxide-RGO [Ni(OH)2–MnO2–RGO] ternary hybrid spherical powders were fabricated as electrode materials [95]. 4.2. Asymmetric Hybrid Capacitors The development of asymmetric capacitors for improving the energy and power density of electrochemical capacitors has been extensively studied. Combining the advantages of long-term cycling, a fast and reversible nonfaradaic negative electrode (−), and a high-capacitive positive faradaic electrode (+), asymmetric capacitors should fulfill the requirements of high energy and power density [96–98]. To fabricate asymmetric capacitors, Mn–Ni–Co ternary oxide (MNCO) nanowires were synthesized by a simple hydrothermal method [99]. The exceptional performance of these capacitors was a result of their nanowire architecture, which provided a large reaction surface area and fast ion and electron transfer. An asymmetric hybrid capacitor with high energy density was assembled successfully by employing an MNCO nanowire (+)//carbon black (−) cell. Figure 13 shows an image of an asymmetric hybrid capacitor and the improved electrochemical performance of an asymmetric cell. Figure 13. (a) Schematic of a representative asymmetric cell. With permission from [100]; Copyright 2014, The Royal Society of Chemistry. (b) Ragone plots of the as-fabricated asymmetric MNCO//carbon black, symmetric MNCO//MNCO, and symmetric carbon black//carbon black electrochemical capacitors. With permission from [99]; Copyright 2012, American Chemical Society. a) b) S pring Metal plate /Spring guide Cathode material Anode material PTFE Separator Stainless steel cell Power density / kW kg-1 Energy density / Wh kg-1 a) S pring Metal plate /Spring guide Cathode material Anode material PTFE Separator Stainless steel cell b) Power density / kW kg-1 Energy density / Wh kg-1 a) b) Energy density / Wh kg-1 Figure 13. (a) Schematic of a representative asymmetric cell. With permission from [100]; Copyright 2014, The Royal Society of Chemistry. (b) Ragone plots of the as-fabricated asymmetric MNCO//carbon black, symmetric MNCO//MNCO, and symmetric carbon black//carbon black electrochemical capacitors. With permission from [99]; Copyright 2012, American Chemical Society. In another study, MnO2–PEDOT nanotubes (mPNTs) were tested in asymmetric hybrid cell configurations, without using binders or conductive fillers, where RGO–CNFs were employed as an electric double-layer electrode material [100]. Surface redox reactions between the transition metal oxides and PEDOT resulted in the desired pseudocapacitance. Moreover, PEDOT has a nano-scale hierarchical structure, which provides a large effective surface area and efficient charge transfer. CNFs were intercalated between the RGO sheets to prevent the restacking of the sheets and to increase the nonfaradaic charge storage. The asymmetric MnO2–mPNTs (+)//RGO–CNFs (−) cell also exhibited superior specific capacitance, cycling stability, and coulombic efficiency as compared to symmetric cells comprising the MnO2–mPNT/RGO–CNF combination. 4.1. Coupling EDLC and Pseudocapacitive Materials These materials have a highly porous nanostructure, relatively high specific surface area, and well-defined Nickel- and manganese-based oxides/hydroxides have high theoretical specific capacitance. They are also inexpensive, naturally abundant, and environmentally friendly. Metal–oxide and graphene–oxide hybrid capacitors have many outstanding properties. Nickel hydroxide-manganese dioxide-RGO [Ni(OH)2–MnO2–RGO] ternary hybrid spherical powders were fabricated as electrode materials [95]. These materials have a highly porous nanostructure, relatively high specific surface area, and well-defined 922 Nanomaterials 2015, 5 spherical morphology. In addition, the synergetic effect of Ni(OH)2, MnO2, and RGO resulted in significantly enhanced specific capacitance of the electrode materials composed of these novel Ni(OH)2–MnO2–RGO ternary hybrid spheres 4.2. Asymmetric Hybrid Capacitors Furthermore, the capacitive performance of the 923 Nanomaterials 2015, 5 asymmetric MnO2–mPNT (+)//RGO–CNF (−) cells were examined in terms of the weight ratio of the positive/negative electrode materials. As Figure 14 shows, the capacitance of the asymmetric MnO2–mPNT//RGO–CNF cell decreased and the coulombic efficiency increased with increasing weight of the RGO–CNFs. The capacitive performance of the asymmetric cells was, therefore, highly dependent on the weight ratio of the electrode [101,102]. Figure 14. Effect of electrode weight: (a) Representative charge/discharge curves measured at 0.1 A·g−1; (b) Specific capacitances; and (c) Coulombic efficiencies measured at different current densities. With permission from [100]; Copyright 2014, The Royal Society of Chemistry. 0.0 0.5 1.0 1.5 2.0 0 10 70 80 90 100 Sym. RGO-CNFs MnO-mPNTs//RGO-CNFs 2:1 MnO-mPNTs//RGO-CNFs 1:1 MnO-mPNTs//RGO-CNFs 1:2 MnO-mPNTs//RGO-CNFs 1:5 Coulombic efficiency (%) Current density (A g 1) 0.0 0.5 1.0 1.5 2.0 0 10 20 30 40 50 60 70 Sym. RGO-CNFs MnO-mPNTs//RGO-CNFs 2:1 MnO-mPNTs//RGO-CNFs 1:1 MnO-mPNTs//RGO-CNFs 1:2 MnO-mPNTs//RGO-CNFs 1:5 Capacitance (F g 1) Current density (A g 1) Sym. RGO-CNFs MnO-mPNTs//RGO-CNFs 2:1 MnO-mPNTs//RGO-CNFs 1:1 MnO-mPNTs//RGO-CNFs 1:2 MnO-mPNTs//RGO-CNFs 1:5 0 50 100 150 200 250 300 350 0.0 0.2 0.4 0.6 0.8 1.0 Voltage (V) Time (s) a) b) c) 0.0 0.5 1.0 1.5 2.0 0 10 20 30 40 50 60 70 Sym. RGO-CNFs MnO-mPNTs//RGO-CNFs 2:1 MnO-mPNTs//RGO-CNFs 1:1 MnO-mPNTs//RGO-CNFs 1:2 MnO-mPNTs//RGO-CNFs 1:5 Capacitance (F g 1) Current density (A g 1) b) Sym. RGO-CNFs MnO-mPNTs//RGO-CNFs 2:1 MnO-mPNTs//RGO-CNFs 1:1 MnO-mPNTs//RGO-CNFs 1:2 MnO-mPNTs//RGO-CNFs 1:5 0 50 100 150 200 250 300 350 0.0 0.2 0.4 0.6 0.8 1.0 Voltage (V) Time (s) a) 0.0 0.5 1.0 1.5 2.0 0 10 70 80 90 100 Sym. RGO-CNFs MnO-mPNTs//RGO-CNFs 2:1 MnO-mPNTs//RGO-CNFs 1:1 MnO-mPNTs//RGO-CNFs 1:2 MnO-mPNTs//RGO-CNFs 1:5 Coulombic efficiency (%) Current density (A g 1) c) b) ) c) ure 14. Effect of electrode weight: (a) Representative charge/discharge curves measured Figure 14. Effect of electrode weight: (a) Representative charge/discharge curves measured at 0.1 A·g−1; (b) Specific capacitances; and (c) Coulombic efficiencies measured at different current densities. With permission from [100]; Copyright 2014, The Royal Society of Chemistry. Nanomaterials 2015, 5 A 3D hierarchical graphene/PPy aerogel (GPA) was fabricated using GO and 1D PPy nanotubes [106]. The as-formed GPAs exhibited low density, large specific surface area, and high compressive strength, which are highly desired properties in porous materials. Moreover, the 1D nanotube geometry facilitated electron transport and allowed relatively larger surface areas than nanorods and nanowires. The aerogel contributed to the low density and formation of pore structures. The double-pore structure (between graphene–graphene sheets and in PPy) had a large surface area and exhibited outstanding electrochemical performance. Such 3D porous nanostructures have advantages in terms of the facile diffusion of electrolyte ions into and out of the inner region of the active materials and effective surface-area utilization [107]. Thus, it is expected that the 3D electrode structure of graphene-based hybrid capacitors will show a synergetic effect caused by materials hybridization and a unique hierarchical structure [108,109]. Another interesting example is a composite of PPy nanowires intercalated with RGO sheets (Figure 16) [110]. The intercalated PPy nanowires prevent restacking of the graphene sheets and allow the formation of an open 3D architecture for electrolytes. The combination of conducting polymers and graphene has led to rapid development of high capacitance for flexible electrochemical capacitors [111,112]. Figure 16. Scheme showing the synthetic route of PPy/RGO composites. With permission from [110]; Copyright 2014, American Chemical Society. GO sheet RGO sheet V2O5 PPy Pyrrole Reduction Reduction RGO sheet GO sheet Figure 16. Scheme showing the synthetic route of PPy/RGO composites. With permission from [110]; Copyright 2014, American Chemical Society. 4.3. 3D Nanostructured Graphene-Based Capacitors 4.3. 3D Nanostructured Graphene-Based Capacitors 3D nanostructured hybrid materials, with better interfacial contact and volume utilization, have stimulated the development of several energy-efficient technologies via in situ polymerization of aniline onto a porous CNF/GO template (Figure 15). Such materials can be used for the facile fabrication of 3D PANI-modified CNF/GO hybrid electrodes, with the template imparting excellent conductivity and flexibility to the electrodes [103]. Specifically, CNFs significantly reduce the aggregation degree of GO, thereby forming a porous structure, which in turn substantially improves the utilization surface of GO. The in situ facial deposition process results in 3D hierarchically nanostructured PANI/CNF/GO composite electrodes with unique acanthine-style PANI nanowires covering the CNF/GO supports. PPy and other conducting polymers and metal oxides can also be used in hybrid capacitors [104,105]. CNF/GO/PANI Aniline Growth Figure 15. Formation mechanism of the CNF/GO/PANI film. With permission from [103]; Copyright 2013, American Chemical Society. GO CNF CNF/GO/PANI Aniline Dispersion Filtration Growth Aniline Growth Dispersion CNF/GO/PANI CNF GO Figure 15. Formation mechanism of the CNF/GO/PANI film. With permission from [103]; Copyright 2013, American Chemical Society. 924 4.4. Summary The electrochemical performance of pseudocapacitive conducting polymers and metal oxides can be improved through combinations with carbon materials. Table 3 summarizes the electrode materials for hybrid capacitors and their electrochemical performance. Vigorous on-going research and development are aimed at improving both the energy and power densities. However, the following issues should be further considered in the practical application of hybrid capacitors: the (i) different charge/discharge rates of EDLCs and pseudocapacitive materials and (ii) unstable charge/discharge cycling performance. 925 Nanomaterials 2015, 5 Nanomaterials 2015, 5 Table 3. Hybrid capacitor materials and electrochemical performance. Table 3. Hybrid capacitor materials and electrochemical performance. Materials (1) Electrode System (2,3) Electrolyte Current Density or Scan Rate Potential Range (V) Specific capacitance (F·g−1) Ref. 4.4. Summary NiO/graphene Two 1 M NaOH 7–20 A·g−1 1.5 130–440 13 Porous carbon/ Fe2O3 nanoparticles Three 1 M Na2SO3 0.5–10 A·g−1 1.0 119–235 14 α-Fe2O3/Graphene Three 1 M Na2SO4 3–10 A·g−1 1.0 98–306 15 Ni graphene aerogels Two 6 M KOH 2–20 A·g−1 1.0 186–366 85 CoO carbon nanoflakes Three 2 M KOH 10 mA·cm−2 0.75 476 86 Faradaic CNTs Three 0.5 M H2SO4 3–100 mV·s−1 0.9 75–260 87 Nanoporous CuO/active carbon Two 3 M KOH 1–10 A·g−1 1.4 54–72 88 Co(OH)2/graphene Three 6 M KOH 2–10 A·g−1 0.7 356–532 89 PANI/RGO Three 1 M H2SO4 0.45 A·g−1 0.8 431 90 PANI/CNF Three 1 M H2SO4 0.3–10 A·g−1 1.2 400–557 91 PANI/N-doped CNTs Three 0.1 M Na2SO4 50 mV·s−1 1.0 250 92 PPy/graphene Two 1 M H2SO4 0.1 A·g−1 1.0 277 94 Ni–Mn–RGO Two 1 M KOH 2–10 A·g−1 1.6 724–1985 95 3D carbon/CoNi3O4 asymmetric Two 3 M KOH 1–100 mA·cm−2 1.8 42–64 96 N-doped carbon/PANI asymmetric Two 1 M Na2SO4 0.5–20 A·g−1 1.1 75–113 97 WO3/PPy nanowire asymmetric Three 3 M NaOH 0.7–7 mA·cm−2 0.6 250–800 98 Mn–Ni–Co oxide nanowire/RGO asymmetric Three 6 M KOH 1–20 A·g−1 0.5 404–638 99 PEDOT/ROG/CNF asymmetric Two 1 M H2SO4 0.1–2 A·g−1 1.0 50–60 100 MnO2/GO asymmetric Two 1 M Na2SO4 0.1–2 A·g−1 2.0 41–84 101 RGO/MnO2 asymmetric Three 1 M Na2SO4 0.1–1 A·g−1 1.5 217–243 102 CNF/GO/PANI Two 1 M H2SO4 2 A·g−1 0.8 479 103 Ni/graphene/CNT Two 6 M KOH 0.2–1.0 A·g−1 0.8 100–105 104 Graphene/PANI nanorods Three 1 M H2SO4 1–8 A·g−1 0.7 836–1665 106 3D CoMoO4/graphene Three 2 M KOH 1.5–85 A·g−1 0.9 1101–2741 108 Graphene/CNT/Mn Two 2 M Li2SO4 1.9 A·g−1 1.6 1108 109 PPy nanowire/RGO Two PVA–H2SO4 gel 1–20 A·g−1 0.8 361–434 110 Porous graphene/PANI Two 1 M H2SO4 1–8 A·g−1 0.8 458–864 111 Porous graphene/PANI Two 1 M H2SO4 0.5–10 A·g−1 0.7 362–385 112 (1) The term “asymmetric” is given when the capacitor cell has a cathode and an anode consisting of different materials, namely asymmetric electrode configuration. (2) The “Two” indicates capacitor cells composed of two electrodes (anode/cathode), separator, and electrolyte, containing symmetric or asymmetric electrode configurations. (3) The “Three” indicates electrode measuring Nanomaterials 2015, 5 Nanomaterials 2015, 5 hierarchical nanoarchitectures with both micro- and macro-pores, in hope of fabricating better electrochemical capacitors. Although they are capable of providing high power for long cycles, EDLCs have a relatively low energy density. Therefore, future development is projected to still focus on the optimization of existing electrode materials and the creation of new materials for achieving energy densities approaching those of batteries. Pseudocapacitors have significantly higher faradaic capacitance than double-layer capacitance. In general, conducting polymers and metal oxides render pseudocapacitance via a faradaic reaction. Enhancing the surface area of electrode materials is still an important factor in pseudocapacitors, as it is with EDLCs. In most cases, the faradaic reaction for pseudocapacitance occurs at the surface of the electrode materials. Nanostructured pseudocapacitive materials with increased surface area, therefore, have been developed and their morphology has been precisely tailored. Typically, fibrous and tubular materials exhibit higher capacitance than other morphologies. Combining different pseudocapacitive nanomaterials can result in excellent electrodes with improved electrochemical performance. However, the excellent performance of pseudocapacitors tends to degrade quickly upon cycling. Various hybrid capacitors have been demonstrated as attractive alternatives to only EDLCs or only pseudocapacitors. The drawbacks of conventional EDLCs and pseudocapacitors, such as limited energy density and poor cycling stability, can be overcome by employing rational hybrid systems of pseudocapacitor (faradaic)-like and EDLC (nonfaradaic)-like electrodes, thereby producing a high working voltage and capacitance. There is growing demand for the development of electrochemical capacitors whose performance exceeds that of batteries. Nanomaterial-based hybrid capacitors have the potential to fulfill these requirements. As a result, it is expected that future extensive research into nanomaterial-based electrochemical capacitors will offer great potential for the construction of next-generation energy storage devices. In particular, the development of nanomaterial-based wearable or flexible high-performance electrochemical capacitors is expected in the near future. In this review, we have focused on nanostructured electrode materials. However, in addition to electrode materials, several factors such as electrolytes, separators, and binders also significantly influence the performance of electrochemical capacitors. First, different types of electrolytes, which are based on aqueous solutions, organic solvents, and ionic liquids, have been used in electrochemical capacitors. In general, aqueous gel electrolytes operate within a narrow potential range with a maximum of 1 V, leading to low cell voltage, which in turn limits energy and power densities. 5. Outlook Exploration of better electrode materials has been extensive over the few past decades. First, the surface area of the electrode/electrolyte interface must be increased in order to enhance the charge storage capacity in EDLCs. The use of a porous electrode with a higher specific surface area may increase the specific capacitance of an EDLC. Since ion desolvation occurs in pores smaller than the solvated ions, increased capacitance can be obtained from electrode materials with sub-nano-size pores. Meso- or macro-pores ensure the accessibility of ions. Thus, there have been many attempts to develop 926 Nanomaterials 2015, 5 Moreover, water evaporation during the operation of these electrolytes over a wide temperature range negatively affects the performance and long-term stability of the devices. Ionic liquid gels, in contrast, are thermally stable, nonvolatile, nonflammable, and nontoxic over a wide potential range of 3.5 V. Sometimes, redox mediators can be introduced into the electrolyte to facilitate the redox reaction, thereby increasing the pseudocapacitance [113]. Likewise, electrochemical capacitors mostly use solution-phase electrolytes, which are prone to leaking. Recently, flexible, wearable electrochemical capacitors have been of particular interest as a mobile power supply for future flexible electronics. However, electrolyte leaking can be problematic in these flexible or wearable devices. All solid-state electrochemical capacitors, in which electrolyte gels function as the separator, can be an alternative to circumvent electrolyte leaking. Nonconductive polymers such as polyvinylidene fluoride and polytetrafluoroethylene are widely used as binders to construct electrodes. These polymers facilitate good adhesion between electrode materials 927 Nanomaterials 2015, 5 or between electrode materials and current collectors. However, intrinsically, they are insulators and thus function as resistances that degrade the electrode performance. To circumvent this problem, conductive fillers have been introduced into the insulating polymers. However, it is difficult to achieve uniform dispersion of the fillers in the polymer matrix for obtaining desirable properties. Notably, an intrinsically conductive solution-processable PANI binder has very recently been prepared, in which PANI was treated with three organic additives as a ternary dopant [114]. The resulting PANI binders had abundant oxygenated functional groups such as sulfonate and hydroxyl groups, which facilitated ion transport. The polar groups of the additives provide adhesive properties via hydrogen-bonding or dipole–dipole interactions. The substituted alkyl chain may further afford adhesive properties to nonpolar surfaces through van der Waals interactions. Consequently, the PANI binders showed good conductivity and adhesive properties without the use of additional conductive fillers or heat treatment. Figure 17 shows the performance of a PANI binder in detail. PANI as a binder was found to significantly enhance the electrode performance of pseudocapacitive (PPy nanospheres and PANI nanofibrils) and EDLC (carbon black) nanomaterials. There is a still need for developing alternative efficient electrode binders capable of improving the device performance. Figure 17. Cont. 400 g 1) QB-PANI PVDF 600 g 1) QB-PANI PVDF 40 50 g 1) QB-PANI PVDF 0 500 1000 1500 0.0 0.2 0.4 0.6 0.8 5 wt% PVDF QB-PANI E (V vs. Nanomaterials 2015, 5 Ag/AgCl) Time (s) 0 1000 2000 3000 4000 0.0 0.2 0.4 0.6 0.8 10 wt% PVDF QB-PANI Time (s) 0.0 0.4 0.8 -3.0 -1.5 0.0 1.5 3.0 5 wt% PVDF QB-PANI I (mA g 1) E (V vs. Ag/AgCl) 0.0 0.4 0.8 -3.0 -1.5 0.0 1.5 3.0 10 wt% PVDF QB-PANI E (V vs. Ag/AgCl) 0 2000 4000 6000 0.0 0.2 0.4 0.6 0.8 5 wt% PVDF QB-PANI E (V vs. Ag/AgCl) Time (s) 0 2000 4000 6000 0.0 0.2 0.4 0.6 0.8 10 wt% PVDF QB-PANI Time (s) -0.4 0.0 0.4 0.8 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 10 wt% PVDF QB-PANI I (mA g 1) E (V vs. Ag/AgCl) -0.4 0.0 0.4 0.8 -0.2 -0.1 0.0 0.1 0.2 15 wt% PVDF QB-PANI E (V vs. Ag/AgCl) 0 50 100 150 200 250 300 0.0 0.2 0.4 0.6 0.8 15 wt% PVDF QB-PANI Time (s) 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0.8 10 wt% PVDF QB-PANI E (V vs. Ag/AgCl) Time (s) -0.3 0.0 0.3 0.6 0.9 -0.8 -0.4 0.0 0.4 0.8 1.2 10 wt% PVDF QB-PANI E (V vs. Ag/AgCl) -0.3 0.0 0.3 0.6 0.9 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 PVDF QB-PANI I (mA g 1) E (V vs. Ag/AgCl) 5 wt% 100 nm a) c) d) f) g) b) e) h) i) j) k) l) 100 nm 100 nm 0 500 1000 1500 0.0 0.2 0.4 0.6 0.8 5 wt% PVDF QB-PANI E (V vs. Ag/AgCl) Time (s) 0 1000 2000 3000 4000 0.0 0.2 0.4 0.6 0.8 10 wt% PVDF QB-PANI Time (s) -0.3 0.0 0.3 0.6 0.9 -0.8 -0.4 0.0 0.4 0.8 1.2 10 wt% PVDF QB-PANI E (V vs. Ag/AgCl) -0.3 0.0 0.3 0.6 0.9 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 PVDF QB-PANI I (mA g 1) E (V vs. Ag/AgCl) 5 wt% 100 nm a) d) g) -0.3 0.0 0.3 0.6 0.9 -0.8 -0.4 0.0 0.4 0.8 1.2 10 wt% PVDF QB-PANI E (V vs. Ag/AgCl) -0.3 0.0 0.3 0.6 0.9 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 PVDF QB-PANI I (mA g 1) E (V vs. Ag/AgCl) 5 wt% d) 0 500 1000 1500 0.0 0.2 0.4 0.6 0.8 5 wt% PVDF QB-PANI E (V vs. Nanomaterials 2015, 5 5 wt% 10 wt% 0 100 200 300 400 Capacitance (F g 1) QB-PANI PVDF 5 wt% 10 wt% 0 150 300 450 600 Capacitance (F g 1) QB-PANI PVDF 10 wt% 15 wt% 0 10 20 30 40 50 Capacitance (F g 1) QB-PANI PVDF j) k) l) 5 wt% 10 wt% 0 150 300 450 600 Capacitance (F g 1) QB-PANI PVDF k) 5 wt% 10 wt% 0 100 200 300 400 Capacitance (F g 1) QB-PANI PVDF j) 10 wt% 15 wt% 0 10 20 30 40 50 Capacitance (F g 1) QB-PANI PVDF l) Figure 17. Electrode binder performance of an intrinsically conductive PANI glue: SEM Figure 17. Electrode binder performance of an intrinsically conductive PANI glue: SEM images of nanoparticle electrodes prepared with a 10 wt.% PANI binder content: (a) PPy images of nanoparticle electrodes prepared with a 10 wt.% PANI binder content: (a) PPy nanospheres, (b) PANI nanofibrils, and (c) carbon black. Representative CV curves and galvanostatic charge–discharge curves of the electrodes recorded at a scan rate of 25 mV·s−1 and a current density of 0.1 A·g−1, respectively, in 1 M sulfuric acid: (d and g) PPy nanospheres, (e and h) PANI nanofibrils (5 wt.% and 10 wt.% binder contents), and (f and i) carbon black electrodes with 10 wt.% and 15 wt.% binder. Gravimetric discharge capacitances calculated from the charge–discharge curves are presented in the histograms: (j) PPy nanospheres, (k) PANI nanofibrils, and (l) carbon black. With permission from [114]; Copyright 2014, The Royal Society of Chemistry. images of nanoparticle electrodes prepared with a 10 wt.% PANI binder content: (a) PPy nanospheres, (b) PANI nanofibrils, and (c) carbon black. Representative CV curves and galvanostatic charge–discharge curves of the electrodes recorded at a scan rate of 25 mV·s−1 and a current density of 0.1 A·g−1, respectively, in 1 M sulfuric acid: (d and g) PPy nanospheres, (e and h) PANI nanofibrils (5 wt.% and 10 wt.% binder contents), and (f and i) carbon black electrodes with 10 wt.% and 15 wt.% binder. Gravimetric discharge capacitances calculated from the charge–discharge curves are presented in the histograms: (j) PPy nanospheres, (k) PANI nanofibrils, and (l) carbon black. With permission from [114]; Copyright 2014, The Royal Society of Chemistry. Additionally, the performance of electrochemical capacitors also depends on the microarrangement of the electrode materials in the cell. Nanomaterials 2015, 5 For example, capacitor applications are often constrained by volume rather than by weight. Unfortunately, nanomaterial-based electrochemical capacitors typically exhibit low volumetric capacitance. Therefore, increasing the packing density of nanomaterials inside the electrode is one of the critical issues in electrochemical capacitor research. In summary, there are key challenges to achieving advances in electrochemical cap (i) control of the 3D structure of electrode materials in the nanometer regime, (ii) battery-like hybrid capacitors with both high energy and power densities, (iii) flexible, all-solid-state devices, (iv) use of intrinsically conductive binders or no binder, (v) increased volumetric capacitance. In conclusion, the electrochemical capacitor market continues to grow and expand into a greater number of applications. Continuous research on electrode materials, cell assembly, and entire cell systems should yield electrochemical capacitors that exhibit the excellent properties of both conventional capacitors and batteries and might ultimately facilitate the emergence of new types of energy storage devices. Nanomaterials 2015, 5 Ag/AgCl) Time (s) 0 1000 2000 3000 4000 0.0 0.2 0.4 0.6 0.8 10 wt% PVDF QB-PANI Time (s) g) 100 nm a) b) 100 nm 0 h) b) 0.0 0.4 0.8 -3.0 -1.5 0.0 1.5 3.0 5 wt% PVDF QB-PANI I (mA g 1) E (V vs. Ag/AgCl) 0.0 0.4 0.8 -3.0 -1.5 0.0 1.5 3.0 10 wt% PVDF QB-PANI E (V vs. Ag/AgCl) e) 3 e) 0 2000 4000 6000 0.0 0.2 0.4 0.6 0.8 5 wt% PVDF QB-PANI E (V vs. Ag/AgCl) Time (s) 0 2000 4000 6000 0.0 0.2 0.4 0.6 0.8 10 wt% PVDF QB-PANI Time (s) h) 2 i) c) c) 100 nm f) -0.4 0.0 0.4 0.8 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 10 wt% PVDF QB-PANI I (mA g 1) E (V vs. Ag/AgCl) -0.4 0.0 0.4 0.8 -0.2 -0.1 0.0 0.1 0.2 15 wt% PVDF QB-PANI E (V vs. Ag/AgCl) 0 50 100 150 200 250 300 0.0 0.2 0.4 0.6 0.8 15 wt% PVDF QB-PANI Time (s) 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0.8 10 wt% PVDF QB-PANI E (V vs. Ag/AgCl) Time (s) f) i) nm Figure 17. Cont. QB-PANI 928 0 Nanomaterials 2015, 5 Figure 17. Electrode binder performance of an intrinsically conductive PANI glue: SEM images of nanoparticle electrodes prepared with a 10 wt.% PANI binder content: (a) PPy nanospheres, (b) PANI nanofibrils, and (c) carbon black. Representative CV curves and galvanostatic charge–discharge curves of the electrodes recorded at a scan rate of 25 mV·s−1 and a current density of 0.1 A·g−1, respectively, in 1 M sulfuric acid: (d and g) PPy nanospheres, (e and h) PANI nanofibrils (5 wt.% and 10 wt.% binder contents), and (f and i) carbon black electrodes with 10 wt.% and 15 wt.% binder. Gravimetric discharge capacitances calculated from the charge–discharge curves are presented in the histograms: (j) PPy nanospheres, (k) PANI nanofibrils, and (l) carbon black. With permission from [114]; Copyright 2014, The Royal Society of Chemistry. References 1. Faraji, S.; Ani, F.N. The development supercapacitor from activated carbon by electroless plating—A review. Renew. Sustain. 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https://openalex.org/W4291432424	https://snv63.ru/2309-4370/article/download/109857/84099	Russian	null	Confessional identity and modern religious practices of the Chuvash	Samarskij naučnyj vestnik	2,022	cc-by	6,370	Ягафова Е.А. Конфессиональная идентичность и современные религиозные практики чувашей УДК 394.2 DOI 10.55355/snv2022112215 УДК 394.2 Статья поступила в редакцию / Received: 08.02.2022 Статья принята к опубликованию / Accepted: 27.05.2022 Статья поступила в редакцию / Received: 08.02.2022 Статья принята к опубликованию / Accepted: 27.05.2022 Самарский государственный социально-педагогический университет (г. Самара, Российская Федерация) Аннотация. В данной статье рассматривается соответствие декларируемой конфессиональной идентично- сти православных и некрещеных чувашей актуальным религиозным практикам, распространенным в обеих группах. В работе выявлены особенности ритуальных практик, показана их роль в формировании конфесси- ональной идентичности группы и ее членов. В соответствии с задачами проанализированы конкретные риту- альные практики, существующие в различных конфессиональных средах, охарактеризовано состояние рели- гиозности отдельных локальных групп православных и некрещеных чувашей. Работа основана на полевых материалах автора, собранных в сельских населенных пунктах Самарской области, Республик Татарстан и Башкортостан и Чувашской Республики в 2020–2021 гг. Результаты исследования показали, что религиозные практики обеих групп не соответствуют в полной мере декларируемой религиозной идентичности. В практи- ках православных чувашей сохраняется существенный пласт воззрений, предписаний и поведенческих прак- тик, восходящих к традиционной религии, а у некрещеных чувашей присутствуют христианские элементы, проникшие в ходе взаимодействия с православным окружением, что позволяет определить религиозность этих групп в одних случаях как синкретизм, в других – как двоеверие. Расхождение конфессиональной иден- тичности с актуальными религиозными практиками обусловлено частичным размыванием конфессиональ- ных «границ» вследствие религиозной конверсии, трансформации ритуальных практик, а также по причине восприятия сообществами обеих религиозных систем как общего этнокультурного наследия. р р у ур Ключевые слова: чуваши; конфессиональная идентичность; религиозные практики; православие; некре- щеные чуваши; ритуалы; обряды; этноконфессиональная группа; традиционная религия; чувашская вера; трансформация; синкретизм; двоеверие. Ягафова Е.А. Ягафова Е.А. Ягафова Е.А. Самарский государственный социально-педагогический университет (г. Самара, Российская Федер CONFESSIONAL IDENTITY AND MODERN RELIGIOUS PRACTICES OF THE CHUVASH © 2022 © 2022 Iagafova E.A. Iagafova E.A. Samara State University of Social Sciences and Education (Samara, Russian Federation) Abstract. The paper discusses the conformity of the declared confessional identity of the Orthodox and unbap- tized Chuvash with actual religious practices common for both groups. The paper reveals features of ritual practices, shows their role in the formation of the confessional identity of the group and its members. In accordance with the tasks, specific ritual practices that exist in various confessional environments are analyzed, the state of religiosity of individual local groups of the Orthodox and unbaptized Chuvash is characterized. The research is based on the aut- hor’s field materials collected in rural settlements of the Samara Region, the Republics of Tatarstan and Bashkorto- stan and the Chuvash Republic in 2020–2021. The results of the study showed that the religious practices of both groups do not fully correspond to the declared religious identity. In the practices of the Orthodox Chuvash, a signifi- cant layer of views, prescriptions and behavioral practices dating back to the traditional religion is preserved, while the unbaptized Chuvash have Christian elements that have penetrated in the course of interaction with the Orthodox environment, which makes it possible to determine the religiosity of these groups, in some cases, as syncretism, in others as dual faith. Differences in confessional identity with current religious practices are due to the partial blurring of confessional «borders» as a result of religious conversion, transformation of ritual practices, and perception by communities of both religious systems as a common ethno-cultural heritage. Keywords: Chuvash; confessional identity; religious practices; Orthodoxy; unbaptized Chuvash; rituals; rite; ethno- confessional group; traditional religion; Chuvash faith; transformation; syncretism; dual faith. Исторические науки Исторические науки Анализ данных Как показывают результаты исследования по пра- вославным приходам, абсолютно все опрошенные признали себя православными верующими, однако доля реально участвующих в ритуальных практиках оказалась значительно ниже. Так, в Девлезеркино храм в своем селе посещают регулярно (еженедель- но) только около 5% и 86,9% – время от времени, в Кош-Елге – 9 и 33%, в Мусирмы – 12 и 80% соответ- ственно. Значительная часть жителей (от 50 до 76% в Девлезеркино, от 12 до 25% в Кош-Елге и от 16 до 15% в Мусирмы) заказывают панихида по родствен- никам, однако в Кош-Елге большинство опрошен- ных (от 55 до 80%) вообще не заказывают никаких треб. На исповеди и у причастия в течение года было не более трети опрошенных: в Девлезеркино – 33 и 34%, в Кош-Елге – 25 и 33%, в Мусирмы – 23 и 35% соответственно. Более половины жителей (54% в Девлезеркино, 57% в Кош-Елге и 52% в Мусирмы) не исповедаются вообще. При этом значительная часть жителей (88% в Девлезеркино, по 74% в Кош- Елге и Мусирмы) совершают денежные пожертвова- ния в пользу церкви, хранят дома религиозную (хри- стианскую) литературу (80%, 70% и 80% соответ- ственно), ритуальные предметы: нательные кресты (95%; 86%; 98%), иконы (99%; 89%; 100%), емкости со святой водой (98%; 83%; 84%), отмечают дома православные праздники (99%; 89%; 96%), в т.ч. Рождество (66%; 32%; 44%), Крещение (66%; 6%; 12%), Пасху (74%; 68%; 70%), Троицу (12%; 46%; 34%). Таким образом, декларируемая религиозность православных чувашей основана лишь частично на участии в церковной жизни, но в основном опосре- дована материальными атрибутами и праздниками [9; 10; 12]. Целью данной работы является выявление осо- бенностей ритуальных практик православных и не- крещеных чувашей и их роли в формировании кон- фессиональной идентичности группы и ее членов. В задачи исследования входит проанализировать кон- кретные ритуальные практики, существующие в раз- личных конфессиональных средах, и проследить на примере некоторых из них проявления конфессио- нальной идентичности, охарактеризовать состояние религиозности отдельных локальных групп право- славных и некрещеных чувашей. Статья основана на полевых материалах автора, собранных в ходе экспедиций 2020–2021 гг. в Самар- ской области, Республике Татарстан и Чувашской Республике. Православные чуваши исследованы в трех приходах, представляющих три разных района расселения чувашей: Чувашскую Республику – с. Му- сирмы Урмарского района [8; 9], Самарскую область – с. Девлезеркино Челно-Вершинского района [10; 11], Республику Башкортостан – с. Кош-Елга Бижбу- лякского района [12]. ф Конфессиональная идентичность и современные религиозные практики чувашей науки действия и другие формы религиозного поведения, так и их понимание и толкование членами сообществ. действия и другие формы религиозного поведения, так и их понимание и толкование членами сообществ. в религиозной жизни. При этом, как и в целом в со- временном российском обществе [1; 2], на индиви- дуальном уровне наблюдается расхождение между декларируемой конфессиональной идентичностью и вовлеченностью в ритуальные практики. Эту тенден- цию можно объяснить «развитием индивидуализма и субъективизма в области религиозных верований и практик» [3, с. 108]. Однако аналогичный вопрос возникает и в отношении всего религиозного сооб- щества: в какой степени бытующие в той или иной конфессии ритуальные практики соответствуют де- кларируемой ими конфессиональной идентичности? Таким образом, проблема соотношения религиозной идентичности и практик может быть рассмотрена и на групповом уровне. В таком ключе изучаемая про- блема рассматривалась в последние десятилетия в ра- ботах А.Н. Крылова [4], Е.А. Кублицкой [5], С.Д. Ле- бедева [6], Т.С. Прониной [1; 2], С.В. Рыжовой [7], С.В. Трофимова [3] и др. Исследование базируется преимущественно на качественных методах: применялся целевой отбор информантов в соответствии с целями и задачами исследования: в каждом селении были записаны глу- бинные интервью с 3–5 местными жителями. В пра- вославных приходах, кроме того, были проведены полуформализованные интервью с жителями села: в Девлезеркино и Кош-Елге было опрошено по 100 че- ловек, в Мусирме – 50 человек. В работе представле- ны данные этого опроса после соответствующей ста- тистической обработки. Постановка проблемы, методология, цели и задачи исследования вославие, а меньшинство осталось в прежней, чуваш- ской, вере чăваш тěнě. По оценочным данным, чис- ленность второй группы оценивается в 3–3,5 тыс. че- ловек и в последнее время стремительно сокращается. вославие, а меньшинство осталось в прежней, чуваш- ской, вере чăваш тěнě. По оценочным данным, чис- ленность второй группы оценивается в 3–3,5 тыс. че- ловек и в последнее время стремительно сокращается. Выделение этих групп среди чувашей обусловле- но декларируемой конфессиональной идентичностью, которую члены обеих групп признают либо в силу отчетливо выраженных религиозных убеждений и исполняемых ритуальных практик, либо по причине участия в обрядах в составе коллектива (сельской общины, семейно-родственной группы), либо «по традиции», восходящей к истории конкретной семьи и рода, но без проявлений определенной активности Samara Journal of Science. 2022. Vol. 11, iss. 2 Исследование религиозности в современном об- ществе невозможно без детального изучения кон- кретных религиозных практик конфессиональных групп, для которых характерны не только деклари- руемая идентичность, но также реальное поведение и его интерпретация членами сообщества. Православ- ные («крещеные» тěне кěнě, крешěн) и некрещеные (тěне кěмен, «истинные» – чăн чăваш) чуваши – две основные этноконфессиональные группы народа, сфор- мировавшиеся в ходе его христианизации в XVII – начале XX в. При этом большинство восприняло пра- Выделение этих групп среди чувашей обусловле- но декларируемой конфессиональной идентичностью, которую члены обеих групп признают либо в силу отчетливо выраженных религиозных убеждений и исполняемых ритуальных практик, либо по причине участия в обрядах в составе коллектива (сельской общины, семейно-родственной группы), либо «по традиции», восходящей к истории конкретной семьи и рода, но без проявлений определенной активности 245 Исторические науки Ягафова Е.А. Ягафова Е.А. Конфессиональная идентичность и современные религиозные практики чувашей Анализ данных Селения находятся в различ- ных в точки зрения этноконфессиональной ситуации условиях: моноэтничного православного окружения (Мусирмы), полиэтничного, преимущественно пра- вославного, соседства (Девлезеркино) и полиэтниче- ской и поликонфессиональной среды (Кош-Елга). Общим для всех приходов является историческая традиция приходской жизни (в Девлезеркино с сере- дины XVIII в. [13, л. 381 об.], в других селениях – с 1880-х гг.) [14; 15], которая в с. Мусирмы не преры- валась. Самарский научный вестник. 2022. Т. 11, № 2 В приведенных выше показателях заметны рас- хождения по селам, которые свидетельствуют, во-пер- вых, о большей степени приобщенности девлезер- кинцев к жизни прихода и церкви, что обусловлено более длительным периодом существования прихода в Девлезеркино по сравнению с приходами в сс. Кош- Елга и Мусирмы и, таким образом, более продолжи- тельной православной традицией, формировавшейся в близком соседстве с русскими и мордовскими се- лениями, в отличие о Кош-Елги, располагавшейся преимущественно в мусульманском татаро-башкир- ском окружении, и моноэтничного окружения с. Му- сирмы. Во-вторых, разница в показателях указывает и на локальные варианты празднично-обрядового ком- плекса: Крещение является престольным праздником с. Девлезеркино и поэтому отмечалось с размахом, с гостеваниями родственников, а Троицу и приурочен- Общины некрещеных чувашей изучались в 9 се- лениях в двух регионах – в с. Старое Афонькино Шенталинского района [16], с. Чувашское Урметьево Челно-Вершинского района [17], с. Староганькино Похвистневского района Самарской области [18], с. Абрыскино, с. Салдакаево, с. Якушкино Нурлат- ского района [19], с. Савгачево и с. Сидулово-Ерык- ла Аксубаевского района [20], с. Новое Ильмово Че- ремшанского района Республики Татарстан [21]. Методология исследования основана на понима- нии религиозных практик как совокупности «интер- претаций и действий, совершаемых людьми в связи с их верованиями, их религиозным опытом и/или их взаимодействием с религиозными институтами» [22]. В этой связи в работе анализируются как ритуальные 246 Ягафова Е.А. Исторические Исторические науки р науки ный к нему день поминовения Ҫимӗк отмечают в Кош-Елгеи Мусирмы, как и в большинстве чуваш- ских сел региона. Выделение этих праздников из множества других (анкетируемым было предложено отметить по уровню осведомленности и участия в праздновании 29 православных праздников) обу- словлено тем, что в основе этих и некоторых других отмеченных в ходе опроса обрядов, таких как Сыр- ная седмица (Масленица), Благовещение Пресвятой Богородицы, Вознесение Христово, День Св. Нико- лая Чудотворца (Никола летний), День св. Анализ данных апостолов Петра и Павла (Петров день), День памяти пророка Илии (Ильин день), Покров Пресвятой Богородицы, День Михаила Архангела (Михайлов день) и др., со- пряжены с традиционным празднично-обрядовым ка- лендарем чувашей, включавшим новогодний цикл Çӗнӗ çул с гаданиями и хождениями ряженых, мас- леницу Çăварни, весенне-летние и осенние поми- нальные обряды, хороводы и гуляния молодежи Уяв / Вăйă, празднование окончания весенних полевых работ Акатуй, осенние благодарственные моления и обычаи зимних гостеваний во время т.н. «престоль- ных праздников», а также множество соответствую- щих запретов и правил поведения участников. С од- ной стороны, по мере распространения христианства чувашские обряды оказались приурочены к датам православного календаря, а с другой стороны, право- славные праздники наполнялись «языческим» содер- жанием. Сформировавшийся в XVIII – начале XX в. «православно-языческий» синкретизм православных чувашей продолжает бытовать и в начале XXI в. Од- ним из показательных примеров является отношение жителей с. Девлезеркино к празднику Петров день, который в анкетах отметили всего 2%, в то время как празднуемый в этот день, 12 июля, проводы Уяв – 63%. В Девлезеркино, как и в других чувашских се- лах юга Закамья, проводы Уяв – главный летний праздник. По этой же причине восстановленный в 2015 г. храм был освящен в честь св. Петра и Павла, хотя исторически местная церковь именовалась Тро- ицкой. В Мусирмы на фоне православных и чуваш- ских праздников выделяется Акатуй – также локаль- ная традиция данной территориальной группы чува- шей [10–12]. рядах (Юпа, Мункун ватти, Çимěк, Кěр сăри), сов- местно проводят гостевания (рет) в праздники [16]. рядах (Юпа, Мункун ватти, Çимěк, Кěр сăри), сов- местно проводят гостевания (рет) в праздники [16]. Подобная картина религиозной жизни характерна и для других конфессионально-смешанных селений: например, Староганькино, Савгачево, Якушкино. Об- щие интересы в хозяйственной деятельности, быту, родственные связи сближают жителей села и позво- ляют им преодолевать (часто сознательно игнориро- вать) конфессиональные различия. Однако если одна часть жителей ограничивается соучастием в религи- озной жизни, но при этом четко различает «свои», т.е. православные, обычаи от обычаев соседей-языч- ников и сохраняет устойчивое православное самосо- знание, то другая часть чувашей-христиан, связан- ных родством с некрещеными, признает догматику и ритуальную практику обеих религий, демонстрируя, таким образом, пример двоеверия. Православные чу- ваши вместе с некрещеными отмечают чувашский Мункун, но также и православную Пасху, поминают предков на Çимĕк в четверг, но и в субботу перед Троицей, празднуют престольные праздники и т.д. Подобное поведение они не рассматривают как на- рушение «нормы»: «Вĕсене туни çылăх мар! (Прове- дение этих обрядов [Е.Я.] не является грехом!)» [18–20]. Анализ данных д р д [ ] р ) [ ] Наиболее зримо нарушение конфессиональных «границ» представлено в похоронно-поминальных ритуалах. Как правило, крещеные проводят помино- вение своих некрещеных родственников по «чуваш- скому обычаю» (чăваш йăлипе). Одной из его осо- бенностей является юпа – ритуал установления на- могильного столба. В общинах некрещеных чувашей он считается обязательным и проводится даже в том случае, если человек умер вдали от родных мест [16; 21]. Другим ритуалом, однозначно идентифицируе- мым с чувашской традицией, является хывни (возда- яние): кусочки еды складывают в общую посуду и вместе с напитками, также собранными в отдельный сосуд, выносят из дома и выбрасывают в особое ме- сто, где обычно никто не ходит; в ходе поминок этот ритуал проделывают неоднократно. Поминая креще- ных предков, такой ритуал уже не проводят. Однако при этом православные произносят те же слова, об- ращенные к предкам, что и некрещеные чуваши во время поминок: «Тутлăхлă çӳрĕр!» (Будьте сыты!), «Умăрта пултăр!» (Пусть будет перед вами!), «Вă- хăтлă килсе вăхăтлă кайăр!» (Вовремя приходите и вовремя уходите!), «Эпир сире асăнатпăр, эсир пире ан асăнăр!» (Мы вас поминаем, но вы о нас не вспо- минайте!) [17; 18]. Samara Journal of Science. 2022. Vol. 11, iss. 2 шей [10 12]. Религиозные практики православных чувашей вариативны на уровне локальных групп и даже селе- ний из-за различных условий приобщения населения к христианству. К примеру, в двух соседних селах, Девлезеркино и Чувашское Урметьево, расположен- ных на расстоянии 7 километров друг от друга, на- блюдается разная картина религиозной жизни, пото- му что в первом православие утвердилось в XVIII– XIX вв., в том числе и благодаря местному храму, а в соседнем селе до настоящего время существует не- многочисленная община некрещеных чувашей, с кото- рыми православные взаимодействуют в быту, по ра- боте, а также в семейно-родственном кругу [11; 17]. Аналогичная картина наблюдается в расположен- ных также по соседству друг с другом сс. Салейкино и Старое Афонькино. В то время как салейкинцы по- сещают храм, придерживаются православных обыча- ев и осуждают некрещеных афонькинцев, православ- ные чуваши в Старом Афонькине вместе с некреще- ными участвуют в общесельских обрядах (Учук, Сěрен, Çăмăр чӳк), в похоронах и поминальных об- Религиозные практики православных чувашей вариативны на уровне локальных групп и даже селе- ний из-за различных условий приобщения населения к христианству. Анализ данных но для определенной группы жителей конфессио- нально-смешанных селений, приобщенных в силу обстоятельств рождения, воспитания и последующей религиозной конверсии к разным традициям. но для определенной группы жителей конфессио- нально-смешанных селений, приобщенных в силу обстоятельств рождения, воспитания и последующей религиозной конверсии к разным традициям. р р р р Участие неофитов в ритуальных практиках раз- ных конфессий обусловлено пониманием и призна- нием позитивной направленности обеих религиоз- ных систем, их нацеленностью на «лучшее». Из ин- тервью с Л.В. Моляновой, 1963 г., жительницей с. Ста- роганькино, православной: «Лайăха шанса, пурте лайăх пултăр, тесе, чиркĕве çӳретĕп. Тĕне кĕмен пул- сан та, пирĕн анне те лайăхшăн, тесе, кĕл тăваччĕ, лайăха шанмалла теччĕ» (В надежде на лучшее, что- бы во всем было хорошо, хожу в церковь. Моя мама, хоть и некрещеная была, молилась за все хорошее, говорила, что надо надеяться на лучшее) [18]. Не- офиты, как правило, убеждены в том, что ценност- ные основания чувашской религии и христианства близки, что, вероятно, позволяет им время от време- ни возвращаться в ритуальное пространство «чуваш- ской веры» с уверенностью в правильности своих действий. Данный концепт является одним из важ- ных факторов восприятия ими православия. Однако не менее значимыми представляются и социальные причины религиозной конверсии некрещеных чува- шей, чаще вызванной семейными обстоятельствами и желанием сохранить конфессиональное единство семьи. В одних случаях мотивом крещения выступа- ет желание жить в одной вере с супругом: «Мăшăрпа пĕр верăпа пурăнмалла тесе кĕтĕм» (Крестилась, чтобы жить в одной вере с супругом); «Упăшкаран уйрăлма кирлĕ мар вĕт. Вилсен, упăшкаран уйрăм пытараççĕ» (Нельзя ведь разлучаться с супругом. Когда умру, могут похоронить на разных кладбищах) [17; 18]. Будучи конфессиональным меньшинством в своем селе и округе, некрещеные чуваши вынужде- ны вступать в браки с православными, а затем и кре- ститься [17]. В других случаях идея общей для всех членов семьи религии, побуждающая их к креще- нию, распространяется и на детей, особенно в крити- ческие моменты жизни – болезни, несчастные слу- чаи, которые чуваши, как некрещеные, так и право- славные, склонны интерпретировать как наказание за отсутствие конфессионального согласия в семье [17]. Таким образом, смена религиозной принадлежности некрещеных чувашей может рассматриваться, с од- ной стороны, как частное явление, связанное с опре- деленными событиями в жизни конкретного челове- ка, но с другой стороны, как тренд в религиозном поведении малочисленного конфессионального со- общества, связанного множеством социальных свя- зей с численно преобладающим православным окру- жением. Анализ данных К примеру, в двух соседних селах, Девлезеркино и Чувашское Урметьево, расположен- ных на расстоянии 7 километров друг от друга, на- блюдается разная картина религиозной жизни, пото- му что в первом православие утвердилось в XVIII– XIX вв., в том числе и благодаря местному храму, а в соседнем селе до настоящего время существует не- многочисленная община некрещеных чувашей, с кото- рыми православные взаимодействуют в быту, по ра- боте, а также в семейно-родственном кругу [11; 17]. Ситуация двоеверия типична для поведения нео- фитов, воспитанных в чувашских традициях в семьях некрещеных чувашей. Чувашские обряды и обычаи они воспринимают как более понятные и близкие, чем православные. Из интервью с К.Г. Бондаревой, 1951 г.р., жительницей с. Чувашское Урметьево, пра- вославной: «Эпĕ хам крешĕн пулсан та мана чăваш йăлисем темшĕн çывăхрах. Мĕншĕн тесен атте те, анне те, çывăх тăвансем те пурте чăвашсем пулнă» (Хотя я сама крещеная, но чувашские обычаи поче- му-то мне ближе. Потому что и отец, и мать, близкая родня – все были чувашами). При этом информант посещает время от времени церковь, читает религи- озную литературу, хранит иконы в божнице, однако признается, что никаких панихид не заказывает, а молится сама дома [17]. Подобное поведение типич- Samara Journal of Science. 2022. Vol. 11, iss. 2 Аналогичная картина наблюдается в расположен- ных также по соседству друг с другом сс. Салейкино и Старое Афонькино. В то время как салейкинцы по- сещают храм, придерживаются православных обыча- ев и осуждают некрещеных афонькинцев, православ- ные чуваши в Старом Афонькине вместе с некреще- ными участвуют в общесельских обрядах (Учук, Сěрен, Çăмăр чӳк), в похоронах и поминальных об- 247 Исторические Ягафова Е.А. Конфессиональная идентичность и современные религиозные практики чувашей но для определенной группы жителей конфессио- нально-смешанных селений, приобщенных в силу обстоятельств рождения, воспитания и последующей религиозной конверсии к разным традициям. резать. Его так и (чув. Питрав так дующий день в че печем пироги, ва Ягафова Е.А. ф Конфессиональная идентичность и современные религиозные практики чувашей науки резать. Его так и называют – «Петровский баран» (чув. Питрав таки. – Е.Я.) и режут накануне. На сле- дующий день в честь Бога (чув. Турра асăнса. – Е.Я.) печем пироги, варим суп. Потом сами же съедаем. Если нет барана, то режем бройлеров <…> На Ильин день (Илен) вместе с семьей едим мед. Еще я на каж- дый праздник тесто ставлю. Кто ни придет, могу угостить <…> Говорят, раньше Казанский не отме- чали. Однажды все пошли в этот день работать, а ко- гда домой вернулись, оказалось дома сгорели. Анализ данных С тех пор у нас этот праздник называют «Огненным праздником» (чув. Вут праçникĕ. – Е.Я.) и в этот день не работают <…> Летнего Николу (Çуллахи Микула) у нас называют «Днем отдыха лошадей» (чув. Лаша кантармалли кун. – Е.Я.). С этого дня начинаются игрища (чув. Вăйă. – Е.Я.) и продолжа- ются до Петрова дня <…> На Благовещение нельзя ходить к людям. Нельзя никому ничего давать – если хотя бы одну вещь дашь, будешь весь год отдавать. И на Егорьев день (чув. Якур кунĕ. – Е.Я.) нельзя от- давать <…> На Покров нужно пироги печь. Я этот обычай соблюдаю, пеку пироги с капустой, морко- вью, тыквой, картошкой. На Крещение нужно печь колобки (чув. Йăва. – Е.Я.), на Пасху – красить яйца, на Петров день – печь пироги с ягодами <…> На Крещение мы всегда святки делали, гадали <…> На Вербное воскресенье вербу ломаем, а потом этими ветками скотину провожаем в стадо» [18]. резать. Его так и называют – «Петровский баран» (чув. Питрав таки. – Е.Я.) и режут накануне. На сле- дующий день в честь Бога (чув. Турра асăнса. – Е.Я.) печем пироги, варим суп. Потом сами же съедаем. Если нет барана, то режем бройлеров <…> На Ильин день (Илен) вместе с семьей едим мед. Еще я на каж- дый праздник тесто ставлю. Кто ни придет, могу угостить <…> Говорят, раньше Казанский не отме- чали. Однажды все пошли в этот день работать, а ко- гда домой вернулись, оказалось дома сгорели. С тех пор у нас этот праздник называют «Огненным праздником» (чув. Вут праçникĕ. – Е.Я.) и в этот день не работают <…> Летнего Николу (Çуллахи Микула) у нас называют «Днем отдыха лошадей» (чув. Лаша кантармалли кун. – Е.Я.). С этого дня начинаются игрища (чув. Вăйă. – Е.Я.) и продолжа- ются до Петрова дня <…> На Благовещение нельзя ходить к людям. Нельзя никому ничего давать – если хотя бы одну вещь дашь, будешь весь год отдавать. И на Егорьев день (чув. Якур кунĕ. – Е.Я.) нельзя от- давать <…> На Покров нужно пироги печь. Я этот обычай соблюдаю, пеку пироги с капустой, морко- вью, тыквой, картошкой. На Крещение нужно печь колобки (чув. Йăва. – Е.Я.), на Пасху – красить яйца, на Петров день – печь пироги с ягодами <…> На Крещение мы всегда святки делали, гадали <…> На Вербное воскресенье вербу ломаем, а потом этими ветками скотину провожаем в стадо» [18]. Анализ данных В этом случае «сердечную палочку» чĕре калакĕ и юпа ставят одновременно [16–18]. ден до домашнего приготовления каши с последую- щим молебном в поле, на месте его традиционного проведения, с участием 2–3 пожилых жительниц се- ла [16]. На примере трансформации ритуальных практик в Старом Афонькине можно отметить общие тенден- ции в религиозной жизни данного конфессионально- го сообщества: сокращение числа участников и сни- жение уровня сакральности существующих ритуалов и, как следствие, редукцию / исчезновение обрядов. Следует также отметить заметное, по сравнению с концом 1990-х гг., снижение уровня компетентности членов и даже лидеров общин в ритуальной практи- ке. Они нередко используют рукописные тексты мо- литв, а не зачитывают их наизусть, иногда обраща- ются к опубликованным текстам [16; 19]. Информан- ты часто говорят о том, что они не знают многого из тех обычаев, которых придерживались их отцы и де- ды. Из интервью с Р.В. Рыбаковой, 1949 г.р., жи- тельницей с. Старое Афонькино: «Учуксем пĕтрĕç ĕнтĕ халь. Ăна тăвакансем ялта темиçе çын кăна юлчĕç: эпĕ тата 4–5 çын. Урăх çук <…> Учука та çерçи чӳкне те ăна çак йăласене çав тери ĕненекен- сем тунă. Ун пек çынсем наверно пирĕн атте- аннесем пулнă. Вĕсем юлашкисем пулнă, малалла уже урăхларах ĕненекеннисем тытăннă. Хальхи çынсем учука ĕлĕкхи çынсем пек ĕненмеççĕ» (Моления учук исчезли в наши дни. Тех, кто проводит их, осталось 4–5 человек. Больше нет <…> И учук, и воробьиный чук проводили очень верующие люди. Такими были, наверно, наши отцы и матери. Они были последни- ми, после них уже были по-другому верующие. Со- временники в учук уже так не верят) [16]. Сокращение сроков и изменение ритуала, в це- лом, обусловлены трансформацией религиозной об- щины, в первую очередь, разделением ее на две ча- сти – местную и проживающую вне села. Разобще- ние родственников препятствует традиционному по- рядку проведения и гарантированному участию всех из них в ритуалах. Совмещение же по срокам похо- рон и Юпа позволяет родственникам присутствовать на этих двух значимых ритуалах. В этих условиях допускается изменение содержания обряда, который в настоящее время, по мнению его участников, про- водят «лениво»: «Халь юпана эпир наянла тăваппăр. Чĕре калакĕпе юпине вилнĕ çынна пытарнă кунах лартаппăр» (Сейчас юпа мы проводим лениво. Сер- дечную палочку и юпа ставим в один день, на похо- ронах) [17]. Вариативность проявляется и в календарной об- рядности: в одних селах сохраняются отдельные об- ряды, в других они исчезли еще в последние десяти- летия XX в. Анализ данных Если в Старом Афонькине ежегодно проводится ритуал вызывания дождя Çумăр чӳк, а в Новом Ильмово ему предшествовал в 2021 г. Учук с жертвоприношением, то в Староганькино моление о дожде в последний раз проводили в 2014 г. После 8- летнего перерыва летом 2021 г. в Абрыскино прове- ли Учук и Çумăр чӳк. Çумăр пăтти (дословно «каша дождя») варили и в соседних селах Якушкино и Сал- дакаево, где некрещеные также проживают совместно с православными. Именно засушливое лето 2021 г. побудило жителей и других чувашских сел Закамья вспомнить обычаи предков: «Çумăр çуккипе ăнтан кайрăмăр та тăвас терĕмĕр» (Отсутствие дождей сильно озадачило нас, решили провести) [16; 19; 21]. Таким образом, даже в отсутствие постоянной прак- тики многие ритуалы сохраняют свою актуальность и при необходимости восстанавливаются, но при этом нарушается общая традиция, все более варьи- рующая по отдельным селениям. Аналогичная картина наблюдается и в других се- лах. В Чувашском Урметьеве Учук с жертвоприно- шением перестали проводить с середины 1970-х гг., домашние моления (Чӳклеме, Карта пăтти и др.) в середине 1980-х гг., Сĕрен исчез в конце 2000-х гг. Как утверждают местные жители, «по старинному обычаю сейчас проводить сложно, потому что стари- ки, знавшие обычаи, все поумирали» («Ĕлĕкхилле тума халь хĕн, мĕншĕн тесен йăласене лайăх пелекен тата тытса пыраканĕ ват куккасем йăлт вилсе пĕтрĕç»). Местное население не владеет или владеет в ограниченном объеме информацией о местах моле- ний Киремет и других сакральных локусах местно- сти (например, йĕрĕх вырăнĕсем), порядке проведе- ния ключевых обрядов, таких как Чӳклеме, Учук, Сĕрен [17]. Samara Journal of Science. 2022. Vol. 11, iss. 2 Аналогичная тенденция наблюдается в целом в традиционной обрядности, а касательно вопроса о соотношении декларируемой идентичности и реаль- ных практик означает их расхождение. Так, в послед- ние десятилетия даже в селениях некрещеных чува- шей с устойчивой религиозной традицией (напри- мер, в с. Старое Афонькино) наблюдается отход от выполнения даже значимых с точки зрения местных жителей ритуалов, таких, как Сěрен, Учук. Значи- мость в данном случае определяется последствиями, как позитивными, так и негативными, которые могут произойти в случае невыполнения ритуала. В част- ности, Сěрен в Старом Афонькине не проводился ни в 2020-м, ни в 2021 гг., хотя жители полагают, что это чревато для села пожарами и болезнями. Учук, масштабно проводившийся в селе еще в последние десятилетия XX – начале XXI в. Анализ данных Однако конверсия, часто формальная, не ме- няет отношения неофитов к традициям предков, при- знаваемых, по-прежнему, «своими», что и проявля- ется в двоеверии. Двоеверие проявляется в отношении неофитов к православным традициям, в частности к праздникам, Таким образом, современные религиозные прак- тики православных чувашей вариативны, в разной степени, в зависимости от локальных групп, насы- щены языческой семантикой и содержанием, что позволяет в одних случаях характеризовать их как проявление православно-языческого синкретизма, а в других – как двоеверие. Вариативность в ритуальных практиках характер- на и для некрещеных чувашей, что позволяет типо- логизировать их религиозность как «языческую», т.е. сохраняющую основные ритуалы «чувашской веры» (например, в Сидулово-Ерыкле), или как «языческо- православную», в которой традиционные верования и обряды переплелись с православными (как в д. Ста- роганькино, Чувашское Урметьево). Степень сохран- ности традиционных ритуалов различается по локу- сам селений. Вот как это выглядит, например, в про- ведении одного ритуала – ритуала установления стол- ба Юпа. Самарский научный вестник. 2022. Т. 11, № 2 Традиционно намогильные столбы чуваши стави- ли раз в год, осенью, в месяц юпа (октябрь). Однако в XX в. обычай трансформировался, и в большинстве селений, возможно, под влиянием православия, его приурочили к поминовению на 40-й день. Такой по- рядок сохраняется в некоторых селениях и сегодня (с. Новое Ильмово, Абрыскино) и проводится при убывающей луне до 40-го дня [21], но обязательно в нечетный день после смерти – 35-й, 37-й или 39-й (Сидулово-Ерыкла) [20]. В селах Нурлатского района придерживаются традиции в выборе дня – ритуал проводится вечером либо в пятницу, либо в воскре- сенье (Якушкино) [19]. Во второй половине XX в., особенно в течение последних двух десятилетий, произошла существенная трансформация ритуала, коснувшаяся сроков его проведения, формы и со- держания. В ряде селений (Старое Афонькино, Чу- вашское Урметьево, Староганькино) Юпа проводит- Двоеверие проявляется в отношении неофитов к православным традициям, в частности к праздникам, которые воспринимаются ими сквозь призму «язы- ческих» традиций и ассоциируются с подготовкой к празднику, жертвоприношением, приготовлением ри- туальных блюд, трапезой, правилами поведения, в том числе запретами. Вот как это выглядит, к приме- ру, в с. Староганькино. Из интервью с Л.В. Моляно- вой: «К Петрову дню (Питрав) стараемся барана за- 248 Самарский научный вестник. 2022. Т. 11, № 2 Ягафова Е.А. Исторические науки ся в день похорон: после того, как гроб с покойным увозят на кладбище, оставшиеся участники обряда заносят в дом деревянный столб юпа, поминают по- койного за столом и, забрав столб, отправляются на кладбище. Заключение Подводя общие итоги исследования, следует от- метить, что актуальные религиозные практики обеих групп не соответствуют в полной мере декларируе- мой религиозной идентичности. В практиках право- славных чувашей сохраняется существенный пласт воззрений, предписаний и поведенческих практик, восходящих к традиционной религии чăваш тěнě: например, такие ритуалы, как «проводы души» Юпа, воздаяние покойным хывни и др. У некрещеных чу- вашей также присутствуют христианские элементы, проникшие в ходе взаимодействия с православным окружением: например, празднование христианских праздников, проведение поминок на 9-й и 40-й дни и др. Такие элементы в обрядности позволяют опреде- лить религиозность этих групп в одних случаях как синкретизм, в других – как двоеверие. Особенно за- метно «смешение» разных традиций в контактных зонах – в конфессионально-смешанных селениях, где религиозные границы проходят не только внутри се- ления, но часто и в семьях. В обрядовый календарь некрещеных чувашей вошли православные праздники. Например, осеннее моление Чӳклеме в Чувашском Урметьево проводят накануне Николы зимнего (Хĕллехи Микула), кото- рый в селе считается престольным праздником [17]. В Староганькино обе конфессии празднуют День св. Дмитрия Солунского (Мĕтри Селенски), Троицу и Пасху. Из интервью с М.И. Ирисбаевой, 1942 г.р., жительницей с. Староганькино: «Пĕрле тăваппăр. Вара эпĕ те вĕсен Троицине хутшăнап. Уйăрса тă- маппăр <…> Çапла, икĕ мункун тăваппăр. Эпĕ юнкун чăваш мăнкунне тăватăп пулсан, вĕсем вырсарникун крешĕнсен мункунне тăваççĕ (Вместе справляем. И я в праздновании Троицы участвую. Не делимся <…> Так, две Пасхи справляем: я в среду – чувашскую, крещеные в воскресенье свою) [18]. Примечательно, что местные чуваши не видят в подобном поведении, праздновании как чувашских, так и православных, «русских», праздников нарушения религиозной нор- мы, а скорее считают «нормой» следование традици- ям обеих конфессий: «Эпир чăваш йăлисене те, вырăс йăлисене те пĕрне те сиктерсе хăвармастпăр. Çылăх пулмасть вăл, çапăçса-вăрçăшса çӳрени мар» (Мы и чувашские обычаи, и русские обычаи не про- пускаем. В этом нет греха, это не ругаться и драться) [17]; «Тăваппăр ĕнтĕ, вăл та праçник вĕт. Эпир вĕсе- не крешĕнсен, чăвашсен тесе уйăрман. Что крешĕ- нĕн, что чăвашăн, пуриншĕн те праçник пĕрре. Мĕн уйăраççĕ вĕсем? Крешĕнĕ вĕт чăвашĕнчен пулса кай- нă. Малтан вĕсем пурте чăваш пулса çуралнă, кай- ран кăна тĕне кĕнĕ. Пуриншĕн те Турă пĕрре, йăли- сем кăна уйрăм» (Справляем, праздник ведь. Мы их не разделяем: кряшенские или чувашские. Что чу- вашские, что кряшенские – для нас все одно – празд- ники. Что они делят? И крещеные ведь от чувашей пошли. Вначале они все родились чувашами, потом только крестились. Для всех Бог один, только обы- чаи разные) [18]. Заключение Расхождения конфессиональной идентичности с актуальными религиозными практиками обусловле- ны неустойчивостью конфессиональных «границ» вследствие религиозной конверсии, трансформации ритуальных практик. Они стали возможны также по- тому, что в обеих группах допускают возможность исполнения ритуальных действий или даже их при- знают частью «своей» традиции: православные чува- ши считают «языческие» элементы «наследием пред- ков», некрещеные принимают христианские обычаи как не противоречащие этому наследию, как часть об- щей с православными чувашами этнической культуры. ф Конфессиональная идентичность и современные религиозные практики чувашей науки Таким образом, религиозные практики некреще- ных чувашей претерпели существенные изменения в сторону сокращения числа участников, редукции об- рядности, снижения компетентности членов общины в догматике и ритуалах; в них органично вошли от- дельные элементы православной празднично-обрядо- вой культуры. ритуал вызывания дождя, проводившийся обычно вслед за общесельским молением Учук, был воспри- нят исключительно православными, в то время как Учук организовывали некрещеные. В настоящее вре- мя оба ритуала исчезли, но крещеные проводят мо- лебны возле креста, установленного на месте быв- шей церкви [17]. Процессы трансформации ритуальных практик затронули и такую консервативную сторону религи- озной жизни, как похоронно-поминальные обычаи и обряды. Например, некрещеные чуваши в Старогань- кино вслед за православными поминают на 9-й и 40- й дни, при том, что уже в день похорон совершают Юпа и «провожают душу» покойного [18]. Местные жители считают, что это происходит вследствие «смешения» некрещеных с крещеных («Тĕрĕссине каласан, эпир чăвашсем халĕ крешĕнсемпе хутшăнса кайнипе крешĕнленсе пыратпăр» – По правде говоря, мы, чуваши, смешавшись с крещеными, постепенно сами становимся кряшенами) [17]. Вместе с право- славными некрещеные участвуют в проведении еже- годных поминальных дней, которые приурочены к христианским праздникам: весной – к православной Пасхе, летом – к Троице, осенью – к празднику Ка- занской Божьей Матери (4 ноября). В первых двух случаях они проводятся в четверг перед соответ- ствующим праздником, а осенью – также в четверг или пятницу, но за одну-две неделю до него. Анализ данных (в последний раз – в 2001 г.) и состоявший из жертвоприношения 5 видов домашних животных и птицы, продолжительного моления и грандиозной трапезы с участием боль- шинства жителей села, в том числе и крещеных, све- Знатоками обычаев и организаторами коллектив- ных ритуалов в них являются, как правило, 2–3 че- ловека, еще 5–6 человек оказывают посильную фи- нансовую и организационную помощь в проведении общесельских молений, остальные, в лучшем случае, жертвуют деньги и присутствуют на них, но могут и отказаться от участия. Во всех селах в организации и проведении коллективных молений или семейно- родовых ритуалов участвуют и крещеные – род- ственники, коллеги, знакомые. В отдельных случаях именно православные выступают знатоками обычаев некрещеных и, как, например, в Староганькине, по- могают организовать в соответствии с ними похоро- ны и поминки некрещеных жителей [18]. Интерес- ный пример трансляции «языческих» ритуалов в пра- вославную среду отмечен в Чувашском Урметьеве: 249 Исторические Исторические науки Ягафова Е.А. Конфессиональная идентичность и современные религиозные практики чувашей ритуал вызывания дождя, проводившийся обычно вслед за общесельским молением Учук, был воспри- нят исключительно православными, в то время как Учук организовывали некрещеные. В настоящее вре- мя оба ритуала исчезли, но крещеные проводят мо- лебны возле креста, установленного на месте быв- шей церкви [17]. Таким образ ных чувашей пр сторону сокраще рядности, сниже в догматике и р дельные элемент вой культуры. Самарский научный вестник. 2022. Т. 11, № 2 Samara Journal of Science. 2022. Vol. 11, iss. 2 Конфессиональная идентичность и совр 8. Полевые материалы автора (ПМА). 2020 г. Чуваш- ская Республика, Урмарский район, с. Мусирмы. 17. ПМА. 2021. Самарская область, Челно-Вершин- ский район, с. Чувашское Урметьево. 18. ПМА. 2021. Самарская область, Похвистневский район, с. Староганькино. 9. ПМА. 2021 г. Чувашская Республика, Урмарский район, с. Мусирмы. р р 19. ПМА. 2021. Республика Татарстан, Нурлатский район, с. Абрыскино, с. Салдакаево, с. Якушкино. 10. ПМА. 2020. Самарская область, Челно-Вершин- ский район, с. Девлезеркино. 11. ПМА. 2021. Самарская область, Челно-Вершин- ский район, с. Девлезеркино. 20. ПМА. 2021. Республика Татарстан, Аксубаевский район, с. Савгачево, с. Сидулово-Ерыкла. 12. ПМА. 2020. Республика Башкортостан, Бижбу- лякский район, с. Кош-Елга. 21. ПМА. 2021. Республика Татарстан, Черемшан- ский район, с. Новое Ильмово. 22. Агаджанян А.С., Русселе К. Как и зачем изучать современные религиозные практики? // Религиозные прак- тики в современной России: сб. ст. М.: Новое издатель- ство, 2006. С. 11–32. 13. Российский государственный архив древних ак- тов. Ф. 1355. Оп. 1. Д. 1874. 14. История церкви [Электронный ресурс] // http:// gov.cap.ru/home/73/www/msh/myweb2/ictoriy.htm. 15. Космо-Дамианский храм (с. Кош-Елга, Бижбуляк- ский р-н РБ) [Электронный ресурс] // Православные ли- ки России, стран ближнего и дальнего зарубежья. http:// likirussia.ru/content/view/204. Исследование выполнено при финансовой под- держке РФФИ в рамках проекта № 20-09-00127 «Религиозные практики чувашей: традиции и их трансформация (конец XX – первые десятилетия XXI в.)». 16. ПМА. 2021. Самарская область, Шенталинский район, с. Старое Афонькино. Список литературы: 1. Пронина Т.С. Религия в структуре идентичности современных россиян // Вестник Ленинградского госу- дарственного университета им. А.С. Пушкина. 2014. Т. 2, № 4. С. 189–200. 1. Пронина Т.С. Религия в структуре идентичности современных россиян // Вестник Ленинградского госу- дарственного университета им. А.С. Пушкина. 2014. Т. 2, № 4. 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https://openalex.org/W4322733394	https://www.frontiersin.org/articles/10.3389/fpubh.2023.1137489/pdf	English	null	Mechanisms influencing the factors of urban built environments and coronavirus disease 2019 at macroscopic and microscopic scales: The role of cities	Frontiers in public health	2,023	cc-by	12,183	TYPE Original Research PUBLISHED 28 February 2023 DOI 10.3389/fpubh.2023.1137489 TYPE Original Research PUBLISHED 28 February 2023 DOI 10.3389/fpubh.2023.1137489 TYPE Original Research PUBLISHED 28 February 2023 DOI 10.3389/fpubh.2023.1137489 OPEN ACCESS EDITED BY Yibin Ao, Chengdu University of Technology, China REVIEWED BY Kayode Oshinubi, Northern Arizona University, United States Xiaowei Li, Xi’an University of Architecture and Technology, China CORRESPONDENCE Jun Wu wujuntj@tcu.edu.cn Lei Wang wanglei2021@tju.edu.cn †These authors have contributed equally to this work and share ﬁrst authorship SPECIALTY SECTION This article was submitted to Environmental health and Exposome, a section of the journal Frontiers in Public Health RECEIVED 04 January 2023 ACCEPTED 02 February 2023 PUBLISHED 28 February 2023 CITATION Zhang L, Han X, Wu J and Wang L (2023) Mechanisms inﬂuencing the factors of urban built environments and coronavirus disease 2019 at macroscopic and microscopic scales: The role of cities. Front. Public Health 11:1137489. doi: 10.3389/fpubh.2023.1137489 COPYRIGHT © 2023 Zhang, Han, Wu and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS OPEN ACCESS EDITED BY Yibin Ao, Chengdu University of Technology, China REVIEWED BY Kayode Oshinubi, Northern Arizona University, United States Xiaowei Li, Xi’an University of Architecture and Technology, China CORRESPONDENCE Jun Wu wujuntj@tcu.edu.cn Lei Wang wanglei2021@tju.edu.cn Longhao Zhang1†, Xin Han2†, Jun Wu1† and Lei Wang3 1School of Architecture, Tianjin Chengjian University, Tianjin, China, 2Department of Landscape Architecture, Kyungpook National University, Daegu, Republic of Korea, 3School of Architecture, Tianjin University, Tianjin, China †These authors have contributed equally to this work and share ﬁrst authorship In late 2019, the coronavirus disease 2019 (COVID-19) pandemic soundlessly slinked in and swept the world, exerting a tremendous impact on lifestyles. This study investigated changes in the infection rates of COVID-19 and the urban built environment in 45 areas in Manhattan, New York, and the relationship between the factors of the urban built environment and COVID-19. COVID-19 was used as the outcome variable, which represents the situation under normal conditions vs. non-pharmacological intervention (NPI), to analyze the macroscopic (macro) and microscopic (micro) factors of the urban built environment. Computer vision was introduced to quantify the material space of urban places from street-level panoramic images of the urban streetscape. The study then extracted the microscopic factors of the urban built environment. The micro factors were composed of two parts. The ﬁrst was the urban level, which was composed of urban buildings, Panoramic View Green View Index, roads, the sky, and buildings (walls). The second was the streets’ green structure, which consisted of macrophanerophyte, bush, and grass. The macro factors comprised population density, trafc, and points of interest. This study analyzed correlations from multiple levels using linear regression models. It also efectively explored the relationship between the urban built environment and COVID-19 transmission and the mechanism of its inﬂuence from multiple perspectives. / p COPYRIGHT © 2023 Zhang, Han, Wu and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. COVID-19, urban built environment, relevance, street view images, computer vision, deep learning COVID-19, urban built environment, relevance, street view images, computer vision, deep learning 1. Introduction Novel pneumonia caused by coronavirus 2019 (COVID-19), which leads to severe acute respiratory syndrome, has been raging worldwide for nearly 3 years since December 2019 (1). The speed of transmission of the virus, its infectiousness, and the number of mutations have been the most unprecedented in human medical history. Large cities and metropolitan areas have been the areas most aﬀected by the spread of the virus, exacerbated by the areas’ dense population distribution (2). To strictly control the rate of COVID-19 transmission and to reduce the rates of infection and deaths, countries have adopted non-pharmaceutical interventions (NPIs) including urban lockdown, home Frontiers in Public Health 01 frontiersin.org Zhang et al. Zhang et al. 10.3389/fpubh.2023.1137489 10.3389/fpubh.2023.1137489 10.3389/fpubh.2023.1137489 United States, Lin et al. (27) in China, and Boterman (28) in the Netherlands. Meanwhile, the relationship between urban building density and COVID-19 lacks elucidation and is subject to a certain degree of controversy (29). A few studies demonstrate that no correlation exists between building density and COVID-19 after omitting certain confounding factors (28). During the COVID-19 pandemic and under government NPIs, the wellbeing of residents living in high-density areas was negatively correlated with living density due to changes in the scope of life and lifestyle behaviors. However, this compact urban form leads to relatively easy access to urban healthcare resources, which could improve the health status of residents (18, 30). isolation, controlled social distancing, and travel restrictions (3, 4). Ever since it reared its ugly head, COVID-19 has attracted substantial attention from the global community, and various studies on COVID-19 have emerged accordingly. A majority of the studies have focused on the related factors of sociodemographics and the urban built environment. The results vary from two diﬀerent research perspectives. From the sociodemographic perspective, the risk of COVID- 19 infection is much higher for the elderly and children than it is for young and middle-aged adults (5–7). In addition, the degree of economic development across regions may exert an impact on the transmission rate of COVID-19 (8, 9). For example, the availability of health insurance has been highly correlated with the spread of COVID-19 (10). Low-income areas, especially older communities with low levels of income, have been more susceptible to COVID-19 infection (11, 12), all of which have been related to regional economic development. 1. Introduction Using logistic regression models, several studies have demonstrated that levels of regional literacy are also associated with the prevalence of COVID-19 (13). Other factors have also been correlated with COVID-19 transmission, such as blood type, respiratory disease, and chronic diseases. Additionally, personal habits have been associated with COVID- 19 transmission (14, 15). The strict implementation of NPI against COVID-19 has proven eﬀective in mitigating the spread of the virus (16, 17). In the post-pandemic era, the patterns of behavior in daily life have changed due to reliable NPIs implemented by governments (18). Under the inﬂuence of NPIs, the range of activities of urban residents has been signiﬁcantly reduced, thereby rendering them increasingly dependent largely on the surroundings of their homes, the natural urban environment, and the built urban environment (19). The surroundings of urban homes and the built environment have exerted a direct eﬀect on the physical and mental health of urban residents (20). Simultaneously, low- density neighborhoods, large homes, developed urban residential surroundings and infrastructure, rich urban greenery, and large urban green spaces can greatly enhance the life satisfaction and wellbeing of residents under COVID-19 NPIs (21). From the perspective of the urban built environment alone, diﬀerent factors in the urban built environment may have an impact on the spread and transmission of COVID-19 during an epidemic pandemic. For example, public transportation (31) and points of interest (POI) (32), among others, are generally considered to exhibit a positive association with COVID-19 transmission. When the outbreak was in its emergent stage, public transportation was considered the main method of COVID- 19 transmission. Therefore, many governments advised urban residents to avoid public transportation as much as possible while introducing corresponding NPIs and limiting the range of activities of residents. In addition, they frequently urged urban residents to use multiple modes of transportation, such as self- driving, walking, and cycling (33). The results of analyses using multiscale geographically weighted regression suggested that the high availability of medical resources around a community could eﬀectively inhibit the spread of COVID-19 (7). Through structural equation modeling and categorical regression modeling, other analyses demonstrated high-quality housing and high-quality green space as being negatively associated with the spread of COVID- 19 (10, 34). Green spaces around large residential areas exerted an inhibitory eﬀect on the deterioration of urban health and wellbeing due to COVID-19 (30). 2.2.1. Google street view images The study obtained urban street panorama images from Google Maps to reﬂect the physical characteristics of the urban environment. Factors related to the urban environment were extracted from these images as evaluation indexes of the urban environment. To improve the representativeness of the physical features and environmental factors of the urban environment, the study created a collection point for every 100 m on all urban roads in the study area. A total of 67,025 collection points were set up to collect images with each collection point having one image based on a 90◦view. Moreover, the study collected four images for each collection point to synthesize the panoramic streetscape images, which reached 268,100 images. The images were cleaned according to the availability of data, and all images were collected from Google Street View (GSV) to analyze the physical characteristics of the city and extract the factors of the urban environment. By appropriately establishing the parameters for image retrieval, the images captured both sides and frontal images of the street. This image acquisition covered all roads in Manhattan. Figure 2 provides a demonstration of the acquisition of the GSV images. 1. Introduction Figure 1 describes the study area and its road network. As the center of the metropolitan area, a major outbreak of COVID-19 is likely to spread rapidly to other areas of the metropolis and continue to expand outward. Thus, understanding the relationship between the spread of COVID-19 and the factors of the urban built environment is an important aspect for urban decision-makers in mitigating the spread of the disease and in developing openness measures. 1. Introduction However, most of the community-level studies at this stage have used administrative boundaries to delineate the selection of variables, and the disadvantage of this method of variable selection is that it does not reﬂect the actual Frontiers in Public Health 02 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 activities of residents. In this regard, Li et al. used structural equation modeling to reveal the relationship between commercial vitality and transportation infrastructure on the increase in the number of conﬁrmed cases, and innovatively used buﬀer zones to extract urban built environment factors around conﬁrmed cases (37). Wang et al. used walking circles at diﬀerent times to investigate the correlation between urban built environment and community level spatial distribution (38). By extracting the established environmental factors in both spatial dimensions and examining the correlation between these factors and the prevalence of COVID-19, the issue of the transmission mechanism of COVID- 19 before and after the implementation of community-level NPI measures was then analyzed. Studies at this stage ignore the lack of multi-level studies on the mechanisms of the urban micro-built environment inﬂuencing the spread of COVID-19. Whether the urban street green environment and urban street spatial quality have an impact on the spread of COVID-19 has not been explored, and the impact of urban built environment on the long-term trend and overall trend of COVID-19 has not been considered comprehensively. In this study, based on the study of the inﬂuence mechanism between the macroscopic built environment and COVID-19, the inﬂuence mechanism between the microscopic built environment and COVID-19 was considered at multiple levels using Google Street View panoramic street view images. The impact of urban built environment on the long- term trend and overall trend of COVID-19 is investigated using multiple variables, and the inﬂuence mechanism of urban built environment on COVID-19 is examined at multiple levels (macro level and micro level) and multiple dimensions (time dimension). The results of the study can provide a basis and reference for governmental decision makers to formulate more reasonable NPI policies to slow down the spread of COVID-19 during pandemic periods. The results of the study may provide a reference solution to control the spread and spread of the virus, and the results may provide eﬀective recommendations to contain potential respiratory disease outbreaks. visit New York City each year signiﬁcantly contribute to the risk of COVID-19 transmission and routes of transmission. 1. Introduction Notably, the risk of infection and transmission rates were high for neighborhoods with high levels of community convenience (35). Integrating all patients with COVID- 19 into high-grade urban hospitals is unrealistic because hospital capacity is far from adequate for treating such a large number of patients at the burgeoning stage of a pandemic such as COVID- 19. Moreover, the risk of collapse of urban public healthcare is prevalent as demonstrated by the collapse of public healthcare to varying degrees in various countries during the COVID-19 outbreak. Therefore, a community-level system for identifying and isolating individuals with infection is essential to the response to COVID-19 (36). From the perspective of the urban built environment, the impact of the urban built environment on COVID-19 is extremely important in addition to socio-demographic factors, which has been conﬁrmed by many studies. The relationship between COVID-19 transmission and population density is relatively controversial. Previous studies demonstrate that the incidence and transmission of COVID-19 are higher in densely populated areas with high population contact (22). A comparison of the results of linear regression models from 182 countries points to a positive association between population density and COVID-19 transmission (23). In contrast, the results of structural equation modeling at the city level illustrate that population density is negatively associated with COVID-19 transmission in Tehran (24). This result is interesting, where a few studies argue that urban population density is non-signiﬁcantly correlated with the spread of COVID-19 (25). The relationship between urban population density and the transmission rate of COVID-19 is complex. Thus, the various responses of governments and urban residents to the pandemic across nations may lead to diﬀerent results, which are reasonably explained by the ﬁndings of Hamidi et al. (26) in the In summary, several questions can be elicited from the inﬂuence of the urban built environment on the spread of COVID- 19: (I) how the macroscopic urban built environment and the microscopic urban built environment have an impact on the spread of COVID-19 in the urban built environment; and (II) what is the impact of the macroscopic urban built environment and the microscopic built environment on the incidence and lethality of COVID-19. 2. Data The study used datasets from Cityscape, ADE20K, and S-S-G-S to train the neural network model, a dataset open to researchers at the Mercedes-Benz R&D Center and Darmstadt University of Technology and published in the 2016 Clean Vehicle Rebate Project. The dataset was collected from 50 cities in Germany and nearby countries, including street scenes in spring, summer, and autumn. Diﬀerent annotators with 96 and 98% pixel consistencies repeatedly annotated the 30 selected data after omitting categories that could be annotated as unclear. The drawback was that the segmentation dataset contained 33 classes, whereas the validation dataset was composed of only 19 semantic segmentation classes because the data volume of a few classes was very sparse. The ADE20K dataset is intended for Scene Understanding, which was opened by the Massachusetts Institute of Technology (MIT) in 2016 and can be used, for instance, in semantics and part segmentation. Using image information for Scene Understanding and parsing, the dataset consists of 27,000 images from Scene Understanding (an open dataset released by Princeton University in 2010) and Places (an open dataset by MIT released in 2014). The ADE20K contains Frontiers in Public Health frontiersin.org 2.1. Research region New York City is considered the ﬁrst epicenter of the COVID- 19 outbreak in the United States. It has a population of ∼8.51 million (as of 2017) and an area of ∼1,214 km2 (including the sea). With an average of 28 people per square mile, New York City is the main international maritime, airport, and ﬁnancial metropolis of the United States and has ﬁve boroughs under its jurisdiction, namely, Brooklyn, Queens, Manhattan, the Bronx, and Staten Island. Manhattan is the most densely populated and smallest of the ﬁve boroughs of New York City, which translates to a very high population and housing density when compared with those of other boroughs in New York City. Manhattan is described as the economic and cultural center of the United States and is home to New York’s central business district, which houses the headquarters of most Fortune 500 companies and the headquarters of the United Nations. Thus, the nearly 50 million tourists who 03 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 FIGURE 1 Study area: (A) Map of the United States; (B) New York County; and (C) Manhattan road network. FIGURE 1 Study area: (A) Map of the United States; (B) New York County; and (C) Manhattan road network. York State, 20,011 were derived from New York City. Over time and with the introduction of various restrictive policies and concerted national eﬀorts to combat the outbreak, the spread of COVID-19 decelerated. Moreover, the outbreak appeared to be moving in a positive direction with the advent of COVID-19 vaccines. more than 3,000 object classes, which greatly compensates for the shortcomings of the Cityscape dataset. The S-S-G-S dataset was constructed by Zhang et al. (39) in 2022 and is mainly used for the analysis of urban vegetation communities. A neural network model trained using this dataset can classify and visualize the structure of urban street vegetation communities. S-S-G-S diﬀers from the Cityscape and ADE20K datasets in that it is directed toward the analysis of urban greenery. The study mainly used the trained DeepLabV3+ neural network model to extract urban features at the micro level. Moving forward to late December 2021, a variant of COVID-19 (omicron) is once again ravaging New York State with a record- breaking 21,908 cases detected in New York State on December 18, 2021, alone. Moreover, an alarming spike in cases was noted in several highly vaccinated neighborhoods in Manhattan. 2.1. Research region With 7-day positivity rates exceeding 10% in more than 10 areas of New York City from December 10 to 16, 2021, Manhattan, once again, clearly became a hotbed of COVID-19 transmission. A total of 790.87 cases were identiﬁed per 100,000 people, and an extremely alarming rate was noted in speciﬁc Manhattan neighborhoods as of December 24, 2021. Greenwich Village and SoHo reported 2,850 conﬁrmed cases per 100,000 people, and Chelsea reached 2,400 conﬁrmed cases per 100,000 people. Nevertheless, no pandemic hotspot in the nation could compare to the dire outbreak in Greenwich Village. Frontiers in Public Health frontiersin.org 2.3. COVID-19 dataset The ﬁrst conﬁrmed case of COVID-19 was reported in Manhattan on March 1, 2020. At the time, the number of conﬁrmed cases of COVID-19 in the entire United States was only 76. However, as of March 25, 2020, the number of conﬁrmed COVID- 19 cases in the United States spiked to 69,008, and the number of deaths reached 1,045, such that COVID-19 rampaged through the country at a rate of 10,000 per day for three consecutive days. However, according to the Centers for Disease Control and Prevention, nearly 50% of all conﬁrmed cases in the United States as of March 25, 2020, are in New York State, which establishes it as the epicenter of the outbreak. Out of the 33,006 cases diagnosed in New The COVID-19 case data in the study were derived from the publication by NYC Health (https://www1.nyc.gov/site/doh/ index.page), which included cumulative totals since the COVID- 19 outbreak in New York City. The Department of Health (DOH) deﬁned the ﬁrst case of COVID-19 as the one conﬁrmed on February 29, 2020. In addition, the DOH recommended the 04 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 FIGURE 2 Demonstration of the acquisition of GSV images. FIGURE 2 Demonstration of the acquisition of GSV images. FIGURE 2 Demonstration of the acquisition of GSV images. FIGURE 2 Demonstration of the acquisition of GSV images. according to ZIP codes using ZCTA. MODZCTA geography combines census blocks with small populations to provide stable estimates of population size for rate calculations. The visualization is available on the website of NYC Health, which also open- sources the case data (https://github.com/nychealth/coronavirus- data#geography-zip-codes-and-zctas). In this manner, accessing appropriate data is easy for researchers. avoidance of interpreting the daily changes in these ﬁles as 1- day data due to the discrepancy between the date of the event and the date of reporting. The internal division of the study area was divided according to the Modiﬁed ZIP Code Tabulation Areas (MODZCTA). NYC Health uses MODZCTA to report information according to geographic location. However, several issues emerge when mapping data reported based on ZIP codes because they do not designate a single area but a collection of points that compose the route of mail delivery. Moreover, a few buildings and non- residential areas were frequently assigned unique ZIP codes. To address these issues, the DOH uses ZIP Code Tabulation Areas (ZCTA) to convert ZIP codes into area units. Frontiers in Public Health 3.3. Micro-scale factors of urban built environment In this study, micro-level urban built environment speciﬁcally refers to the direct perception of the features of the urban landscape by pedestrians. Many studies demonstrate that computer vision combined with panoramic urban streetscape images can extract the features of the urban built environment and evaluate the urban built environment at the street level. This tendency proves that computer vision has gradually entered the scope of urban research. The current study selects the network model open- sourced by Chen et al. (43) in 2018, which pertains to a semantic segmentation network based on the DeepLabV3+ neural network model. The study made this selection for two reasons. The ﬁrst is that the DeepLabV3+ neural network model is the latest version in the DeepLab series, which modiﬁes VGG16 to introduce null convolution in DeepLabV1. The Atrous Spatial Pyramid Pooling (ASPP) model is designed in DeepLabV2; DeepLabV3 combines. The model proved its accuracy by outperforming mainstream deep learning algorithms (such as SegNet and PSPNet) in performance evaluation competitions such as the PascalVOC and Cityscapes benchmark tests in 2012. The second is that DeepLabV3+ features a better recognition eﬀect compared with other mainstream deep learning models in the interpretation of urban scenes. The reason is that the model is designed for analyzing urban scenes, such that it exhibits certain advantages compared with those of other models when recognizing green structures in urban streets. The third is that the model uses DeepLabV3 as an encoder to generate the features of arbitrary dimensions using Atrous Convolution and adopts the ASPP strategy to use multiple eﬀective sites with upsampling to achieve multiscale feature extraction. Moreover, it uses a cascade decoder to recover boundary detail information. Depthwise Separable Convolution is also used to reduce the number of parameters to further improve the accuracy and speed of the segmentation algorithm. This study selects the panoramic green view rate, the green structure of urban streets, buildings, roads, walls, and sky visibility to represent the micro-scale features of the urban built environment (Figure 5). According to MODZCTA, the Manhattan area of New York City, United States, was divided into 68 areas. After data ﬁltering, the study identiﬁed 45 valid areas, and a ﬁshing net was generated within the study area for a total of 1,551 grids. These grids will be used for analysis and spatial cells. 2.3. COVID-19 dataset The United States Census Bureau developed ZCTA geography to map data reported This study used Manhattan, New York in the United States as the study area and created a ﬁshing network according to the 68 zones of MODZCTA to compare the mechanisms between the factors of the urban built environment and COVID-19 transmission in diﬀerent zones and to investigate the reason Manhattan became the center of the pandemic many times during the outbreak. Frontiers in Public Health 05 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 10.3389/fpubh.2023.1137489 3.3. Micro-scale factors of urban built environment The study calculated the CCC and CCR of the 45 independent areas and averaged them according to the ﬁshing nets to reﬂect the overall number of cases in Manhattan. In addition, by calculating and visualizing the average of the number of valid POIs and environmental factors of the urban streetscape within the grids, the study intends to better establish the relationship of the urban built environment at the macro- and micro-levels to COVID-19. Figure 3 presents the visualization results of CCC and CCR in the study area. 3.1. Outcome variable: COVID-19 The outcome variables of the study were the number of conﬁrmed and suspected cases of COVID-19 [COVID_CASE_COUNT (CCC)] and the incidence of conﬁrmed and suspected cases of COVID-19 per 100,000 people [COVID_CASE_RATE (CCR)] in Manhattan, New York, United States. The diﬀerence between CCC and CCR is that CCR is a longer-term trend than CCC, and the relationship between the factors of urban built environment and COVID-19 under NPIs can be determined by comparing with CCC. 3.2. Macro-scale: Factors of urban built environment The study selected only three aspects, namely, density, diversity, and traﬃc, from the 5D’s model framework (40, 41) for the evaluation of the factors of the built urban environment and the physical characteristics of the city at the macro level. In terms of density, the study used urban population density as an evaluation indicator. In the evaluation index of diversity, the study selected the data on POIs to measure the diversity of the urban environment. A POI consists of ﬁne-grained data that comprehensively reﬂect accurate information on urban land use. The POI data used in the study were downloaded from OpenStreetMap (OSM) and reclassiﬁed according to the basic functions of the city after the data were screened, which included the omission of irrelevant, duplicate, and empty data. The study obtained 16,003 valid entity POIs for Manhattan, which were classiﬁed using C·M·E·P·R (Table 1). The C·M·E·P·R classiﬁcation, as a method of classifying urban POIs on the basis of built-up characteristics, categorizes urban POIs according to urban functions such as commerce, healthcare, education, public services, and entertainment. Moreover, the POIs were classiﬁed according to C·M·E·P·R. The valid POIs were mapped to the ﬁshnet grid of the study area, and the entropy score of the POI data per grid was calculated to determine diversity (42), which is calculated as follows: 3. Methodology 3.1. Outcome variable: COVID-19 The formula pi is the proportion of the ith type of POI, and n is the total number of all POI types in the ﬁshing grid. In turn, it better reﬂects the inﬂuence relationship between the urban built environment and COVID-19. Figure 4 provides the visualization results of macro factors of the urban built environment. frontiersin.org 3.4. Statistical analysis The study conducted a four-step statistical analysis, namely: (i) Pearson’s correlation analysis of the CCC and CCR data using all data on the macro- and micro-level factors of the urban built environment, respectively. (ii) Z standardization of two independent variables. The z-score can transform two or more sets of data into unitless z-scores, which renders data standards uniform and, thus, improves Mix Index = − Xn i= 1 pilnpi. Frontiers in Public Health 06 frontiersin.org frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 FIGURE 3 Visualization of CCC and CCR in the study area. (A) CCC Visualization in Manhattan, New York. (B) CCR Visualization in Manhattan, New York. GU 3 Visualization of CCC and CCR in the study area. (A) CCC Visualization in Manhattan, New York. (B) CCR Visualization in Manhattan, New York. Visualization of CCC and CCR in the study area. (A) CCC Visualization in Manhattan, New York. (B) CCR Visualization in Manhattan, New York. ation of POI types according to C·M·E·P·R and corresponding categories in the original OpenStreetMap (OSM) dataset. TABLE 1 Classiﬁcation of POI types according to C·M·E·P·R and corresponding categories in the original OpenStreetMap (OSM) dataset. POI types Categories in the OSM dataset Count Percentage C Restaurants, beverages, malls, markets, stores, various shops, greengrocers, hairdressers, vendors, cinemas, car dealerships, car rentals, etc. 8,997 56.22% M Chemist, clinic, dentist, doctor, hospital, optician, pharmacy, veterinary, etc. 630 3.94% E College, kindergarten, library, playground, school, university, etc. 357 2.23% P Governmental organization, social group, communal facilities, ﬁnancial facilities, convenience, camera surveillance, etc. 5,331 33.31% R Scenic spot, park, open square, tourist attraction, theater, viewpoint, etc. 688 4.30% C, commercial; M, medical; E, education; P, public service; R, recreation. C, commercial; M, medical; E, education; P, public service; R, recreation. be called multicollinearity. In this case, the results of parameter estimation are no longer valid; thus, the current study uses variance inﬂation factor (VIF) to test for potential multicollinearity between the macro- and micro- level independent variables (Table 2). VIF is calculated as follows: data comparability and weakens data interpretation. The z- standardization formula is as follows: Z = X −µ σ , Where µ is the mean and σ is the standard deviation. (iii) The existence of a degree of correlation (approximate covariance) between the explanatory variables can also VIF = 1 1 −R2 , Frontiers in Public Health 07 Frontiers in Public Health 07 frontiersin.org Zhang et al. Frontiers in Public Health 3.4. Statistical analysis 10.3389/fpubh.2023.1137489 GURE 4 isualization of macro-scale factors of urban built environment. (A) Commercial, (B) education, (C) public, (D) medical, (E) recreation, and (F) POP. Visualization of macro-scale factors of urban built environment. (A) Commercial, (B) education, (C) public, (D) medical, (E) recreation, and (F) POP. Where R2 denotes goodness-of-ﬁt or the determination coeﬃcient of linear regression and describes the percentage of explanatory variables in the regression equation. The results indicate the absence of covariance for all independent variables, VIF values are <5, and all factors can be included in the linear regression model. hypothesis that there was heteroscedasticity. To address these concerns, the study employed the robust regression method. 4.1. Pearson’s correlation analysis Lastly, data at diﬀerent levels with various dependent variables were included in the ordinary least squares (OLS) model. Furthermore, the study employed the White and BP tests to verify whether or not heteroscedasticity exists in the data, to test the original hypothesis that there was no heteroscedasticity in the model, to conﬁrm whether or not the results rejected the original hypothesis, and to determine if there was a rejection of the original This study used Pearson correlation analysis to examine the correlations between CCC and CCR and 12 macro-level urban built environment (Public, Education, Commercial, Medical, Recreation, Airports, Bus Station, Bus Stop, Ferry, Railway, Taxi, and POP) and 8 micro-level urban built environment (i.e., 08 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 FIGURE 5 Visualization of micro-scale factors of urban built environment. (A) PVGVI, (B) bus stop, (C) road, (D) macrophanerophytes, (E) bush, and (F) grass. B ildi R d W ll M h h G B h i h CCC h 0 001 l l f i iﬁ Al h h h d Visualization of micro-scale factors of urban built environment. (A) PVGVI, (B) bus stop, (C) road, (D) macrophanerophytes, (E) bush, and (F) grass. Sky, Building, Road, Wall, Macrophanerophyte, Grass, Bush, and PVGVI) in Manhattan, New York, USA, respectively, using Pearson’s correlation coeﬃcient (PCC) to indicate the strength of the correlations. with CCC at the 0.001 level of signiﬁcance. Although the study noted no correlation among Commercial, Medical, Airports, Bus Station, Railway, Tax, and CCC, their PCC values are close to 0, and all p-values are >0.05. Figure 4C illustrates that Public, Education, Commercial, Medical, Bus Stop, Railway, Taxi, and POP have signiﬁcant positive correlations with CCR, where Commercial (PCC = 0.26, p < 0.001), Medical (PCC = 0.11, p < 0.001), Bus Stop (PCC = 0.16, p < 0.001), Railway (PCC = 0.10, p < 0.001), and POP (PCC = 0.092, p < 0.001) demonstrated showed signiﬁcance at the 0.001 level, which indicate a signiﬁcant positive correlation with CCC. Lastly, the study found no correlation among Recreation, Airports, Ferry, and CCR. Figures 6A, 7A depict the relationship between CCC and CCR and macro-level factors, where CCC presents a signiﬁcant negative correlation with Public (PCC = −0.12, p < 0.001), Recreation (PCC = −0.16, p < 0.001), and Ferry (PCC = −0.077, p < 0.01). Moreover, the study observes a signiﬁcant negative correlation between Public and Recreation. 4.1. Pearson’s correlation analysis Both correlations indicate signiﬁcance at the 0.001 level. Education (PCC = 0.11, p < 0.001), Bus Stop (PCC = 0.13, p < 0.001), and POP (PCC = 0.30, p < 0.001) displayed signiﬁcant positive correlations Frontiers in Public Health 09 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 FIGURE 6 Correlation coefcient between urban built environment and CCC Pearson. (A) Correlation coefcient between macro urban built environment and CCC Pearson. (B) Correlation coefcient between micro urban built environment and CCC Pearson. p < 0.05, p < 0.01, *p < 0.001. FIGURE 6 Correlation coefcient between urban built environment and CCC Pearson. (A) Correlation coefcient between macro urban built environment and CCC Pearson. (B) Correlation coefcient between micro urban built environment and CCC Pearson. p < 0.05, p < 0.01, p < 0.001. FIGURE 6 Correlation coefcient between urban built environment and CCC Pearson. (A) Correlation coefcient between macro urban built environment and CCC Pearson. (B) Correlation coefcient between micro urban built environment and CCC Pearson. p < 0.05, p < 0.01, p < 0.001. IGURE 6 Correlation coefcient between urban built environment and CCC Pearson. (A) Correlation coefcient between macro urban built env CCC Pearson. (B) Correlation coefcient between micro urban built environment and CCC Pearson. p < 0.05, p < 0.01, p < 0.00 10 Frontiers in Public Health frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 FIGURE 7 Correlation coefcient between urban built environment and CCR Pearson. (A) Correlation coefcient between macro urban built environment and CCR Pearson. (B) Correlation coefcient between micro urban built environment and CCR Pearson. p < 0.05, p < 0.01, p < 0.001. FIGURE 7 Correlation coefcient between urban built environment and CCR Pearson. (A) Correlation coefcient between macro urban built environment and CCR Pearson. (B) Correlation coefcient between micro urban built environment and CCR Pearson. p < 0.05, p < 0.01, p < 0.001. IGURE 7 Correlation coefcient between urban built environment and CCR Pearson. (A) Correlation coefcient between macro urban built env CCR Pearson. (B) Correlation coefcient between micro urban built environment and CCR Pearson. p < 0.05, p < 0.01, *p < 0.00 11 Frontiers in Public Health frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 TABLE 2 Summary statistics of all variables in Manhattan, New York, United States (n = 1,551). TABLE 2 Summary statistics of all variables in Manhattan, New York, United States (n = 1,551). 4.1. Pearson’s correlation analysis Variables (unit) Min Max Mean SD VIF (Z-score) Dependent variable COVID Case Count (CCC) (N) 0 27,410 13814.951 6340.799 COVID Case Rate (CCR) (N) 0 54364.28 30825.563 7509.91 Independent variables Macro-scale built environment Public service (N) 0 97 3.437 6.911 1.296 Education (N) 0 5 0.23 0.576 1.029 Commercial (N) 0 63 5.801 8.736 1.417 Medical (N) 0 18 0.406 1.055 1.287 Recreation (N) 0 14 0.444 1.016 1.244 Airports (N) 0 1 0.001 0.025 1.001 Bus station (N) 0 2 0.008 0.098 1.041 Bus stop (N) 0 8 0.932 1.275 1.175 Ferry (N) 0 2 0.004 0.072 1.005 Railway (N) 0 3 0.102 0.348 1.144 Taxi (N) 0 2 0.006 0.088 1.043 POP (N) 0 5722.99 1042.108 1033.205 1.167 Micro-scale built environment Sky View Factors (SVF) (%) 0 0.279 0.038 0.042 1.224 Building (%) 0 0.45 0.186 0.109 2.245 Road (%) 0 0.504 0.253 0.131 1.999 PVGVI (%) 0 0.529 0.111 0.104 1.791 Wall (%) 0 0.459 0.058 0.077 1.256 Street greening structure Macrophanerophytes (%) 0 0.59 0.114 0.098 1.667 Bush (%) 0 0.399 0.016 0.032 1.518 Grass (%) 0 0.234 0.012 0.029 1.496 (i). All VIF values were standardized using z-score; (ii). Min., minimum; Max., maximum; SD, standard deviation; N, number; %, Percentage; (iii). Concerning the problem that the minimum value of the micro-scale factors of the built environment is 0, the reason is that during panoramic streetscape crawling, certain indoor images will be crawled, which leads to the minimum value of certain micro-scale factors at 0. At the same time, model recognition errors were noted, but the sample size is very small, which will not inﬂuence the results. (i). All VIF values were standardized using z-score; (ii). Min., minimum; Max., maximum; SD, standard deviation; N, number; %, Percentage; (iii). Concerning the problem that the minimum value of the micro-scale factors of the built environment is 0, the reason is that during panoramic streetscape crawling, certain indoor images will be crawled, which leads to the minimum value of certain micro-scale factors at 0. At the same time, model recognition errors were noted, but the sample size is very small, which will not inﬂuence the results. Figures 6B, 7B present the relationship of CCC and CCR to micro-level factors, where positive correlations were noted among Building (PCC = 0.15, p < 0.001), Road (PCC = 0.26, p < 0.001), and CCC, and all of them show. 4.1. Pearson’s correlation analysis The study found negative correlations among Wall, Grass, Bush, PVGVI, and CCC, where Grass (PCC = −0.18, p < 0.001) and Bush (PCC = −0.15, p < 0.001) at the 0.001 level of signiﬁcance, which indicates a signiﬁcant negative correlation with CCC, whereas no correlation was found between Sky and Macrophanerophytes to CCC. Figure 4D points to a positive correlation among Building, Road, Wall, and CCR at the 0.001 level of signiﬁcance, which indicates a signiﬁcant negative correlation with CCR. The correlation between Macrophanerophytes, Grass, Bush, PVGVI, and CCR was all negative at the 0.001 level of signiﬁcance, among which the PCC value of Macrophanerophytes was −0.5, which extremely exceeded the other variables and indicates a signiﬁcant negative correlation with CCR. Frontiers in Public Health frontiersin.org 4.2. Robust regression model The OLS linear regression of CCC and CCR as outcome variables resulted in four models. Macro- and micro-level factors of the urban built environment were separately included as variables in the models to determine the relationship of CCC and CCR to Frontiers in Public Health 12 frontiersin.org 10.3389/fpubh.2023.1137489 Zhang et al. TABLE 3 Results of the white and BP tests. Table 4 depicts the correlation between Model 1 with CCC as the dependent variable and 20 factors of the urban built environment as the independent variables. It uses robust regression analysis (M-estimation) to construct the correlation between the variables of urban built environment and COVID-19. The study ﬁnds that the macro-level factors, Education, Commercial, POP, and Bus Stop, exert a signiﬁcant positive inﬂuence on the relationship between the urban built environment and COVID- 19. The correlation coeﬃcient of POP was 0.297, which exceeded all other variables. In particular, Commercial is the only factor that exerts a signiﬁcant positive eﬀect on CCC and CCR as the dependent variables for both regression models. The regression coeﬃcient of Public is −0.255 with a p-value of 0.004, which is more signiﬁcant than the other variables. 4.2. Robust regression model White heteroscedasticity test BP heteroscedasticity test X2 P X2 P White test and BP test results of CCC and macro urban built environment 91.844 0.034 24.380 0.018 White test and BP test results of CCC and micro-level urban built environment 182.923 0.000 120.011 0.000 Results of white test and BP test of CCR and macro urban built environment 182.923 0.034 120.011 0.000 Results of white test and BP test of CCR and micro-level urban built environment 435.371 0.000 273.668 0.000 White heteroscedasticity test BP heteroscedasticity test X2 P X2 P White test and BP test results of CCC and macro urban built environment 91.844 0.034 24.380 0.018 White test and BP test results of CCC and micro-level urban built environment 182.923 0.000 120.011 0.000 Results of white test and BP test of CCR and macro urban built environment 182.923 0.034 120.011 0.000 Results of white test and BP test of CCR and micro-level urban built environment 435.371 0.000 273.668 0.000 The micro-level factors that displayed signiﬁcant negative eﬀects in the micro-urban built environment were signiﬁcantly higher; Building, Wall, Grass, Bush, and PVGVI exerted signiﬁcant negative eﬀects on CCC, where Grass obtained a regression coeﬃcient of −0.357 and a p-value of 0.000, which were higher than those of the other variables in the same model in terms of signiﬁcance and regression coeﬃcient. PVGVI and Grass exhibited a signiﬁcant negative eﬀect relationship for Models 1 and 2. The regression coeﬃcient for Grass was higher in Model 1 than that in Model 2; however, the signiﬁcance of both Models is the same (p-values = 0.000). Road and Macrophanerophytes exerted a signiﬁcant positive eﬀect on CCC; both p-values were 0.000, which is higher than the other variables in terms of signiﬁcance, except for Grass, which is equal. the independent variables at diﬀerent levels. The equation for OLS linear regression is as follows: Y = Xβ + ε, Where Y is the dependent variable, X denotes the matrix of explanatory variables, β represents the vector of coeﬃcients, and ε is the vector of random error terms. The variables were included in the OLS model for the White and BP tests. Table 3 presents the results. In the case of heteroscedasticity, the study conducted the White and BP tests to verify the original hypothesis, that is, no heteroscedasticity exists in the model. 4.2. Robust regression model Table 3 illustrates that both tests reject the original hypothesis at p < 0.05, which indicates that heteroscedasticity exists in the model. Table 5 presents the results of the robust regression analysis for Model 2 with CCR as the dependent variable and the 20 urban built environment factors as the independent variables. The ﬁnding indicates that Public and Commercial show a signiﬁcant positive relationship with CCR at the macro level, whereas POP indicates a signiﬁcant negative relationship with CCR. Public and POP produced the opposite results for both models (Model 1: Public: regression coeﬃcient = −0.255, POP: regression coeﬃcient = 0.297; Model 2: Public: regression coeﬃcient = 0.07, POP: regression coeﬃcient = −0.088). Table 3 suggests that heteroscedasticity exists in the regression data, and the conclusions obtained by the commonly used OLS regression estimation method may be biased because it considers the minimized residual sum of squares as a criterion. Therefore, it also considers anomalous data. In this case in the model regression considered for robust regression analysis (M-estimation), the study uses the Huber robust method with the following formula: The micro-level factors Sky, Building, and Wall presented a signiﬁcant positive relationship with CCR, whereas Macrophanerophytes, Grass, and PVGVI pointed to a signiﬁcant negative relationship with CCR. PVGVI is more signiﬁcant in Model 2 than it was in Model 1 (Model 1: PVGVIp = 0.023; Model 2: PVGVIp = 0.000). Macrophanerophytes present opposite results in Models 1 and 2 (Model 1: regression coeﬃcient = 0.213; Model 2: regression coeﬃcient = −0.047). n X i=1 ρ P 1 2 i aT i X −L = min n X i=1 ρ P 1 2 I aT i X −L , where a real function ρ deﬁned in a one-dimensional Euclidean space R is selected for the independent identically distributed equal precision model, such that aT i denotes the row vector of the design matrix; X is the extreme value solution; and P represents the weight of the corresponding observation or observation error. 5. Discussion Tables 4, 5 depict Models 1 and 2, respectively. The diﬀerence between the models is the use of CCC and CCR as the dependent variables, respectively. CCR can be used to illustrate the long-term trend of COVID-19, which could help in analyzing the impact of NPIs on the relationship between the urban built environment and COVID-19. Alternatively, CCC can be used for analyzing the relationship between the impact of a pure urban built environment and COVID-19. This study investigated the relationship between the factors of the urban built environment and COVID-19 using robust regression analysis (M-estimation) based on solving the heteroscedasticity of the OLS regression model. The study categorized urban built environment into two dimensions, namely, macro and micro, in two urban spatial dimensions, where Frontiers in Public Health 13 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 TABLE 4 Model 1: Robust regression results of CCC and urban built environment (n = 1,551). Regression coefcient SD t p 95% CI R2 Adjusted R2 F Constant 0.423 0.02 21.266 0.000∗∗ 0.384 to 0.462 0.193 0.183 F(20, 1,530) = 18.302, p = 0.000 Macro urban built environment Public −0.255 0.088 −2.884 0.004∗∗ −0.428 to −0.082 Education 0.141 0.049 2.885 0.004∗∗ 0.045 to 0.236 Commercial 0.116 0.055 2.107 0.035∗ 0.008 to 0.224 Medical −0.013 0.107 −0.122 0.903 −0.222 to 0.196 Recreation −0.235 0.085 −2.747 0.006∗∗ −0.402 to −0.067 POP 0.297 0.037 8.001 0.000∗∗ 0.224 to 0.369 Traﬃc factors Airports −0.06 0.217 −0.278 0.781 −0.485 to 0.365 Bus station 0.026 0.115 0.228 0.82 −0.199 to 0.251 Bus stop 0.138 0.038 3.612 0.000∗∗ 0.063 to 0.212 Ferry −0.363 0.155 −2.351 0.019∗ −0.666 to −0.060 Railway −0.09 0.051 −1.765 0.078 −0.189 to 0.010 Taxi 0.029 0.128 0.225 0.822 −0.222 to 0.280 Micro-level urban built environment Sky 0.053 0.043 1.237 0.216 0.384 to 0.462 PVGVI −0.05 0.022 −2.276 0.023∗ −0.094 to −0.007 Building −0.145 0.037 −3.891 0.000∗∗ −0.218 to −0.072 Road 0.141 0.032 4.374 0.000∗∗ 0.078 to 0.204 Wall −0.115 0.041 −2.8 0.005∗∗ −0.196 to −0.035 Macrophanerophytes 0.213 0.05 4.259 0.000∗∗ 0.115 to 0.311 Grass −0.357 0.058 −6.136 0.000∗∗ −0.470 to −0.243 Bush −0.198 0.08 −2.481 0.013∗ −0.355 to −0.042 Dependent variable: CCC; ∗p < 0.05, ∗∗p < 0.01. macro-level factors include variables related to urban traﬃc, and micro-level factors pertain to urban green structures. density. 5. Discussion Residents in these areas must travel long distances to obtain essential resources, where long-distance travel implies increased chances of contact with strangers and COVID-19 infection. In summary: (i) high population density increases the likelihood of human contact, which facilitates the spread of the virus. However, with the implementation of NPIs, residents could only move within a small area; thus, the virus could not spread among areas. (ii) Areas with high-density populations typically have relatively well-developed infrastructure to provide convenient and timely treatment for residents, which, thereby, inhibits the spread of NPI (26, 29). In particular, under strict NPIs, the outdoor activities of residents are restricted, which eﬀectively inhibits the spread of the virus in high-density areas (24). However, at the CCC level, the government for areas with high population density and high commercial activities performs better in terms of pandemic control and detection than did areas with low population density with a higher detection rate than that of areas with low population density. This ﬁnding results in a higher number of conﬁrmed and suspected cases compared with those of areas with low population density. Frontiers in Public Health 5.1. COVID-19 and urban built environment The study used the relationship between the number (CCC) and incidence (CCR) of conﬁrmed and suspected cases of COVID- 19 per 100,000 people in Manhattan, United States, as an entry point for the factors of the urban built environment. However, in the regression analysis with CCR as the dependent variable, POP exhibited a signiﬁcant negative eﬀect on CCR. In analyzing this entirely contradictory result, the study considered the eﬀect of Commercial, which exerted a signiﬁcant positive eﬀect on CCC and CCR but with diﬀerent factors at 0.116 and 0.041, respectively. In other words, residents can obtain necessities in a small area after the outbreak of a potential pandemic, and NPIs are better compared with those in areas with low population 14 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 TABLE 5 Model 2: Robust regression results of CCR and urban built environment (n = 1,551). Regression coefcient SD t p 95% CI R2 Adjust R2 F Constant 0.557 0.006 93.445 0.000∗∗ 0.545 to 0.568 0.283 0.273 F(20, 1,530) = 30.130, p = 0.000 Macro urban built environment Public 0.07 0.026 2.63 0.009∗∗ 0.018 to 0.122 Education −0.006 0.015 −0.416 0.677 −0.035 to 0.023 Commercial 0.041 0.017 2.473 0.013∗ 0.008 to 0.073 Medical 0.006 0.032 0.198 0.843 −0.056 to 0.069 Recreation 0.044 0.026 1.706 0.088 −0.007 to 0.094 POP −0.088 0.011 −7.893 0.000∗∗ −0.110 to −0.066 Traﬃc factors Airports −0.031 0.065 −0.47 0.639 −0.158 to 0.097 Bus station 0.028 0.034 0.819 0.413 −0.039 to 0.095 bus stop 0.012 0.011 1.066 0.287 −0.010 to 0.035 Ferry −0.032 0.046 −0.688 0.492 −0.123 to 0.059 Railway −0.01 0.015 −0.65 0.516 −0.040 to 0.020 Taxi 0.049 0.038 1.263 0.207 −0.027 to 0.124 Micro-level urban built environment Sky 0.029 0.013 2.27 0.023∗ 0.004 to 0.055 PVGVI −0.063 0.007 −9.421 0.000∗∗ −0.076 to −0.050 Building 0.079 0.011 7.074 0.000∗∗ 0.057 to 0.101 Road 0.009 0.01 0.882 0.378 −0.010 to 0.027 Wall 0.069 0.012 5.574 0.005∗∗ 0.045 to 0.093 Macrophanerophytes −0.047 0.015 −3.123 0.002∗∗ −0.076 to −0.017 Grass −0.115 0.017 −6.594 0.000∗∗ −0.149 to −0.081 Bush 0.023 0.024 0.952 0.341 −0.024 to 0.070 Dependent variable: CCR; ∗p < 0.05, ∗∗p < 0.01. At the same time, control and control eﬀorts are correspondingly lower due to the lower population density, which results in an increased number of cases without data. The situation of no data collection. Frontiers in Public Health 5.1. COVID-19 and urban built environment In future studies, we will apply multiscale geographically weighted regression models with added potential factors to calibrate the existing models for further accuracy in the analysis given that more data are available at the city level. became negligible; instead, many schools were requisitioned for the isolation of patients, which exerted a positive eﬀect on the control of the outbreak after lockdowns. Alfano et al. (44) demonstrated that the premature opening of schools increased the number of COVID- 19 cases in Italy. This result suggests that during an outbreak, the government should implement strict NPIs in schools while ensuring equity in education. The higher the PVGVI, the farther away from the city center, the lower the population density, and the less space and medium for virus transmission and corresponding inhibitory eﬀects on virus transmission. Macrophanerophytes exerted a signiﬁcant positive eﬀect on CCC, whereas Bush and Grass exerted a signiﬁcant negative eﬀect on CCC when analyzed from the perspective of the green structure of urban streets. The reason for this phenomenon may be that in densely populated areas with developed commercial activities, the green structure is relatively homogeneous and shows a single- tree state. Conversely, areas with a rich green structure have correspondingly low population density and more homogeneous commercial activities, which can be analyzed in combination with macro-level POP and Commercial. 5.2. Research values A series of recommendations for the results of the study have the following applications: (i) they can be applied at the level of prevention of widespread spread of COVID-19 in cities to minimize the risk of infection and the rate of virus transmission among urban residents by exploring the mechanisms of inﬂuence of the built environment and COVID-19. Eﬀective control of virus transmission was achieved at the early stage of the outbreak. (ii) Based on the results of the study, government oﬃcials and policy makers can better formulate more reasonable NPI policies to prevent widespread infection and cross-infection and reduce the risk of infection among urban residents, while ensuring the wellbeing, health and comfort of urban residents. (iii) The study uses Google Street View panoramic street view images to extract and quantify urban micro built environment factors from the macro built environment and the micro built environment, respectively, to explore the impact of COVID-19 at the urban street level, and the results provide a data base for future urban renewal. This enables cities to play a more important role in facing the trend of COVID-19 epidemic normalization. 5.1. COVID-19 and urban built environment The number of bus stops tends to be proportional to population density; the higher the population density, the higher the number of bus stops. Essentially, bus stops are places where urban residents are most likely to come into contact with strangers. A high frequency of contact with strangers implies an increased chance of infection. In general, public transportation infrastructure that increases population contact is considered a key factor in the spread of infectious diseases (25). Thus, a range of eﬀective measures should be taken to limit the spread of disease in public transport, including limiting passenger density, increasing the frequency of services, and reserving tickets. Other low-carbon and environmentally friendly active transportation modes, such as walking and bicycling, should also be encouraged. to unfamiliar environments. Thus, the virus is likely to spread through public facilities before the introduction of corresponding NPIs. The number of public facilities in areas with high population density far exceeds that in areas with low population density, such that corresponding transmission rates and probability of transmission are also higher. On the contrary, at the CCC level, the frequency of the use of public facilities is suppressed due to NPIs, and residents will voluntarily reduce their frequency of use of public facilities when they are aware of a potential pandemic. This scenario indirectly leads to a negative correlation between CCC and the Public with a regression index of −0.235 over other factors. Schools tend to be places where pedestrian traﬃc is high, no less than in commercial areas, and the interaction between students and teachers may accelerate the spread of the virus. When students are infected with the virus at school, NCPI can easily infect family members through parent–child interaction, and the spread of the virus within colleges and universities is typically diﬃcult to reasonably control. As the implementation of NPIs led to school closure, the correlation between schools and CCR Similarly, at the public level, the results of CCC and CCR indicate a clear contradiction. From the CCR level, the higher the use of public facilities, the higher the probability of exposure Frontiers in Public Health 15 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 10.3389/fpubh.2023.1137489 Manhattan were not available or could not be speciﬁcally mapped within each study area, such as household income structure, demographics, gender, underlying disease status, occupation, and ethnic composition, which have been noted in previous studies to be associated with 2019 coronavirus disease transmission. Frontiers in Public Health 5.1. COVID-19 and urban built environment At the same time, within the time point of the COVID-19 pandemic, the lives of residents were frequently restricted by various NPIs, which resulted in extremely complex and confusing life activities and social relationships. Thus, the study selected only 20 variables, which indicates the exclusion of other potential variables such as the density of foot traﬃc in the region. Previous studies demonstrated that individual behaviors exerted an eﬀect on the spread of COVID-19; however, such variables are statistically unavailable, relatively diﬃcult to obtain, and more diﬃcult to collect in the ﬁeld due to various policy restrictions imposed by NPIs. The absence of such variables may have led to certain anomalies in the results of the study. Moreover, the eﬀect of spatial autocorrelation cannot be avoided despite the multilevel and multidimensional considerations. Thus, future studies should consider additional aspects and potential variables to explore the relationship between the factors of the urban built environment and COVID-19. This, data on COVID-19 published by NYC Health provided substantial support to various urban studies on COVID-19. However, the published information on the number of cases is, in fact, incomplete due to the lack of statistical data on the number of cases due to the current pandemic policy implemented in the United States. Thus, certain individuals contracted COVID-19 but displayed no symptoms (asymptomatic) due to the lack of assurance of the detection rates of COVID-19 in the population. Moreover, individuals with the infection were not sampled for nucleic acids; thus, they remained unaware of their COVID-19 infection, which rendered their network and range of activities and transmission of the virus virtually uncontrollable and unavoidable. Possible non- linear eﬀects of the variables in this study. The starting point of the robust regression is still based on the processing method of linear data, but the principle of adopting the method should be considered when processing the experimental data, and if the data have non- linear eﬀects, the experimental data can be made permutation substitution so that they are transformed into a linear functional relationship for the test. In future studies, we will apply multiscale geographically weighted regression models with added potential factors to calibrate the existing models for further accuracy in the analysis given that more data are available at the city level. 5.1. COVID-19 and urban built environment Manhattan were not available or could not be speciﬁcally mapped within each study area, such as household income structure, demographics, gender, underlying disease status, occupation, and ethnic composition, which have been noted in previous studies to be associated with 2019 coronavirus disease transmission. At the same time, within the time point of the COVID-19 pandemic, the lives of residents were frequently restricted by various NPIs, which resulted in extremely complex and confusing life activities and social relationships. Thus, the study selected only 20 variables, which indicates the exclusion of other potential variables such as the density of foot traﬃc in the region. Previous studies demonstrated that individual behaviors exerted an eﬀect on the spread of COVID-19; however, such variables are statistically unavailable, relatively diﬃcult to obtain, and more diﬃcult to collect in the ﬁeld due to various policy restrictions imposed by NPIs. The absence of such variables may have led to certain anomalies in the results of the study. Moreover, the eﬀect of spatial autocorrelation cannot be avoided despite the multilevel and multidimensional considerations. Thus, future studies should consider additional aspects and potential variables to explore the relationship between the factors of the urban built environment and COVID-19. This, data on COVID-19 published by NYC Health provided substantial support to various urban studies on COVID-19. However, the published information on the number of cases is, in fact, incomplete due to the lack of statistical data on the number of cases due to the current pandemic policy implemented in the United States. Thus, certain individuals contracted COVID-19 but displayed no symptoms (asymptomatic) due to the lack of assurance of the detection rates of COVID-19 in the population. Moreover, individuals with the infection were not sampled for nucleic acids; thus, they remained unaware of their COVID-19 infection, which rendered their network and range of activities and transmission of the virus virtually uncontrollable and unavoidable. Possible non- linear eﬀects of the variables in this study. The starting point of the robust regression is still based on the processing method of linear data, but the principle of adopting the method should be considered when processing the experimental data, and if the data have non- linear eﬀects, the experimental data can be made permutation substitution so that they are transformed into a linear functional relationship for the test. frontiersin.org Acknowledgments This work used the DeepLabV3+ model. This model was opened source by Chen et al. (43). We thank Chen for the model. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 5.3. Research limitations The study draws preliminary conclusions on the relationship between the urban built environment and COVID-19 transmission, which focused on the relationship between CCC and CCR as the independent variables and the inﬂuence of the urban built environment. The correlation between the urban built environment and COVID-19 transmission was determined using Robust regression analysis (M-estimation). The major ﬁndings are summarized as follows: This study has its limitations. First, the data published by NYC Health are divided according to the MODZCTA, where individual buildings are designated unique zip codes in several instances. This tendency can exert a confounding eﬀect on the data, and although the study screened a few of the confounding factors at certain levels, this data-level confounding continues to exist. Moreover, although the study sample was expanded according to ﬁshnet divisions, the original sample only comprises 45 areas, which is not representative of all areas in the United States. Second, other demographic data for (i) Education, Commercial, POP, and Bus Stop exerted a signiﬁcant positive relationship with CCC at the macro (i) Education, Commercial, POP, and Bus Stop exerted a signiﬁcant positive relationship with CCC at the macro 16 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 datasets. We will continue to upload new datasets and optimize the datasets in the future. Our research team based on Python language, Pytorch deep learning framework, DeepLabV3+ neural network used in our research, the code can be downloaded from our GitHub website (https://github.com/muteisdope/Model.git). level. Public and Commercial displayed a signiﬁcant positive relationship with CCR. Public and Recreation have a signiﬁcant negative relationship with CCC. POP has a signiﬁcant negative relationship with CCR. level. Public and Commercial displayed a signiﬁcant positive relationship with CCR. Public and Recreation have a signiﬁcant negative relationship with CCC. POP has a signiﬁcant negative relationship with CCR. (ii) Macrophanerophytes, Grass, and PVGVI have a signiﬁcant negative eﬀect on CCR. Road and Macrophanerophytes have a signiﬁcant positive eﬀect on CCC. Sky, Building, and Wall have a signiﬁcant positive eﬀect on CCR. Funding This article is supported by the Research on Rural Ecological Landscape Creation Model of the National Key R&D Program of the 13th Five-Year Plan (2019YFD1100402) and Research on Cultural Heritage Design of Tianjin Urban Landscape-Tianjin Art Science Planning Project (C18083). Conﬂict of interest The recommendations may serve as a reference for solutions for other cities at the level of controlling the transmission and spread of the virus. Meanwhile, the ﬁndings may provide valid suggestions for curbing potential outbreaks of respiratory diseases. However, the applicability of the variables is limited and does not reﬂect the regional economic level, demographic, and other sociodemographic characteristics of the city due to the limitations of this study and the data sources. Therefore, the generalizability of the results should be carefully considered. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Author contributions LZ and XH: conceptualization and writing—original draft. LZ and JW: resources. LW and JW: supervision. LW: validation. All authors contributed to the article and approved the submitted version. Data availability statement (iii) Medical, Airports, Bus Station, Railway, and Taxi do not exert any inﬂuence on the relationship between CCC and CCR at the macro- and micro-levels of the urban built environment. (iii) Medical, Airports, Bus Station, Railway, and Taxi do not exert any inﬂuence on the relationship between CCC and CCR at the macro- and micro-levels of the urban built environment. (iii) Medical, Airports, Bus Station, Railway, and Taxi do not exert any inﬂuence on the relationship between CCC and CCR at the macro- and micro-levels of the urban built environment. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material. The current COVID-19 situation remains severe, and predicting the direction of the pandemic is diﬃcult. To cope with more severe pandemic situations, this study provides several recommendations for urban built environments in the context of its results. First, the government should provide easy access to essential resources for urban residents within a controlled range, reduce the frequency of long-distance travel, save travel costs, reduce unnecessary human contact, and control the medium of transmission to reduce the speed and eﬃciency of the virus transmission. Second, for areas with high population density and commercial activities, strict NPIs should be implemented, such that if a potential outbreak occurs, then the area can quickly and adequately mobilize favorable resources to eﬀectively control the outbreak. Third, the frequency of use of public facilities should be controlled. Although urban public transportation is an important part of the future low-carbon city, it continues to play an important role in the spread of the virus at this stage. In addition, the number of passengers should be controlled, their health status should be strictly tested, and safe social distancing should be observed to eﬀectively control the spread of the virus. The government can promote and introduce incentives to encourage residents to use other modes of travel. Fourth, schools or educational settings were found to be at risk during outbreaks of COVID-19 due to their dense population and foot traﬃc; thus, a series of strong measures should be taken such as distance teaching or a limited number of people in schools. Author’s note The S-G-S-S datasets used in this study can be downloaded and used from our GitHub site (https://github.com/muteisdope/S-G-S- S-Dataset.git), allowing users to modify, upload, and optimize the Frontiers in Public Health Frontiers in Public Health 17 frontiersin.org Zhang et al. 10.3389/fpubh.2023.1137489 References GIS-based spatial modeling of COVID- 19 incidence rate in the continental United States. Sci Total Environ. (2020) 728:138884. doi: 10.1016/j.scitotenv.2020.138884 29. Liu L. Emerging study on the transmission of the Novel Coronavirus (COVID-19) from urban perspective: evidence from China. Cities. (2020) 103:102759. doi: 10.1016/j.cities.2020.102759 8. Aycock L, Chen X. 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https://openalex.org/W2800430008	https://zenodo.org/records/2281030/files/article.pdf	French	null	Afdunstningsmätningar i Pyhäjärvi invid Tammerfors åren 1912 och 1913	Geografiska annaler	1,919	public-domain	1,288	LITTERATUR. E. BLOMQVIST, AfdunsIningsmdiningar i PyhJadrzi invid Tammerfors dren 1912 och 1913. Meddelanden fdrn Hydrografiska byran. III. Helsingfors 1917. E. BLOMQVIST, AfdunsIningsmdiningar i PyhJadrzi invid Tammerfors dren 1912 och 1913. Meddelanden fdrn Hydrografiska byran. III. Helsingfors 1917. Les observations sur l'dvaporation des lacs sont, comme on le sait, assez difficiles. 11 est donc d'une grande importance d'en avoir pour diff6rentes regions de la terre. Le travail en question nous fournit d'importants renseignements sur l'6vaporation des lacs en Finlande. Une station d'observation avait et 6 6tablie dans la partie sud-est de Pyhajdrvi & Toppari, SW. de Tammerfors. On mesura l'evaporation et la temp6rature aussi bien a terre que dans l'eau et outre cela l'humidit6 de l'air, l'insolation (at l'aide de termo- metres a insolation et d'hdliographes) et la direction et la force du vent. Dans la partie opposee du lac, dans le Sotka str6m, on fit des mesures sur l'evaporation du lac et sur l'eau tombde a terre. Au milieu du lac, on mesura aussi l'6vaporation pendant les jours calmes et on la mesura de meme dans le marecage de Hylkiinkorpi pendant un certain temps. Pour les mesures de l'6vaporation, on employa ' Toppari l'evaporo- metre de Wild, un vase de t6le cylindrique enfonce dans le sol, un vase semblable enfonce dans l'eau de la baie pres du rivage et un vase enregistreur du meme genre enfonce dans l'eau du lac. Dans le Sotka str6m, on mesura a l'aide de vases semblables, avec et sans appareil enregistreur. Au milieu du lac, l'appareil enregistreur 6tait fix6 sur un radeau et c'est un appareil du mrme genre qui fut employe aussi pour les mesures dans le marecage. L'appareil de Wild 6tait au debut sans abri, mais il fut plus tard muni d'une couverture legere. Les appareils enregistreurs furent egalement munis de couvertures semblables. Les dvaporomhtres enfonces dans l'eau etaient prot6g6s contre les ondes par des cloisons de bois. p Les mesures effectuees montrent qu'a l'aide de l'dvaporomrtre de Wild on a obtenu des valeurs beaucoup plus grandes qu'avec le vase enfoncd dans le sol. Ensuite, les vases enfonc6s dans l'eau du lac ont donne des valeurs plus grandes que le vase place at terre. ningsmätningar i Pyhäjärvi invid Tammerfors åren 1912 och 1913 by E. Blomqvist Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Wiley and Swedish Society for Anthropology and Geography are collaborating with JSTOR to digitize, preserve and extend access to Geografiska Annaler. This content downloaded from 128.235.251.160 on Fri, 12 Dec 2014 14:25:28 PM All use subject to JSTOR Terms and Conditions This content downloaded from 128.235.251.160 on Fri, 12 Dec 2014 14:25:28 PM All use subject to JSTOR Terms and Conditions LITTERATUR. Les r6sultats sont donc trbs divergents de ceux obtenus par les mesures allemandes, qui donnaient des valeurs plus grandes pour les vases a terre que pour ceux enfonces dans l'eau et les valeurs les plus petites 5 l'aide de l'dvaporometre de Wild. I1 est h remarquer aussi que, des evaporometres finlandais, ceux qui 6taient enfonces dans l'eau ont donn6 des valeurs plus grandes que ceux places a terre, quoique ceux-lE fussent couverts mais pas ceux-ci. La comparaison entre les valeurs obtenues pour 1'evaporation du mar6cage et les valeurs du lac montre un exc6dent de 35 % dans le premier cas. Si l'on compare les valeurs finlandaises "t celles qui ont 6t obtenues pour le lac du Hjilmaren pendant la m~me p6riode, on constate que les premieres 6taient de beaucoup les plus grandes. En somme les rdsultats de ces mesures confirment l'irr6gularit6 qui les distingue. This content downloaded from 128.235.251.160 on Fri, 12 Dec 2014 14:25:28 PM All use subject to JSTOR Terms and Conditions 261 LITTERATUR L'auteur examine ensuite, t l'aide de la methode de corr6lation, la relation entre l'vaporation et certains facteurs metdorologiques specialement en ce qui concerne l'humidit6 de l'air. Le resultat est que l'evaporation d'une surface d'eau est assez proportionnelle a la difference entre la tension maxima de la vapeur d'eau a la temperature de l'eau de la surface et l'humidit' absolue de l'air. Une etude sur la relation entre l'dvaporation et la pression barom6trique et la force du vent a donne des coefficients de correlation tr's petits. S'il tait -tabli que l'vaporation d'une surface de terre soit proportionnelle a la difference entre la tension maxima de la vapeur d'eau at la temperature du termometre mouille et l'humidite absolue au meme moment, il serait facile de calculer la marche annuelle de l'dvaporation d'un bassin, pour leque on connait l'dvaporation annuelle " l'aide des mesures sur l'eau tombde et le d6bit l dvaporation l aide d6bit. Quelques calculs que nous avons faits, il y a quelques annees, sur la corr6lation entre l'evaporation du lac Hjalmaren et quelques facteurs met6orologiques, notamment l'humidite relative et absolue ainsii que la difference psychrometrique, ont donne les plus grands coefficients pour la correlation entre l'dvaporation et la dite difference psychrometrique. psychrometrique. L'auteur compare aussi les valeurs obtenues 5 l'aide de l'evaporometre de Wild et at l'aide des autres appareils. 1 Since 19o8 no fewer than three Danish expeditions have visited N. E. Greenland, in consequence of which the results of the Denmark Expedition have been completed and corrected in certain districts. This content downloaded from 128.235.251.160 on Fri, 12 Dec 2014 14:25:28 PM All use subject to JSTOR Terms and Conditions LITTERATUR. II constate que les premieres sont en corr6lation 'troite avec les valeurs obtenues " l'aide des vases places a terre mais assez peu avec les valeurs obtenues a l'aide des vases enfoncies dans l'eau. Le resultat ne tend guere a prouver la possibilite de remplacer les mesures de l'evaporation des lacs par les mesures qu'on peut effectuer "a terre plus facilement. AXEL WALLEN T. P. KOCH, Survey of North-East Greenland. Danmark-Expeditionen til Gronlands NO-kyst, 1906-i90o8. Bind VI No. 2. Saertryk av >>Meddelelser om Gronland> XLVI. Kobenhavn, 1916, Sid. 51-468, pl. VI-VII, 149 fig. The external fortunes of the Denmark Expedition, with the tragic death of Mylius- Erichsen, are well known, and have previously been mentioned in the periodical ,,Ymer". The above-mentioned work contains the scientific account of the geographical operations of the Expedition. Expedition. The Expedition went out with extremely great aims before it; and, thanks to the self-sacrificing energy of the members, the most important of them were attained. Their sphere of investigation embraced the whole of north-eastern Greenland between Haystack and Cape Bridgman, situated between 7 5 and 83'/2' N. Lat. forming over iooo km in length and comprising more than one sixth of the circumference of Greenland. The southern part of the region had previously been roughly mapped from the sea by the Duke of Orleans ,,Belgica" Expedition, and the northern-east part had been visited by Peary in 1893--95 and in i900oo. The contours of the whole region have now been laid out by the Denmark Expedition and the maps of the previous expedition have been corrected.' Beyond the general map-work, the program of the Expedition included making detailed maps of the regions round the winter-quarters and other regions inter- esting to science, determining the geographical coordinates of the winter-quarters, and making investigations into refraction. These last investigations consisted in the establishment This content downloaded from 128.235.251.160 on Fri, 12 Dec 2014 14:25:28 PM All use subject to JSTOR Terms and Conditions
https://openalex.org/W4280508631	https://www.zora.uzh.ch/id/eprint/219302/1/ZORA_s12936_022_04166_x.pdf	English	null	Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris	Malaria journal	2,022	cc-by	10,240	Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2022 © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris , Helene ; Hernández-Lara, Carolina ; Kubacki, Jakub ; Borel, Nicole ; Albini, Sarah ; Valkiūnas, Gedimin DOI: https://doi.org/10.1186/s12936-022-04166-x Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-219302 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 Intern ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Pendl, Helene; Hernández-Lara, Carolina; Kubacki, Jakub; Borel, Nicole; Albini, Sarah; Valkiūnas, Gediminas (2022). Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected road- kill fieldfare Turdus pilaris. Malaria Journal, 21:148. DOI: https://doi.org/10.1186/s12936-022-04166-x Pendl et al. Malaria Journal (2022) 21:148 https://doi.org/10.1186/s12936-022-04166-x Malaria Journal Open Access Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris Helene Pendl1, Carolina Hernández‑Lara2, Jakub Kubacki3, Nicole Borel4, Sarah Albini5 and Gediminas Valkiūnas2* Abstract Background: Species of Plasmodium (Haemosporida, Plasmodiidae) are remarkably diverse haemoparasites. Infor‑ mation on genetic diversity of avian malaria pathogens has been accumulating rapidly, however exo‑erythrocytic development of these organisms remains insufficiently addressed. This is unfortunate because, contrary to Plasmo- dium species parasitizing mammals, the avian malaria parasites undergo several cycles of exo‑erythrocytic develop‑ ment, often resulting in damage of various organs. Insufficient knowledge on the exo‑erythrocytic development in most described Plasmodium species precludes the understanding of mechanisms of virulence during avian malaria. This study extends information on the exo‑erythrocytic development of bird malaria parasites. Methods: A roadkill fieldfare (Turdus pilaris) was sampled in Switzerland and examined using pathologic, cytologic, histologic, molecular and microbiologic methods. Avian malaria was diagnosed, and erythrocytic and exo‑erythro‑ cytic stages of the parasite were identified using morphologic characteristics and barcode DNA sequences of the cytochrome b gene. The species‑specific characteristics were described, illustrated, and pathologic changes were reported. Results: An infection with Plasmodium matutinum lineage pLINN1 was detected. Parasitaemia was relatively low (0.3%), with all erythrocytic stages (trophozoites, meronts and gametocytes) present in blood films. Most growing erythrocytic meronts were markedly vacuolated, which is a species‑specific feature of this parasite’s development. Phanerozoites at different stages of maturation were seen in leukocytes, macrophages, and capillary endothelial cells in most organs examined; they were particularly numerous in the brain. Like the erythrocytic meronts, growing phanerozoites were markedly vacuolated. Conspicuous exo‑erythrocytic development and maturation in leucocytes suggests that this fieldfare was not adapted to the infection and the parasite was capable to escape from cellular immunity. Conclusions: This is the first report of exo‑erythrocytic development of the malaria parasite lineage pLINN1 during single infection and the first report of this lineage in the fieldfare. The findings of multiple phanerozoites in brain, skeletal muscle, and eye tissue in combination with signs of vascular blockage and thrombus formation strongly sug‑ gest an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slowly moving Correspondence: gediminas.valkiunas@gamtc.lt 2 Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania Full list of author information is available at the end of the article Correspondence: gediminas.valkiunas@gamtc.lt 2 Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania Full list of author information is available at the end of the article Necropsy, cytology, histology Necropsy was performed following a standardized proto- col [15] approximately two hours after collection. A full organ set was preserved comprising samples from the cardiovascular (heart, heart blood), the respiratory (lung, air sacs), gastrointestinal-hepatic (oropharynx, crop, proventriculus, ventriculus, intestine, pancreas, cloaca, liver), urogenital (kidney, ovary, oviduct), endocrine (thyroid gland), hematopoietic (spleen, bone marrow, thymus), musculo skeletal (pectoral and femoral mus- cles, femoral bone, skull bone), and central nervous and sensory system (all parts of the brain including the eye- balls). Samples were split into halves with one part frozen at − 20° for virology/molecular diagnostics and the other part fixed in buffered formalin for histology (Formafix™ Switzerland AG, Stationsstr. 3, CH 8335 Hittnau). Small samples were preserved in formalin only. To date, the exo-erythrocytic development remains non-described or fragmentarily known in most avian haemosporidian parasite species. This is unfortunate because, contrary to malaria parasites of humans and other mammals, avian Plasmodium species undergo several cycles of exo-erythrocytic merogony and can markedly damage organs before they acquire the ability to infect red blood cells. Furthermore, secondary exo- erythrocytic development can be initiated by merozo- ites from erythrocytic meronts [5–7]. This complicates understanding of the development of avian malaria par- asites in avian hosts and makes disease prognoses to be speculative even during low chronic malaria parasitaemia [8, 9]. Numerous experimental observations show that the secondary exo-erythrocytic meronts (phanerozo- ites) might lead to severe disease and even mortality of birds [5, 6, 10, 11]. However, most experimental studies deal with non-adapted host-parasite associations, which could bias the understanding of the true virulence of the same pathogen in natural populations. Information on the exo-erythrocytic development of haemosporidian parasites in naturally infected birds would be a valuable supplement to experimental observations, but currently remains insufficient in wildlife [4, 12, 13]. This study pro- vides first information on a natural infection with Plas- modium matutinum (genetic lineage pLINN1) in a wild Cytologic samples were taken from fresh material with swab-roll imprint preparation from conjunctiva, cloaca, air sacs, and intestinal contents, with blood film tech- nique from fluidy parts of heart and lung, with imprints or scrape-squash preparations from liver, lung, spleen, kidney, myocardium, feather quills, brain, thyroid gland, pectoral and femoral muscle. The imprint preparations of the brain were taken from the material protruding from the skull fractures close to the third eyelids. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pendl et al. Malaria Journal (2022) 21:148 Page 2 of 13 car. Further studies on exo‑erythrocytic stages of haemosporidian parasites are pivotal to understand the true level of populational damage of avian malaria in wild birds. ar. Further studies on exo‑erythrocytic stages of haemosporidian parasites are pivotal to understand populational damage of avian malaria in wild birds. Keywords: Plasmodium matutinum, pLINN1, Birds, Exo‑erythrocytic development, Virulence, Roadkil fieldfare (Turdus pilaris). This parasite lineage is com- mon in European species of thrushes (Turdidae) and fly- catchers (Muscicapidae), and it seems to be transmitted across the Holarctic [13, 14]. Case history On September 7th, 2021, one of the authors (HP) wit- nessed a roadkill of a fieldfare (Turdus pilaris) close to a nature reserve in the Canton Zug, Switzerland (47° 13’ 38.4" N, 8° 24’ 21.4" E). The bird flew frontally into the grille of a slow-moving car and fell into the road ditch in front of the author. First examination confirmed the death of the bird with multiple fractures visible at the frontal part of the head and the thorax. Permission of collection of the carcass was given by telephone by the Office of Forestry and Game of the Canton Zug. Background Avian malaria pathogens of the genus Plasmodium (Plas- modiidae, Haemosporida) are cosmopolitan, with over 50 species described [1] and many more different genetic lineages determined (MalAvi database, http:// 130. 235. 244. 92/ Malavi, accessed February 2022). As these para- sites are present in the peripheral blood circulation, they are easy to access for morphologic and genetic studies, which during the past 25 years resulted in a prominent increase of knowledge on various aspects of their genetic diversity as well as geographic and host distribution [2]. Development of sensitive and easy to use genetic mark- ers considerably improved opportunities for pathogen diagnostics and ecologic research. However, information on an important part of the life cycle of malaria para- sites and related haemosporidians (Haemosporida)–the exo-erythrocytic development–still remains at an early stage. This is particularly true for wild birds due to the difficulties in accessing these stages for research, which requires animal dissection, direct investigation of organs and application of histologic techniques [3, 4]. Necropsy, cytology, histology Drops of heart and lung blood were dried and stored on filter paper for molecular diagnostics. Cytologic samples were stained with a one-step Wright-Giemsa-Protocol and Pendl et al. Malaria Journal (2022) 21:148 Page 3 of 13 (Plasmodium sp.) and negative (nuclease-free ddH2O) controls were included. PCR products were run on a 2% agarose gel to check for positive amplifications, which were sequenced from 3’ and 5’ ends with Big Dye Ter- minator V3.1 Cycle Sequencing Kit and ABI PRISMTM 3100 capillary sequencing robot (Applied Biosystems, Foster City, CA, USA). Cytochrome b mitochondrial gene sequences (479 bp) quality and presence of mixed infections (double peaks) was assessed using SnapGene Viewer 5.2.4 software (Insightful Science, San Diego, CA, USA, www. snapg ene. com; accessed on 20 November 2021). Lineage identification was carried out by BLAST searches in MalAvi [22] and GenBank databases with Megablast algorithm (www. ncbi. nlm. nih. gov/ genba nk/; accessed on 10 October 2021). Obtained DNA sequence information was compared with results of microscopic parasite identification. (Plasmodium sp.) and negative (nuclease-free ddH2O) controls were included. PCR products were run on a 2% agarose gel to check for positive amplifications, which were sequenced from 3’ and 5’ ends with Big Dye Ter- minator V3.1 Cycle Sequencing Kit and ABI PRISMTM 3100 capillary sequencing robot (Applied Biosystems, Foster City, CA, USA). Cytochrome b mitochondrial gene sequences (479 bp) quality and presence of mixed infections (double peaks) was assessed using SnapGene Viewer 5.2.4 software (Insightful Science, San Diego, CA, USA, www. snapg ene. com; accessed on 20 November 2021). Lineage identification was carried out by BLAST searches in MalAvi [22] and GenBank databases with Megablast algorithm (www. ncbi. nlm. nih. gov/ genba nk/; accessed on 10 October 2021). Obtained DNA sequence information was compared with results of microscopic parasite identification. mounted with Entellan New™ [16]. Formalinized sam- ples were embedded in paraffin, and histologic sections of 2 μm thickness were prepared in Hematoxylin-Eosin (HE), Periodic-Acid-Shift (PAS), and Prussian Blue (PB) stain according to standard techniques at the Fachpraxis für Tierpathologie, Hartelstraße 30, D-80689 Munich, Germany (https:// www. tierp athol ogie- muenc hen. de). Histopathologic and cytopathologic evaluation of the samples was carried out with an Olympus BX41 light microscope under × 40, × 100 and × 1000 magnifica- tion. West Nile Virus, Usutu Virus Additional PCRs for West Nile Virus (WNV) and Usutu Virus were run on RNA extracts from frozen liver samples. WNV reverse transcriptase real-time PCR was carried out according to a modified protocol by Eiden et al. [25] (modifi- cation and validation thereof done at the Institute of Virology and Immunology, Mittelhäusern) detecting WNV lineage 1 and 2 genomes and including an internal amplification con- trol (eGFP-PCR) [25, 26]. The 25 µl reaction mix contained 12.5 µl “2x QuantiTect Probe RT-PCR Master Mix” (Qiagen), 400nM of forward primer WNV_Eiden_mod_F (5‘AGA AGT TCG TCT GCG TGA GC3’), 400nM of reverse primer WNV_Eiden_mod_R (5‘GCC CTC CTG GTT TCY TAG A3’) and 200nM of probe WNV_Eiden_mod_P (5‘FAMTGA CAA ACT TAG TAG TGT TTG TGA GGATT-TAMRA3’), 0.25 µl “QuantiTect RT Mix” (Qiagen) and 5 µl sample RNA. Usutu Virus reverse transcriptase real-time PCR was essentially done as described in [27]. All primers and probe were purchased from Microsynth AG, Balgach, Switzerland. All PCR reactions were run on a “7500 Fast RealTime PCR System” (Thermo Fisher Scientific), with the standard cycle Necropsy, cytology, histology Photographic documentation was performed with a ProgRes® C10 Plus digital camera and ProgRes® Captive- Pro v2.8.8 imaging software from Jenoptik Optical Sys- tems GmbH, Germany. Microbiology Mi bi l Microbiology diagnostics to detect possible concurrent infections were performed at the Vetsuisse Faculty, Uni- versity of Zurich. Microscopic examination of blood films Examination of stained films from heart and lung blood was performed using an Olympus BX51 light microscope equipped with an Olympus DP12 digital camera and Olympus DP-SOFT imaging software. Per blood film, 100 microscope fields were scanned at high magnifica- tion (× 1000) to prepare images of parasites and estimate parasitaemia intensity of erythrocytic stage. The latter was determined by counting the actual number of para- sites in 2000 erythrocytes and expressed as percentage according to [17]. Phanerozoite parasitaemia intensity was estimated using the similar methodology. Mainly, the actual number of single observed phanerozoites, which were seen after screening of a portion of blood smear containing 2000 erythrocytes, were counted. Morpho- logic identification of the parasite species was carried out on blood films at high magnification according to [6]. Exoerythrocytic stages were measured using ImageJ 1.53a software (National Institutes of Health, Bethesda, MD, USA, https:// imagej. nih. gov/ ij/ USA; accessed on 21 October 2021) [18]. Voucher parasite preparations con- taining blood stages (accession number of blood slides 49394NS and 49395NS) and tissue meronts (accession numbers of cytologic and histologic preparations 49396– 49403 NS) were deposited at Nature Research Centre, Vilnius. Chlamydiaceae d Extracted DNA from liver, heart, brain, lung, spleen, kid- ney and ventriculus were screened for Chlamydiaceae infections using the 23 S rRNA Chlamydiaceae-specific real-time PCR, resulting in an amplicon of 111 base pairs and using primers Ch23S-F, Ch23S-R and probe Ch23S- p [23]. Internal positive controls included enhanced green fluorescent protein (eGFP) [24]. Next generation sequencing for whole virome Frozen organ samples were pooled in four batches for Next Generation Sequencing (NGS) with batch 1 con- taining lungs and heart, batch 2 containing liver, batch 3 containing ventriculus, and batch 4 containing intestines. The organ samples were prepared according to previously established ViroScreen protocol at the Virology Insti- tute of the University of Zurich, Switzerland [28]. Briefly, 30 mg of homogenized by scalpel organs were diluted in 270 µl PBS and homogenized in the TissueLyser (Qiagen) for 2 min at 20 Hz. Then, the samples were enriched for viral nucleic acid, amplified by sequence-independent single primer amplification and sequencing libraries have been constructed. Sequencing was performed at Func- tional Genomics Center Zurich (ETH, Zurich, Switzer- land) on the Illumina NovaSeq machine in a 2 × 150 bp read length run. The raw sequencing reads (from 2.7 to 18.7 million reads per sample) were quality controlled and aligned in reference guided analysis and de novo assembly pipelines as described previously [29]. In sum- mary, quality-controlled reads were aligned to an inhouse database containing 61,620 complete viral genomes downloaded from the NCBI database, and assembled using megahit (version 1.1.3) with multiple k-mers [30] and metaspades (v3.12.0) [31]. Bacteriological culture Thawed frozen liver tissue was cultured using tryptone soy broth, Columbia agar with 7% sheep blood and bro- mothymolblue-lactose agar (Oxoid / Thermo Fisher Sci- entific, Waltham, MA, USA), incubated aerobically for 48 h at 37 °C. DNA extraction, PCR and sequencing for malaria parasite molecular characterization DNA was extracted from the drops of heart and lung blood and re-thawed tissue samples from the frozen retained samples stored on filter paper using an ammo- nium acetate protocol [19]. Then, a standard nested PCR protocol was applied to identify the cytochrome b line- age [20, 21]. Primers HaemNFI/HaemNR3 and HaemF/ HaemR2, as well as parameters of PCR, were the same as those described in the original protocol. Positive Pendl et al. Malaria Journal (2022) 21:148 Page 4 of 13 Page 4 of 13 protocol: 30 min at 48 °C, 10 min at 95 °C, then 45 times: 15 s at 95 °C, 1 min at 53 °C, 1 min at 70 °C. All samples were ana- lysed in duplicates. protocol: 30 min at 48 °C, 10 min at 95 °C, then 45 times: 15 s at 95 °C, 1 min at 53 °C, 1 min at 70 °C. All samples were ana- lysed in duplicates. the cloaca were slightly soiled with yellowish, opaque fluid identical with the liquid content of the cloaca. Inter- nal examination revealed a longitudinal rupture of the heart and multiple ruptures in the lungs with prominent amounts of blood dispersed into the surrounding cavi- ties. The intestines were filled with yellowish, opaque, seromucous fluid. Spleen, liver, and kidney were slightly enlarged and swollen. Serosal surfaces were thin, clear, and translucent. The brain showed signs of prominent haemorrhage and tissue disintegration in the frontal area of the cerebrum as well as haemorrhages in the occipito- basal part of the cerebellum suggesting an additional con- tre coup lesion. Parasitology l Cytologic and histologic examinations revealed intracel- lular stages of P. matutinum in many tissues, with lung and brain being the most affected organs with a dissemi- nated to diffuse distribution pattern. Lesser amounts of parasitic stages in oligofocal distribution were detected in the myocardium, the Pecten oculi, and skeletal mus- cle tissue (periocular, pectoral, and femoral muscle). Low parasitic load, often only detectable as single find- ings in cytologic preparations, was seen in peripheral blood, spleen, bone marrow, gastrointestinal tract, liver, and kidney. Parasites were easily detected in cytologic preparations, whereas in histologic preparations a clear visualization of the parasites often was hampered by the relative thickness of the tissue section compared to the monolayer of the cytologic specimens. Multiple scanning of the same view in various planes of focus was necessary for visualization and particularly difficult in haemic cells. Description of erythrocytic stages Parasitaemia was 0.3%. Most erythrocytic stages of P. matutinum were seen in thin films prepared from heart and lung blood with rare additional findings in the bone marrow. PCR diagnostics detected the lineage pLINN1 of this parasite (GenBank accession OL653715) and con- firmed the morphologic identification. A single haemos- poridian infection was present. Necropsy The bird was a female fieldfare with adult plumage and in slight hypertrophy of pectoral muscle condition (grade 2–3 at a semiquantitative scale from 0 = cachectic to 4 = obese) [32]. The inner secondary feathers were not grown to full-length most likely due to seasonal moult- ing. Search for ectoparasites was negative. The bird weighed 79 g, which is slightly underweight according to data available online (Birds of Switzerland, https:// www. vogel warte. ch/ en/ birds/ birds- of- switz erland/ field fare, accessed 10 January 2022). Rigor mortis was absent. External examination revealed multiple open fractures of the sternum and the frontal skull between the eyes with prominent haemorrhages and brain tissue protruding externally from under the third eyelids. Feathers around p p Trophozoites (Fig. 1a, b), growing (Fig. 1c–e) and mature (Fig. 1f) erythrocytic meronts and gametocytes (Fig. 1g, h) were seen. These were present mainly in mature erythrocytes, but trophozoites were also seen in immature, polychromatic erythrocytes. Multiple infec- tion of one erythrocyte with several growing parasites was common (Fig. 1a, b, d). Each early trophozoite pos- sessed a prominent nucleus and a readily visible cen- trally located vacuole within the cytoplasm (Fig. 1a). In growing trophozoites, the amount of nuclear material and cytoplasm was slightly increased, and two small vacuoles and a pigment granule were visible (Fig. 1b). Page 5 of 13 Pendl et al. Malaria Journal (2022) 21:148 Pendl et al. Malaria Journal (2022) 21:148 Growing meronts were characterized by prominent nuclei and cytoplasm with several readily visible vacu- oles (Fig. 1c, e). With increasing maturation, the size of the nuclei, the amount of cytoplasm and the number of vacuoles decreased, but the number of pigment granules increased. Mature meronts contained up to 30 merozo- ites. Pigment granules were gathered in a solid mass, and vacuoles were absent (Fig. 1f). Mature gametocytes were roundish, contained prominent nuclei and roundish or slightly oval pigment granules. These stages were often present in enucleated erythrocytes (Fig. 1g, h). Other morphologic details of blood stages coincided with for- mer parasite descriptions [6, 14] and are not repeated here. phanerozoites were significantly bigger than erythrocytic meronts (compare Fig. 1d, e and p–s). Interestingly, large (up to 47 μm in length), elongate, nearly mature phan- erozoites also were seen in the circulation (Fig. 1t, u). Some of them were phagocytized by mononuclear cells (Fig. 1w). Necropsy Such stages normally occur in endothelial cells of capillaries (see Fig. 2a–e). Like in the erythrocytic meronts, the young phanero- zoites developing in immune cells were markedly vacu- olated and possessed prominent nuclei and abundant amounts of cytoplasm (Fig. 1i, k, m–o). The conspicuous, mainly circular vacuoles were variable in size with the largest vacuoles reaching 1.5 μm in diameter. The amount of the cytoplasm and size of nuclei decreased as parasites matured, and vacuoles were absent in mature phanerozo- ites (Fig. 1j, l). Mature merozoites were roundish or oval bodies with a readily visible portion of cytoplasm and centrally located nuclei (Fig. 1v). They were 1.6 ± 0.2 μm in biggest diameter. (See figure on next page.) Fig. 1 Blood stages of Plasmodium (Haemamoeba) matutinum (lineage pLINN1) from a roadkill fieldfare Turdus pilaris: erythrocytic trophozoites (a, b), erythrocytic meronts (c–f), macrogametocyte (g), microgametocyte (h), phanerozoites (i–u, w) and phanerozoic merozoites (v). Multiple infection of same host cell with several parasites was common (a, b, d). Erythrocytic meronts often contained prominent vacuoles (c, e). Mature gametocytes often enucleated infected erythrocytes (g, h). Developing (i, k) and mature (j) phanerozoites in mononuclear leucocytes; note the prominent cytoplasm, nuclei, and vacuoles (i, k) and numerous mature phanerozoic merozoites, each containing a prominent nucleus and cytoplasm (j). Mature phanerozoite in a granulocyte (l); note the numerous nearly mature merozoites. Developing phanerozoites in mononuclear leucocytes (m-o); note the prominent vacuoles, which numbers increase as the parasites increase in size (compare m with n, o). Extracellular phanerozoites in heart blood at different stages of maturation (p-s); note that vacuolization of the cytoplasm decreases in maturing phanerozoites (compare p, q with r, s). Extracellular developing (t) and nearly mature phanerozoite (u), which are normally located in endothelial cells of capillaries (see Fig. 2b) but were washed out from the capillaries and present in the heart blood as free bodies; note numerous developing merozoites and still adjacent host‑cell nucleus. Mature phanerozoic merozoites (v); note oval shape of the parasites containing prominent nuclei and cytoplasm. Phanerozoite phagocytized by a mononuclear leucocyte (w) indicating an immune reaction against free phanerozoites intra vitam; note the degenerating nuclei and cytoplasm of the affected parasite. All images taken from cytologic preparations of heart blood in Wright Giemsa stain, except for the images m and o (cytologic imprint of the lung in Wright Giemsa stain). Simple arrows vacuoles, triangle arrowheads parasite nuclei, simple arrowheads pigment granules, simple wide short arrows developing merozoites, triangle wide arrowheads host cell nuclei, simple wide long arrows parasites, simple wide arrowheads mature merozoites. Scale bars 10 μm Description of exo‑erythrocytic stages In addition to erythrocytic stages (see description above), numerous phanerozoites (Fig. 1i–u) at different stages of maturation and mature merozoites (Fig. 1j, v) were detected in blood films prepared from heart and lung blood. Phanerozoite parasitaemia intensity was approxi- mately 0.1%. Apart from few granulocytes (Fig. 1l), the majority of phanerozoites were seen in mononuclear cells morphologically most likely pertaining to the mon- onuclear-phagocyte-system (MPS) of monocytes and macrophages (Fig. 1i–k, m–o). The largest phanerozoites reached 20 μm in diameter (Fig. 1k, n, o). Furthermore, extracellular roundish phanerozoites (Fig. 1p–s) and free mature phanerozoic merozoites (Fig. 1v) were common in the blood films, most likely resulting from either arti- ficial destruction of host cells during blood film prepa- ration or true rupture intra vitam. Their morphology was similar to equivalent stages located intracellularly. However, phanerozoites can be readily distinguished due to absence of pigment granules, which develop only in erythrocytic stages in malaria parasites. Additionally, Elongate phanerozoites (Fig. 2a–f, h–k) were exclu- sively seen in capillary endothelial cells of affected organs. They were particularly numerous in brain (Fig. 2a–f) and lung tissue with up to five phanerozoites per × 400 view field. Moderate (histology) to focally high (cytol- ogy) numbers were visible in the myocardium and pec- toral muscle (Fig. 2k). The parasite followed the shape of capillaries, which often were completely blocked by large parasites (Fig. 2a–e, i–k). Both roundish and elongate phanerozoites were seen in lungs and kidneys (Fig. 2j, l). Elongate young phanerozoites were markedly vacuo- lated with prominent nuclei and conspicuous amounts of cytoplasm. Vacuolization, nuclear size, and cytoplasmic amount decreased as the parasites matured. This is par- ticularly well visible in Fig. 2a, which shows two adjacent elongate phanerozoites at different stages of maturation. Vacuoles were seen in some nearly mature phanerozoites Pendl et al. Malaria Journal (2022) 21:148 Page 6 of 13 Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Pendl et al. Malaria Journal (2022) 21:148 Page 7 of 13 was seen in histologic sections of lung, liver, skeletal muscle, smooth muscle of the gizzard, as well as myo-, epi- and pericardium. The poorly demarcated infiltrates were present as perivascular cuffs or showed multifocal to disseminated interstitial distribution. In addition, the lung was moderately congested with mild fluid accumu- lation in aerated spaces and multifocal, mild to moderate perivascular oedema in beginning organization. Description of exo‑erythrocytic stages A single, small, well-demarcated granuloma surrounding small nematode cross-sections was present. (Fig. 2f). Blockage of vascular lumina with congestion (Fig. 2d) up to thrombus formation with cellular disin- tegration, nuclear fragmentation and fibrin deposition (Fig. 2c, g) was observed in brain, lung, myocardium, and skeletal muscle. For the first time in an avian malaria case, phanero- zoites were detected in endothelial cells of eye tissues. Mature stages were present in the endomysium of an oculomotoric skeletal muscle fibre (Fig. 2h) and within the Pecten oculi (Fig. 2i). In the spleen, prominent proliferation and nuclear degeneration (possibly due to beginning autolysis) was seen in the ellipsoidal reticular cells of the Schweiger-Sei- del sheaths (SSS). The surrounding periarteriolar white pulp (PWP) contained a prominent amount of plasma cells, many of which also showed mild signs of autolysis/ necrosis. There was a diffuse accumulation of golden- brown to blackish-brown pigment, which was mostly located intracellularly and partially birefringent under polarization. Search for parasitic structures revealed only very few intra-erythrocytic stages in cytology and was negative in histology. Cytology, Histology, Microbiology A differential leukocyte count performed on 200 blood cells in the heart blood film revealed 69% mononuclear cells, 25% heterophilic granulocytes, and 6% eosinophilic granulocytes. The mononuclear cells consisted of both small and large lymphocytes as well as monocytes, all of which showed cytomorphologic features of reactiv- ity such as increased cytoplasmic basophilia, vacuola- tion, bleb formation and a rather smooth delicate nuclear chromatin pattern. This similarity of morphology par- ticularly hampered the differentiation of equally sized large lymphocytes and monocytes, which therefore were categorized in one mononuclear cell portion. The eryth- roid line was characterized by a moderate left shift with regular appearance of intravascular mitoses and dispro- portionately high numbers of very immature stages with round cell shape, high nucleo-cytoplasmic (N/C) ratio and deep basophilic cytoplasm. A corresponding left shift of the erythroid line in the bone marrow was pre- sent. In addition to the above-mentioned phagocytosis of parasitic stages, erythrophagocytosis by monocytes was regularly seen. A severe mononuclear infiltration was seen in the cytologic preparations of lung tissue with par- ticularly high numbers of larger cells with cytologic char- acteristics of plasma cells, large lymphocytes and cells of the monocyte/macrophage system. Mild nematodiasis (Capillaria spp.) and cestodiasis with a single longitudinal section of each helminth was found in the intestinal lumen with mild signs of lym- phoplasmacytic reactivity in the villous cores of the adja- cent intestinal mucosa. Molecular testing for Chlamydiacaeae, West Nile and Usutu Virus, and bacterial culture from liver tissue was negative. In the NGS, neither in the reference align- ment to a database containing 61,620 complete viral genomes nor in de novo assembly viral reads/contigs were detected. Fig. 2 Phanerozoites of Plasmodium (Haemamoeba) matutinum in endothelial cells of brain (a–f), eye (h, i), lungs (j), pectoral muscle (k) and kidney (l). Two phanerozoites in different stages of maturation in a capillary of the frontal telencephalon (a); note that the younger phanerozoite (top) contains more vacuoles, larger nuclei and more cytoplasm than the nearly mature phanerozoite (bottom). Maturing phanerozoite in a capillary of the frontal telencephalon (b); note that the parasite completely blocks the capillary. Four phanerozoites at different stages of maturation in capillary endothelial cells of the frontal telencephalon (c); note the signs of cellular disintegration and nuclear fragmentation, whose exclusive occurrence in close neighbourhood to phanerozoites supports vascular blockage as cause of disintegration. Two phanerozoites in different stages of maturation in capillary endothelial cells of the molecular layer of the cerebellum (d); congestion of erythrocytes is visible. Maturing phanerozoite in a capillary endothelial cell of the mesencephalon (e). Mature phanerozoite in the frontal telencephalon (f); note that vacuolization is still visible in the maturing parasite. Fibrinoid microthrombosis in a capillary of the cerebellum molecular layer (g). Phanerozoite in a capillary endothelial cell of the endomysium of an oculomotoric skeletal muscle fibre, which is adjacent to the ocular bulb (h). Phanerozoite within a capillary endothelial cell of the Pecten oculi (i). Phanerozoite in capillaries of lung (j) and pectoral muscle (k); note the closely located nuclei of host cells. Phanerozoite in the kidney (l); note the markedly vacuolated cytoplasm. Images taken from cytologic imprints in Wright‑Giemsa stain (a–c, f, l) and histologic preparations in hematoxylin & eosin stain (d, e, g–k). Simple arrows vacuoles, triangle arrowhead nuclei of developing phanerozoites, triangle wide arrowheads host cell nuclei, simple wide arrows developing merozoites, triangle arrow microthrombus Scale bars 10 μm (See figure on next page.) Discussion Addition- ally, erythrocytic merogony is markedly synchronized, with meront maturation peaking in the morning and the cycle of the merogony being close to 24 h [5, 6, 34]. Vec- tors of the lineage pLINN1 are currently unknown, but likely to be Culex mosquitoes, as they were shown to be competent vectors of unidentified lineages of P. matuti- num in America and Europe [11, 35, 36]. This parasite lineage was found in naturally infected Culex mosqui- toes in Italy [37], however it remains to be proved that this parasite lineage completes sporogony and develops sporozoites in these mosquitoes. Conspicuous circular vacuoles were described in phan- erozoites and/or erythrocytic meronts of P. matutinum, P. giovannolai, Plasmodium griﬃthsi, Plasmodium lutzi and Plasmodium tejerai, which belong to the subgenus Haemamoeba [5, 6]. Similar vacuoles have been reported in zygotes, ookinetes, early oocysts and gametocytes of many species of haemosporidian parasites belonging to the families Plasmodiidae, Haemoproteidae, Leucocyto- zoidae and Garniidae [6, 14, 40, 41]. The origin and func- tion of such vacuoles, however, remains insufficiently understood. It is believed that they contain material, which plays a role in energy metabolism and is involved in the lipid metabolism of actively growing parasites. Similar to other fatty structures, this material might be washed out during alcohol fixation leaving a vacuole-like space in stained samples [6, 7, 14, 42]. Further studies are needed to elucidate the true nature of these struc- tures. Absence of vacuoles in mature phanerozoites in P. matutinum suggests that vacuolization is a feature of immature, growing phanerozoites. To date, a primary exo-erythrocytic development has not been described for any strain of P. matutinum [5, 6, 14]. This paper describes for the first time a secondary exo-erythrocytic cycle with characteristic development of phanerozoites after a natural infection in a wild bird for the lineage pLINN1 during single natural infection. The observed morphology and location of the exo-eryth- rocytic meronts were indistinguishable from those seen in canaries (Serinus canaria) experimentally infected with unknown lineages of P. matutinum isolated from the Common blackbird and redwing in Italy [5, 6, 10] and the Common blackbird in Switzerland [11]. Previous stud- ies reported the presence of phanerozoites in reticulo- endothelial cells of brain, liver, spleen, kidneys, lungs, heart muscle and bone marrow. According to our case report this list must be complemented with ocular struc- tures and skeletal muscle. Discussion The key results of this study comprise (i) the first report and description of exo-erythrocytic development of the P. matutinum lineage pLINN1 during single infection, (ii) the first report of this infection in the fieldfare, and (iii) Mild to moderate lymphohistiocytic to lymphoplas- macytic inflammation with signs of lymphoid necrosis Pendl et al. Malaria Journal (2022) 21:148 Page 8 of 13 Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Pendl et al. Malaria Journal (2022) 21:148 Page 9 of 13 Page 9 of 13 presence of conspicuous vacuolization in the cytoplasm of immature phanerozoites, the development of usu- ally > 100 merozoites in mature phanerozoites with up to > 300 merozoites in the largest phanerozoites. The latter feature together with the presence of phanerozoites in brain tissue distinguishes P. matutinum from the mor- phologically similar malaria parasite Plasmodium giovan- nolai, which also parasitizes species of the genus Turdus in Europe [5, 6, 38, 39] but remains non-characterized molecularly [13]. It is worth to mention that numerous elongate phanerozoites were seen in blood films (Fig. 1u). This suggests that mature large phanerozoites might be washed out from fixed tissues into the circulation dur- ing intense P. matutinum infection, but this observation remains speculative due to possible mechanical impact. However, G. Valkiūnas (unpublished, pers. obs.) has observed occasionally similar structures in blood films of Common blackbirds, whose were naturally infected with Plasmodium sp. in Europe, indicating that such process might occur naturally and may be worth more attention of researchers. The biological meaning of this phenom- enon remains unclear. the first report of phanerozoite development in ocular structures, suggesting that this infection contributes to avian road kills due to impaired vision. Plasmodium matutinum (pLINN1) is common in palearctic birds with a broad geographic distribution. Molecular characterization was developed using blood stages of the parasite isolated from the Thrush nightin- gale (Luscinia luscinia) [14]. This lineage is particularly common in species of the genus Turdus in Europe with the Common blackbird (Turdus merula) being the most common host [13]. This study expands the range of natu- ral avian hosts for this malaria infection by the fieldfare, a migratory bird species with broad palearctic distribution [33]. Blood stages of P. matutinum can be readily distin- guished due to marked vacuolization of the cytoplasm in trophozoites and growing meronts (Fig. 1a–e). Discussion Furthermore, massive infection of circulating leucocytes and tissue macrophages was seen for the first time during this infection. Himmel et al. [43] investigated the occurrence of vari- ous haemosporidian infections in a large sample of Eura- sian blackbirds and song thrushes (Turdus philomelos) whose were found dead in Austria. Co-infections of vari- ous haemosporidians predominated in these samples, and numerous new lineages of Plasmodium parasites were found. It was shown that P. matutinum (pLINN1) often caused high exo-erythrocytic meront intensi- ties in various organs. With the presence of cytomeres in maturing exo-erythrocytic meronts and the absence of conspicuous vacuolization in most of the illustrated Characteristic features of the phanerozoite devel- opment of P. matutinum include the development of roundish phanerozoites in large mononuclear cells and occasionally granulocytes, the presence of elongate phan- erozoites in endothelial cells of capillaries in various organs with particular prominence in brain and lung; the Pendl et al. Malaria Journal (2022) 21:148 Page 10 of 13 Page 10 of 13 exo-erythrocytic meronts in P. matutinum (pLINN1) two unusual characters of the exo-erythrocytic stages were reported. These features were not observed (cytomeres) or not characteristic (absence of vacuoles in growing meronts) in our study, which was based on a single P. matutinum (pLINN1) infection. These observations were in accordance with former studies dealing with this para- site morphospecies [5, 6, 10, 11, 35]. It is difficult to rule out that some of the described exo-erythrocytic stages, which were attributed to pLINN1 [43] might belong to other Plasmodium lineages, which could occur in co- infection. These observations raise questions for future research on exo-erythrocytic development of avian Plas- modium species, particularly in regard of the presence of cytomeres in developing exo-erythrocytic meronts of these pathogens. So far, cytomeres were not observed in tissue stages of avian malaria parasites [1, 5–7, 43]. an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slow driving car. Impairment of vascular perfusion of the Pecten oculi due to blockage by endothelial phanerozoites may have a direct impact on the eye function itself. The Pecten oculi is a unique structure of the avian eye composed of mul- tiple capillaries and larger blood vessels surrounded by pigment cells. It is assumed to serve as a nutritive organ for the avascular retina and to balance the intraocular microenvironment by regulation of pressure, pH, and physical stability of the vitreous body [45–51]. Discussion To prove this hypothesis, however, ophthalmologic examinations intra vitam would have been necessary. y Plasmodium matutinum (pLINN1) lineage is com- mon in wild birds in Austria [4, 13] and pathogenic for local endemic birds in New Zealand [12, 52], where it was probably introduced together with their Turdus host species. The same lineage was recently reported to cause lethal malaria in captive African penguins Spheniscus demersus and Lovebirds Agapornis roseicolli in Italy [37, 53], and Atlantic puffins Fratercula arctica in Switzer- land [54]. These and other exotic to Europe bird species likely are non-adapted to pLINN1 infection. It is worth to note that – similar to this case in the fieldfare—parasi- taemia was low during most reported mortalities, which raises suspicion of tissue damage by exo-erythrocytic stages as cause of death. Exo-erythrocytic merogony in various organs is reported to be the most striking his- tologic lesion in pet and aviary birds and is particularly prominent in non-adapted hosts [55]. Furthermore, it is considered to be key pathogenic stage in experimental infections of naïve poultry flocks with Plasmodium durae, Plasmodium gallinaceum, and Plasmodium octamerium [56]. Mortality in most of these cases was caused by cer- ebral dysfunction due to the early and prominent devel- opment of exo-erythrocytic phanerozoites in endothelial cells. The brain capillaries were occluded by the swol- len endothelial cells, preventing normal blood flow, and causing anoxic conditions resulting in clinical symptoms resembling cerebral stroke. Likewise, the foci of degener- ation and necrosis of single fibres seen in the cardiac and skeletal muscle in close proximity to blocked and deterio- rated vessels are considered a sequela of local ischaemia. Different strains of P. matutinum showed differences in virulence when inoculated to domestic canaries [5, 6, 36, 44]. High virulence and mortality were described in canaries after experimental exposure to Italian and Swiss strains [11, 38], whereas American strains seemed to be less aggressive with frequent recovery of the birds [5, 36]. Experimental inoculation with infected blood showed that domestic canaries were susceptible to a strain of pLINN1 isolated from the Thrush nightingale (Luscinia luscinia), but parasitaemia was low and mortality was not observed [14]. The virulence of this parasite in wild birds remains insufficiently understood. Corradetti et al. [10] reported the death of one redwing (Turdus iliacus) after experimental infection with an unknown lineage of P. Discussion matutinum and speculated that the stress of prolonged captivity keeping might have impaired the host parasite balance. This study shows that natural infection of the lin- eage pLINN1 is virulent and pathogenic in the free-living fieldfares without relationship to captivity stress. As the parasites matured and produced merozoites in immune cells (Fig. 1j), immune evasion from cellular immunity was suspected. The prominent presence of phanerozoite stages in several organs in comparison to the rather low parasitaemia further suggests that the examined fieldfare was not adapted to P. matutinum (pLINN1). The finding of large, nearly mature phanerozoites in the peripheral blood may represent an artificial contamination of the samples, as the blood films were taken from traumati- cally ruptured heart and lung tissue. Phagocytosis of such phanerozoites by monocytic cells, however, indicate an immune reaction intra vitam against phanerozoite laden endothelial cells, which are washed out from capillaries into the circulation during massive infections. Pulmonary oedema is one of the key findings of avian malaria in captive birds and could also be confirmed for the fieldfare. Right ventricular hypertrophy (RVH) due to hypoxic pulmonary arterial hypertension is a well-docu- mented sequela of Plasmodium species and Aegyptianella pullorum infections in poultry. It is caused by hypoxic pulmonary arterial vasoconstriction as a response to anaemia [56, 57]. The moderate left shift of the erythroid line seen in the heart blood and the bone marrow of the fieldfare indicates an increased erythropoietic activity, Multiple phanerozoites in samples of brain, skeletal muscle, and eye tissue, in combination with signs of vas- cular blockage and thrombus formation raise suspicion of Pendl et al. Malaria Journal (2022) 21:148 Pendl et al. Malaria Journal (2022) 21:148 Page 11 of 13 Page 11 of 13 as the nature of this cell type differs from mammals and remains not fully understood in avian species [60–63]. Prominent exo-erythrocytic development, phagocytosis of phanerozoites seen in circulating monocytes and con- current successful maturation of phanerozoites within leukocytes raises suspicion of both an active antiparasitic immune response and immunoevasive mechanisms play- ing a role in the pathogenesis of this malaria case. As this report is based on a single natural infection of unknown history with a co-infection with helminths, these con- siderations remain speculative. Targeted immunologi- cal studies under experimental conditions would be of interest to clarify a true correlation of these findings to an infection with P. Discussion matutinum, to characterize the anti- parasite response profile of the host immune system as reported for other apicomplexan pathogens [64], and to elucidate possible immunoevasive strategies which allowed the parasite to escape from cellular immunity. whose key trigger is peripheral tissue oxygen deficiency. Although the values for the PCV were not measured, these findings strongly indicate hypoxia most likely caused by anaemia and/or tissue malperfusion due to the diffuse blockage of capillary beds by phanerozoites. Liver and spleen were only slightly enlarged, inflam- matory infiltrates were mild to moderate, and parasitic stages and pigment granules were few to absent. Conse- quently, the organs also did not show a blackish pigmen- tation frequently reported in literature. This discoloration results from haemozoin accumulation in macrophages. Haemozoin is birefringent and negative on Prussian blue stain for iron, while haemosiderin is golden-brown and stains positively with Prussian blue [58]. Both pigments were present in low numbers. Mild gastrointestinal worm infections are a common finding in necropsies of wild birds with usually little impact on the general body condition and health. Com- pared to the prominent lesions in brain, heart, and lung related to the Plasmodium infection, the low-grade hel- minthiasis seen in the intestine and the lung was consid- ered of subordinate importance for the fatal roadkill. The concurrent seromucous diarrhoea, however, speaks for clinically manifest gastrointestinal disease. It may have been caused by the intestinal nematodiasis with or with- out an undetected gastrointestinal microbial co-infection and was possibly facilitated by the malarial infection [59]. This study shows that P. matutinum (pLINN1) is an aggressive malaria parasite, which can develop even in immune cells and is dangerous for non-adapted wild birds. This study supports formerly fragmental obser- vations that avian Haemamoeba malaria parasites can develop and produce merozoites in monocytes and mac- rophages [5, 6]. The true role of this infection as a poten- tial threat for wildlife bird populations remains to be investigated. Millions of birds are killed on roads due to collisions with vehicles each year [65]. This study shows that severe malaria infections likely contribute to such mortalities. Interestingly, examination of roadkill juvenile chaffinches Fringilla coelebs revealed exceptionally high (up to 7%) parasitaemia of Haemoproteus species in comparison to the same age bird species, which were mist-netted at the same area [6]. This indicates possible involvement of avian haemoproteosis in road mortalities. Discussion This is not unexpected due to recent findings of megalomeronts of Haemoproteus parasites in brain of naturally infected birds [3]. This limited available information suggests that avian haemosporidian infections are worth more attention as agents of avian diseases. The described case of severe P. matutinum (pLINN1) malaria in the field- fare emphasizes the importance of further studies on exo-erythrocytic stages of haemosporidian parasites as potential underestimated cause of fatal disease in wild bird populations in general, and in roadkill in particular. Clarification of a possible concurrent microbial dis- ease with special emphasis on West Nile Virus, Usutu Virus, and Chlamydiaceae was of particular interest, as the inflammatory patterns and organ distribution of lesions seen corresponded to patterns of these diseases described in literature [55]. Testing for microbial co-infections was negative in the extraintestinal compartment, and NGS was negative for both the extraintestinal and the intestinal compartment. This shows that the moderate to prominent inflammatory infiltrates observed in several organs outside the gastro- intestinal tract were related to the malaria infection. Like many parasites of the phylum Apicomplexa, infec- tions with Plasmodium species trigger a predominantly mononuclear inflammatory reaction consisting of vari- ous portions of lymphocytes, plasma cells, and mac- rophages/histiocytes. Depending on the host immunity and the pathogenicity of the lineages the inflammatory lesions vary from mild to dramatic. Severe reactions may be mistaken for lymphoid neoplasia [55]. The findings in the SSS and PWP of the spleen suggest a prominent reac- tion of the highly phagocytic reticular ellipsoid associated cells and an increased reactivity of the B-cells in the sur- rounding peri ellipsoid lymphocytic sheath. Interpreta- tion of the eosinophilia seen in the heart blood is limited Availability of data and materials Voucher preparations of blood and tissue stages of P. matutinum (pLINN1) were deposited at Nature Research Centre, Vilnius. The data generated during this study are included in this published article. 15. Chitty J. Sample taking and basic clinical pathology. In: Chitty J, Monks D, editors. BSAVA Manual of avian practice. Gloucester: British Small Animal Veterinary Association; 2018. pp. 172–86. 16. Pendl H, Kreyenbühl K. Clinical pathology. In: Poland G, Raftery A, editors. BSAVA Manual of backyard poultry medicine and surgery. Gloucester: British Small Animal Veterinary Association; 2019. pp. 85–104. References 1. Valkiūnas G, Iezhova TA. Keys to the avian malaria parasites. Malar J. 2018;17:212. 1. Valkiūnas G, Iezhova TA. Keys to the avian malaria parasites. Malar J. 2018;17:212. 2. Santiago‑Alarcon D, Marzal A. Avian malaria and related parasites in the tropics: ecology, evolution and systematics. Berlin: Springer Nature; 2020. 3. Duc M, Ilgūnas M, Kubiliūnaitė M, Valkiūnas G. First report of Haemopro- teus (Haemosporida, Haemoproteidae) megalomeronts in the brain of an avian host, with description of megalomerogony of Haemoproteus pastoris, the blood parasite of the Common starling. Anim (Basel). 2021;11:2824. 3. Duc M, Ilgūnas M, Kubiliūnaitė M, Valkiūnas G. First report of Haemopro- teus (Haemosporida, Haemoproteidae) megalomeronts in the brain of an avian host, with description of megalomerogony of Haemoproteus pastoris, the blood parasite of the Common starling. Anim (Basel). 2021;11:2824. 4. Himmel T, Harl J, Matt J, Weissenböck H. A citizen science‑based survey of avian mortality focusing on haemosporidian infections in wild passerine birds. Malar J. 2021;20:417. 4. Himmel T, Harl J, Matt J, Weissenböck H. A citizen science‑based survey of avian mortality focusing on haemosporidian infections in wild passerine birds. Malar J. 2021;20:417. 5. Garnham PCC. Malaria parasites and other Haemosporidia. Oxford: Black‑ well; 1966. 5. Garnham PCC. Malaria parasites and other Haemosporidia. Oxford: Black‑ well; 1966. 6. Valkiūnas G. Avian malaria parasites and other Haemosporidia. Boca Raton: CRC; 2005. 6. Valkiūnas G. Avian malaria parasites and other Haemosporidia. Boca Raton: CRC; 2005. 7. Atkinson CT, Thomas NJ, Hunter DB. Parasitic diseases of wild birds. Ames: Wiley‑Blackwell; 2008. 8. Ilgūnas M, Bukauskaitė D, Palinauskas V, Iezhova TA, Fragner K, Platonova E, et al. Patterns of Plasmodium homocircumﬂexum virulence in experi‑ mentally infected passerine birds. Malar J. 2019;18:174. 9. Vanstreels RET, de Angeli Dutra D, Ferreira‑Junior FC, Hurtado R, Egert L, Mayorga LF, et al. Epidemiology, hematology, and unusual morphologi‑ cal characteristics of Plasmodium during an avian malaria outbreak in penguins in Brazil. Parasitol Res. 2019;118:3497–508. Conclusions This study reports an active natural P. matutinum (line- age pLINN1) infection in the fieldfare. The exo-eryth- rocytic development of the pLINN1 infection was described for the first time. It consisted of phanerozoites, Pendl et al. Malaria Journal (2022) 21:148 Pendl et al. Malaria Journal (2022) 21:148 Page 12 of 13 which matured in circulating immune cells (monocytes/ macrophages and granulocytes) as well as in capillary endothelial cells of multiple organs. Conspicuous vacu- olization of the cytoplasm in phanerozoites and erythro- cytic meronts is a characteristic feature of the merogony during P. matutinum infection. 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https://openalex.org/W1983024157	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0035173&type=printable	English	null	APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing	PloS one	2,012	cc-by	11,416	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: K.M. was funded by a grant of the French National Research Agency (ANR-05-BLAN-0072-01). Additional funding was provided by the CNRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pascal.genschik@ibmp-cnrs.unistra.fr ¤ Current address: Vivocell Biosolutions GmbH, Graz-Andritz, Austria ¤ Current address: Vivocell Biosolutions GmbH, Graz-Andritz, Austria been shown to bind and recruit substrates. More recently, another subunit of the APC/C, APC10 has also been identified as a part of a catalytic module together with APC2 and CDH1 and to be directly involved in the substrate recognition step and poly- ubiquitin chain extension [6,7,8]. Katia Marrocco1, Marie-Claire Criqui1, Je´roˆ me Zervudacki1, Gregory Schott1,2, Herfried Eisler1¤, Aude Parnet1, Patrice Dunoyer1, Pascal Genschik1* Katia Marrocco1, Marie-Claire Criqui1, Je´roˆ me Zervudacki1, Gregory Schott1,2, Herfried Eisler1¤, Aude Parnet1, Patrice Dunoyer1, Pascal Genschik1* 1 Institut de Biologie Mole´culaire des Plantes, Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 2357, Conventionne´ avec l’Universite´ de Strasbourg, Strasbourg, France, 2 Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Abstract Background: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. Conclusions/Significance: Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4. Citation: Marrocco K, Criqui M-C, Zervudacki J, Schott G, Eisler H, et al. (2012) APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing. PLoS ONE 7(4): e35173. doi:10.1371/journal.pone.0035173 Editor: Miguel A. Blazquez, Instituto de Biologı´a Molecular y Celular de Plantas, Spain Editor: Miguel A. Blazquez, Instituto de Biologı´a Molecular y Celular de Plantas, Spain Received December 23, 2011; Accepted March 9, 2012; Published April 24, 2012 Received December 23, 2011; Accepted March 9, 2012; Published April 24, 2012 Copyright: 2012 Marrocco et al. PLoS ONE \| www.plosone.org The APC10 subunit of the APC/C complex physically and specifically interacts with DRB4 Mutations in different subunits of APC/C led to female gametogenesis defects (typically at the first mitosis) presumably due to the inability to degrade mitotic cyclins. In addition, the APC3b subunit has also been implicated in postembryonic differentiation at the meristems [15], whereas CCS52A, plant CDH1 orthologues, have been shown to control endoreduplication in rosette leaves and meristem maintenance in roots [16,17]. So far, only substrates of APC/C that regulates the cell cycle progression have been identified in plants. However, we have shown in a previous work that APC/C remains active in post-mitotic plant cells and that APC/C hypomorphic mutant lines exhibit severe developmental abnormalities such as disorga- nized vascular tissue, indicating a role for APC/C in plant vasculature development and organization [18]. More recently, Zheng et al. identified APC/C as a dual integrator controlling both Cyclin B1;1 (CYCB1;1) degradation and transcriptional regulation during male gametogenesis [19]. This work highlighted that APC/C is necessary to recruit RNA polymerase II to MIR159 promoters as in apc/c mutants the level of miR159 is reduced which leads to an accumulation of DUO1 transcripts and therefore of CYCB1;1 transcripts. APC10 is a core APC/C subunit that participates in the recruitment of substrates, though this is mainly achieved by APC/ C co-factors belonging to the CDC20/FIZZY or CDH1/FIZZY- RELATED families [4]. In Arabidopsis, six CDC20 genes and three CDH1 genes (called CCS52A1, CCS52A2 and CCS52B) have been predicted [14], and a recent study has demonstrated that in the CDC20 family only two isoforms, AtCDC20.1 and AtCDC20.2, seem to be functional [28]. We then tested by yeast two-hybrid and GST pull-down assays whether DRB4 interacts with these different co-factors. From this analysis, we conclude that DRB4 only interacts with APC10, but not with any of the other co-factor tested (Figure 1A and B). On the other hand, DRB4 belongs to a small multigenic family with five members in Arabidopsis, named DRB1 to 5 [23]. Therefore we tested whether APC10 is also able to interact with other members of this protein family. Using the yeast two-hybrid assay and BiFC, we could only detect an interaction between APC10 and DRB4 (Figure 1C and D, and data not shown). Moreover, this interaction was localized in the nucleus, in agreement with previously published sub-cellular localizations for DRB4 and APC10 [23,29]. The APC10 subunit of the APC/C complex physically and specifically interacts with DRB4 Expression analysis of APC/C members in mammals has revealed that this complex is not only expressed in dividing cells [10]. Contrary to CDC20, CDH1 is also expressed in differen- tiated cells such as neurons. It has been shown that APC/CCDH1 drives cell differentiation in muscles through the degradation of Skp2 and Myf5 [11]. More surprisingly, APC/C has been shown to have a crucial role in post-mitotic neurons at different levels like axonal growth and patterning. SnoN and Id2 are two nuclear proteins identified as targets of the APC/C in these processes, as in CDH1-depleted neurons, both proteins are stabilized [9]. In Drosophila, APC/C acts at the pre-synaptic level controlling synaptic size by targeting Liprin-/ for degradation [12]. Whereas in C. elegans, a recent study has shown that APC/C acts at the post- synaptic level controlling the number of glutamate receptor by targeting GluR1, a subunit of AMPA receptors, for degradation [13]. In order to identify novel APC/C substrates, a yeast two-hybrid screen was performed using APC10/DOC1 as the bait to screen a three week-old Arabidopsis seedlings library. Among 134 clones that were obtained, forty-two were re-confirmed as putative interactors after plasmid retransformation. Among them, the clone API2 (APC10 Interactor 2) was fished nine times in this screen and sequencing revealed that its cDNA corresponds to the At3g62800 gene encoding DRB4 (for Double-stranded RNA Binding protein 4). The full-length cDNA of DRB4 was amplified and cloned into the yeast two-hybrid vector allowing us to further confirm the interaction with APC10 (Figure 1A). To validate this interaction obtained in yeast, we performed in vitro pull down assays. For this purpose, we generated a GST protein-fusion with APC10 that was expressed in E. coli and subsequently purified. The DRB4 protein labelled with [35S] methionine was produced in vitro. These assays showed that DRB4 was pulled down with GST-APC10, but not with GST alone (Figure 1B). Finally, we also validated this protein interaction in planta by bimolecular fluorescence complementation (BiFC) experiments. Plasmids YN-APC10 and YC-DRB4 were co- bombarded into etiolated mustard hypocotyls, and a strong YFP signal was observed in the nucleus of examined cells (Figure 1C). In Arabidopsis, all different subunits of APC/C have been identified [14] and several studies support a key role of APC/C in the regulation of cell cycle [3]. The APC10 subunit of the APC/C complex physically and specifically interacts with DRB4 From these results we conclude that the interaction between APC10 and DRB4 is specific and occurs in the nuclei of plant cells. In order to identify new substrates of APC/C, we performed a yeast two-hybrid screen using APC10/DOC1 subunit as a bait. Among all putative interactors that were obtained, we focused our attention on a protein, which belongs to the Double-stranded RNA Binding protein (DRB) family, named DRB4. DRB proteins associate with Dicer-like proteins (DCLs) and are involved, for instance, in the biogenesis of miRNA or trans-acting siRNA (tasiRNA) in plants [20,21,22]. DRB4 contains two dsRNA binding motifs (dsRBD1 and 2) in its N-terminal half [23]. It interacts with DCL4, one of the four Dicer-like proteins present in Arabidopsis, to generate 21 nt-long sRNAs (such as tasiRNA, DCL4-dependent miRNAs or viral derived siRNAs) from endogenous or exogenous dsRNAs [21,22,24,25,26]. In a recent work, DRB4, and more precisely its dsRBD2 motif, was shown to be essential for DCL4 activity in vitro [27]. Here, we show that APC10 specifically interacts with DRB4, that both genes have similar expression profiles and that DRB4 protein overaccumu- lates in apc10 hypomorphic mutants. All together this work provides novel insights into APC/C functions outside of the cell Introduction The ubiquitin-26S proteasome system (UPS) is the major regulator to control the abundance of key factors and enzymes in all eukaryotes [1]. In higher plants, the UPS plays a central role in cell cycle regulation, hormone signalling, development, chromatin regulation or response to environmental stresses among others [2,3]. Targets of the UPS are first poly-ubiquitylated by the sequential action of three enzymes (E1s, E2s and E3s) and then degraded by the 26S proteasome. The E3 enzymes (also called Ubiquitin protein Ligases) play the central role in this mechanism as they specifically recognise and select substrates. The Anaphase Promoting Complex/Cyclosome (APC/C) is a conserved multi- subunit E3 ligase, composed of at least 11 core subunits and a co- activator protein from the CDC20/FIZZY or CDH1/FIZZY- RELATED families [4,5]. APC2 and APC11 constitute the catalytic module of the enzyme, whereas CDC20 and CDH1 have The APC/C is a key regulator of the cell cycle transitions that especially acts at the metaphase to anaphase transition and at the exit from mitosis [5]. During prometaphase, spindle-assembly- checkpoint proteins such as MAD2 and BUBR1 are activated at kinetochores and inhibit by sequestrating the APC/CCDC20. In metaphase, when all kinetochores are attached to microtubules, APC/CCDC20 becomes activated and promotes the degradation of the anaphase inhibitor PDS1/SECURIN and thereby activates the protease separase. Separase then cleaves cohesin complexes and initiates sister-chromatid separation. After anaphase, APC/ CCDH1 mediates the final degradation of mitotic B-type cyclins which leads to Cyclin-Dependent Kinase 1 (CDK1) inactivation as PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e35173 1 April 2012 \| Volume 7 \| Issue 4 \| e35173 APC/C-Mediated Degradation of DRB4 cycle, and connects two important regulatory pathways, selective protein degradation and RNA silencing. well as many other cell cycle regulators such as Plk1, Aurora kinases, Tpx2, BUB1 or CDC20 among others and thus enables exit from mitosis [5]. Moreover during G1, the APC/C remains active and plays critical roles in maintaining G1 phase and controlling the onset of DNA replication, thus protecting chromosomal integrity [9]. The second dsRNA binding domain of DRB4 is necessary for its interaction with APC10 To further understand how APC10 interacts with DRB4, we generated deleted versions of DRB4 that were tested in yeast two- hybrid assays. First, we observed that DRB4 full-length is able to dimerize (Figure 2). Moreover, our results showed that the same domain of DRB4 that is responsible for its dimerization is also required for its interaction with APC10. Thus the interaction between both proteins is lost with the deleted versions D2 and D3 of DRB4, which lack the two dsRBDs, but is maintained when only the first dsRBD is absent (deletion D1). This suggests that the second dsRBD is necessary for this interaction. However, this domain alone (deletion D7) does not interact with DRB4 or April 2012 \| Volume 7 \| Issue 4 \| e35173 PLoS ONE \| www.plosone.org 2 APC/C-Mediated Degradation of DRB4 Figure 1. Specific interaction between APC10 and DRB4 occurs in vitro and in vivo. (A) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. DRB4 was fused to the binding-domain (BD) whereas APC10, CDC20-1/-2/-3/-4 and CCS52A1/A2/B were fused to the activation domain (AD). Empty BD and AD vectors were used as negative controls. (B) [35S]methionine-labelled DRB4 was incubated with recombinant GST-APC10, GST-CDC20-1/-2/-3 or GST alone. After several washes, proteins were affinity-purified on glutathione-Sepharose beads, and loaded on an acrylamide gel. The pulled-down proteins were analyzed by autoradiography. The same result was obtained in three independent experiments. (C) Bimolecular fluorescence complementation showed APC10 and DRB4 interaction in planta. Recombinant YN-APC10 and YC-DRB1/2 or 4 were co-bombarded together with a NLS-CFP construct into 4 day-old mustard seedlings. Fluorescence was observed using an E800 fluorescence microscope. YN and YC alone were used as negative controls. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. A strong YFP signal was observed in the nucleus of 91% of examined cells (64/70). No fluorescence signal was obtained after bombardment with the following plasmid combinations YN-+YC-DRB4 and YN-APC10+YC- or with YN-APC10+YC-DRB1 and YN-APC10+YC-DRB2. (D) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. APC10 was fused to the BD and DRB1/2/3/4 and 5 were fused to the AD. Empty BD and AD vectors were used as negative controls. doi:10.1371/journal.pone.0035173.g001 Figure 1. Specific interaction between APC10 and DRB4 occurs in vitro and in vivo. (A) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. APC10 and DRB4 exhibit similar expression patterns To further investigate the relevance of the APC10-DRB4 interaction, we compared the expression profiles of both genes in different plant tissues. It was previously described by using GUS reporter lines, that DRB4 was specifically expressed in the vasculature, the root and the shoot apical meristem [30]. Thus, we generated Arabidopsis transgenic lines expressing the GUS gene under the control of the APC10 promoter region. A 1500 bp region upstream of the ATG in addition to the first exon, the first intron and the beginning of the second exon of APC10 was cloned in frame with the GUS reporter gene. Stable transgenic lines were generated and twenty independent lines were selected for a unique copy of the transgene. Five representative lines were further analyzed in details. Staining for detection of GUS activity revealed a strong expression in vascular tissues in leaves, hypocotyls and The second dsRNA binding domain of DRB4 is necessary for its interaction with APC10 DRB4 was fused to the binding-domain (BD) whereas APC10, CDC20-1/-2/-3/-4 and CCS52A1/A2/B were fused to the activation domain (AD). Empty BD and AD vectors were used as negative controls. (B) [35S]methionine-labelled DRB4 was incubated with recombinant GST-APC10, GST-CDC20-1/-2/-3 or GST alone. After several washes, proteins were affinity-purified on glutathione-Sepharose beads, and loaded on an acrylamide gel. The pulled-down proteins were analyzed by autoradiography. The same result was obtained in three independent experiments. (C) Bimolecular fluorescence complementation showed APC10 and DRB4 interaction in planta. Recombinant YN-APC10 and YC-DRB1/2 or 4 were co-bombarded together with a NLS-CFP construct into 4 day-old mustard seedlings. Fluorescence was observed using an E800 fluorescence microscope. YN and YC alone were used as negative controls. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. A strong YFP signal was observed in the nucleus of 91% of examined cells (64/70). No fluorescence signal was obtained after bombardment with the following plasmid combinations YN-+YC-DRB4 and YN-APC10+YC- or with YN-APC10+YC-DRB1 and YN-APC10+YC-DRB2. (D) Yeast two-hybrid analyses were performed by mating on non-selective (-LW) and selective (-LWA) media. APC10 was fused to the BD and DRB1/2/3/4 and 5 were fused to the AD. Empty BD and AD vectors were used as negative controls. doi:10.1371/journal.pone.0035173.g001 APC10, suggesting that dsRBD2 is necessary but not sufficient for either DRB4 dimerization or its interaction with APC10. roots, and in the shoot apical meristem (Figure S1). Surprisingly for a gene involved in cell cycle function, its expression was not detected in meristematic tissue within the apical region of root tips. However, this observation is in agreement with a recent expression analysis of APC10 [29], and is also consistent with the expression profile of APC6, another APC/C subunit [31]. Our results together with published data show that APC10 and DRB4 exhibit overlapping expression profiles. PLoS ONE \| www.plosone.org DRB4 is a novel substrate of the APC/C complex Northern blot analysis (upper panels) of DRB4 mRNA accumula- tion in flower extracts from Col-0 and two independent transgenic lines expressing DRB4 under the control of the strong 35S promoter (referred hereafter as lines DRB4 OE-4 and -27). Accumulation of EF1a mRNA is used as a loading control. Western blot analysis (lower panel) performed using an antibody directed against DRB4. The asterisk indicates a non-specific cross-reacting band that can also be used as a loading control. Pictures of 5 week-old Col-0 and DRB4 OE-27 plants. (B) Three week-old DRB4 OE-27 seedlings were incubated in liquid medium supplemented or not with 100 mM MG132. After 4 h and 6 h of incubation, seedlings were collected and total protein extracted. Western blot was performed using antibodies against DRB4 or TSN as a loading control. (C) Western blot analysis of DRB4 accumulation in flower extracts from Col-0, drb4, dcl4, DRB4 OE-27 line, RNAi APC10-38 and RNAi APC6-20 lines. Coomassie staining was used as a loading control (LC). doi:10.1371/journal.pone.0035173.g003 Figure 3. DRB4 over-accumulates after MG132 treatment and in APC/C RNAi lines. (A) Characterization of DRB4 overexpressing lines. Northern blot analysis (upper panels) of DRB4 mRNA accumula- tion in flower extracts from Col-0 and two independent transgenic lines expressing DRB4 under the control of the strong 35S promoter (referred hereafter as lines DRB4 OE-4 and -27). Accumulation of EF1a mRNA is used as a loading control. Western blot analysis (lower panel) performed using an antibody directed against DRB4. The asterisk indicates a non-specific cross-reacting band that can also be used as a loading control. Pictures of 5 week-old Col-0 and DRB4 OE-27 plants. (B) Three week-old DRB4 OE-27 seedlings were incubated in liquid medium supplemented or not with 100 mM MG132. After 4 h and 6 h of incubation, seedlings were collected and total protein extracted. Western blot was performed using antibodies against DRB4 or TSN as a loading control. (C) Western blot analysis of DRB4 accumulation in flower extracts from Col-0, drb4, dcl4, DRB4 OE-27 line, RNAi APC10-38 and RNAi APC6-20 lines. Coomassie staining was used as a loading control (LC). doi:10.1371/journal.pone.0035173.g003 Figure 3. DRB4 over-accumulates after MG132 treatment and in APC/C RNAi lines. (A) Characterization of DRB4 overexpressing lines. DRB4 is a novel substrate of the APC/C complex DRB4 loss-of-function mutations exhibit a zippy phenotype with elongated and downward curled leaves due to reduced accumu- lation of TAS3 tasiRNAs [21,22]. First, we decided to generate Arabidopsis lines that over-accumulate DRB4 protein to evaluate whether this would also compromise normal plant development. The full-length cDNA of DRB4 was cloned downstream of the CaMV 35S promoter and Arabidopsis plants were transformed with this construct. Thirty independent lines were selected and PLoS ONE \| www.plosone.or PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e35173 3 APC/C-Mediated Degradation of DRB4 Figure 2. Mapping of interaction domains between APC10 and DRB4. (A) Schematic representation of DRB4 deletion constructs generated and fused to the activation domain (AD). Interaction with full-length APC10 or DRB4 fused to the binding-domain (BD) was then scored by yeast two-hybrid assays (see B). A summary of three independent assays is indicated on the right. +, interactions scored based on growth on selective media. 2, no growth on selective media. (B) One of the three yeast two-hybrid assays between DRB4 or APC10 and DRB4 deleted versions. After mating, yeast was grown at 28uC for 3 days on non-selective (2LW) or strong selection (2LWA) media. Empty AD and BD vectors were included as negative controls. doi:10.1371/journal.pone.0035173.g002 27 line to treat three week-old seedlings with the proteasome inhibitor MG132 and samples were collected after 4 and 6 hours Figure 2. Mapping of interaction domains between APC10 and DRB4. (A) Schematic representation of DRB4 deletion constructs generated and fused to the activation domain (AD). Interaction with full-length APC10 or DRB4 fused to the binding-domain (BD) was then scored by yeast two-hybrid assays (see B). A summary of three independent assays is indicated on the right. +, interactions scored based on growth on selective media. 2, no growth on selective media. (B) One of the three yeast two-hybrid assays between DRB4 or APC10 and DRB4 deleted versions. After mating, yeast was grown at 28uC for 3 days on non-selective (2LW) or strong selection (2LWA) media. Empty AD and BD vectors were included as negative controls. doi:10.1371/journal.pone.0035173.g002 Figure 3. DRB4 over-accumulates after MG132 treatment and in APC/C RNAi lines. (A) Characterization of DRB4 overexpressing lines. DRB4 is a novel substrate of the APC/C complex Northern blot analysis (upper panels) of DRB4 mRNA accumula- tion in flower extracts from Col-0 and two independent transgenic lines expressing DRB4 under the control of the strong 35S promoter (referred hereafter as lines DRB4 OE-4 and -27). Accumulation of EF1a mRNA is used as a loading control. Western blot analysis (lower panel) performed using an antibody directed against DRB4. The asterisk indicates a non-specific cross-reacting band that can also be used as a loading control. Pictures of 5 week-old Col-0 and DRB4 OE-27 plants. (B) Three week-old DRB4 OE-27 seedlings were incubated in liquid medium supplemented or not with 100 mM MG132. After 4 h and 6 h of incubation, seedlings were collected and total protein extracted. Western blot was performed using antibodies against DRB4 or TSN as a loading control. (C) Western blot analysis of DRB4 accumulation in flower extracts from Col-0, drb4, dcl4, DRB4 OE-27 line, RNAi APC10-38 and RNAi APC6-20 lines. Coomassie staining was used as a loading control (LC). doi:10 1371/journal pone 0035173 g003 Figure 2. Mapping of interaction domains between APC10 and Figure 2. Mapping of interaction domains between APC10 and DRB4. (A) Schematic representation of DRB4 deletion constructs generated and fused to the activation domain (AD). Interaction with full-length APC10 or DRB4 fused to the binding-domain (BD) was then scored by yeast two-hybrid assays (see B). A summary of three independent assays is indicated on the right. +, interactions scored based on growth on selective media. 2, no growth on selective media. (B) One of the three yeast two-hybrid assays between DRB4 or APC10 and DRB4 deleted versions. After mating, yeast was grown at 28uC for 3 days on non-selective (2LW) or strong selection (2LWA) media. Empty AD and BD vectors were included as negative controls. doi:10.1371/journal.pone.0035173.g002 Figure 2. Mapping of interaction domains between APC10 and DRB4. (A) Schematic representation of DRB4 deletion constructs generated and fused to the activation domain (AD). Interaction with full-length APC10 or DRB4 fused to the binding-domain (BD) was then scored by yeast two-hybrid assays (see B). A summary of three independent assays is indicated on the right. +, interactions scored based on growth on selective media. 2, no growth on selective media. (B) One of the three yeast two-hybrid assays between DRB4 or APC10 and DRB4 deleted versions. After mating, yeast was grown at 28uC for 3 days on non-selective (2LW) or strong selection (2LWA) media. Effects of DRB4 over-accumulation on heterochromatic siRNAs In the tasiRNA pathway, miRNA-loaded RISC mediate the initial cut of non-coding TAS precursor transcripts [32,33,34,35]. This, in turn, promotes the tasiRNA biogenesis cascade through conversion by RDR6 of the cleaved precursors into long-dsRNA that is processed by DCL4 into 21 nt-long tasiRNAs in a process that also requires DRB4 [32,36,37,38,39]. Consequently, tasiRNA targets over-accumulate in both dcl4 and drb4 mutants [22]. Therefore, we decided to test the accumulation of tasiRNA and their targets in the DRB4 OE line and APC/C hypomorphic lines that also over-accumulate DRB4, as well as miR173, which is the miRNA triggering the synthesis of TAS1 tasiRNA. However, we could not detect a difference in the accumulation of TAS1 tasiRNA (Figure 4A), miR173 (Figure 4A) or TAS3 tasiRNA (data not shown) in the DRB4 OE-27 line nor in APC/C hypomorphic lines compared to wild type plants, whereas 21-nt tasiRNA level was significantly reduced in drb4 and absent in dcl4 mutants (Figure 4A). Probing for miR159 accumulation revealed that the DCL1- dependent miRNA pathway was also not affected in any of the tested mutants (Figure 4A). It is also known that some miRNAs derive from DCL4-mediated processing of pri-miRNA precursors [40]. We therefore tested the accumulation of miR822 – a DCL4- dependent miRNA - in our different backgrounds. We were not able to detect any difference in the accumulation of miR822 in the DRB4 OE-27 line nor in APC/C hypomorphic lines compared to wild type plants (data not shown). It was recently published that DRB2 and DRB4 have antagonistic impact on RNA polymerase IV (polIV)-dependent siRNA (p4-siRNAs) levels [40]. Indeed, loss of DRB2 resulted in increased accumulation of p4-siRNAs, whereas drb4 mutant exhibited reduced p4-siRNAs levels, although the extent of this reduction was variable. Moreover, transgenic plants overexpress- ing DRB2 mimicked drb4 mutants, and exhibited reduced p4- siRNA levels. Based on those findings, we decided to monitor the accumulation of a set of 24 nt heterochromatic siRNAs in our hypomorphic APC10 or APC6 lines together with the DRB4- overexpressing plants. In agreement with previous results [40], TR2258 and siRNA02, two 24 nt-long siRNAs produced from polIV-dependent loci [41] were down-regulated in dcl4 and drb4 mutants (Figure 6). However, their accumulation in our transgenic lines was not affected compared to wild type plants suggesting that over-accumulation of DRB4 does not impact p4-siRNAs levels. DRB4 is a novel substrate of the APC/C complex Empty AD and BD vectors were included as negative controls. doi:10.1371/journal.pone.0035173.g002 doi:10.1371/journal.pone.0035173.g003 27 line to treat three week-old seedlings with the proteasome inhibitor MG132 and samples were collected after 4 and 6 hours of treatment. Total proteins were extracted and western blots were performed using antibodies against DRB4 and RNA binding protein Tudor-SN (TSN) as a loading control (Figure 3B). The increase of DRB4 protein accumulation observed in MG132- treated samples, but not in mock-treated seedlings, suggests that DRB4 protein is degraded by the 26S proteasome. analyzed at the molecular level for DRB4 expression. Figure 3A presents the results of two independent lines, accumulating either somewhat similar (line DRB4 OE-4) or strongly increased (line DRB4 OE-27) DRB4 levels in comparison to wild-type control plants. It is noteworthy that endogenous DRB4 transcripts are difficult to detect on RNA gels. In contrast with drb4 null mutation, over accumulation of DRB4 did not produce any obvious phenotypic alterations compared to wild type during the whole plant development (Figure 3A and data not shown). Based on the above result, we reasoned that plants defective for APC/C activity should accumulate more DRB4 protein than wild- type plants. Loss-of-function mutations in APC/C subunits lead to early arrest during gametogenesis in Arabidopsis, therefore to test our hypothesis, we used our previously established hypomorphic RNAi lines for two APC/C subunits, APC6 and APC10, showing reduced APC/C activity [18]. Western blot analysis performed on flower extracts revealed an over-accumulation of DRB4 in both If DRB4 is a substrate of the APC/C, it should be degraded by the 26S proteasome. Endogenous DRB4 protein can easily be detected in plant inflorescences (Figure 3A), but not in seedlings, a plant material in which the proteasome activity can be pharmacologically inhibited. Therefore we used the DRB4 OE- PLoS ONE \| www.plosone.org April 2012 \| Volume 7 \| Issue 4 \| e35173 4 APC/C-Mediated Degradation of DRB4 APC10 and APC6 hypomorphic RNAi lines compare to wild-type plants. This increased accumulation of DRB4 in genetic backgrounds where APC/C activity is reduced strongly suggests that DRB4 is indeed a genuine new target of the APC/C. upper panels, respectively). As expected, increased levels of viral RNA were detected in drb4. However, in plants over-accumulating DRB4, viral RNA accumulation and vsiRNA patterns were similar to those detected in wild-type plants suggesting that overexpression of DRB4 does not significantly affect the antiviral RNA silencing pathway. Effects of DRB4 over-accumulation on heterochromatic siRNAs We next checked the accumulation of 24 nt siRNAs SimpleHAT2 and siRNA1003, two sRNAs that require, in addition to polIV, the activity of RNA polymerase V (polV) for their production [41,42]. Interestingly, these polIV/polV-dependent siRNAs were not affected by dcl4 or drb4 mutations but showed a slight but consistent reduction in hypomorphic APC10 or APC6 lines (Figure 6). However, as this decrease in their accumulation was not observed in our DRB4 overexpressing line, it suggests that this apparent down-regulation is not directly related to the increased levels of DRB4 in APC10/APC6 hypomorphic mutants. Using quantitative-PCR (qPCR), we also analyzed the accu- mulation of TAS1 and TAS3 targets in the different genetic backgrounds (Figure 4B). The upper panel shows the level of endogenous APC10, APC6 and DRB4 transcripts in the different lines. The levels of APC10 and APC6 are reduced in their respective hypomorphic lines, whereas the transcript level of DRB4 is unchanged in these lines. With the exception of a slight but significant increase of transcript level for TAS targets in drb4 and dcl4 mutants, no significant difference was observed in DRB4 OE- 27 and APC/C hypomorphic lines. Therefore, from these experiments we can conclude that over-accumulation of DRB4 does not affect the tasiRNA pathway. Discussion DRB4 is the first non-cell cycle APC/C complex substrate in plants DRB4 over-accumulation does not affect the siRNA pathway Effects of DRB4 over-accumulation on heterochromatic siRNAs PLoS ONE \| www.plosone.org DRB4 is the first non-cell cycle APC/C complex substrate in plants p For many years, the APC/C complex was only known as the major E3 ubiquitin ligase regulating the cell cycle. It is just only recently that new functions of APC/C have been described in the organization, size and growth of differentiated tissues and cells, such as neurons [9,11,12,13]. In these processes several new APC/ C targets have been identified. Here, we describe for the first time a substrate of the plant APC/C which is not involved in the regulation of cell cycle. Our results have shown that DRB4 interacts specifically with APC10, is able to homodimerize and that the 2nd dsRBD is necessary for this interaction as well as for DRB4 homodimerization. Interestingly, this domain seems to be very important for DRB4 function as it is essential for dsRNA binding and for DCL4 interaction and activity [27]. By western blot assays it was also shown that DRB1 (also known as HYL1) is able to homodimerize and even to heterodimerize with other DRB proteins, though these interactions were weaker than with DCL1 [23]. Moreover, a recent study has highlighted an antagonistic role of DRB2 and DRB4 on RNA polymerase IV-dependent siRNA levels [40]. Hence, it was shown that in drb2 mutant the level of p4- siRNA is significantly increased, whereas in drb4 or in a DRB2 over-accumulating line this level is reduced. These results support that DRB proteins are part of multiple protein complexes with different functions. Moreover, maintaining proper DRB proteins accumulation might be of importance as these proteins, through Viral infection is not affected by DRB4 over-accumulation Viral infection is not affected by DRB4 over-accumulation The antiviral RNA silencing pathway also requires DCL4 and DRB4 for production of viral-derived siRNA (vsiRNA). It was previously shown that in drb4 and dcl4 mutants, accumulation of those 21-nt vsiRNAs were reduced or absent, respectively, and that, concomitantly, accumulation of 22-nt and 24-nt vsiRNAs was strongly increased [30]. To test the effect of DRB4 over- accumulation on viral infection, DRB4 OE lines were infected with modified Tobacco rattle virus (TRV-PDS) where the RNA2- encoded 2b and 2c sequences were replaced by a fragment of the Arabidopsis phytoene desaturase (PDS) gene. This virus triggers the appearance of a strong photobleaching phenotype in infected tissues due to virus-induced gene silencing (VIGS) of the PDS [24]. Figure 5A illustrates the photobleaching phenotype in wild type, drb4 and DRB4 OE plants 14 days post-inoculation. The extent and consistency of VIGS was not altered in DRB4 OE lines compared to WT- or drb4-infected plants. Northern blot analysis was then performed on RNA extracted from infected leaves using a PDS probe to detect viral RNA and vsiRNA (Figure 5B, the two April 2012 \| Volume 7 \| Issue 4 \| e35173 PLoS ONE \| www.plosone.org 5 APC/C-Mediated Degradation of DRB4 Figure 4. Over-accumulation of DRB4 does not affect trans-acting siRNA biogenesis and their targets accumulation. (A) Northern blot analysis of various sRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. Trans-acting siRNA and miRNA accumulation were detected using a TAS1 tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (B) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against APC10, APC6, DRB4 and three target genes of the tasiRNA pathway (TAS1 target, ARF3 and ARF4) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each. doi:10.1371/journal.pone.0035173.g004 APC/C-Mediated Degradation of DRB4 Values are normalized to miR159 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1. doi:10.1371/journal.pone.0035173.g005 Figure 6. Reduced APC/C activity slightly affect polIV/polV- dependent heterochromatic siRNA accumulation. Northern blot analysis of polIV-dependent (siRNA02, TR2258) or polIV/polV-depen- dent (siRNA1003, simpleHAT) siRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. miR173 accumulation is used here as a loading control. Values are normalized to miR173 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1. doi:10.1371/journal.pone.0035173.g006 Figure 5. Viral infection of drb4 and DRB4 OE line. (A) Pictures from infected plants with the TRV-PDS virus, 14 days post-infection (dpi). Scale bar: 1 cm. (B) Northern blot analysis of TRV-PDS viral RNA or viral-derived siRNA accumulation in Col-0, drb4 and DRB4 OE-27 line using a PDS specific probe. TAS1 tasiRNA accumulation was detected using a siRNA255 probe. miR159 accumulation was used here as a loading control. Values are normalized to miR159 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1. doi:10.1371/journal.pone.0035173.g005 early step of the pathway. While in this study a clear reduction of miR159 in apc mutants was observed, in our case we could not detect any change in the miR159 level between wild type and APC hypomorphic lines (Figure 4). This discrepancy can be explained by the fact that we used different tissues to analyze sRNA accumulation and that the effect of APC/C on miR159 might be specific to pollen development. homodimerization or heterodimerization, could compete for binding to dicer proteins and thus modulate their activity. g p y In plants, the APC/C has only been involved in cell cycle regulation and endoreduplication [15,16,17,18,43,44,45]. Here, we identified a new substrate of the APC/C complex, which is not involved in the regulation of the cell cycle but in siRNA biogenesis pathways. A recent work has also shown a link between APC/C and miRNA biogenesis pathway during male gametophyte development [19]. Analyses of apc8-1 and apc13-2 mutants revealed that APC/C was necessary for tricellular pollen development and that it was involved in the recruitment of RNA polymerase II to the miR159 promoter. In fact, the level of miR159 was reduced in these apc mutants leading to an increase in DUO1 expression. Figure 4. Over-accumulation of DRB4 does not affect trans-acting siRNA biogenesis and their targets accumulation. (A) Northern blot analysis of various sRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. Trans-acting siRNA and miRNA accumulation were detected using a TAS1 tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (B) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against APC10, APC6, DRB4 and three target genes of the tasiRNA pathway (TAS1 target, ARF3 and ARF4) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each. doi:10.1371/journal.pone.0035173.g004 APC/C-Mediated Degradation of DRB4 DUO1 being a transcription factor activating CYCB1;1, they observed an increased expression of this cyclin in these mutants, which defines the APC/C complex as a factor regulating both transcription and degradation of CYCB1;1. Though it is still unclear if APC/C acts directly or indirectly on the Pol II recruitment to miR159 promoter, this work established a role of the APC/C in sRNA biogenesis and most likely at a very Figure 4. Over-accumulation of DRB4 does not affect trans-acting siRNA biogenesis and their targets accumulation. (A) Northern blot analysis of various sRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. Trans-acting siRNA and miRNA accumulation were detected using a TAS1 tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (B) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against APC10, APC6, DRB4 and three target genes of the tasiRNA pathway (TAS1 target, ARF3 and ARF4) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each. doi:10.1371/journal.pone.0035173.g004 APC/C-Mediated Degradation of DRB4 (A) Pictures from infected plants with the TRV-PDS virus, 14 days post-infection (dpi). Scale bar: 1 cm. (B) Northern blot analysis of TRV-PDS viral RNA or viral-derived siRNA accumulation in Col-0, drb4 and DRB4 OE-27 line using a PDS specific probe. TAS1 tasiRNA accumulation was detected using a siRNA255 probe. miR159 accumulation was used here as a loading control. Values are normalized to miR159 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1. doi:10.1371/journal.pone.0035173.g005 Figure 6. Reduced APC/C activity slightly affect polIV/polV- dependent heterochromatic siRNA accumulation. Northern blot analysis of polIV-dependent (siRNA02, TR2258) or polIV/polV-depen- dent (siRNA1003, simpleHAT) siRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. miR173 accumulation is used here as a loading control. Values are normalized to miR173 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1. doi:10.1371/journal.pone.0035173.g006 Figure 6. Reduced APC/C activity slightly affect polIV/polV- dependent heterochromatic siRNA accumulation. Northern blot analysis of polIV-dependent (siRNA02, TR2258) or polIV/polV-depen- dent (siRNA1003, simpleHAT) siRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. miR173 accumulation is used here as a loading control. Values are normalized to miR173 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1. doi:10.1371/journal.pone.0035173.g006 Figure 5. Viral infection of drb4 and DRB4 OE line. (A) Pictures from infected plants with the TRV-PDS virus, 14 days post-infection (dpi). Scale bar: 1 cm. (B) Northern blot analysis of TRV-PDS viral RNA or viral-derived siRNA accumulation in Col-0, drb4 and DRB4 OE-27 line using a PDS specific probe. TAS1 tasiRNA accumulation was detected using a siRNA255 probe. miR159 accumulation was used here as a loading control. Values are normalized to miR159 and are expressed as a ratio relative to the wild-type Col-0, which was arbitrarily set to 1. doi:10.1371/journal.pone.0035173.g005 Figure 5. Viral infection of drb4 and DRB4 OE line. (A) Pictures from infected plants with the TRV-PDS virus, 14 days post-infection (dpi). Scale bar: 1 cm. (B) Northern blot analysis of TRV-PDS viral RNA or viral-derived siRNA accumulation in Col-0, drb4 and DRB4 OE-27 line using a PDS specific probe. TAS1 tasiRNA accumulation was detected using a siRNA255 probe. miR159 accumulation was used here as a loading control. Figure 4. Over-accumulation of DRB4 does not affect trans-acting siRNA biogenesis and their targets accumulation. (A) Northern blot analysis of various sRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. Trans-acting siRNA and miRNA accumulation were detected using a TAS1 tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (B) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against APC10, APC6, DRB4 and three target genes of the tasiRNA pathway (TAS1 target, ARF3 and ARF4) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each. doi:10.1371/journal.pone.0035173.g004 APC/C-Mediated Degradation of DRB4 Figure 4. Over-accumulation of DRB4 does not affect trans-acting siRNA biogenesis and their targets accumulation. (A) Northern blot analysis of various sRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. Trans-acting siRNA and miRNA accumulation were detected using a TAS1 tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (B) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against APC10, APC6, DRB4 and three target genes of the tasiRNA pathway (TAS1 target, ARF3 and ARF4) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each. doi:10.1371/journal.pone.0035173.g004 Figure 4. Over-accumulation of DRB4 does not affect trans-acting siRNA biogenesis and their targets accumulation. (A) Northern blot analysis of various sRNA accumulation in Col-0, drb4, dcl4, DRB4 OE27, RNAi APC10-38 and RNAi APC6-20 lines. Trans-acting siRNA and miRNA accumulation were detected using a TAS1 tasiRNA255, a miR159 specific probe and a miR173 specific probe, respectively. APC10 and APC6 probes were used to score the accumulation of siRNA targeted against APC10 and APC6 genes in their respective RNAi lines. U6 was used for the loading control. (B) Quantitative real-time PCR reactions were performed on total RNA from the same background depicted in (A). Specific primers against APC10, APC6, DRB4 and three target genes of the tasiRNA pathway (TAS1 target, ARF3 and ARF4) were used. RNA levels were normalized to that of Actin2 (At3g18780) and then to the value of the wild-type plants, which was arbitrarily set to 1. Error bars represent standard deviation from 2 independent experiments involving triplicate PCR reactions each. doi:10.1371/journal.pone.0035173.g004 April 2012 \| Volume 7 \| Issue 4 \| e35173 PLoS ONE \| www.plosone.org 6 APC/C-Mediated Degradation of DRB4 early step of the pathway. While in this study a clear reduction of miR159 in apc mutants was observed, in our case we could not d t t h i th iR159 l l b t ild t d APC Figure 5. Viral infection of drb4 and DRB4 OE line. Over-accumulation of DRB4 does not alter plant development Our results revealed a strong over-accumulation of DRB4 protein in APC hypomorphic mutants. These mutants exhibit a number of developmental alterations [18], which could however be attributed to a failure to degrade many other proteins beside DRB4. Indeed, DRB4 protein over-accumulation in transgenic plants did not result in any obvious phenotype compared to wild type during plant development nor at a molecular level on tasiRNA accumulation. This result is even more surprising, as it was showed that DRB2 over-expressing lines show a phenotype that copies a drb4 mutant at molecular and morphological levels [40]. One possibility to explain the lack of phenotype in DRB4 OE lines is that in the tasiRNA pathway, different steps involve different actors which could also be limiting factors. For instance, PLoS ONE \| www.plosone April 2012 \| Volume 7 \| Issue 4 \| e35173 7 PLoS ONE \| www.plosone.org APC/C-Mediated Degradation of DRB4 DRB4 interacts with DCL4 to cleave long dsRNA into 21-nt small siRNAs. Thus, though the level of DRB4 is increased, if this is not also the case for DCL4 and/or its RNA targets, it may not affect sRNA production. However, we cannot exclude the possibility that the regulation of DRB4 protein turnover by the APC/C may become important in specific conditions such as in response to some stresses or environmental changes. DRB4 interacts with DCL4 to cleave long dsRNA into 21-nt small siRNAs. Thus, though the level of DRB4 is increased, if this is not also the case for DCL4 and/or its RNA targets, it may not affect sRNA production. However, we cannot exclude the possibility that the regulation of DRB4 protein turnover by the APC/C may become important in specific conditions such as in response to some stresses or environmental changes. were also cloned into a GatewayTM entry vector and then transferred by recombination into yeast two-hybrid vectors for the interaction tests. The primers used to generate the deletions are the following: deletion 1 (GGATCCGAGGGAATTGATGTTG CC/GATATCTTATGGCTTCACAAGACG), deletion 2 (GGA TCCTCGAACCAGACCGGA/GATATCTTATGGCTTCAC AAGACG), deletion 3 (GGATCCGGTATGAAGATGAACAT TGC/GATATCTTATGGCTTCACAAGACG), deletion 4 (GG ATCCATGGATCATGTATACAAAGGTC/GATATCATCA GTAGTTAGTGCACTAAG), deletion 5 (GGATCCATGGAT- CATGTATACAAAGGTC/GATATCGTTCCCATTTTTGA- TATCTCATG), deletion 6 (GGATCCATGGATCATGTATA- CAAAGGTC/TGGACTTTGTGGCGTCAA) and deletion 7 (G GATCCGAGGGAATTGATGTTGCC/GATATCGTTCCCA TTTTTGATATCTCATG). The UPS: another level of control for the sRNA biogenesis pathway DRB4 over-expressing line and other mutant lines DRB4 over-expressing line and other mutant lines DRB4 full-length cDNA was cloned by LR recombination into pK2GW7 vector [55] which allows its expression under the control of the CaMV 35S promoter. Arabidopsis plants were stably transformed using ‘floral dip’ method [54] and 30 independent lines were selected for a unique copy of the transgene. drb4-1 and dcl4-2 mutants were previously described [21,36]. The generation and molecular characterization of APC10 and APC6 hypomorphic lines (RNAi APC10 and RNAi APC6) were previously published [18]. GST Pull-Down E. coli (BL21) was transformed with the different constructs or the empty vector and grown at 37uC until A600 = 0.5. Synthesis of glutathione S-transferase (GST) fusion proteins was induced by addition of 20% Arabinose, and the bacteria were incubated for an additional 4 h at 28uC. Bacteria were harvested, resuspended in extraction buffer (150 mM NaCl/1 mM EDTA/100 mM Tris, pH 7.5/1 mM DTT/0.5% NP40/10 mM b-glycerine phos- phate/1 mM NAF/1 mM PMSF) plus a protease inhibitor mixture (Roche), and sonicated. GST-tagged proteins were isolated by affinity chromatography on glutathione-Sepharose beads (GE Healthcare). [35S]methionine-labelled DRB4 was produced by coupled in vitro transcription and translation of pGBKT7–DRB4 using a TNT-Quik kit (Promega). The transla- tion product was incubated for 4 h at 4uC with the beads. After washing with extraction buffer, immobilized proteins were taken up in gel-loading buffer, and subjected to SDS/PAGE. Total Yeast two-hybrid assays Yeast two-hybrid assays were performed using Saccharomyces cerevisiae haploid strains PJ69-4a and PJ69-4 alpha (MATa/alpha, trp1-901, leu2-3, 2112, ura3-52, his3-200, gal4D, gal80D, LYS::- GAL1-HIS3, GAL2-ADE2, met2::GAL7-lacZ) [56]. The yeast two- hybrid was performed using cross-mating assays, as described in [57]. Diploid yeasts were selected on synthetic defined (SD)/- Leu/-Trp (SD-LW) medium, whereas interactions were selected on SD/-Leu/-Trp/-Ade (-LWA) medium. The UPS: another level of control for the sRNA biogenesis pathway Intensive research on the RNA silencing field over the past decade has brought to light the existence of several layers of regulation on its machinery [46]. Additionally to transcriptional regulation, several studies have shown that both sRNAs and their associated proteins can be posttranscriptionally or posttranslation- ally modified at multiple steps of the pathway. The 39-end of sRNA are highly heterogeneous and has been shown to be subjected to various modifications. Thus, the Arabidopsis methyl transferase, HEN1 adds a methyl group to the 29-OH at the 39- end of sRNAs which protects them from uridylation and degradation [47]. In mammals, miRNAs have been shown to be adenylated and this modification would affect both their stability and their activity [48]. Materials and Methods Constructs and primers APC10 promoter analysis A fragment containing 1.5 kb upstream of the ATG, the first exon, the first intron and the begining of the second exon of APC10 gene was cloned into pMDC163 vector [53] to trigger the expression of the bGlucuronidase (GUS) reporter gene. Arabidopsis plants were stably transformed using the ‘floral dip’ method [54] and 20 independent lines were selected for a unique copy of the transgene. Proteins associated to sRNA and involved in their biogenesis are also subjected to posttranslational modifications. In human, Drosha, an enzyme required for pri-miRNA processing, was found phosphorylated and that this protein modification was essential for its localization into the nucleus [49]. TRBP is a human dsRNA-binding protein, which has also been shown phosphorylated [50]. This modification enhances TRBP stability and consequently increases the level of its associated protein Dicer. However, in our DRB4 over-accumulating plants, we failed to detect a significant increase in the level of its associated dicer, DCL4 (data not shown). This indicates that an over-accumulation of the dsRNA-binding protein does not necessarily lead to an over- accumulation of its dicer partner. Argonaute2 (Ago2) is another player of the sRNA pathway found to be modified. Human Ago2 can be hydroxylated at a proline residue, which has a role on its stability and its localization to the processing bodies [46]. Moreover it has been shown that mouse Ago2 is ubiquitylated and targeted for degradation by the 26S proteasome [51]. This process is mediated by Lin41, a stem cell-specific E3 ubiquitin ligase that directly interacts with Ago2. In our study, we revealed another connection between the UPS and the sRNA regulatory pathways by showing that a player of the sRNA biogenesis is regulated through this degradation machinery. While we cannot exclude that the APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, it is also conceivable that this E3 ubiquitin ligase has some basic housekeeping functions in maintaining precise cellular-protein concentrations and homeostasis of key regulatory proteins. We thank Malek Alioua for technical assistance for q-PCR. Total RNA was extracted using TRIzol (Invitrogen), precipi- tated with isopropanol and redissolved in 50% formamide. Northern analyses of low and high molecular weight RNA were performed with 15 and 5 mg of total RNA, respectively, as described in [58]. Radio-labelled probes for detection of APC10, APC6 and TRV-PDS were made by [a-32P]dCTP random priming reactions, whereas DNA oligonucleotides complementary GUS Staining For GUS staining, the plant material was fixed in 80% acetone on ice for 20 minutes and then washed two times with 0.05 M Phosphate buffer. Plants were then immersed in the enzymatic reaction solution (0.05 M Phosphate Buffer/0.005% Triton/ 0.5 M ferricyanide/0.5 M ferrocyanide/10 mM EDTA/1 mM 5-bromo-4-chromo-3-indolyl b-d-glucuronide) and incubated overnight at 37uC in the dark. The plant material was cleared with ethanol washes and examined under a light microscope (E800, Nikon). Western Blot Total protein were extracted from three week-old seedlings or floral buds and analyzed by SDS-PAGE followed by Western blot using 1:10000 polyclonal DRB4 antibody [22]. Viral Infection TRV-PDS viral transcripts were amplified by inoculation to Arabidopsis thaliana leaves. Sap was subsequently extracted from those leaves at 10 dpi (1 g tissue/1 ml 5 mM NaP PH 7.5) and used to inoculate Arabidopsis rosette (about 5 weeks old, prior to bolting). Infected leaves were collected at 14 dpi and subjected to molecular analyses (Northern blots). In planta Bimolecular Fluorescence Complementation (BiFC) Total RNA was extracted from leaves using TRIzol (Invitro- gen), precipitated with isopropanol and redissolved in water. To remove traces of genomic DNA in RNA samples, equal amounts of total RNA were treated with DNase (Promega) that was subsequently heat inactivated. cDNAs were synthesized by reverse transcription using High Capacity cDNA reverse Transcription Kit (Applied BiosystemsTM). The cDNAs were then analyzed by the SYBRH Green-Based Detection system (Applied Biosystems) for real-time qPCR. The following primers were used: APC10 (AGGAGAAGTTTGAAGAACATGGA/TGCACAACAACAA- TAACTAATCTG), APC6 (GCTTGGCTTACACTTACCATT TGC/AATCAACCCCGTTCTGACATTC), DRB4 (AGTGTC TTCACCATTTCAAAGAGA/CTCTCTCATTCGCATACAC TGG), TAS1 target (GACATTGGGCGCATGTGG/AGCCT- CAATCACTTCTCCTTTCA), ARF3 (TGGTCCCAAGAGAA GCAGG/TCCACCATCCGAACAAGTG), ARF4 (GCCGCTG AAGATTGTTTTGCTC/AGTAGATGCCTCCTTGGTTGA CC). Mustard (Sinapis alba L.) seeds were sown on four layers of moist filter paper in Plexiglass boxes and cultivated for 4 days in darkness at 25uC. For transformation and BiFC experiments, etiolated seedlings were fixed to standard glass microscope slides with surgical adhesive (B-400 Secure Adhesive; Factor II Inc., Lakeside, AZ, USA). The split-YFP and CFP constructs (5 mg of each plasmid) were introduced into mustard hypocotyl cells with gold particles bombardment, as described in [52]. After transformation, mustard seedlings were placed vertically in sterilized water and kept at 25uC in darkness overnight prior to microscope analysis. Images were recorded with an E800 fluorescence microscope (Nikon) by using CFP- and YFP-specific filters. 10. Gieffers C, Peters BH, Kramer ER, Dotti CG, Peters JM (1999) Expression of the CDH1- associated form of the anaphase-promoting complex in postmitotic neurons. Proc Natl Acad Sci U S A 96: 11317–11322. Supporting Information Figure S1 APC10 expression profile. A fragment compris- ing 1.5 kb of promoter region, the first exon, the first intron and the beginning of the second exon of APC10 was cloned in frame upstream of the GUS reporter gene. Several independent transgenic lines were selected and GUS staining was performed on 20 day-old seedlings. The pictures are representative of the expression profiles observed for most of the lines. Scale bar: 100 mm. (TIF) 11. Li W, Wu G, Wan Y (2007) The dual effects of Cdh1/APC in myogenesis. FASEB J 21: 3606–3617. MG132 treatment Seeds from DRB4 OE line were sown on MS medium and grown for three weeks. Seedlings were then transferred into MS liquid medium and a sample was taken and frozen for time 0. Medium was then supplemented or not with MG132 (100 mM) and seedlings were incubated at room temperature for several hours. Author Contributions Conceived and designed the experiments: KM PD PG. Performed the experiments: KM MCC JZ GS HE AP. Analyzed the data: KM MCC PD PG. Contributed reagents/materials/analysis tools: KM JZ PD PG. Wrote the paper: KM PD PG. Conceived and designed the experiments: KM PD PG. Performed the experiments: KM MCC JZ GS HE AP. Analyzed the data: KM MCC PD PG. Contributed reagents/materials/analysis tools: KM JZ PD PG. Wrote the paper: KM PD PG. 7. Carroll CW, Enquist-Newman M, Morgan DO (2005) The APC subunit Doc1 promotes recognition of the substrate destruction box. Curr Biol 15: 11–18. 5. Peters JM (2006) The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol 7: 644–656. 8. da Fonseca PCA, Kong EH, Zhang Z, Schreiber A, Williams MA, et al. (2011) Structures of APC/CCdh1 with substrates identify Cdh1 and Apc10 as the D- box co-receptor. Nature 470: 274–278. 6. Carroll CW, Morgan DO (2002) The Doc1 subunit is a processivity factor for the anaphase-promoting complex. Nat Cell Biol 4: 880–887. p 9. Eguren M, Manchado E, Malumbres M (2011) Non-mitotic functions of the Anaphase-Promoting Complex. Sem Cell Dev Biol 22: 572–578. Northern Blot We thank Malek Alioua for technical assistance for q-PCR. 2. Viestra RD (2009) The ubiquitin–26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol 10: 385–397. Constructs and primers Full-length cDNA from APC10, CDC20-1 to -4, CDH1.1 to 1.3 and DRB1 to 5 were amplified and cloned into a GatewayTM entry vector. cDNAs were then cloned into different destination vectors according to the interaction assay. The pGAD-T7-GW and pGBT9-GW (Clonetech and modified with the GatewayTM technology from Invitrogen) were used for yeast two-hybrid screen and pair-wise interactions, the pDEST15 (Invitrogen) was used for GST pull-down assay, and the pMAV-YN-GW and pMAV-YC- GW [52] were used for BiFC assay. 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https://openalex.org/W2779661227	https://biblio.ugent.be/publication/8554720/file/8559730	English	null	The Mark, the Thing, and the Object: On What Commands Repetition in Freud and Lacan	Frontiers in psychology	2,017	cc-by	12,183	The Mark, the Thing, and the Object: On What Commands Repetition in Freud and Lacan Gertrudis Van de Vijver1, Ariane Bazan2 and Sandrine Detandt2 In Logique du Fantasme, Lacan argues that the compulsion to repeat does not obey the same discharge logic as homeostatic processes. Repetition installs a realm that is categorically different from the one related to homeostatic pleasure seeking, a properly subjective one, one in which the mark “stands for,” “takes the place of,” what we have ventured to call “an event,” and what only in the movement of return, in what Lacan calls a “thinking of repetition,” conﬁrms and ever reconﬁrms this point of no return, which is also a qualitative cut and a structural loss. The kind of “standing for” Lacan intends here with the concept of repetition is certainly not something like an image or a faithful description. No, what Lacan wishes to stress is that this mark is situated at another level, at another place, it is “entstellt,” and as such, it is punctually impinging upon the bodily dynamics without rendering the event, without having an external meta-point of view, but cutting across registers according to a logics that is not the homeostatic memory logics. This paper elaborates on this distinction on the basis of a confrontation with what Freud says about the pleasure principle and its beyond in Beyond the Pleasure Principle, and also takes inspiration from Freud’s Project for a Scientiﬁc Psychology. We argue that Lacan’s theory of enjoyment takes up and generalizes what Freud was after in Beyond the Pleasure Principle with the Wiederholungszwang, and pushes Freud’s thoughts to a more articulated point: to the point where a subject is considered to speak only when it has allowed the other, through discourse, to have impacted and cut into his bodily pleasure dynamics. Keywords: Freud, Lacan, repetition compulsion, jouissance, fort-da, beyond the pleasure principle, representation, dopamine Edited by: Edited by: Pierre-Henri Castel, Centre National de la Recherche Scientiﬁque (CNRS), France Reviewed by: Fabian Fajnwaks, Paris 8 University, France Stéphane Thibierge, Université Paris Diderot Paris 7, France Reviewed by: Fabian Fajnwaks, Paris 8 University, France Stéphane Thibierge, Université Paris Diderot Paris 7, France Correspondence: Gertrudis Van de Vijver gertrudis.vandevijver@ugent.be Ariane Bazan ariane@ulb.ac.be Correspondence: Gertrudis Van de Vijver gertrudis.vandevijver@ugent.be Ariane Bazan ariane@ulb.ac.be Specialty section: This article was submitted to Psychoanalysis and Neuropsychoanalysis, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Psychoanalysis and Neuropsychoanalysis, a section of the journal Frontiers in Psychology Received: 19 September 2017 Accepted: 11 December 2017 Published: 22 December 2017 Citation: Van de Vijver G, Bazan A and Detandt S (2017) The Mark, the Thing, and the Object: On What Commands Repetition in Freud and Lacan. Front. Psychol. 8:2244. doi: 10.3389/fpsyg.2017.02244 HYPOTHESIS AND THEORY published: 22 December 2017 doi: 10.3389/fpsyg.2017.02244 INTRODUCTION It is well into his life as a practicing psychoanalyst that Freud wished to come to a ﬁrmer theoretical grounding of the clinical observation that people do not necessarily want to get rid of their suﬀering or their symptoms. Against therapeutic eﬀorts of all kinds, people time and again repeat, even cannot not but repeat, what makes them suﬀer. This is what Freud means by Wiederholungszwang, the compulsion to repeat, situated, so he says, beyond the pleasure principle. In the text with the same name, Beyond the Pleasure Principle (Freud, 1920/1955), he explores the theoretical underpinnings of this clinical phenomenon. The idea he defends here is that the principle of 1See, for instance, the end of II, where Freud speaks of tendencies beyond the pleasure principle, “more primitive than it and independent of it” (Freud, 1920/1955, p. 17), or at the end of III, where he states that the compulsion to repeat is “more primitive, more elementary, more instinctual than the pleasure principle which it over-rides” (Freud, 1920/1955, p. 23). We note the unhappy translation of triebhafter into more instinctual – the translation of Trieb by drive would have been more accurate. Luckily, this is remediated in the new translation from the upcoming Revised Standard Edition, edited by Mark Solms. A critical and annotated version of Beyond the Pleasure Principle has meanwhile been published in Psychoanalysis and History (Freud, 1920/2015). 3cf. Freud (1900/1953, The Interpretation of Dreams, p. 461) and Freud (1920/1955, Beyond the Pleasure Principle, II, pp. 14–17). The game concerns a young child (Freud’s grandson) that used to play with his toys in such a way that he makes them disappear out of his sight, pronouncing then the long sound o-o-o. One day, while lying in his cradle, the child plays with his a bobbin, throwing it over the edge, pronouncing o-o-o again, and then, pulling the bobbin back in his little bed, triumphantly greets it with a-a-a. The two sounds are interpreted by Freud as expressing respectively fort and da, away and back. TO THINK WITH THE OBJECT: FREUD AND LACAN INTERPRETING THE CHILDREN’S GAME “fort-da” p g q In this paper, we propose to clarify what is at stake in Lacan’s diagnosis of a “conceptual intrusion” in Freud’s text. We argue that Lacan’s theory of the signiﬁer and of enjoyment basically takes up and generalizes what Freud was after in Beyond the Pleasure Principle with the Wiederholungszwang, and that, notwithstanding the overt diﬀerences in style – Freud being more versed into biological metaphors and concepts, Lacan more into logical and topological formalizations – it is not the case that Lacan’s theory of the signiﬁer with its focus on formalization is far removed from the apparently more bodily concerns of Freud. On the contrary, Lacan pushes Freud consequently to the point where the act of speaking itself is shown to involve an ineliminable place of the speaking other, while also having a subversive impact on what constitutes the homeostatic bodily pleasure dynamics. In Beyond the Pleasure Principle, Freud re-discusses the fort-da children’s game3 to make it clear that the compulsion to repeat is not just to be equated with the repetition of painful events. Both the compulsion to repeat and the repetitive children’s games are related to the dynamics of excitations and their discharge, that is, to the pleasure dynamics. But they are so in a diﬀerent way. p y y y As early as Freud (1895/1966), proposes in his Project for a Scientiﬁc Psychology that homeostasis, the process that seeks the state of minor tension, is the default mode of mental functioning. This means that the mental system seeks in the ﬁrst place to get rid of tension, that is, it is after the restauration of a previous state of less tension. Freud acknowledges of course that there is no mental functioning on that basis alone, because the mental apparatus also needs to be able to retain tension for a suﬃcient timespan, and it needs to be able to do this in organized ways, otherwise there would be no way of acting eﬀectively in the surrounding world.4 The reality principle is what captures this requirement of retaining tension in order to adequately act and maintain oneself in the world. Both the pleasure principle and the reality principle, however, are eventually seeking a decrease in tension.5 This paper has two parts. In the ﬁrst part, we explain what Lacan means with the idea that we are thinking with the object. Citation: December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org 1 The Mark, the Thing, and the Object Van de Vijver et al. repetition. To this end, we draw on Freud’s clinically interpreted anecdote of fort-da, and on Lacan’s re-interpretation of it in his seminars The Four Fundamental Concepts of Psychoanalysis (Lacan, 1963–1964/1973) and La Logique du Fantasme (Lacan, 1966–1967/2017). In the second part, we clarify and critically discuss how the use of signiﬁers introduces a radical cut organized around the limit points of the pleasure principle. Here, we depart from the distinction Freud introduces in his Project for a Scientiﬁc Psychology between understanding and judging, having it correspond, respectively, with the realm of representational, grasping bodily movements and the functioning of the mark, seen as a precursor and initiator of the properly subjective realm, with, between both, a relation of fundamental contingency or arbitrariness that serves as the ground for the compulsion to repeat. repetition, and not the pleasure principle, is the most basic module of mental life, grounded in the drives. Freud is bold in his clinical aﬃrmation – yes, the basic module of mental life is the compulsion to repeat, and not the pleasure principle1 – but he clearly does struggle to articulate the Wiederholungszwang in relation to the pleasure principle. In Logique du Fantasme, Lacan (1966–1967/2017) invites us to consider that Beyond the Pleasure Principle constitutes a “conceptual intrusion” in Freud’s work. He insists: “Do we really measure what is at stake here?” To him, the Wiederholungszwang, articulated in terms of jouissance, enjoyment, constitutes a genuine break with the pleasure principle, a contradiction even with what Freud would have thought until then to be the module of the functioning of the mental system, namely homeostasis, that holds that living substances always seek the state of minor tension. To Lacan, there is no doubt about the fact that the pleasure principle reissues homeostasis for mental life: the mental system, in as far as it is ruled by the pleasure principle, “echoes,” “repeats,” “redoubles” organic, homeostatic requirements.2 2“Quand Freud introduit pour la première fois, dans son Jenseits à lui, l’Au- delà du principe du plaisir, le concept de répétition comme du forçage, Zwang, répétition, Wiederholung – cette répétition est forcée: Wiederholungszwang – quand il l’introduit pour donner son état déﬁnitif au statut du sujet de l’inconscient, mesure-t-on bien la portée de cette intrusion conceptuelle? Si elle s’appelle ‘au-delà du principe du plaisir,’ c’est précisément en ceci qu’elle rompt avec ce qui, jusque-là, lui donnait le module de la fonction psychique, à savoir cette homéostase qui fait écho à celle que nécessite la substance de l’organisme, qui la redouble et la répète et qui est celle que, dans l’appareil nerveux isolé comme tel, il déﬁnit par la loi de la moindre tension. Ce qu’introduit la Wiederholungszwang est nettement en contradiction avec cette loi primitive, celle qui s’était énoncée dans le principe du plaisir” (Lacan, 1966–1967/2017, séance XI, p. 134). 4That corresponds to the function of the ego. The ego cannot function but as a set of ‘permanently activated neurons,’ i.e., neurons that can retain excitation “Thus the ego is to be deﬁned as the totality of the ψ cathexes, at a given time, in which a permanent component is distinguished from a changing one” (cf. Freud, 1895/1966, Project for a Scientiﬁc Psychology, p. 323). 5That is why they can be said to be both operating in function of the death drive, as Freud explicitly admits in Beyond the Pleasure Principle (Freud, 1920/1955), VII, pp. 62 ﬀ. In “The economic problem of masochism” (Freud, 1924/1962), however, 4That corresponds to the function of the ego. The ego cannot function but as a set of ‘permanently activated neurons,’ i.e., neurons that can retain excitation “Thus the ego is to be deﬁned as the totality of the ψ cathexes, at a given time, in which a permanent component is distinguished from a changing one” (cf. Freud, 1895/1966, Project for a Scientiﬁc Psychology, p. 323). Frontiers in Psychology \| www.frontiersin.org TO THINK WITH THE OBJECT: FREUD AND LACAN INTERPRETING THE CHILDREN’S GAME “fort-da” This is important to come to clarity about his account of the signiﬁer as participating in the dynamics of pleasure and December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org Frontiers in Psychology \| www.frontiersin.org 2 The Mark, the Thing, and the Object Van de Vijver et al. The question is, of course, how much tension the mental system will or should be capable of retaining, and why or how it will do so. This matter is at the core of the Freudian distinction between the pleasure principle and the compulsion to repeat. In discussing the fort-da game, Freud stresses that the child, by staging the presence and the disappearance of his toys, compensates for the anxiety and the pain that the absence of the mother is provoking in him. The game repeats the supposedly painful event, which suggests that there is no immediate relief of tension involved. Freud, however, does not hesitate to say that the indemniﬁcation at stake involves a direct beneﬁt. It concerns a diﬀerent kind of pleasure though, one stemming from another source (Freud, 1920/1955, p. 17). The diﬀerence, so Freud explains, has to do with the fact that the child succeeds in being less passively subjected to what he experiences, has found ways to actively master, that is, to bind excitations through the throwing away and pulling back of his bobbin and the repetitive “o-a,” fort-da. In this way, discharge is enabled, while a distance is created with drive satisfaction. The use of “o-a” – that Lacan refers to as signs, marks – is what makes the mastering drive independent of whether the memory was itself pleasurable or not (Ibidem, pp. 16–17).6 However, even if the child’s mental functioning is independent of the initial objects of satisfaction, even if the use of signiﬁers installs the pleasure dynamics at another level, at another place, there is in Freud’s viewpoint on mental life still an “echoing,” “repeating,” “redoubling,” as Lacan states, of organic, homeostatic requirements. The module of mental life is homeostasis, that is, the pleasure principle. Lacan’s (1963–1964/1973, p. 60) comments on the fort-da experiment in The Four Fundamental Concepts of Psychoanalysis are revealing for the questions that concern us here. when dealing with signiﬁers, or, with Freud, to talk of pleasure as stemming from “another source,” is in agreement with the Freudian idea that memory and consciousness are two diﬀerent and mutually exclusive systems: opening the possibility of using signiﬁers is opening the possibility of memory and immediately closes oﬀtheir presence in consciousness. Thirdly, it is worth noting in passing that Freud speaks in the context of the use of signiﬁers, as in theater plays, of enjoyment, Genuβ (Freud, 1920/1955, p. 17) – the subject enjoys the commemoration of painful events in the play – a thing that will be of importance in the Lacanian viewpoint on enjoyment. Freud equates death drive with the Nirwanaprinciple, the pleasure principle and the reality principle as its modiﬁed form, with the representatives of libido. TO THINK WITH THE OBJECT: FREUD AND LACAN INTERPRETING THE CHILDREN’S GAME “fort-da” Lacan is not siding with Freud’s clinically based distinction between the repetition at stake in the children’s game – still situated under the heading of the pleasure principle, albeit distinguished from mere drive satisfaction – and the “real” repetition he wishes to consider as lying beyond the pleasure principle, i.e., the compulsion to repeat. On the contrary, he considers the fort- da game as an instantiation of what constitutes mental life at heart, ruled, that is, by repetition, and, therefore, not ruled by a pleasure principle that echoes homeostasis. In this sense, repetition, also coined as enjoyment by Lacan, is in his view not “natural,” not obeying to what instincts or needs command in terms of pleasurable discharge.7 To Lacan, what counts in repetition, and what to him is illustrated in the fort-da game, is the attachment to that which stays the same, namely, the signiﬁers, “o-a,” fort-da, endlessly repeated.8 That the repetitive use of “o-a” takes place in an apparently signifying relation to a multiplicity of toys, in a variety of situations, is not what counts in the ﬁrst place – it rather risks to distract us from its genuine signiﬁcance. As a matter of fact, to consider the fort-da as a stamp of some or other event – the absence of the mother, for one, as Freud suggests – is too quickly complicit with a semantic-representational account and thereby misses the real point.9 According to Lacan, following here Wallon, the child is 6Several comments are in order here. Firstly, there is one clear passage in Jenseits in which Freud speaks of the importance of the trait (the Zug) in relation to repetition: “This ‘perpetual recurrence of the same thing’ causes us no astonishment when it relates to active behavior on the part of the person concerned and when we can discern in him an essential character-trait (Characterzug) which always remains the same and which is compelled to ﬁnd expression in a repetition of the same experiences.” We are much more impressed by cases in which the subject appears to have a passive experience, over which he has no inﬂuence, but in which he meets with a repetition of the same fatality (Freud, 1920/1955, Beyond the Pleasure Principle, II, p. 22, italics original). TO THINK WITH THE OBJECT: FREUD AND LACAN INTERPRETING THE CHILDREN’S GAME “fort-da” While, in the context of the discussion of fort- da, Freud stresses in the ﬁrst place “the use of the object” and talks of pleasure stemming ‘from another source,’ Lacan explicitly interprets the “o-a” as signs or marks, in line with the passage we mentioned [for instance in Logique du Fantasme (Lacan, 1966–1967/2017), XI, p. 136 (italics original)], where he writes: “(. . .) identité signiﬁcante du ‘plus’ ou du ‘moins’ comme signe de ce qui doit être répété [character-trait (. . .) which is compelled to ﬁnd expression in a repetition],” and also in The Four Fundamental Concepts (Lacan, 1963–1964/1973), V, p. 54, where he discusses automaton and tuchè: “(. . .) l’insistance des signes à quoi nous nous voyons commandés par le principe du Plaisir.” That Lacan speaks of signs might be confusing, in as far as the sign here is purely formal and is deﬁned in terms of oppositions. As such, it is what can be understood by the functioning of the signiﬁer. Clearly, what Lacan intends is something diﬀerent from more traditional (philosophical) accounts of the sign where it refers to something for someone (Peirce), or from cognitive views according to which the sign is what identiﬁes stimuli on external grounds. With Lacan, following Freud, a sign or a mark is constituted by parameters or characteristics holding from within the subjective realm itself. As we explain further in this paper, Lacan speaks here of signs in reference to the Einzige Zug, which is the symbolic mark, the ﬁrst signiﬁer, that indicates that something was lost and cannot but be repeatedly searched for. By initiating that movement of repetition at the level of the signiﬁers, the little human being grafts himself upon the other, and inscribes himself in what Lacan calls le champ de l’Autre, the ﬁeld of the Other (cf. Lacan, 1966–1967/2017, XVII, pp. 224–225/p. 136). Even if the distinction between the ﬁrst and the second signiﬁer, the Einzige Zug (the “sign”) and the “proper” signiﬁer can only be made from within the realm of “signiﬁcance,” the realm of the functioning of the signiﬁer, it is nevertheless relevant to logically distinguish that ﬁrst moment, the one in which something merely indicates that something was lost without being part of a diﬀerential system of signiﬁers. Of course, this is a mythical moment, set apart logically. Frontiers in Psychology \| www.frontiersin.org 12Freudian pleasure, then, is not an aﬀect in the common sense meaning of the word (namely, a hedonic or agreeable feeling). Freudian pleasure is relief, not delight. Therefore, it is not concerned with valence as aﬀects are. Without going into details, we are inclined to see aﬀect, in the common sense, as far less determining and orienting for behavior than the drive system and as not organized around the adequacy of the act (for more details, see Bazan and Detandt, 2013, 2015; Bazan et al., 2016; Detandt, 2016). the dynamics of the body, and that understands representations as motor forms that correspond to the central imagery that arises from action intentions that did not completely lead to discharge (see Jeannerod, 1994, p. 201; Bazan, 2007, pp. 125–126). The point we wish to make in relation to the above passage, is that in repetition, it is not the content that counts, but the form. As Lacan indicates, the child jumps toward the “o-a” in a movement of bridging what appeared as a lack in his vicinity. To interpret this “o-a” as a “representation” referring to the mother leaving the room, expected to come back through the door, adds too much and too quickly elements of content. What Lacan wishes to indicate, clearly in line with Freud – who also, in Beyond the Pleasure Principle, notes that the child is not in panic at the moment the mother leaves the room – is that it concerns a throw and pull-movement, i.e., a movement of the acting subject, doubled, over-written by a phoneme sequence, “o-a,” a signiﬁer which is ﬁrst and foremost a motor form. For more details (see Bazan and Van de Vijver, in preparation). Understanding and Judging We know that pleasure is seen by Freud as discharge of tension; it is a temporary, ﬂoating, and partial suspension of displeasure.12 Tension – displeasure, if not trauma – constitutes the background against which pleasure has to be thought. We also know that as long as we live, there is, structurally, the encounter with unpleasantly high levels of tension (Freud, 1895/1963, 1895/1966; Lacan, 1963–1964/1981). Within this setting, the ﬁrst air entering the respiratory system, the ﬁrst milk entering the digestive system, can likely be called traumatic experiences. What exactly is at stake in these experiences? So, in sum, Freud grounds the pleasure principle in the possibility of discharge, and quite logically considers the child’s game as a successful kind of discharge. What to him lies beyond the pleasure principle, has to do with those occasions where discharge appears to be problematic or radically impossible, such as in traumatic neuroses or in the phenomena of negative transference. To Lacan, however, this clinically observed distinction risks to miss the essential point, namely that in the What Freud writes in his Project for a scientiﬁc psychology is relevant here. Freud makes a distinction between understanding and judging, that he grounds in the idea that the complex of what surrounds the child, the fellow human being in the ﬁrst place, falls apart into two components, one which “makes an impression by its constant structure and stays together as a Thing, while the other can be understood by the activity of memory – that is, can be traced back to information from [the subject’s] own body” (Freud, 1895/1966, p. 331, italics original). So, to understand, is to ﬁnd relief in and through the proper bodily movements, that is, to succeed in grasping something (com- prehensio), so that, by one’s own means or not, an eﬀective handle is found on the basis of which discharge becomes possible. To judge, on the other hand, refers to something that resists understanding, a thing that for that reason “stays together as a Thing” and impresses by its constant structure, and that is to be covered and bridged by other means, with what Freud refers to as traits or marks (Züge). TO THINK WITH THE OBJECT: FREUD AND LACAN INTERPRETING THE CHILDREN’S GAME “fort-da” In other words, the child does not constitute its mental life on the basis of, for instance, a “representation” referring to the mother leaving the room, expecting that she will come back at some point through the same door.10 It is in the vicinity where the lack makes itself directly felt that the play with the bobbin and the utterance of “o-a” take place. As the bobbin, the “o-a” is actually the little thing that is detachable from him while being still retained, the little thing on the basis of which the infant explores and expands his universe in a movement of self-mutilation – throwing the thing, part of his own movements, away, and thereby bridging the abyss created by the absence of what was in his vicinity a moment before “It is with his object that the child jumps over the borders of his territory changed in wells and that he begins the incantation” (Lacan, 1963–1964/1973, p. 60, our translation).11 The little subject of the fort-da, successfully ﬁnding discharge through the act of repetitively pronouncing “o-a,” is in the repetitive movements he initiates with his bobbin and covers with “o-a”. signifying procedures whereby the child, or any speaking being for that matter, deals with absence and presence, there is a structural loss, a structural impossibility that inescapably emerges with the use of signiﬁers, with the use of “o-a.” This structural loss is not disconnected from the issue of discharge, and thus of pleasure, but does initiate another domain, obeying a diﬀerent logics, a logics ruled by repetition. In order to make this clear, we have to explain how the use of the ﬁrst signiﬁer is connected to the pleasure dynamics, or rather, how it cuts with that dynamics and how it gives rise to the functioning of a new domain. To that end, we need to reconstruct and articulate in more detail how the child is caught into his movements, and how he ﬁnds an orientation on that basis. TO THINK WITH THE OBJECT: FREUD AND LACAN INTERPRETING THE CHILDREN’S GAME “fort-da” Unless explicitly speciﬁed, we shall from here on speak of signiﬁers in order to avoid confusion between the philosophical use of the concept of sign and the psychoanalytical one. Secondly, to suggest that “another level” is initiated 7The use of terms such as “natural” is never unproblematic. In this context, we take it that Lacan intends natural as opposed to cultural. We do not want to open the philosophical discussion on nature/nurture here, but we do want to stress that the opposition is perhaps less straightforward than Lacan seems to suggest. We will argue further on that even if the compulsion to repeat is seen to be categorically distinguished from the pleasure principle grounded in a homeostatic dynamics, this does not mean that there are no biological constraints to be taken into account. As a matter of fact, what we intend to show is that the compulsion to repeat, even if it does not follow homeostatic principles, does have a logic that can be said to be biologically anchored and is clearly linked to precise biological constraints (Bazan and Detandt, 2013). 8This is perfectly in line Freud’s “perpetual recurrence of the same thing,” stated in Jenseits, albeit not in the passage where we ﬁnd fort-da. See footnote 6 for the full quote. 9The term representation (as well as meaning, content, . . .) is extremely tricky. It is not the place here to unfold its various (mainly philosophical) traps and potentialities, but in relation to Lacan, it can be said that the representational realm mostly goes hand in hand with meaning, content, semantics, all of the imaginary order, against which he warns time and again. This is not per se the most adequate or the most interesting option though. We explain further on an alternative viewpoint on representation, one that is more radically embedded in December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org 3 The Mark, the Thing, and the Object Van de Vijver et al. vigilant for what it experiences as a lack exactly next to him, in his vicinity, not where the mother left the room and where he could expect her to come back. Frontiers in Psychology \| www.frontiersin.org Understanding and Judging To Freud, that is what judgment does: it corresponds to the impossibility of ﬁnding adequate movements that would lead to a grasp of the complex (understanding), and constitutes a cut with it by approaching the 10Here, is an example of how tricky the term representation can be. It is here used between inverted commas to highlight its traditional philosophical sense, i.e., something that stands for something for someone: the child fears that the mother, whom he saw leaving through the door, will perhaps not come back. Following our viewpoint on representation, and following also Lacan and Wallon in the interpretation of the case, we would say that the activated motor-pattern of the eyes while the mother left the room constitutes the representational structure, formal in nature, and that it is exactly that pattern that is repeated in the play with the bobbin and the accompanying “o-a”: the movement of throwing the bobbin away and pulling it back repeats the movement of something being at one point in the vicinity and at another point leaving a void in its absence. 11In line with Aristotle, Lacan will add here that man not only thinks with his object, he is, as a subject, where the object is put into practice This probably refers to Aristotle’s (1984) idea that the mind is “none of the things existing in actuality before thinking” (De Anima iii 4, 429a24). In other words, thinking is nothing in actuality in abstraction of the form that thinks. Or still, our thinking is only with our objects of thought, that is, the forms. And that is precisely what is at stake here, and what we explain further on: “o-a” are formal objects that correspond to, or “stand for” actions tied together into motor packages that, in their opposite nature, organize what can be called subjective life. December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org 4 The Mark, the Thing, and the Object Van de Vijver et al. our view, to what Freud called the “Triebrepräsentanz,”17 that is, the point where the subject allowed for the fact of “being taken by surprise” and that opens the possibility of returning to that point. The Thing, the Mark, and Primary Judgment g What happens in feeding, is that the child, most commonly, ﬁnds by itself the voluntary sucking movements that will contribute to the feeding.14 The act is what is ﬁrst, with its motivational point – from where, why, for what reason it is undertaken – left unfathomable from within the system that undertakes it.15 As a consequence of the sucking movement, milk enters the system. That is, for the system, a surprise. That the movement of sucking, undertaken, so to speak, “out of nowhere,” would lead to milk entering, was not foreseen and could not be foreseen. The ﬁrst milk that enters the system comes as a surprise, and cannot but come as a surprise; it constitutes an event: the milk is an external, a priori hostile element entering the system. However proximate the entering of milk with the sucking movements is, both are, for the system concerned, disconnected, in the sense that there is nothing in the act of sucking that is connected with milk: their relation is contingent. Also the fact that the sucking brings a certain relief simply related to the sucking itself, is initially disconnected from the milk and does not diminish the surprising eﬀect of the latter. The event of milk entering the system for the ﬁrst time is inscribed as a mark, but it is not understood – the milk “stays together as a Thing.” Our hypothesis is therefore that what is marked is ﬁrst and foremost the event itself: the mark is the point, the punctual point expressing and inscribing the bodily surprise.16 The mark corresponds, in What happens then with the undertaken movement that has made the entering of milk possible, and what about the relief to which it eventually contributed, or not? What role does it play in this “preliminary subjectivation”? Clearly, this movement, or cluster of movements, is of no help in understanding the event, but, being proximate, it gets linked to it, contingently but no less ﬁrmly. It is this link, inherently contingent but factually proximate, that, in our view, lies the ground for the further articulation of subjectivity, and of which Lacan will say that it is the ground for repetition. Understanding and Judging We consider this “being taken by the event in the form of a mark” as the ﬁrst step in the process of subjective positioning, the ﬁrst or primary judgment, corresponding to what Freud calls “Bejahung,” or what with Lacan becomes “Bejahung pure, primitive” (Lacan, 1955–1956/1981, p. 95) or “Bejahung primaire” (Lacan, 1966, p. 387). It does involve the position of the subject, albeit in a very preliminary and inviting sense: the fact that the trait was inscribed as a mark of the event, witnesses to a subjective choice – the little human let himself be surprised by the milk entering, it could as well have chosen not to drink. It therefore opens subjectivation as a task, an agenda.18 However, it is important to note that this logical time of being struck by surprise, is not exclusive to human beings, but, as we will explain further on, has to be supposed in vertebrates in general too. complex through a trait, a mark. A judgment is thereby “entstellt” with regard to understanding, it installs another realm, another domain.13 We propose to apply this schema to the dialectics between pleasure and displeasure and to what lies beyond. The potentiality of this move is twofold: on the one hand, it contributes to anchoring the functioning of the mark into the dynamics of pleasure and displeasure, approaching it, so to speak, “from below,” and, on the other hand, it allows to ground the (f)act of speaking in the obstacles and the impossibilities the human being encounters speciﬁcally in and through the motor patterns and their potentiality to lead to discharge. Let us return to our examples, the entering of milk in particular. 13For a more extensive Lacanian discussion of the Thing, la Chose (see Lacan, 1959–1960/1986; Lew, 2014). 17There can be a hesitation between Triebrepräsentanz and Triebrepräsentant, the ﬁrst referring to the function of taking-the-place-of (“tenant-lieu”), the second to the taking-the-place-of itself. We wish to stress in the ﬁrst place the notion of Repräsentanz. We shall see further on the delicate status of this point. For an extensive discussion of this concept (see Tort, 1966/2016), and for a subtle and pertinent “mise au point,” primarily in relation to the functional interpretation of the Repräsentanz (see Lew, 1983). also the dopamine release corresponding with disrupting, aversive and traumatic events (Bazan and Detandt, 2015), taken together as dopamine release ‘marking’ the unexpected event, independent of its valence (Bazan et al., 2016), and leading to a physiological registration known as ‘incentive sensitization.’ 18It would be possible and relevant to further elaborate on this issue in terms of alienation, as Lacan himself does all along in Logique du Fantasme. The subject (of the unconscious) is in “the part that is lost”; it is the subject of “je ne pense pas,” and that part is what shows itself “by surprise.” In relation to surprise, Lacan refers to Theodor Reik as the sole analyst having stressed its importance in relation to the unconscious (Reik, 1935/1976; cf. Lacan, 1966–1967/2017, VII, p. 92, Logique du Fantasme). 14Our schema is also applicable to what happens in breathing, but it is diﬀerent in the sense that the child, most commonly, ﬁnds by itself, through voluntary breathing movements, the adequate act that creates a relief of tension. In breathing – in contrast, e.g., with feeding – there is no constitutive need to for a contribution of the other, nor is there a diﬀerence in timing between ﬁnding the grasping movement (the breathing movement) on the one hand, and the satisfaction of the drive. We chose for the example of feeding, because it allows us to more straightforwardly articulate the diﬀerent moments we wish to distinguish here. Frontiers in Psychology \| www.frontiersin.org 15This structurally missed step and the ways to retroactively recover it, whereby it is identiﬁed as the cause of our acting, is, actually, the ground for the hypothesis of the unconscious. 20It is certainly not a genetically identiﬁable moment, as it is a moment of historization, indicating the registration of the contingency of a subject’s history in the form of a marked event. We do however think (as indicated in footnote 16) that there is a physiological correlate to this marking, in the form of a dopamine release, probably a dopamine spike. Note that if biological correlates can be situated at precise moments, their mental realization is dynamic, hence the use of the term ‘logical moment’ instead of ‘chronological moment’ (see also Bazan and Detandt, 2017). The Thing, the Mark, and Primary Judgment In other words, the fact that milk enters the digestive system and eventually brings relief is secondary to the eﬀect of the event as such – and it is the latter, not the former, that induces the repetition.24 realm, namely the realm of movements that can become representational. The adjacent movement thus carries both sides: it refers to the Repräsentanz, with the mark taking the place of the Thing and having the potentiality to elicit Vorstellungen, representations – these two diﬀerent logical moments reﬂect Freud’s inaugural distinction between judgment at the one hand and comprehension at the other. Beyond that ﬁrst “sticky moment” where the Repräsentanz remains a pure potentiality – which is, actually, a logical moment, not a genetically identiﬁable moment20 – a call for other types of movements is launched, intentionally directed grasping movements this time, that are eﬀectuated as a return to what escaped comprehension. Indeed, the ﬁrst sucking is a sucking to discharge the sucking tension, but if it is followed by the event of the milk coming in21, and if it is thus given the weight of the mark, the subject can choose to have the next sucking as an intentionally directed movement, to grasp – i.e., to get – the milk. In this way, the Repräsentanz is what elicits representational – i.e., mental – activity. This subjective representational work is a work of understanding, of com-prehension, or at least, it is an attempt to understand, to grasp. This work is constituted on the basis of the marked adjacent movements – marks that are, as we have argued, the marks of a non-understanding, of a limit to understanding, that is, a limit to the possibility of grasping something, a limit to making that something (the milk entering as a surprise) into an object. What has to be further elaborated, therefore, is what this movement of “thinking of return” exactly involves, how it is marking speciﬁcally the human being as a speaking being, with representations becoming genuinely signiﬁers, and what, if anything, constitutes, in this context, the diﬀerence with the compulsion to repeat. In order to further unfold this, we need to turn to the status of the object, its relation to the possibility and the meaning of discharge, as well as to the role of the fellow human being in this fabric of pleasure and enjoyment. The Thing, the Mark, and Primary Judgment Very much in line with this, Lacan will consider the mark as the ﬁrst signiﬁer, S1, corresponding to Freud’s unitary trait, the “Einzige Zug,” the symbolic mark that constitutes an event by indicating a cut with the level of what is being marked (Lacan, 1966–1967/2017, p. 135). The S1 enables the primary judgment that has the form of an aﬃrmation (Bejahung): it marks that there was an event that struck the body. However, to Lacan, the S1 has to be called symbolic already: in order to be called a mark at all, it intrinsically demands to be deployed and ever re-deployed through the articulation of representations that engage with other signiﬁers.22 If the mark would not have elicited the subject to a return, it would not be an Einzige Zug, an S1. S2 then stands for the chain of signiﬁers that aim at a return to the ﬁrst signiﬁer in an attempt to grasp or understand the initial moment of surprise, and in this sense corresponds to the representational activity which Freud refers to as the com-prehensio. Lacan speaks here of a “thinking The Thing, the Mark, and Primary Judgment What we propose here is that the adjacent movement, being only contingently linked to the ungraspable Thing of which the subject is factually experiencing the eﬀects of surprise, this adjacent movement indicates and covers, “stands for,” the ungraspable Thing. Freud himself is speaking of Vorstellungsrepräsentanz, a term that caused a huge confusion among psychoanalysts and scholars.19 Freud sometimes identiﬁes the Triebrepräsentanz with the Vorstellungsrepräsentanz, and there is something understandable at this. We would be inclined to say that the ﬁrst adjacent movement, contingently sticking to the mark, so to speak, also already belongs to another 16This event is, so we think, beyond the mechanical part of it, the explosion of the sugar receptors in the mouth massively and suddenly highly activated. As we have discussed elsewhere, a physiological marker is proposed for this surprise in the form of a release of dopamine at the level of the nucleus accumbens, i.e., the dopamine peak indicating unexpected reward (Bazan and Detandt, 2013) but 19It was for instance translated as “représentant représentatif” (Laplanche and Pontalis) or by Lacan as “tenant lieu de la représentation” (see Tort, 1966/2016; Lew, 1983, for a discussion). December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org 5 The Mark, the Thing, and the Object Van de Vijver et al. of return,” a “thinking of repetition” (Lacan, 1966–1967/2017, p. 135).23 The articulation of this representational realm, its structuring, is properly symbolic, constituted of representations, but it is, meanwhile, very much anchored in the body, determined by what is, along a subjective history, being “accepted and inscribed” as a mark and what is contingently adjacent to it as an undertaken movement. In other words, what initiates the repetition of these actions is not the possible reward or relief they might bring about. What causes repetition is the fact that the action is being linked to the event, the event being constituted by surprise. Note that the marking, with an adjacent movement being contingently linked up with it, is independent from whether the event was painful (ﬁrst air coming into the lungs) or rewarding (ﬁrst milk entering the mouth cavity). 22Here, we easily jump from representations to signiﬁers. However, we propose that both are logically equivalent. Indeed, a representation (see footnote 7) is thought as a motor activation rest of an action intention that could not be discharged in actual motor activation; a signiﬁer, then, is merely the application of this logic to the act of speaking, i.e., to articulatory phoneme motor patterns. Both representations in general, and signiﬁers speciﬁcally, are thus motor potentialities, i.e., forms, without any determined content or meaning. 23See here Lacan’s discussion of the children’s play with “o-a.” To Lacan, from the moment the child uses “o” (S1), we cannot but add to it the diﬀerential “a” (S2), revealing the moment of S1 in isolation as a mythical, logical moment. He calls therefore the S1 symbolic, and states that there is from thereon no grounding to be looked for in the similarities or diﬀerences between objects such as toys, bobbins, mothers, to identify what a mark is. Nothing of this sort is hidden in the plays of Freud’s grandson that would justify the use of “o-a,” as nothing of this sort would justify the marked surprise eﬀect in the child. According to Lacan, a unitary trait identiﬁes something – in our interpretation, an event – but it is only through the repetition, in the diﬀerential play with other signiﬁers, that an event genuinely becomes a subjective event. 24The ﬁrst events in life frequently lead to the repetition of actions crucial for our survival, but our intuition is that this is at the bottom of it a matter of chance, not of teleology. E.g., a pigeon that made a wing movement before receiving a grain (Skinner, 1948), will from then on also repeat that wing movement because it had been registered as what had to be repeated, even if it had nothing to do with its survival chance (https://www.youtube.com/watch?v=8uPmeWiFTIw). 21See footnote 16. Frontiers in Psychology \| www.frontiersin.org The Signiﬁer Inscribed in a Basic Non-attunement of Actions and Needs: The Role of the Other So, it is from within the repetition compulsion that relief of tension becomes possible – it is not the relief of tension that is the ground for repetition. a Thing” and resists understanding, that the child is launched, here again, for an endless ‘thinking of return,’ a re-elaboration of his ﬁrst vocalizations, in an attempt to grasp after all that which entered his system as a surprising event and with regard to which it did not succeed in articulating the appropriate adequate actions. We therefore agree with Lacan when he states that the child thinks with his object, which means that he subjectivizes through the handlings with his object. We also agree with him (cf. his reading of “o-a”), that the use of signiﬁers, and the linguistic, signifying practices at large have to be understood along the same lines: a signiﬁer is handled as an object, the object perhaps, on the basis of which the child explores and expands his subjective universe. We have explained in the previous part how these handlings are articulated in terms of a failure in grasping the Thing, how the marks in a sense “take over,” or at least initiate a new realm of being, the realm properly constituted by signiﬁers (which is the Other, in Lacan’s terms). We remind here that the signiﬁer is a form, a motor-pattern, only contingently linked to what brings discharge – indeed, it marks precisely what was not understood and could not be brought back to memories of the proper body. Repetition or enjoyment, a “thinking of return,” as Lacan calls it, is therefore, for the human being, intrinsically bound up with the nature of the signiﬁer. The child, in the same movement of adopting the signiﬁer that is oﬀered to him as a formal potentiality by the other, inscribes himself in a universe of vocalizations where it is structurally impossible to grasp the Thing. It is from there on condemned to run after the Thing, to commemorate what can be called, perhaps, a moment of exquisite subjectivity – the structurally escaping moment of having been struck by surprise. The Signiﬁer Inscribed in a Basic Non-attunement of Actions and Needs: The Role of the Other By structurally missing this point because of the fact that the Thing is situated at another level and cannot be brought back to bodily understanding, the subject endlessly, repetitively, runs after “the facts”: it repeats the marks in themselves, and strives for understanding after all, attempts to make the Thing into an object, to bring it back to proper and directed body movements that bring discharge. Both realms, however much intertwined they are, are disconnected realms, only contingently bound up. As we saw with the “fort-da” game, the child produces the ﬁrst signiﬁers “out of nowhere,” or at least, these signiﬁers cannot be grounded in the distinctions between his toys, between the mother or the father being absent or present. There is no way back from signiﬁers to meanings25: the relation between form and content is neither innately, nor naturalistically grounded. As we saw with the ﬁrst mark, the Einzige Zug: signiﬁers emerge at the point where a “naturalistic” grounding – an adequate grasping of the object, leading to discharge – reaches a limit. Or perhaps more correctly: the use of the signiﬁer indicates that a limit was reached, indicates that the bodily movements were inadequate. g f p It is often said, from within a psychoanalytical setting, that what drives the human being is not the satisfaction of needs. We agree with this. In line with what we elaborated in relation to higher vertebrates, however, we consider that the structural non-attunement of needs and actions holds in the same way for human beings, and that it is intrinsically related to the way in which vertebrate bodies are constituted. This allows us now to address the question of the speciﬁcity of the human being, as a speaking being, from a slightly diﬀerent angle. It is true, indeed, as Freud already highlighted in his Project for a Scientiﬁc Psychology, that the human child is born in a conﬁguration of helplessness, which implies that the fellow human being plays a role that is structurally of the utmost importance in the constitution of his subjective world. Let us return to our example of the milk. Up until this point we have brought the scenario as if what is crucially at stake for the child is the sucking. 25This is an ironic reference to Russell’s (1905) “On Denoting,” where he says, albeit from a diﬀerent angle, but with, in our view, the same stakes at play, that there is no backward road from denotations to meanings. The Signiﬁer Inscribed in a Basic Non-attunement of Actions and Needs: The Role of the Other Let us return once more to the question of what happens in the deployment of directed actions by the subject, knowing that it must have been historically struck by the event, accompanied by the experience of the adjacent movement contingently linked to it. We know that all vertebrates capable of action have to cope with an initial non-attunement of actions and needs. The reason for this is structural. Vertebrates are characterized by a double body: an inner, invertebrate sack-like body with all the big vegetative systems (respiration, digestion, excretion, etc.) and a “newly invented” outer body constituted by a skeleton and striated muscles (see Bazan, 2007). While needs arise in the inner, December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org 6 The Mark, the Thing, and the Object Van de Vijver et al. invertebrate body, the speciﬁc actions for the satisfaction of these needs are outer body actions. The structural non-attunement between actions and needs resides in the fact that it is not a priori clear what outer body action could constitute a response to what the inner body needs. Even if this gap is less prominent in most vertebrates as compared to humans (e.g., little horses get on their feet and move toward the mother nipple in the span of hours after birth), the idea is nevertheless that, even in animals, this instinct-encouraged movement has to be sanctioned by a mark (a dopamine-release) to be registered as a movement with a high potential for repetition, and that therefore, independently of instincts, the body registers the history of (contingent) events. Once a speciﬁc action has been linked to an event, what drives to repeat this action, is disconnected from the drive satisfaction itself. Indeed, the relief caused by the satisfaction of an internal body need is only contingently connected to what the external body succeeded to develop as an action. The relief, as the tension itself, is a serendipitous addendum, a by-product, important for survival, but not determinative for what drives repetitive behavior. No matter what the outcome, the child will not stop the endless repetition, the sucking, or, as Freud stressed, the endless uttering of “o-o-o” and then “a-a-a.” From the moment the child accepted and marked the event, it is driven by the repetition compulsion to grasp. Frontiers in Psychology \| www.frontiersin.org The Signiﬁer Inscribed in a Basic Non-attunement of Actions and Needs: The Role of the Other It is a space where contingency, or rather, arbitrariness between form and content reigns: the exchanges between the child and the other, in as far as they are based in signiﬁer exchanges, are ﬁrstly formal exchanges, content being realized in the historical interweaving between those forms, the adjacent bodily movements and the web of directed and intentional actions deployed in their wake. representations corresponding more or less adequately to some or other object out there, are missing the point. A representation is an object, is a motor pattern, and the articulation of the space of motor patterns, being a constraining space, is at the meantime the enabling condition for what counts as an object: the constraint is the possibility.27 This account of the object enables us to address (i) the typically Lacanian idea of the object as bound up with a structural loss – the fact of launching comprehending grasp movements is indicative of, rests on, a step being missed, as we have shown, the step corresponding to a non-understanding, covered by the mark – with the object a that theoretically indicates this ever missed object, (ii) the issue of objective reality, that here refers to successful grasping movements, i.e., movements leading to discharge. Clearly, the issue of discharge is crucial in the constitution of the object. As a matter of fact, only that which can give rise to discharge has a chance to lead to objectiﬁcation. Along these lines, Lacan states that it is impossible to understand what an object is without the dimension of satisfaction, a thing that, to him, largely escaped the philosophical tradition.28 There is, however, a potential confusion in the way in which Lacan uses the term satisfaction, at least in as far as we take it to refer to the satisfaction of needs. We have indeed argued that there is a contingent, arbitrary relation between what brings satisfaction of needs and what is constituted as an object through motor- patterns. We have also argued that object-constitution happens on grounds radically cut from what brings satisfaction of needs. We are indeed speaking of another level, of pleasure “from another source” as Freud states, of objects of enjoyment, as Lacan calls it. In other words, objects of pleasure are not situated at the level of the satisfaction of needs29! The Signiﬁer Inscribed in a Basic Non-attunement of Actions and Needs: The Role of the Other However, due to his helplessness, the repetitive action of the sucking is, per force, supplemented by other actions, e.g., crying, vocalizations, that contingently, but crucially, contribute to realize the conditions within which relief becomes possible. Again, what drives the child to act and repeat its actions is not the possibility of relief in itself, it is the attempt to grasp what initially escaped, namely the surprising event. And in this grasping attempt, the other, as a speaking being, is once more an ineliminable factor. Indeed, a bunch of contingent movements, situated primarily in the realm of vocalizations, that, by surprise, out of nowhere, made a diﬀerence (i.e., brought the mother, the milk, relief), doubles, in a far more whimsical fashion, the logically ﬁrst contingency of the sucking movement. Indeed, a mother, with far more ﬁerceness than, e.g., milk, resists objectiﬁcation, stays together as a Thing. More correctly, it is to the extent that the other “stays together as What both Freud and Lacan note in relation to this repetitive dynamics, a thing that follows logically here, but needs to be December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org Frontiers in Psychology \| www.frontiersin.org 7 The Mark, the Thing, and the Object Van de Vijver et al. stressed time and again, is that the action does not at all need to be adequate to be repeated. This is quite generally what clinical experience conﬁrms: it may be certiﬁed that praying or singing does not stop the earthquake – it is nevertheless repeated. The child’s repetitive “o-a” does not impact on the leaving and the return of the mother, but it is joyfully and victoriously repeated nevertheless. We have defended elsewhere (Bazan et al., 2016) that even if the act is not adequate in grasping the thing, it still is better than sideration or bewilderment; it is the execution in itself that brings relief. Freud will say it is relief “from another source”; Lacan will consider that it radically concerns another domain, the domain of signiﬁers to which the child ﬁnds entrance. And in this domain, the other, the Other, occupies an ineliminable place: without the other/the Other, there would be no “signiﬁcance,” no functioning of the signiﬁers. 27The resemblance with Kantian epistemology is straightforward. Kant’s dictum that “the Thing in itself is not knowable” serves as the starting point for his epistemology: from the moment we talk about objects and objectivity, we talk about what there is “for us,” in our words, what is within the range of the graspable through our motor patterns. Another way of saying the same thing, from within the formalistic tradition in philosophy (Frege in the ﬁrst place) is that the grasping space is a functional space – in line with Kant’s philosophy that articulates the functionalism of Reason. It is from within the functional space that the place is prepared, delineated, circumvented, of what can come to satisfy the function. That place within a functional space, that is the object. In our words: that motor pattern ready to grasp something, that is the object. The formal discussion that is relevant in this regard, is the one on the relation between intension (that deﬁnes the function) and extension (that satisﬁes the function). Lacan works with these distinctions frequently, not in the least in his Seminar XII, Problèmes Cruciaux pour la Psychanalyse (Lacan, 1964–1965/2003). The Signiﬁer Inscribed in a Basic Non-attunement of Actions and Needs: The Role of the Other We propose therefore to reserve the term satisfaction for the level of needs, to talk of objects of pleasure when we are aiming at the object constitution that is related to what brings relief, diminution of tension, and of 26The diﬀerence between Repräsentanz and Repräsentant can once more be brought forward here: however frozen or crystallized the motor pattern is (Repräsentant) it is nevertheless a motor-pattern, hence formal, potentially ready to receive diﬀerent contents, and thus it is also functional in nature (Repräsentanz). The Object as a Coherent Motor Package and the Experience of Satisfaction What is there to say then about the object? We would be inclined to consider an object as a coherent motor package, a bounded set of grasp movements that opens the possibility of discharge. We already said that the Repräsentanz26 “stands for” the event: it might be considered as a crystallization of movement parameters into a solidary whole. It is diﬀerent from what philosophers are traditionally inclined to call an object, though, as it is a contingent whole of movements arbitrarily cut out of a sequence and has no intentional directedness. However, as argued, the motor pattern has the potentiality to launch for a return under the form of directed actions. These actions produce what we would call in the proper sense “mental” representations of what ﬁrst intruded the system and stayed together as a Thing. It are then these “mental representations” that, when executed, can lead to discharge, however partial and temporary that is, and which can, in our view, be genuinely called objects. In other words, the mental, representational inscription amounts to an objectiﬁcation. As this is likely to be the most delicate point of our argumentation, we dare to insist. Firstly, what we call an object or a representation is ﬁrst and foremost a motor pattern, a motor intention, as Jeannerod calls it (Jeannerod, 1994; Bazan, 2007): it is the motor form within which something can be grasped. To address the question of the object, is therefore in the ﬁrst place to ask for the formal arrangement of the space of possible motor patterns. What serves as a ﬁlling up in that space – content, meaning, . . . – is secondary, and does not inform about the arrangement of the space itself. In other words, questions about 28There is no way, dixit Lacan, to conceive of an object without the dimension of satisfaction. With regard to the homeostatic, organic account, he says: “Rien, dans tout cela, qui pousse à la recherche, à la saisie, à la constitution d’un objet. Le problème de l’objet comme tel est laissé intact par toute cette conception organique d’un appareil homéostatique. Il est très étonnant qu’on n’en ait pas jusqu’ici marqué la faille. Freud ici, assurément, a le mérite de marquer, que la recherche de l’objet est quelque chose qui n’est concevable qu’à introduire la dimension de la satisfaction” (Lacan, 1966–1967/2017, p. 156). 28There is no way, dixit Lacan, to conceive of an object without the dimension of satisfaction. With regard to the homeostatic, organic account, he says: “Rien, dans tout cela, qui pousse à la recherche, à la saisie, à la constitution d’un objet. Le problème de l’objet comme tel est laissé intact par toute cette conception organique d’un appareil homéostatique. Il est très étonnant qu’on n’en ait pas jusqu’ici marqué la faille. Freud ici, assurément, a le mérite de marquer, que la recherche de l’objet est quelque chose qui n’est concevable qu’à introduire la dimension de la satisfaction” (Lacan, 1966–1967/2017, p. 156). December 2017 \| Volume 8 \| Article 2244 CONCLUSION In Beyond the Pleasure Principle, Freud seeks to describe and articulate the functioning of the psychic apparatus in situ, that is, anchored in the ways in which human beings sense, move and act. In discussing the issue of Wiederholungszwang, Freud, here perhaps more than anywhere else, starts from the clinical observation of quantities of excitation of which it is not easy, not possible even, for the subject to get rid. That is where to him the disjunction between pleasure and repetition ﬁnds entrance: at the point that cannot be silenced through understanding, the point where pleasure, the possibility of decreasing tension, has come to a limit, the point that in its insistence searches other ways out. Freud’s overt biological phrasing is certainly not a matter of looking to ground the psychical in the biological; it is a matter of cutting the psychical at the correct joints. And this cutting cannot but start from the embarrassment in relation to the body, that is, from the moments and the points where something does not obey the logics of pleasure and lies beyond it as a compulsion to repeat. In sum, we have argued for the inscription of the dynamics of signiﬁers in the structural non-attunement that already exists between actions and needs in mammals, leading to the repetition of actions independently from their being useful or not. Our purpose thereby was not at all to diminish the speciﬁcity of the human condition as a speaking condition. On the contrary, our purpose was thereby to show that we are tempted, time and again, to interpret human behavior too quickly as guided by intentional, consciously guided principles and mechanisms. Signiﬁer repetition is the basic human condition, not intentional behavior! That is what Lacan stresses over and again, linked to the nature and the functioning of the signiﬁer. In this way, Lacan’s viewpoint operates, more explicitly than Freud’s, a categorical shift from the idea that man is or should be guided by what brings satisfaction to his needs, to the idea that man is driven to repeat what was structurally missed. In speaking of a “conceptual intrusion” in relation to the compulsion to repeat, Lacan focuses on what constitutes the mental as a speciﬁc kind of object. In this, he wishes to “ensure,” “faire valoir” Freud (Lacan, 1966– 1967/2017, XIII, p. The Object as a Coherent Motor Package and the Experience of Satisfaction 29It is even questionable whether we can speak of objects of satisfaction; the term “experience of satisfaction” seems more adequate, even if it also demands to be further unfolded, certainly in light of the meaning of the term “experience” in the philosophical tradition. December 2017 \| Volume 8 \| Article 2244 December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org 8 The Mark, the Thing, and the Object Van de Vijver et al. of the mark, in which it is indicated (marked) that something stays together as a Thing exactly to the extent that it is not understood, not grasped through adequate motor-patterns, (iv) that mental representations (signiﬁers), understood as phonemic motor packages, are inscribed into this bodily dynamics of non-attunement, which means that they are particular motor- forms attempting to grasp that from which they are initially and structurally disconnected (the Thing), (v) that this distinction between understanding and judging, combined with the idea that signiﬁers or mental representations are motor-patterns, provides us with a basis to identify processes of repetition (Wiederholungszwang) in terms of repeated attempts situated at the level of the marks, structurally disconnected from what is satisfying at the level of needs, (vi) that the initial helplessness of the infant, together with the subtlety of language, with its (small and ﬂexibly recombinable) phonemic motor-packages oﬀered by the other/the Other, is the means through which the categorical diﬀerence between humans and other vertebrates can be made clinically relevant, and ﬁnally, (vii) that the representational grasping movement corresponds to objectiﬁcation, whereby the object expresses the formal readiness of the representational space, a readiness that can be, in secondary instance, ﬁlled up in various ways, but that, due to the initial non-attunement in which it is grounded, is structurally missing the Thing that it initially marked, leading to an endless compulsion to repeat, without which? There would be no humanity, no culture, no subjective life. enjoyment to indicate the impossibility of relief that the subject is desperately holding on to. It is in relation to this that the exchanges with the fellow human being have to be investigated. Of course, the fellow human being is essential for what is to be called the constitution of objects of pleasure. CONCLUSION 280) in what he was eventually after – the subject of the unconscious – and that is exactly the mental apparatus with as a module the compulsion to repeat. In line with this viewpoint, and taking up Lacan’s revisiting of it in terms of enjoyment, we have argued (i) that the insistence with which subjects repeat is to be grafted upon the structural disconnectedness between what articulates behavior and what satisﬁes needs, (ii) that this structural disconnectedness, this non- attunement, is to be linked to the bodily make up of vertebrates at large, with the basic distinction between an internal body as a source of excitation and an external body as a motoric means of responding to this excitation in an attempt to diminish it, (iii) that it is relevant to introduce here the Freudian distinction between understanding and judging, and to identify understanding with the articulated motor-patterns of the external body that aim at grasping (com-prehending), and the judging with a dynamics The Object as a Coherent Motor Package and the Experience of Satisfaction In providing for the essential means of discharge – carrying out the speciﬁc acts – the other structurally intervenes in the temporality of excitation and discharge of the child, co- determines the identiﬁcation of what counts as an object of pleasure, and in this way also co-determines what lies beyond in terms of enjoyment. That this has its implications for what counts as an experience of satisfaction is evident, but the important thing to note is that it is not the satisfaction that determines the constitution of the objects of pleasure or of enjoyment. Rather, the satisfaction of the need, in this new scheme, is over-written or replaced by the possibility of discharge through grasping movements leading to objectiﬁcation, a possibility that was opened up as a return to the marked event covering that which stayed together as a Thing. The space of satisfaction of needs is thereby subverted into a space whereby the subject is endlessly and repetitively demanding to be recognized at another “level,” the one of subjectivity, expecting from the other to tell him the answer, that is, to bring (to be), for him, the object of relief. Frontiers in Psychology \| www.frontiersin.org REFERENCES Jeannerod, M. (1994). The representing brain: neural correlates of motor intention and imagery. Behav. Brain Sci. 17, 187–245. doi: 10.1017/S0140525X00034026 Lacan, J. (1955–1956/1981). Les Psychoses, Le Séminaire III, éd. M. Jacques-Alain. P i S il Jeannerod, M. (1994). The representing brain: neural correlates of motor intention and imagery. Behav. Brain Sci. 17, 187–245. doi: 10.1017/S0140525X00034026 Aristotle. (1984). “De anima,” in The Complete Works of Aristotle, Vol. 1, ed. J. Barnes (Princeton, NJ: LXXI). Lacan, J. (1955–1956/1981). Les Psychoses, Le Séminaire III, éd. M. Jacques-Alain. Paris: Seuil. Bazan, A. (2007). Des Fantômes Dans la Voix. Une Hypothèse Neuropsychanalytique Sur la Structure de L’inconscient. Collection Voix Psychanalytiques. Montréal, QC: Liber. Lacan, J. (1959–1960/1986). L’Ethique de la Psychanalyse, Le Séminaire. Livre VII. Paris: Seuil. Lacan, J. (1963–1964/1973). Les Quatre Concepts Fondamentaux de la Psychanalyse, Le Séminaire XI, éd. J.-A. Miller. Paris: Seuil. Bazan, A., and Detandt, S. (2013). On the physiology of jouissance: interpreting the mesolimbic dopaminergic reward functions from a psychoanalytic perspective. Le Séminaire XI, éd. J.-A. Miller. Paris: Seuil. Bazan, A., and Detandt, S. (2013). On the physiology of jouissance: interpreting the mesolimbic dopaminergic reward functions from a psychoanalytic perspective. Front. Hum. Neurosci. 7:709. doi: 10.3389/fnhum.2013.00709 Lacan, J. (1963–1964/1981). The Four Fundamental Concepts p f y Book XI, trans. A. Sheridan. New York, NY: W.W. Norton & Company. Front. Hum. Neurosci. 7:709. doi: 10.3389/fnhum.2013.00709 Bazan, A., and Detandt, S. (2015). Trauma and jouissance, a neuropsychoanalytic perpective. J. Cent. Freud. Anal. Res. 26, 99–127. Lacan, J. (1964–1965/2003). Problèmes Cruciaux pour la Psychanalyse, Le Séminaire XII, éd. M. Roussan. Paris: Association Freudienne Internationale. Bazan, A., and Detandt, S. (2017). The grand challenge for psychoanalysis and neuropsychoanalysis: a science of the subject. Front. 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She launched the core-idea of applying Freud’s distinction between judging and understanding to the process whereby a signiﬁer is for the ﬁrst time accepted in a dynamics that was until then a homeostatic December 2017 \| Volume 8 \| Article 2244 9 The Mark, the Thing, and the Object Van de Vijver et al. was intense and the result can be called a common result. SD contributed with her doctoral research on jouissance and the compulsion to repeat, part of the research background that served as a basis for this reﬂective article. pleasure dynamics. The articulation of this idea happened on the basis of already well-advanced research in neuropsychoanalysis on the signiﬁer and on jouissance by AB. She is then second main author. The collaborative work between both these authors Frontiers in Psychology \| www.frontiersin.org December 2017 \| Volume 8 \| Article 2244 REFERENCES Doctoral dissertation, Université Libre de Bruxelles, Bruxelles. Reik, T. (1935/1976). Der Überraschte Psychologe. Über Erraten und Verstehen Unbewusster Vorgänge; Le Psychologue Surpris. Deviner et Comprendre les Processus Inconscients, (trad. Denise Berger). Paris: Denoël. Freud, S. (1895/1963). “On the grounds for detaching a particular syndrome from neurasthenia under the description ‘anxiety neurosis,” in The Standard Edition of the Complete Psychological Works of Sigmund Freud, Vol. 3, trans. J. Strachey (London: The Hogarth Press). Russell, B. (1905). “On denoting,” in Logic and Knowledge, ed. R. C. Marsh (London: George Allen and Unwin), 41–56. g Skinner, B. F. (1948). Superstition’ in the pigeon. J. Exp. Psychol. 38, 168–172. doi: 10.1037/h0055873 Freud, S. (1895/1966). “Project for a scientiﬁc psychology,” in The Standard Edition of the Complete Psychological Works of Sigmund Freud, Vol. 1, trans. J. Strachey (London: The Hogarth Press). Tort, M. (1966/2016). « A Propos du Concept Freudian de ‘Représentant’ (Repräsentanz), Concept and Form: The Cahiers pour l’Analyse and Contemporary Thought. Available at: http://cahiers.kingston.ac.uk/vol05/ cpa5.2.tort.html Freud, S. (1900/1953). “The interpretation of dreams,” in The Standard Edition of the Complete Psychological Works of Sigmund Freud, Vol. 5, trans. J. Strachey (London: The Hogarth Press). Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Freud, S. (1920/1955). “Beyond the pleasure principle,” in The Standard Edition of the Complete Psychological Works of Sigmund Freud, Vol. 18, trans. J. Strachey (London: The Hogarth Press), 1–64. Freud, S. (1920/2015). “Beyond the pleasure principle,” in Psychoanalysis and History, Vol. 17, trans. M. Solms (London: International Psycho-Analytical), 151-204. Copyright © 2017 Van de Vijver, Bazan and Detandt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Freud, S. (1924/1962). “The economic problem of masochism”, in The Standard Edition of the Complete Psychological Works of Sigmund Freud: The Ego and the Id and Other Works, Vol. 19, trans. J. Strachey (London: The Hogarth Press), 155–170. December 2017 \| Volume 8 \| Article 2244 Frontiers in Psychology \| www.frontiersin.org 10
https://openalex.org/W4362424241	https://figshare.com/articles/journal_contribution/Figure_S2_from_Multilevel_Regulation_of_-Catenin_Activity_by_SETD2_Suppresses_the_Transition_from_Polycystic_Kidney_Disease_to_Clear_Cell_Renal_Cell_Carcinoma/22428993/1/files/39875322.pdf	English	null	Figure S4 from Multilevel Regulation of β-Catenin Activity by SETD2 Suppresses the Transition from Polycystic Kidney Disease to Clear Cell Renal Cell Carcinoma	null	2,023	cc-by	824	Figure S2 A B D C E F PTECs Setd2-OE PTECsMYC-OE Cell number (105) Day1 Day2 Day3 0 1 2 3 P=0.0273 P=0.0383 Relative absorbance Day1 Day2 Day3 0.0 0.5 1.0 1.5 2.0 P=0.0243 P=0.0177 Closure (%) 0.4 0.6 0.8 P=0.0356 nvasion ability 0.6 0.8 1.0 1.2 P=0.0079 0hr Setd2-OE PTECsMYC-OE Vector Vector Setd2-OE Setd2fl/fl PTECsMYC-OE Vector PTECsMYC-OE Setd2 mRNA expression Setd2fl/fl Setd2-KO 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0003 Setd2 mRNA expression Vector Setd2-OE 0.0 0.2 0.4 15 20 25 P<0.0001 MYC mRNA expression MYCfl/+ MYC-OE 0.0 0.2 0.4 15 20 25 P<0.0001 MYCStopfl/+ kidney tumor Wild-type Setd2fl/fl Setd2-KO Liver metastases Lung metastases Normal liver Normal lung Ksp1.3/Cre Relative mRNA expression Cdh1 Vim Twist Snail Slug 0.0 0.5 1.0 1.5 2.0 2.5 P=0.0069 P=0.0007 P<0.0001 P=0.0002 P<0.0001 Figure S2 A B D C E F PTECs Setd2-OE PTECsMYC-OE Cell number (105) Day1 Day2 Day3 0 1 2 3 P=0.0273 P=0.0383 Relative absorbance Day1 Day2 Day3 0.0 0.5 1.0 1.5 2.0 P=0.0243 P=0.0177 Closure (%) 0.4 0.6 0.8 P=0.0356 nvasion ability 0.6 0.8 1.0 1.2 P=0.0079 0hr Setd2-OE PTECsMYC-OE Vector Vector Setd2-OE Setd2fl/fl PTECsMYC-OE Vector PTECsMYC-OE Setd2 mRNA expression Setd2fl/fl Setd2-KO 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0003 Setd2 mRNA expression Vector Setd2-OE 0.0 0.2 0.4 15 20 25 P<0.0001 MYC mRNA expression MYCfl/+ MYC-OE 0.0 0.2 0.4 15 20 25 P<0.0001 MYCStopfl/+ kidney tumor Wild-type Setd2fl/fl Setd2-KO Liver metastases Lung metastases Normal liver Normal lung Ksp1.3/Cre Relative mRNA expression Cdh1 Vim Twist Snail Slug 0.0 0.5 1.0 1.5 2.0 2.5 P=0.0069 P=0.0007 P<0.0001 P=0.0002 P<0.0001 Figure S2 Figure S2 B PTECs Setd2 mRNA expression Setd2fl/fl Setd2-KO 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0003 Setd2 mRNA expression Vector Setd2-OE 0.0 0.2 0.4 15 20 25 P<0.0001 MYC mRNA expression MYCfl/+ MYC-OE 0.0 0.2 0.4 15 20 25 P<0.0001 A MYCStopfl/+ kidney tumor Wild-type Setd2fl/fl Setd2-KO Liver metastases Lung metastases Normal liver Normal lung Ksp1.3/Cre B PTECs Setd2 mRNA expression Setd2fl/fl Setd2-KO 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0003 A B Figure S2 A B D C E F G PTECs Setd2-OE PTECsMYC-OE Cell number (105) Day1 Day2 Day3 0 1 2 3 P=0.0273 P=0.0383 Relative absorbance Day1 Day2 Day3 0.0 0.5 1.0 1.5 2.0 P=0.0243 P=0.0177 Wound Closure (%) Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 P=0.0356 Relative invasion ability Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0079 0hr 24hr Setd2-OE Setd2-OE PTECsMYC-OE Vector Vector Setd2-OE Setd2fl/fl PTECsMYC-OE Vector PTECsMYC-OE Setd2 mRNA expression Setd2fl/fl Setd2-KO 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0003 Setd2 mRNA expression Vector Setd2-OE 0.0 0.2 0.4 15 20 25 P<0.0001 MYC mRNA expression MYCfl/+ MYC-OE 0.0 0.2 0.4 15 20 25 P<0.0001 MYCStopfl/+ kidney tumor Wild-type Setd2fl/fl Setd2-KO Liver metastases Lung metastases Normal liver Normal lung r=1 r=0.700, P<0.0001 r=-0.548, P<0.0001 r=-0.480, P<0.0001 r=0 228 P<0 0001 SETD2 VHL CA9 VIM CDH1 TCGA 606 ccRCC samples Ksp1.3/Cre Relative mRNA expression Cdh1 Vim Twist Snail Slug 0.0 0.5 1.0 1.5 2.0 2.5 P=0.0069 P=0.0007 P<0.0001 P=0.0002 P<0.0001 D F Setd2-OE PTECsMYC-OE Cell number (105) Day1 Day2 Day3 0 1 2 3 P=0.0273 P=0.0383 Relative absorbance Day1 Day2 Day3 0.0 0.5 1.0 1.5 2.0 P=0.0243 P=0.0177 Wound Closure (%) Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 P=0.0356 Relative invasion ability Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0079 -OE Setd2-OE Vector Vector PTECsMYC-OE Setd2 mRNA Setd2fl/fl Setd2-KO 0.0 0.2 0.4 0.6 Setd2 mRNA Vector Setd2-OE 0.0 0.2 0.4 15 MYC mRNA MYCfl/+ MYC-OE 0.0 0.2 0.4 15 r=1 r=0.700, P<0.0001 r=-0.548, P<0.0001 r=-0.480, P<0.0001 r=0.228, P<0.0001 r=-0.188, P<0.0001 TCGA 606 ccRCC samples Slug 002 P<0.0001 C Setd2-OE Setd2fl/fl PTECsMYC-OE Relative mRNA expression Cdh1 Vim Twist Snail Slug 0.0 0.5 1.0 1.5 2.0 2.5 P=0.0069 P=0.0007 P<0.0001 P=0.0002 P<0.0001 D Setd2-OE PTECsMYC-OE Cell number (105) Day1 Day2 Day3 0 1 2 3 P=0.0273 P=0.0383 Relative absorbance Day1 Day2 Day3 0.0 0.5 1.0 1.5 2.0 P=0.0243 P=0.0177 Vector D C D C E F G Setd2-OE PTECs Cell number (105) Day1 Day2 Day3 0 1 2 3 P=0.0273 P=0.0383 Relative absorbance Day1 Day2 Day3 0.0 0.5 1.0 1.5 2.0 P=0.0243 P=0.017 Wound Closure (%) Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 P=0.0356 Relative invasion ability Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0079 0hr 24hr Setd2-OE Setd2-OE PTECsMYC-OE Vector Vector Setd2-OE Setd2fl/fl PTECs Vector PTECsMYC-OE r=1 r=0.700, P<0.0001 r=-0.548, P<0.0001 r=-0.480, P<0.0001 r=0.228, P<0.0001 r=-0.188, P<0.0001 SETD2 VHL CA9 VIM CDH1 CDH2 TCGA 606 ccRCC samples Relative mRNA expression Cdh1 Vim Twist Snail Slug 0.0 0.5 1.0 1.5 2.0 2.5 P=0.0069 P=0.0007 P<0.0001 P=0.0002 P<0.0001 F Wound Closure (%) Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 P=0.0356 Relative invasion ability Vector Setd2-OE 0.0 0.2 0.4 0.6 0.8 1.0 1.2 P=0.0079 r r Setd2-OE Setd2-OE PTECsMYC-OE Vector Vector PTECsMYC-OE F E 0hr 24hr Setd2-OE PTECsMYC-OE Vector E G r=1 r=0.700, P<0.0001 r=-0.548, P<0.0001 r=-0.480, P<0.0001 r=0.228, P<0.0001 r=-0.188, P<0.0001 SETD2 VHL CA9 VIM CDH1 CDH2 TCGA 606 ccRCC samples TCGA 606 ccRCC samples G
https://openalex.org/W4387810306	https://www.frontiersin.org/articles/10.3389/fped.2023.1197156/pdf?isPublishedV2=False	English	null	Febrile seizure in children with COVID-19 during the Omicron wave	Frontiers in pediatrics	2,023	cc-by	4,418	TYPE Original Research PUBLISHED 20 October 2023 DOI 10.3389/fped.2023.1197156 Objective: To explore the clinical characteristics and prognosis of febrile seizure in children with COVID-19. Objective: To explore the clinical characteristics and prognosis of febrile seizure in children with COVID-19. Methods: This study is a single-center retrospective cohort study. The cases included febrile seizures in children with COVID-19 admitted to the Renji Hospital from April 7th, 2022 to June 2nd, 2022. We compared children with and without febrile seizures in their clinical characteristics such as sex, age, symptoms, seizure manifestation, COVID-19 severity, and SARS-CoV-2 nucleic acid test results. The children with febrile seizures were followed up by telephone and outpatient service about one month after the nucleic acid turned negative and discharged from the hospital. COPYRIGHT © 2023 Xu, Chen, Zhou, Zhou and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: A total of 585 cases of children with COVID-19 were included in the analysis. There were 15 children (1.8%) with febrile seizures, age from six months to three years old, nine boys (60.0%) and six girls (40.0%). The manifestations of febrile seizures were all generalized tonic-clonic seizures. The median nucleic acid negative conversion time was 11 (IQR:10.75,13) days. Our ﬁrst comparison involved comparing children without underlying diseases; there was no signiﬁcant difference in sex, COVID-19 severity, and clinical manifestations, but there was an age difference (2 vs. 1.3, P = 0.047). There was no difference in SARS-CoV-2 nucleic acid negative time between the two groups (11d vs. 13d, P = 0.128). One child had new clinical manifestations during the follow-up, but his EEG and MRI were normal. Conclusion: Febrile seizure may be children’s primary neurological manifestation of COVID-19. It may occur in children with no history of epilepsy and is not associated with severe illness. The long-term neurological outcomes of these children should be followed up. febrile seizures, COVID-19, Omicron, children, febrile convulsions Pu Xu 1, Xuelian Chen 2, Jianguo Zhou 1, Wenhao Zhou 1 and Laishuan Wang 1* Pu Xu 1, Xuelian Chen 2, Jianguo Zhou 1, Wenhao Zhou 1 and Laishuan Wang 1* Pu Xu 1, Xuelian Chen 2, Jianguo Zhou 1, Wenhao Zhou 1 and Laishuan Wang 1* 1National Health Commission Key Laboratory of Neonatal Diseases, Department of Neonatology, Children’s Hospital of Fudan University, Shanghai, China, 2Department of Obstetrics and Gynecology, Renji Hospital of Shanghai Jiao Tong University School of Medicine, Shanghai, China KEYWORDS febrile seizures, COVID-19, Omicron, children, febrile convulsions 2.2. Data collection Demographic and clinical data of including gender, age, PCR test, infection severity, vaccination status (no data in Table 1), and nucleic acid test negative conversion time, were collected by reviewing the hospital electronic medical records. 2.6. Statistical analysis The study used Microsoft Access 13.0 to create the database and SPSS 20.0 for statistical analysis. The inter-group comparisons of clinical characteristics in the seizure and the non-seizure groups were conducted by chi-square and rank-sum tests. Children of age 0–3 years who had symptomatic COVID-19 infection and without underlying chronic diseases were included in the study. The study patients were divided into two groups: COVID-19 children who had febrile seizures and COVID-19 children who did not have febrile seizure during the course of infection. The disease severity of COVID-19 infection children was classiﬁed based on the WHO COVID-19 Clinical Progression Scale (9), including ﬁve categories: uninfected, mild, moderate, severe, or death. 1. Introduction Children with COVID-19 account for about 10% of all COVID-19 cases and have a less severe illness compared with adult patients (1). The most common symptoms are fever and cough and other symptoms include fatigue, myalgia, nausea and vomiting, abdominal pain, and diarrhea. Most of the symptoms resolve within one week (2, 3). Neurological symptoms varied between new onset seizures, anosmia, ageusia and focal arteriopathy in many studies (4–6). During the Omicron strain epidemic, the number of COVID-19 infections, hospitalization and neurological symptoms were higher than other SARS-CoV-2 variant outbreaks (7, 8). Encephalitis and death have been reported in COVID-19 infected Frontiers in Pediatrics frontiersin.org 01 Xu et al. 10.3389/fped.2023.1197156 3. Results During the study period, a total of 871 children were admitted to Renji Hospital for treatment of symptomatic COVID-19. Of these, 585 children age 0–3 years who did not have underlying diseases were included in the study. Patient demographic and clinical presentations are summarized in Table 1. 2.4. The deﬁnition of nucleic acid conversion children in Hong Kong, Japan, Taiwan and other regions (9). Seizure in children with COVID-19 may be caused by viral encephalitis and brain injury. This study evaluated 585 cases of 0–3 year old young children who were hospitalized for symptomatic COVID-19 infection during the Omicron strain epidemic in Shanghai, China. We identiﬁed 15 children who developed febrile and summarized their clinical characteristics in order to provide a reference for the diagnosis and treatment of seizure in children with COVID-19. The nucleic acid conversion was deﬁned as two consecutive daily negative SARS-CoV2 PCR results. Nucleic acid conversion time was deﬁned as the time duration from the onset of symptoms to the ﬁrst time of the two (ﬁrst negative day) consecutive negative PCR tests. 2.1. Study design and participants This single-center retrospective study was conducted at Renji Hospital (South branch), School of Medicine, Shanghai Jiao Tong University, from April 7, 2022, to June 2, 2022. The hospital was designated treatment of children with COVID-19 infection during the COVID-19 pandemic in Shanghai, China. This study was approved by the ethics committee of the Children’s Hospital of Fudan University (IRB No. 2022–82). 3.1. Clinical presentations of children with seizure during COVID-19 infection There were 15 children, 9 boys and 6 girls, had febrile seizure during the course of infection (Table 1). They were 6 months to 3 years old, with a median age of 2 years (IQR:1.3,2.7). Seven (47%) had mild infection and eight (53%) had moderate infection. All 15 children had fever during the infection. The fevers lasted 2–4 days with peak temperatures of 38–40°C. Other symptoms included cough (n = 9, 60%), runny nose (n = 4, 27%), fatigue (n = 3, 20%), vomiting (n = 1, 7%), no diarrhea and other infection in the course of infection. All 15 children had 1–2 min generalized tonic-clonic seizures while febrile. All of them had only one seizure episode and none of them were treated antiepileptic medication. The median time of nucleic acid negative conversion in the 15 children was 11 (IQR:10.75, 13) days (Figure 1). 2. Method The cutoff point of follow-up was about one month (4–5 weeks) after negative PCR test and the patient was discharged from the hospital. Telephone follow-up: whether there were still COVID-19-related clinical manifestations after discharge, the time and duration of these symptoms, and whether appetite, body weight, sleep and energy changed after discharge. Frontiers in Pediatrics 2.3. Laboratory analysis All cases were laboratory-conﬁrmed as infected with the Omicron variant of SARS-COV-2 by reverse transcriptase- polymerase chain reaction (RT-PCR) nucleic acid test. Nasal swabs were conducted by trained nurses using a standard procedure. Specimens underwent RT-PCR tests for SARS-CoV-2 nucleocapsid gene targets with standardized methods and interpretive criteria. Two SARS-CoV-2 genes, including ORF1ab, and N were detected using SARS-CoV-2 nucleic acid detection, with a cycle threshold of <35 as a positive result, following the manufacturer’s instructions. Frontiers in Pediatrics 02 frontiersin.org Xu et al. 10.3389/fped.2023.1197156 3 2 Comparison between children with and 3 3 Follow up TABLE 1 General data, clinical manifestations and telephone follow-up of children with febrile seizure. Gender Age Severity of illness Nucleic acid conversion time (day) Past history Vaccination COVID-19 related symptoms Convulsive form Telephone follow-up 1 Male 1 year 6 months Mild 14 N N Fever Fever during seizures, 1 general seizure lasting 1–2 min Normal 2 Male 2 years Mild 11 There was one febrile seizure at the age of 1 years and 1 years 10 months. N Fever, Fatigue Fever during seizures, 1 general seizure lasting 1–2 min Normal 3 Female 1 year 3 months Mild 13 N N Fever, Cough, Fatigue Fever during seizures, 1 general seizure lasting 1–2 min Normal 4 Male 3 years Moderate 9 N N Fever Fever during seizures, 1 general seizure lasting 1–2 min Normal 5 Male 3 years Moderate 10 N N Fever Fever during seizures, 1 general seizure lasting 1–2 min Normal 6 Male 8 months Moderate 15 N N Fever, Cough Fever during seizures, 1 general seizure lasting 1–2 min Normal 7 Female 3 years Moderate 8 N N Fever, Cough, Diarrhea Fever during seizures, 1 general seizure lasting 1–2 min Normal 8 Male 2 years Moderate 13 N N Fever Fever during seizures, 1 general seizure lasting 1–2 min Normal 9 Male 5 months Moderate 11 N N Fever, Cough Fever during seizures, 1 general seizure lasting 1–2 min Normal 10 Male 1 years 4 months Moderate 12 N N Fever Fever during seizures, 1 general seizure lasting 1–2 min There is a signiﬁcant increase in night terrors/nocturnal crying. 2.3. Laboratory analysis 11 Female 2 years Mild 11 N N Fever, Cough Fever during seizures, 1 general seizure lasting 1–2 min Normal 12 Male 2 years 8 months Mild 11 N N Fever, Cough Fever during seizures, 1 general seizure lasting 1–2 min Normal 13 Female 1 years 10 months Mild 15 N N Fever, Cough Fever during seizures, 1 general seizure lasting 1–2 min Normal 14 Female 2 years 3 months Mild 9 N N Fever, Fatigue Fever during seizures, 1 general seizure lasting 1–2 min Normal 15 Female 2 years 5 months Moderate 12 N N Fever Fever during seizures, 1 general seizure lasting 1–2 min Normal 3.2. Comparison between children with and without febrile seizure during COVID-19 infection 3.3. Follow-up The parents of 1 Febrile seizure occurred in 252 cases, and the incidence of febrile seizure was 0.84%. We compared our febrile seizure group with children with febrile seizure without COVID-19 in Shanghai, there were no signiﬁcant differences in incidence (1.8% vs. 1.2%, P = 0.095) and gender (P = 0.783) between the two age-matched groups (15). These ﬁndings suggest that febrile seizure is not a common neurological manifestation of COVID-19 infection (14). In another study, the number of children admitted to emergency departments for febrile seizure was signiﬁcantly lower than in previous years due to the habit of wearing masks and social isolation during the COVID-19 epidemic (16). FIGURE 1 Comparison of the time required for each group since the onset of symptoms or the ﬁrst positive nucleic acid. neurological evaluation. No abnormalities were seen on the video EEG and brain MRI. Frontiers in Pediatrics 3.2. Comparison between children with and without febrile seizure during COVID-19 infection 3.3. Follow-up The parents of 1 3.2. Comparison between children with and without febrile seizure during COVID-19 infection 3.3. Follow-up The parents of 1 The parents of 15 children with febrile seizures were followed up by telephone at about one month (4–5 weeks) after negative PCR test. Fourteen children (93%) were reported healthy with no persistent or new COVID symptoms or other health problems after discharge. During the telephone follow- up, the parents of Child #10 (in Table 1) reported that the child had signiﬁcant increase in numbers of night terrors/ crying at night but no seizures, fever, cough, gastrointestinal abnormalities and other symptoms. Two months after discharge, this child was readmitted to our hospital for Demographics and clinical presentations of children in the seizure group and non-seizure group were compared (Table 2). There was no signiﬁcant difference in sex, number of patients in different categories of infection severity and clinical manifestation between the two groups. However, there was a signiﬁcant difference in median age (2 vs. 1.3 years, P = 0.047). There was no signiﬁcant difference in nucleic acid negative conversion time between the two groups (11d vs. 13d, P = 0.128) (Figure 1). Frontiers in Pediatrics 03 frontiersin.org Xu et al. 10.3389/fped.2023.1197156 TABLE 2 Comparison of clinical characteristics between febrile seizure group and non-febrile seizure group. FIGURE 1 Comparison of the time required for each group since the onset of symptoms or the ﬁrst positive nucleic acid. Febrile seizure group (N = 15) Non-febrile seizure group (N = 570) P Gender (%) Male 9 (60.0%) 328 (57.5%) 0.692 Female 6 (40.0%) 242 (42.5%) 0.692 Median age (IQR, year) 2 (1.3, 2.7) 1.3 (0.75, 2) 0.047 Severity of illness (%) Mild 7 (46.7%) 353 (61.9%) 0.141 Moderate 8 (53.3%) 217 (38.1%) 0.141 Severe 0 (0%) 0 (0%) 1 General symptoms (%) Fever 15 (100%) 540 (94.7%) 1 Cough 8 (53.3%) 319 (56.0%) 1 Diarrhea 0 (0%) 77 (13.5%) 0.248 Vomit 1 (6.7%) 61 (10.7%) 1 Fatigue 3 (20.0%) 96 (16.8%) 0.741 multicenter study, Garazzino et al. evaluated 168 children with COVID-19 and reported the prevalence of afebrile and febrile seizures to be 1.8% and 1.2% (3). During 2019.01–2020.12, there were 29,825 cases of febrile children without COVID-19 treated in Shanghai with a sex ratio of 1.4 (17,377 males and 12,448 females). The age ranged from 5 months to 12 years old, with an average of (5.02 ± 1.64) years. frontiersin.org 4. Discussion Febrile seizure is one of the most common neurological disease in infants and young children, which usually occurs from 6 months to 5 years old, and the incidence rate is 2% and 4% in children under ﬁve years old. The prognosis is usually good, although about 1/3 of children are at risk of recurrence (10). The pathogenesis of febrile seizure is not clear and is generally believed to be caused by multiple factors, including, but not limited to, elevated body temperature, viral infection, some vaccinations, family inheritance, etc. al (11). Viral infections, especially those that cause high fever, have been shown to increase neuronal excitability and lower seizure threshold, especially in the immature nervous system (12). Common viruses that cause febrile seizure include human herpesvirus 6, inﬂuenza, adenovirus, parainﬂuenza and chickenpox (13). The age range of 15 coronavirus Omicron variant infection children were from 6 months to 3 years old, which conformed to the typical age range of febrile seizure, and all the seizure were generalized (tonic-clonic) seizure. Fifteen children had only one seizure in the course of the disease, and the duration was 1– 2 min, which was consistent with simple febrile seizure. Some cases have been reported in South Africa and Sweden in children outside the typical age range for febrile seizure (17, 18). Available evidence indicates that novel coronavirus is known to be neuroinvasive and can cause cytokine storms, increasing nerve excitability (19, 20). It has also been suggested that children with COVID-19 may experience hypoxia, metabolic disorders, organ failure or brain damage, all of which may lead to a lower seizure threshold (21) in children with COVID-19, the underlying causes of seizure may be related to fever, encephalitis or childhood multiple system inﬂammatory syndrome (MIS-C), so we should be careful to diagnose febrile seizure. Considering the seriousness We here report 15 cases of novel coronavirus Omicron variant infection with febrile seizure in children, accounting for 1.8% of the children’s cases treated in designated hospitals. A US study showed that 0.5% of children with COVID-19 were diagnosed with febrile seizure, and most of them had no co-infection, with about 9 percent of them requiring intensive care (14). In an Italian 04 frontiersin.org Xu et al. 10.3389/fped.2023.1197156 of children infected with COVID-19, these causes must be considered when children develop seizure. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Funding Supported in part by a grant from the National Key Research and Development Program of China (2021YFC2701800, 2021YFC2701801) and the Shanghai Municipal Science and Technology Major Project (ZD2021CY001). Ethics statement During the follow-up, one child had new health problems, and there was no abnormality in VEEG and cerebral MRI during the follow-up. Among the reported cases, the short-term outcome of nervous system injury in most children is good, but whether there are long-term sequelae remains to be further studied. A large amount of evidence shows that the incidence of post- COVID-19 syndrome is higher in adults (22), but there are few related studies in children. Therefore, it is necessary to follow up those COVID-19 cases for a longer time and the ongoing (uncompleted data) follow up program will give us more evidence. As a retrospective single-center study, the characteristics of COVID-19 children with febrile seizure were reported for the ﬁrst time in mainland China. However, due to the low incidence of febrile seizure, the overall number of cases is small. Based on the existing 15 cases of children, we found that the clinical manifestations of febrile seizure caused by novel coronavirus were similar to those caused by other related viruses, compared with children without febrile seizure without underlying diseases in the same age group. There was no signiﬁcant difference in sex, classiﬁcation, clinical manifestation and viral nucleic acid negative time, and the prognosis was good. Compared with adults, febrile seizure may be the main manifestation of COVID-19 in some children. It may occur even in children who have no history of epilepsy and are not associated with serious illness. Attention should be paid to early identiﬁcation and timely improvement of the relevant nervous system examination and long-term continuous follow-up to verify the impacts on the developing nervous system. During the follow-up, one child had new health problems, and there was no abnormality in VEEG and cerebral MRI during the follow-up. Among the reported cases, the short-term outcome of nervous system injury in most children is good, but whether there are long-term sequelae remains to be further studied. A large amount of evidence shows that the incidence of post- COVID-19 syndrome is higher in adults (22), but there are few related studies in children. Therefore, it is necessary to follow up those COVID-19 cases for a longer time and the ongoing (uncompleted data) follow up program will give us more evidence. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identiﬁable images or data included in this article. 6. Mirzaee SMM, Gonçalves FG, Mohammadifard M, Tavakoli SM, Vossough A. Focal cerebral arteriopathy in a pediatric patient with COVID-19. Radiology. (2020) 297(2):E274. doi: 10.1148/radiol.2020202197 Author contributions LW and WZ: designed the project. PX: wrote the ﬁrst draft and reviewed data from online databases. JZ and XC: edited subsequent drafts of the paper and approved the ﬁnal manuscript. All authors contributed to the article and approved the submitted version. As a retrospective single-center study, the characteristics of COVID-19 children with febrile seizure were reported for the ﬁrst time in mainland China. However, due to the low incidence of febrile seizure, the overall number of cases is small. Based on the existing 15 cases of children, we found that the clinical manifestations of febrile seizure caused by novel coronavirus were similar to those caused by other related viruses, compared with children without febrile seizure without underlying diseases in the same age group. There was no signiﬁcant difference in sex, classiﬁcation, clinical manifestation and viral nucleic acid negative time, and the prognosis was good. Compared with adults, febrile seizure may be the main manifestation of COVID-19 in some children. It may occur even in children who have no history of epilepsy and are not associated with serious illness. Attention should be paid to early identiﬁcation and timely improvement of the relevant nervous system examination and long-term continuous follow-up to verify the impacts on the developing nervous system. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 8. Shi DS, Whitaker M, Marks KJ, Anglin O, Milucky J, Patel K, et al. Hospitalizations of children aged 5–11 years with laboratory-conﬁrmed COVID-19 —COVID-NET, 14 states, March 2020–February 2022. Morb Mortal Wkly Rep. (2022) 71(16):574. doi: 10.15585/mmwr.mm7116e1 7. Marks KJ, Whitaker M, Agathis NT, Anglin O, Milucky J, Patel K, et al. Hospitalization of infants and children aged 0–4 years with laboratory-conﬁrmed COVID-19—COVID-NET, 14 states, March 2020–February 2022. 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https://openalex.org/W4310190332	https://bmchealthservres.biomedcentral.com/counter/pdf/10.1186/s12913-022-08844-z	English	null	Legal advice and care-effective use of care and case management: limits, risks and need for change	BMC health services research	2,022	cc-by	7,038	© The Author(s) 2022, corrected publication 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Introduction An important dimension of care and case managers is to support geriatric patients in obtaining social services in medical, nursing, therapeutic and social fields. To this, they advise and represent their patients. Methods The documentation of patient contacts with case managers of a network of physicians was evaluated. In particular, activities involving legal advice were analysed in detail, compared with the current legal situation in Germany and evaluated. In addition, qualitative expert interviews were conducted. The content and the legal require- ments of legal services law were determined by applying legal interpretation methods (esp. wording, telos, systemat- ics). The results of the evaluation of the documentation were compared with legal requirements. Results Care and case management touches activities in some fields of action without having a legal basis in legal services law. This leads to the fact that these services may not be provided and to - uninsured and uninsurable - liabil- ity risks. Discussion With the introduction of care and case management into standard care, both social law and the Legal Services Act must be adapted to enable the legally compliant use of care and case managers. Otherwise, certain services that are useful for the care of patients may not be provided. Keywords Care and case management, Legal requirements, Social law, Public health, Geriatrics BMC Health Services Research BMC Health Services Research Ruppel et al. BMC Health Services Research (2022) 22:1439 https://doi.org/10.1186/s12913-022-08844-z Open Access Legal advice and care‑effective use of care and case management: limits, risks and need for change Thomas Ruppel1, Max Georg Hügel2, Simone Gloystein1 and Neeltje van den Berg1 Introductionh The project was carried out in Germany, so some aspects, such as occupational profiles and legal frame- works, have a German context. Care and case manage- ment organises and coordinates the care of patients with complex treatment needs and establishes treat- ment pathways for patients. Care and case manage- ment is often used for geriatric patients. They support patients with different care needs and in various living situations to find their way through the jungle of wel- fare state services. Correspondence: Thomas Ruppel kanzlei@gesundheitsrecht.de 1 University of Greifswald, Greifswald, Germany 2 Bucerius Law School, Hamburg, Germany *Correspondence: Thomas Ruppel kanzlei@gesundheitsrecht.de 1 University of Greifswald, Greifswald, Germany 2 Bucerius Law School, Hamburg, Germany For the implementation of the intervention in the following project, health professionals were qualified according to a modular curriculum that was used for the training of case managers. Content was taught on various © The Author(s) 2022, corrected publication 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ruppel et al. BMC Health Services Research (2022) 22:1439 Page 2 of 7 Page 2 of 7 fields of action, such as health and project management, risk identification, creation of clinical pathways, case management or risk management. The qualification pro- gram for the prospective case managers consisted of an online part (40 hours) and a practical training in presence (40 hours). The online training consisted of independent completion of a homework assignment, video lessons, and video tutorials. Participants completed the hands-on training in the form of a one-week classroom workshop. The two training formats were followed by the “extended training in practical everyday life”. The case managers brought relevant experience from their initial training as nurses, therapists or physician assistants. They provided consultation and coordination services on medical, ther- apeutic, nursing and social aspects across occupational groups, sectors and social codes. of the intervention group have been cared for by care and case managers over a period of twelve months. On the basis of an extensive geriatric assessment, gaps in care and individual patient needs and resources were identified. On the basis of the assessment results, indi- vidual treatment plans were drawn up for patients in collaboration with treating general practitioners. The effects of intervention will be evaluated in a controlled design with five intervention and three control regions. The design and methods of the Rubin project have been published [8]. For the analysis of the activities of care and case managers in relation to the legal situation, the docu- mentation of patient contacts with case managers for a network (member of the intervention group) of the RubiN project was evaluated. In particular, the activi- ties related to legal advice were analysed in detail, com- pared with the current legal situation in Germany and evaluated. Management of the often complex care of geriatric patients by care and case managers or other coordinat- ing functions has often positive effects on both the health situation of patients and on the [1] burden [2] of relatives [3]. Through care and case management, tasks in the health care system are better assigned to the right places and at the right times, which for example, also leads to a reduction in the workload of general practitioners [4, 5]. In addition to the evaluations of performance, expert interviews were conducted as open, semi-structured surveys with the project managers of intervention net- works. The results of the performance evaluations were deepened and explained with these interviews. The content and legal requirements of the Legal Ser- vices Act were determined by applying the legal meth- ods of interpretation. Interpretation methods are legal methods used in science and legal practice to inter- pret the contents of norms, to exegete a text. The aim of interpretation is to determine the content, abstract concepts are thereby given a concrete meaning; they include, among others, the methods for interpreting the wording of a norm, for understanding the previous and current norm maker, for the systematic position of a provision in the structure of one and further laws, for achieving the highest possible effectiveness of constitu- tional requirements in lower-ranking legal provisions, etc. These methods are applied in whole or in part in the interpretation of a norm. The [9] results of the documentation evaluations and the expert interviews were compared with legal requirements [10]. Legal lim- its found in this way were interpreted and conclusions were drawn in terms of utilities and jurisprudence. In many cases, it makes organisational sense for care and case managers to work in networks of doctors rather than for individual practices. The care of geri- atric patients in physician or care networks improves, for example, the utilization of services for patients with dementia [6, 7]. Since care and case managers provide advice at the interfaces of medical, social and legal issues, this raises the question of the legal limits of legal advice by care and case managers and a possible need for legal adaptation, since otherwise effective use of care in standard care can- not be guaranteed and would be associated with liability risks for case and care managers. In this paper, those selected activities of case managers in the RubiN project (regional network of care, funded by the Innovation Fund of the G-BA) that are at the inter- face of legal advice and representation, are analysed in terms of content and compared with the current legal situation in Germany. Activities involving legal advice, using the example of RubiNi The project “RubiN -continuous care in regional net- works” is designed as a prospective controlled interven- tion study to investigate whether multi-professional, cross-sectoral and assessment-based car and case man- agement leads to an improvement in the identification, care situation and health status of geriatric patients who still living in their own homes in different regions in Germany. In the RubiN project, geriatric patients Data from the five physician intervention networks were evaluated. In the networks, a total of 3418 patients were recruited for the RubiN- project. After checking the inclusion criteria (age ≥ 70, score from the geriat- ric screening (ANGELINA) ≥ 2 from at least two differ- ent subject complexes and membership of a statutory Ruppel et al. BMC Health Services Research (2022) 22:1439 Ruppel et al. BMC Health Services Research (2022) 22:1439 Page 3 of 7 Ruppel et al. BMC Health Services Research (2022) 22:1439 Table 2 Legal advice content with reference to health insurance (N = 1106 from total 24,028) health insurance) and taking into account revocations granted or other reasons for inactivation (before the start of intervention on the part of the patients), the data of 3059 patients were taken into account in this work. These 3059 patients (1970 were women) were followed up as part of the RubiN project. The patients were on average 81,9 years old. For the 3059 patients, a total of 34,940 contact logs with service documentation are available for the intervention period of 12 months. On average, 11,4 performance records were completed per patient during that time. The minimum number per patient is 1 and the maximum number per patient is 105 service records; 6 service records were completed most frequently. A total of 63,902 consultation contents (Table 1) were documented, of which 3170 consultation contents (Tables 2 and 3) were relevant to a possible violation of the Legal Services Act. Activities involving legal advice, using the example of RubiNi Table 2 Legal advice content with reference to health insurance (N = 1106 from total 24,028) Participation in applications legal area SGB V Number Prescription of physical therapy 232 Prescription physiotherapy 31 Prescription of ergotherapy 37 Speech therapy 12 Aid 175 Rehab sport (partly SGB XI) 46 Hospitalization 64 Geriatric day clinic 67 Specialised geriatric rehabilitation 73 Application for treatment in a geriatric clinic 16 Outpatient nursing service - Medical treatment care 146 Outpatient care service - Domestic work 36 Household help 27 Travel costs 42 Copayment exemption 102 Table 1 first shows the total number of advisory services. Table 2 contains that part of the counselling services that is directly relevant to the topic of legal counsel- ling, namely the involvement of care and case manag- ers in filing of applications, categorised according to the Social Codes V (statutory health insurance) and IX (rehabilitation and participation of people with disabilities). Results of the expert interviews An expert interview is a qualitative research method in which experts in their field are interviewed using guide- lines. It is not about the person themselves, but about their expertise or their way of seeing or acting (insider knowledge) in their field of expertise. For the interviews, topics were collected beforehand, which were then dis- cussed in a flexible framework. This was a semi-struc- tured approach. Five expert interviews were conducted with the project managers of the practice networks par- ticipating as consortium partners in RubiN. In the course of these expert interviews, one practice network said clearly that, from the point of view of the practice net- work and care and case managers there, no legal advice was given; patients were only asked whether living wills and health care proxies existed, where they were stored Some of the services were documented as “applica- tions”. The term “application” represents very hetero- geneous activities: the search for forms, assistance in filling out the forms by care and case managers, and the application process by care and case managers as rep- resentatives of the patients. The involvement in appli- cations in this sense covered a wide variety of service areas. The most common services were “Application for a care degree”, “Application for a prescription for physi- otherapy” and “Application for aids”. Care and case managers were also involved in 89 appli- cations for the determination of severely disabled status and the granting of benefits under the State Law on the Blind and in one appeal procedure. Apart from social law, care and case managers also supported their patients in the drafting of living wills (137 cases), health care pow- ers of attorney (137 cases) and guardianship orders (61 cases). Results of the expert interviews Table 3 Legal advice content with reference to long-term care insurance(N = 2064 from total 24,028) Participation in applications legal area SGB XI Number Daycare 117 Short-term care 78 Preventive care 77 Full inpatient care 50 Home nursing 11 Care level 1107 Outpatient nursing service - Basic care 93 Low-threshold care and relief service 251 Home emergency call 130 Measures to improve the living environment 150 Table 3 Legal advice content with reference to long-term care insurance(N = 2064 from total 24,028) Participation in applications legal area SGB XI Number Daycare 117 Short-term care 78 Preventive care 77 Full inpatient care 50 Home nursing 11 Care level 1107 Outpatient nursing service - Basic care 93 Low-threshold care and relief service 251 Home emergency call 130 Measures to improve the living environment 150 Table 3 Legal advice content with reference to long-term care insurance(N = 2064 from total 24,028) Table 1 Consultation content: (N = 63,902 with 23,518 contacts) Variable Number Medical: 16,867 Nursing: 13,258 Therapeutic: 13,613 Social: 20,164 Table 1 Consultation content: (N = 63,902 with 23,518 contacts) Variable Number Medical: 16,867 Nursing: 13,258 Therapeutic: 13,613 Social: 20,164 Table 1 Consultation content: (N = 63,902 with 23,518 contacts) Variable Number Medical: 16,867 Nursing: 13,258 Therapeutic: 13,613 Social: 20,164 Ruppel et al. BMC Health Services Research (2022) 22:1439 Page 4 of 7 question is not completely clear, but can only be obtained through reflection and evaluation. and whether the family doctor was informed. If no living wills and health care proxies existed, they were advised to do so and provided with information material from the Federal Ministry of Justice. In response to an explicit enquiry, this network of practices stated that, in particu- lar, no advance directives and living wills to tick off had been distributed, nor had such prefabricated precau- tionary documents been jointly completed. The staff had been trained by a local authority, and great care had been taken to ensure that the care and case managers knew their limits. l The “necessity” of a legal examination required by law contains an objective and a subjective component. The subjective component is linked to the expectations of the person seeking legal advice. If he asks specific legal ques- tions, his expectation of receiving a legal service becomes clear. If this is the case, it is a legal service. Results of the expert interviews Objective aspects such as the perception of the general public or the protective purpose of the law (what is the purpose of the law, what exactly is this law supposed to “protect”) are then no longer relevant [12].h The managing director of another practice network stated that the care and case managers would provide advice on all relevant social security codes, in particu- lar on basic security, help with care and the scope of the family insurance fund, but also on matters of tenancy law and housing benefit, and would provide support with applications. The objective component catches in particular the cases in which the person seeking legal advice does not even know that he is asking for legal advice or even expressly wants to do without it. It consists of the val- ues of the usual views of business dealings and the pro- tective purpose of the RDG and is therefore ultimately paramount [13]. In this context, in particular, the express waiver of the protection of the Legal Services Act by the person seeking legal advice is not possible (cf. § 3 RDG). In a third practice network, the interviewee stated in the interview that in the case of living wills (usually a written declaration of intent of a person in the event that he or she is no longer able to express his or her will to doctors or nursing staff) and health care proxies (an instrument of legal precaution which stipulates that the proxy handles financial, organisational and medical pro- cedures on behalf of the person who has granted the power of attorney), reference would be made to the care associations. However, the forms of the Federal Ministry of Justice are also available. If there are problems with fill- ing out the forms, care and case managers also provide support. As a result, it is evident that much of the work of care and case managers is likely to fall under the heading of ‘legal services’. This is indeed supported by the empirical research on which this article is based, as explained in more detail in the following section. Advice and representation in applications for social benefitsh The evaluation of data and of interviews show that care and case managers are involved in a variety of very heter- ogeneous ways in making applications to different service providers. Applying the definition of legal counselling that has been elaborated, these services, both advisory and external representation, are inadmissible for lack of authorisation. This is because, according to the inter- views, care and case managers act as advisors in social law matters, among other things, and support patients in filling out application forms. Such advice and sup- port necessarily presupposes that care and case manag- ers relate the actual circumstances of their patients to the relevant legal bases. A completely schematic application, identical for each individual case, is far from the case in this individual counselling of each patient. Moreover, since patients wanted to have concrete legal questions answered in this counselling and since, in any case, legal transactions regard professional advice in social, tenancy and other fields of law as legal advice (cf. § 11 para. 2 RDG), care and case managers provide a legal service in the sense of § 2 para. 1 RDG in such cases without having the necessary permission in the sense of § 3 RDG. From the activities actually carried out, it can be deduced that expertise was lacking, especially where legal deadlines One practice network reported in the interviews that there had been requests to help with the drafting of wills, this had been refused. Concept of legal adviceh The described counselling and representation activities of the care and case managers are legal services in the sense of § 2 para. 1 of the Legal Services Act (RDG; this regu- lates who and under which circumstances may provide legal services in Germany) and thus subject to registra- tion according to §§ 3, 15b RDG: Legal service is “any activity in concrete third-party matters as soon as it requires a legal examination of the individual case” (Section 2 (1) RDG). The require- ments for the existence of a “legal examination” are quite low, which is already evident from the legislative his- tory, in which alternative proposals such as “extensive legal examination” or “in-depth legal examination” were rejected in favour of the general, simple “legal examina- tion”, which does not require any special characteristic [11]. Legal advice is given when the answer to a legal Ruppel et al. BMC Health Services Research (2022) 22:1439 Page 5 of 7 Page 5 of 7 to pension insurance [16]. The exception regulation in § 1908f exp. 4 BGB with the consultation to precaution- ary powers of attorney applies only to these and only to recognized care associations. The RDG thus applies to all other providers of these services [17]. and formal requirements had to be complied with or where knowledge of special regulations (such as in the case of fictitious authorisations) and court rulings (such as the case law of the supreme court [BGH] on require- ments for living wills) was important. As a result, the payers of the services applied for directly by care and case managers are initially obliged under current law to reject care and case managers - or more precisely: their employer - as representatives of the insured patients, since the service is provided in contra- vention of Section 3 of the RDG, Section 13 (5) SGB X. Procedural acts performed after rejection are invalid, but acts performed up to that point remain valid (reverse conclusion from § 13, Subsection 7, Sentence 2, SGB X) [14]. However, it is obvious that the cost units do not even notice this because the representation is not made clear to the outside; especially if, after legal advice by care and case managers, the insured patient signs the appli- cation himself. Concept of legal adviceh Additional risks of this legal advice are manifold, for example with regard to questions of form and time limits when filing objections or the ignorance of the fictitious approval according to § 13 Para. 3a SGB V, which is especially important in psychotherapy. In the case of this fictitious approval, if the health insurance fund does not decide on an application for benefits within a certain period of time, it is pretended that approval has been granted, the approval is fictitious (faked). In this way, there is a risk that patients’ claims will be lost or that other legal disadvantages will be caused. It remains questionable whether the activity according to § 5 RDG is permitted as an ancillary service to another profession or activity. According to § 5 para. 1 sentence 2 RDG, this is to be assessed “according to its content, scope and factual connection with the main activity, tak- ing into account the legal knowledge required for the main activity”. This is because many professional activi- ties also require the provision of peripheral legal services in order to be able to perform it in accordance with the requirements of the activity. Whether legal advice is necessary as an ancillary service to the job description results in particular from legally regulated job descrip- tions and job descriptions [18]. The decisive factor here is that the legal service is related to a (main) service and that it is only an ancillary service in relation to this [19]. The performance records show that counselling ser- vices take up a large share of the activities of care and case managers. Non-counselling services only accounted for a small proportion, such as referrals to network partners (N = 1355). Delegation services according to Annex 24 of the Bundesmantelvertrag (agreement between the Kas- senärztliche Bundesvereinigung (Federal Association of Statutory Health Insurance Physicians) and the Spitzen- verband Bund der Krankenkassen (National Association of Statutory Health Insurance Funds) to ensure the provi- sion of medical care for insured persons) as physical, del- egated medical services took place in the entire project in only one intervention region (N = 3756); here, the focus was on blood glucose measurements, blood sampling and medication checks.h Discussion and conclusion As comprehensible and sensible as this is from a holis- tic care science perspective, it meets with considerable reservations from a legal perspective. Legally, a partisan lawyer role and a neutral mediator role are categorically mutually exclusive. This is explicitly regulated, for exam- ple, in the Mediation Act (§§ 1 para. 2, 2 para. 3, 3 para. 2 MediationsG), which states that anyone who is or was already acting for a party in the same matter may not act as a neutral and all-partial mediator (extrajudicial media- tor between parties). Anyone who is partisan can no longer be all-partisan or impartial. Care and case managers also provide legal services in their holistic counselling and assistance to patients. There is currently no legal basis for these. However, low-thresh- old access to counselling on social benefit entitlements and living wills and advance directives makes sense for the effective use of care and case management and the achievement of the associated goals. Otherwise, counsel- ling would not be provided at all or would be provided by persons who are legally ignorant or not authorised to do so (relatives, doctors, care services, care support centres). A lawyer’s monopoly for counselling on this scale would presumably lead to the loss of this instrument without replacement rather than to a shift of legal counselling to the legal profession, which in itself is called upon to do this, but which is less easily accessible, especially for geri- atric patients. Further problems of this double role arise from the fact that the function of care and case managers becomes contourless in the application process. If they, as media- tors between the patient and the service provider, are to realise both interests equally, they would logically also have to be part of the communication process of both sides. In this case, however, an application for ben- efits from the patient would already be considered to have been received by the service provider when it is received by the care and case manager (§ 16 para. 2 sen- tence 2 SGB I); conversely, the care and case manager as the patient’s representative would also be authorised to receive authorisations and other declarations with effect for the patient - until a rejection according to § 13 para. 5 SGB X. Discussion and conclusion The care and case managers would thus be responsible both to the service providers and to the patients for any delays in passing on information. Counselling is the main service provided by care and case managers. The classification of legal counselling as an ancillary service could only be considered if one were to differentiate between counselling and legal counselling for care and case managers. This cannot be delineated from the service records; moreover, this differentiation would artificially split the holistic counselling approach of care and case managers. This is all the more true as the medical and social counselling tasks are inextricably linked to counselling on legal claims precisely to enforce the medical and social goals of the patients. The purpose of the RDG, which is to protect against improper legal advice by those not sufficiently qualified for this and those with liability insurance, also argues, in view of the considerable risks described, that the assumption of a legal advisory ancillary service within the meaning of § 5 RDG in addition to counselling as the main service is ruled out. Whenever legal issues are also the subject of counselling by care and case managers, the counselling activity as a whole is subject to authorisation. This leads to a considerable role conflict of care and case managers between their mediator position on the one hand and the role of the “patients advocate” on the other. This responsibility, with the risk of considerable loss of trust and data protection problems on both sides, is problematic. Because with such an involvement of care and case managers in the treatment process, patients can never be sure whose interests the care and case managers are supposed to look after and actually do look after. Therefore, in order to close this gap in the need for counselling, it should be considered - under the condi- tion of sufficient qualification - to allow appropriate legal counselling services and representation in individual cases by care and case managers for their patients. The curriculum of the additional legal qualification must therefore be quite demanding and comprehensive, at least for living wills and advance directives (cf. § 1908f paras. 1 and 4 BGB), in order to do justice to the com- plexity of these fields of counselling. Without such a qualification, care and case managers cannot fulfil the trust placed in them by patients and the situational need for counselling. No suitable exemption rules Apart from lawyers, legal services may also be provided, among others, by way of exception by publicly funded consumer associations, associations of voluntary welfare work pursuant to § 5 SGB XII and recognised associa- tions for the promotion of the interests of disabled per- sons pursuant to § 15 (3) BGG within the scope of their tasks and responsibilities (§ 8 (1) no. 5 RDG). However, these and other exemption provisions - i.e. special per- mits for legal advice by non-lawyers - are not fulfilled in the case of care and case management. In particular, this service is not provided by recognised associations of independent welfare work within the meaning of SGB XII or by associations for the promotion of the interests of persons with disabilities, but is rather closely related to health services provided by health insurance. It is also not a privileged pension consultation in the sense of § 10, Subsection 1, Sentence 1, No. 2, RDG. This excep- tional circumstance always requires a concrete pension reference for consultation to the social security right [15]. When creating the RDG, the legislature deliber- ately decided against the creation of a general social law advisor and deliberately wanted to limit the exception The evaluation of data does not show whether possible legal counselling activities fall under “social” or “medical”, if there was a reference to benefit applications vis-à-vis health insurance funds. In the one (out of five) interven- tion region in which delegation services were provided, 5198 consultations (4515 consultations with patients directly, 556 consultations with relatives (relatives only), 106 joint consultations (patient and relative) and 21 without related information) were recorded with a total of 11,866 consultation contents (medical: 4803; nursing: 1039; therapeutic: 1191; social: 4833) were compared with a total of 3756 medical delegation services. Even in the intervention region where care and case managers also provided delegation services at the same time, coun- selling services still accounted for the majority of ser- vices; where no delegation services were provided, this is the case anyway. Ruppel et al. BMC Health Services Research (2022) 22:1439 Page 6 of 7 Page 6 of 7 Ruppel et al. BMC Health Services Research (2022) 22:1439 Discussion and conclusion Systematic interpretation aims to reveal the content of a norm by attempting to derive the norm’s function from the context with other norms and the overall structure; Pieper, in: Dauses/Ludwigs, Handbuch des EU-Wirtschaftsrechts, work status: 51st EL October 2020, B I marginal no 24. 10. Cf. Bydlinski, Juristische Methodenlehre und Rechtsbegriff, 2nd ed. 1991, p. 395 ff. 11. Cf. Krenzler, in: Krenzler, RDG, 2nd ed. 2017, § 2 marginal no. 16. 12. Krenzler, in: Krenzler, RDG, 2nd ed. 2017, § 2 marginal no. 27. 13. So also von Lewinski, in: Klowait/Gläßer, MediationsG, 2nd ed. 2018, § 2 RDG Rn. 16. 14. Prehn, in: Diering/Timme/Stähler, SGB X, 5th ed. 2019, § 13 marginal no. 34. 15. BT-Drs. 16/3655, p. 64; Schmidt, in: Krenzler, RDG, 2nd ed. 2017, § 10 marginal no. 35. 16. BT-Drs. 16/3655, p. 64; Schmidt, in: Krenzler, RDG, 2nd ed. 2017, § 10 Rn. 33. 17. Cf. For the health care proxy OLG Karlsruhe 23.12.2010 - 4 U 109/10, ZIP 2012, 20. 18. Allemand, in: Henning/Lackmann/Rein, Privatinsolvenz, 2020, § 5 RDG Rn 1. 19. von Lewinski, in: Klowait/Gläßer, MediationsG, 2nd ed. 2018, § 5 RDG Rn. 2. Heymann R, Flessa S, Hoffmann W. Delegation of GP-home visits to quali- fied practice assistants: assessment of economic effects in an ambulatory healthcare centre. BMC Health Serv Res. 2010;10:155. regulating paid legal advice also to care and case manag- ers (§ 10 para. 1 p. 1 no. 2a RDG), the necessary qualifica- tion requirements, personal suitability, liability insurance protection etc. and be implemented, analogous to § 12 RDG already applicable to other professional groups. Accordingly, the person must be of good repute, have liability insurance cover and prove theoretical - compris- ing at least 120 hours (§ 4 RDG) and - at least 2 years - knowledge for the sub-areas in which he or she intends to provide legal services. 5. 5. van den Berg N, Meinke C, Matzke M, Heymann R, Flessa S, Hoffmann W. Delegation of GP-home visits to qualified practice assistants: assessment of economic effects in an ambulatory healthcare centre. BMC Health Serv Res. 2010 Jun 8; 10:155. 6. See Thyrian JR, Michalowsky B, Hertel J, Wübbeler M, Gräske J, Holle B, et al. How Does Utilization of Health Care Services Change in Peo- ple with Dementia Served by Dementia Care Networks? Results of the Longitudinal, Observational DemNet-D-Study. J Alzheimers Dis. 2018;66(4):1609–17. 7. Acknowledgements Not applicable. 8. Gloystein S, Thomé F, Goetz K, Warkentin N, Mergenthal K, Engler F, et al. RubiN - continuous care in regional networks: a study protocol for a prospective controlled trial. BMC Geriatr. 2021;21(1):183. Funding O A 10. Cf. Bydlinski, Juristische Methodenlehre und Rechtsbegriff, 2nd ed. 1991, p. 395 ff. Open Access funding enabled and organized by Projekt DEAL. The authors are funded by the Innovation Fund of the Federal Joint Committee (G-BA) accord- ing to §§ 92a and 92b of the Fifth Book of the German Social Code (SGB V). Funding code 01NVF17029 (RubiN) Regional uninterrupted in the network. 11. Cf. Krenzler, in: Krenzler, RDG, 2nd ed. 2017, § 2 marginal no. 16. 11. Cf. Krenzler, in: Krenzler, RDG, 2nd ed. 2017, § 2 marginal no. 16. 12. Krenzler, in: Krenzler, RDG, 2nd ed. 2017, § 2 marginal no. 27 13. So also von Lewinski, in: Klowait/Gläßer, MediationsG, 2nd ed. 2018, § 2 RDG Rn. 16. 13. So also von Lewinski, in: Klowait/Gläßer, MediationsG, 2nd ed. 2018, § 2 RDG Rn. 16. Availability of data and materials 14. Prehn, in: Diering/Timme/Stähler, SGB X, 5th ed. 2019, § 13 marginal no. 34. 14. Prehn, in: Diering/Timme/Stähler, SGB X, 5th ed. 2019, § 13 marginal no. 34. There is no data in the scientific sense that has any bearing on the back- ground of the article, as it is a legal breakdown of the problem. It is therefore in the nature of things that there is already no data material that could be co-published. However, it is not necessary for the comprehensibility of the content. 15. BT-Drs. 16/3655, p. 64; Schmidt, in: Krenzler, RDG, 2nd ed. 2017, § 10 marginal no. 35. 15. BT-Drs. 16/3655, p. 64; Schmidt, in: Krenzler, RDG, 2nd ed. 2017, § 10 marginal no. 35. g 16. BT-Drs. 16/3655, p. 64; Schmidt, in: Krenzler, RDG, 2nd ed. 2017, § 10 Rn. 33. 16. BT-Drs. 16/3655, p. 64; Schmidt, in: Krenzler, RDG, 2nd ed. 2017, § 10 Rn. 33. 17. Cf. For the health care proxy OLG Karlsruhe 23.12.2010 - 4 U 109/10, ZIP 2012, 20. 17. Cf. For the health care proxy OLG Karlsruhe 23.12.2010 - 4 U 109/10, ZIP 2012, 20. References • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 1. van den Berg N, Meinke C, Heymann R, Fiss T, Suckert E, Pöller C, et al. AGnES: supporting general practitioners with qualified medical practice personnel: model project evaluation regarding quality and acceptance. Dtsch Arztebl Int. 2009. 1. van den Berg N, Meinke C, Heymann R, Fiss T, Suckert E, Pöller C, et al. AGnES: supporting general practitioners with qualified medical practice personnel: model project evaluation regarding quality and acceptance. Dtsch Arztebl Int. 2009. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 2. Robinson TE, Boyd ML, north D, Wignall J, Dawe M, McQueen J, Frey RA, Raphael DL, Kerse N. proactive primary care model for frail older people in New Zealand delays aged-residential care: a quasi-experiment. J Am Geriatr Soc. 2021;69(6):1617–26. 3. Warkentin N, Wilfling D, Laag S, Goetz K. Experiences of family caregivers regarding a community-based care- and case-management intervention. A qualitative study: Health Soc Care Community; 2021. Authors’ contributions 8. Gloystein S, Thomé F, Goetz K, Warkentin N, Mergenthal K, Engler F, et al. RubiN - continuous care in regional networks: a study protocol for a prospective controlled trial. BMC Geriatr. 2021;21(1):183. Dr. Dr. Thomas Ruppel and Dr. Max Hügel wrote the main manuscript text. Simone Gloystein and Prof. Dr. van den Berg prepared all of the figures and did the research. All authors reviewed the manuscript. Data availability section: All data generated or analysed during this study are included in this published article. The authors read and approved the final manuscript. prospective controlled trial. BMC Geriatr. 2021;21(1):183. 9. Systematic interpretation aims to reveal the content of a norm by attempting to derive the norm’s function from the context with other norms and the overall structure; Pieper, in: Dauses/Ludwigs, Handbuch des EU-Wirtschaftsrechts, work status: 51st EL October 2020, B I marginal no 24. Discussion and conclusion Wübbeler M, Thyrian JR, Michalowsky B, Hertel J, Laporte Uribe F, Wolf- Ostermann K, Schäfer-Walkmann S, Hoffmann W. Nonpharmacological therapies and provision of aids in outpatient dementia networks in Germany: utilization rates and associated factors. J Multidiscip Healthc. 2015;8:229–36. Ethics approval and consent to participate Not applicable. 19. von Lewinski, in: Klowait/Gläßer, MediationsG, 2nd ed. 2018, § 5 RDG Rn. 2. 19. von Lewinski, in: Klowait/Gläßer, MediationsG, 2nd ed. 2018, § 5 RDG Rn. 2. Consent for publication Not applicable. Competing interests Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. The authors declare that they have no competing interest The authors declare that they have no competing interests. Received: 20 May 2022 Accepted: 16 November 2022 Received: 20 May 2022 Accepted: 16 November 2022 Declarations 18. Allemand, in: Henning/Lackmann/Rein, Privatinsolvenz, 2020, § 5 RDG Rn 1 18. Allemand, in: Henning/Lackmann/Rein, Privatinsolvenz, 2020, § 5 RDG Rn 1. Discussion and conclusion The legal power of representation should be limited to the initial procedure (application) and not also cover the appeal procedure. The concept of GeriNurses on which the RubiN project is based describes care and case managers as “advocates” and “representatives” of the patient and includes among their tasks (literally) the “independent application in the form of counselling and accompaniment of the Social Code”. According to this, care and case managers should also be so-called gatekeepers and, in particular, check patients’ entitlements in the health system. From the perspective of care science, care and case managers are thus given a double role; they are supposed to be both the patient’s advocate and a neutral mediator between the patient and the service providers, especially the health and long-term care insurance funds. One way to achieve such a design would be to supple- ment the opening clauses for objectively permitted legal advice in section 10, paragraph 1, sentence 1 RDG (e.g. by adding a no. 2a). The authors propose to open the law Ruppel et al. BMC Health Services Research (2022) 22:1439 Page 7 of 7 Heymann R, Flessa S, Hoffmann W. Delegation of GP-home visits to quali- fied practice assistants: assessment of economic effects in an ambulatory healthcare centre. BMC Health Serv Res. 2010;10:155. 5. van den Berg N, Meinke C, Matzke M, Heymann R, Flessa S, Hoffmann W. Delegation of GP-home visits to qualified practice assistants: assessment of economic effects in an ambulatory healthcare centre. BMC Health Serv Res. 2010 Jun 8; 10:155. 6. See Thyrian JR, Michalowsky B, Hertel J, Wübbeler M, Gräske J, Holle B, et al. How Does Utilization of Health Care Services Change in Peo- ple with Dementia Served by Dementia Care Networks? Results of the Longitudinal, Observational DemNet-D-Study. J Alzheimers Dis. 2018;66(4):1609–17. 7. Wübbeler M, Thyrian JR, Michalowsky B, Hertel J, Laporte Uribe F, Wolf- Ostermann K, Schäfer-Walkmann S, Hoffmann W. Nonpharmacological therapies and provision of aids in outpatient dementia networks in Germany: utilization rates and associated factors. J Multidiscip Healthc. 2015;8:229–36. 8. Gloystein S, Thomé F, Goetz K, Warkentin N, Mergenthal K, Engler F, et al. RubiN - continuous care in regional networks: a study protocol for a prospective controlled trial. BMC Geriatr. 2021;21(1):183. 9. References 4. van den Berg N, Heymann R, Meinke C, Baumeister SE, Flessa S, Hoffmann W. Effect of the delegation of GP-home visits on the development of the number of patients in an ambulatory healthcare centre in Germany. BMC Health Serv Res. 2012;12:355 van den Berg N, Meinke C, Matzke M,
https://openalex.org/W4230933307	https://europepmc.org/articles/pmc5320506?pdf=render	English	null	Correction: Corrigendum: Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress	Scientific reports	2,017	cc-by	277	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 7:42453 \| DOI: 10.1038/srep42453 Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan In this Article, Gi-Ming Lai is incorrectly listed as being affiliated with ‘Division of Hematology and Medical Oncology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan’. The correct affiliation is listed below: Corrigendum: Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress cientific Reports 5:10420; doi: 10.1038/srep10420; published online 27 May 2015; updated on 22 February 2017 In this Article, Gi-Ming Lai is incorrectly listed as being affiliated with ‘Division of Hematology and Medical Oncology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan’. The correct affiliation is listed below: In this Article, Gi-Ming Lai is incorrectly listed as being affiliated with ‘Division of Hematology and Medical Oncology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan’. The correct affiliation is listed below: Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei Taiwan This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 7:42453 \| DOI: 10.1038/srep42453
W2895684882.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0204649&type=printable	en		Isotope values of the bioavailable strontium in inland southwestern Sweden—A baseline for mobility studies	PloS one	2,018	cc-by	13,789	RESEARCH ARTICLE Isotope values of the bioavailable strontium in inland southwestern Sweden—A baseline for mobility studies Malou Blank ID1, Karl-Göran Sjögren ID1, Corina Knipper2, Karin M. Frei ID3, Jan Storå4 1 Department of Historical Studies, University of Gothenburg, Gothenburg, Sweden, 2 Curt-Engelhorn Center for Archaeometry gGmbH, Mannheim, Germany, 3 Environmental Archaeology and Materials Science, The National Museum of Denmark, Brede, Denmark, 4 Department of Archaeology and Classical studies, Stockholm University, Stockholm, Sweden a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Blank M, Sjögren K-G, Knipper C, Frei KM, Storå J (2018) Isotope values of the bioavailable strontium in inland southwestern Sweden—A baseline for mobility studies. PLoS ONE 13(10): e0204649. https://doi.org/10.1371/journal. pone.0204649 Editor: Peter F. Biehl, University at Buffalo - The State University of New York, UNITED STATES Received: February 2, 2018 Accepted: September 12, 2018 Published: October 4, 2018 Copyright: © 2018 Blank et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The analyses were financed by The Atlas of Ancient Human Genomes in Sweden, funded by The Swedish Research Council -VR 2013-1905, and The Swedish Foundation for Humanities and Social Sciences, RJ M13-0904:1. malou.blank.backlund@gu.se Abstract The inland area of southwestern Sweden is well known for its well-preserved archaeological animal and human remains dating back to the Mesolithic and Neolithic (10000–4000 and 4000–1700 BC). They allow application of multiple bioarchaeological methods, giving insights into various and complementary aspects of prehistoric human life, as well as economic and social structures. One important aspect concerns human mobility and its relation to social networks and to circulation of objects. Here, strontium isotope analysis plays a crucial role. The present study aims to construct a strontium isotope baseline of southwestern Sweden with considerably greater coverage and higher resolution than previously published data. As the region has been affected by glacial events, the relation between bedrock geology and isotope signals of the bioavailable strontium in such areas is given special attention. We determined strontium isotope ratios for 61 water and five archaeological animal samples, and combined the data with previous measurements of two water and 21 non-domestic faunal samples. The results reveal a complex pattern. Several areas with distinct baseline ranges can be distinguished, although with overlaps between some of them. Overall, the bioavailable strontium isotope signals mirror the basement geology of the region. The highest ratios occur in the geologically oldest eastern parts of the Precambrian terrain, while lower ratios are found in the western part, and the lowest ratios occur in the youngest Paleozoic areas. At the same time, there are minor deviations compared to the underlying bedrock, due to glacial transport, overlying sediments, and local intrusions of younger rocks. The background data set now available allows for more nuanced and detailed interpretations of human and animal mobility in the region, in particular by identification of subregions with differing strontium isotope ratios within the Precambrian province. Also, we can now identify long distance mobility with greater confidence. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 1 / 30 A strontium isotope baseline for inland southwestern Sweden Introduction This study aims to construct a strontium (Sr) isotope baseline of southwestern Sweden to provide a foundation for archaeological studies of human and animal mobility. In general the preservation of bones and teeth in Sweden is poor due to the acidic soils. However, there are areas with favourable geologic conditions for bone preservation, including the sedimentary regions of southwestern Sweden with calcareous soils. Here, human and animal bones dating back to the Mesolithic (10000 to 4000 cal BC) have been found, but the Neolithic (4000 to 1700 cal BC) and later periods are also well represented. In the sedimentary area of Falbygden (Fig 1), one of Northern Europe’s largest concentrations of early Middle Neolithic (3300 to 2800 cal BC) passage graves and Late Neolithic (2350 to 1700 cal BC) gallery graves occurs, producing a remarkable assemblage of well-preserved animal and human remains [1–6]. This also applies to later periods, such as for instance the abundant assembly of skeletons from Varnhem, one of the earliest Christian cemeteries, dating back to 900 cal AD [7]. The clear spatial structure of the geology and the well preserved human and animal bone material makes this area an unusually fruitful case study for investigations combining bioarchaeological and archaeological data in order to understand prehistoric economy and society. Since Falbygden is essentially an”island” of Cambro-Silurian sedimentary rocks surrounded by older Precambrian crystalline rocks, it constitutes an ideal setting for mobility studies by way of isotopic analysis. A clear distinction between the Cambro-Silurian and the Precambrian regions was indicated by previous Sr isotope studies [8, 9]. However, these studies leave several issues regarding the background isotope variation unresolved, due to a rather low number of sample sites. This concerns for instance the variation between and within different Precambrian provinces, the possibility of spatial variation within the Cambro-Silurian region, and the effect of glacial transport of sedimentary rocks into the Precambrian area to the south of Falbygden. This baseline study is part of ongoing research on Middle and Late Neolithic human and animal mobility in western Sweden. Regional networks and contacts with southern Scandinavia and indirectly with continental Europe during the Neolithic can be traced by building traditions, burial practices and imported goods [10–13]. The Middle Neolithic remains have been focus for a number of scientific analyses, including osteology, aDNA, 14C dating, stable isotopes, Sr and sulphur isotopes, etc. [1, 3, 6, 8, 9, 12, 14–17]. Through these studies, the population in Falbygden has been shown to be part of a much larger system of exchange/alliances with surrounding Precambrian regions, involving humans but in particular also cattle. Furthermore, Sr isotope results have indicated an increased human mobility and an increased variation in the isotope ratios during the Late Neolithic [18, 19]. Previous studies suggested that ca 25% of the people buried in Falbygden passage graves originated from areas with Precambrian bedrock, and the variation in the in the Sr isotope signals suggest that several different regions were involved. For cattle, the number of imported animals was even higher (>50%) and the Sr isotope signals indicate more varied and partly different areas of origin from those of humans. At the same time, clear cases of long distance movement seem exceedingly rare, in some contrast to the archaeological evidence. This raises questions about the limitations of mobility studies by isotopic analysis, but also highlights the limitations of our present knowledge about Sr isotope variation in Scandinavia. In order to better understand the functioning of these complex exchanges, we need a better grasp of Sr isotope variation over a larger area, particularly in the Precambrian provinces. This is a prerequisite for more elaborate discussions of different kinds of mobility which still are not addressed sufficiently [20–23]. In combination with other archaeological and PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 2 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 1. Simplified bedrock geology of southern Sweden, by Malou Blank. Based on geological maps from the Geological Survey of Sweden, data (Berggrund 1:1 million). Background map created using data from Esri. Data and maps licensed to University of Gothenburg. 1: Halle and Hunne mountains, 2: Kinnekulle, 3: Falbygden. Investigated area marked with black line. https://doi.org/10.1371/journal.pone.0204649.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 3 / 30 A strontium isotope baseline for inland southwestern Sweden bioarchaeological data, this would involve issues such as the temporal and spatial scale of individual movement, different movement patterns for specific categories of people, the mobility patterns of people versus those of animals, and possible social drivers behind these patterns. Such issues will be discussed in upcoming papers, evaluating isotope data from humans and domestic animals against the background variation presented here. The necessary level of detail of the background data is dependent on the underlying geological complexity, as well as on the research questions asked, and no general rules can be formulated. Numerous environmental samples from archaeological sites as well as from the surrounding areas are needed not only to establish the local bioavailable range but also to consider or rule out potential areas of origin of non-local individuals. A baseline of southwestern Sweden is also important for mobility studies in neighbouring regions. Archaeological human and animal materials from southern Sweden are at present included in several mobility research projects (e.g. [24–27]). Baselines have previously been produced for e.g. the British Isles [28, 29], France [30], Denmark [31–33], the Netherlands [34], Switzerland [35], parts of the North Atlantic regions [36] and in parts of Mesoamerica [37]. In Sweden, background data are only available for a few restricted areas mostly in connection to specific archaeological sites [8, 9, 19, 24, 38–48]. An overview of Sr isotope ratios in southern Sweden has been published by Blank and Knipper [19]. In addition, there is an ongoing study investigating the Sr isotope baseline of the west coast of southern Sweden [25, 26] which will be an important complement to this study. In order to expand the knowledge of Sr isotope spatial variation in western Sweden, we measured a total of 61 water and five animal samples from an area covering 120 x 130 kilometres. These were combined with previous data from two water samples and 21 samples from non-domestic small fauna, to obtain as high resolution as possible (S1 Appendix). Background Strontium isotopes and mobility Strontium isotope analysis has become an important method in provenance studies in a wide range of fields, including archaeology [49– 51], ecology [52], food [53–55] and forensic science [56, 57]. In archaeological research, Sr isotope analysis was introduced by the pioneering work of Ericson [50] and has become a standard method for studying human and animal mobility. In many cases, this method has given us insights into prehistoric socio-dynamics that would not be possible with other methods [9, 18, 58–69]. Strontium is an alkaline earth element which has four naturally occurring isotopes; three stable and one, 87Sr, radiogenic. The ratio between two of them, 87Sr/86Sr, is used in provenance studies. The radiogenic 87Sr is formed by radioactive β-decay of 87Rubidium [70]. Different rocks are therefore characterised by 87Sr/86Sr ratios dependent on the original content of rubidium as well as on the formation age [70, 71]. Bioavailable Sr originates mainly from weathering rock minerals. However, weathering is a process that affects different types of minerals unequally, which in themselves vary in both Sr content and Sr isotope ratios. Bioavailable Sr ranges may therefore vary from those of the parent bedrock as they represent a mixture of Sr ratios from different sources [72, 73]. The element passes from soils and water into the biosphere and food chain and further into the human skeleton with minimal biological isotope fractionation [49, 74, 75]. Hence, the 87Sr/86Sr ratio is effective for identifying human and animal mobility. It is assumed that human and animal Sr isotope signals give a weighted average of the bioavailable 87Sr/86Sr ratios within their home range [49]. Comparing isotope values in for example human tooth enamel, which is formed during childhood, to those of samples that PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 4 / 30 A strontium isotope baseline for inland southwestern Sweden reflect the bioavailable Sr around the archaeological site can thus identify possible movements of individuals and mobility patterns of populations. In addition to weathering rocks, there may also be contributions from precipitation, windblown dust, fertilizers, and sea spray [49, 51]. The impact of such factors has been discussed, but may in many cases be considered not to be large enough to obscure the signal from the local geology. According to Frei and Frei ([32]:157), rainwater does not have measurable impact on the Sr isotope composition of surface water in Denmark. These authors argued that this might be due to the fact that soils in Denmark are partially composed of Sr rich limestone derived material, which are the dominant Sr source in surface water and the low Sr concentration in rainwater. The potential impact of Sr from modern sources, such as fertilizers, to the local Sr isotope baseline ranges has also been debated [31, 49, 76]. Frei and Frei [31] argued that only extremely high amounts of fertilizers that are currently used within the Danish regions would alter the Sr isotope signals of the Danish soils and surface waters. Nevertheless, to avoid the influence of fertilizers as much as possible, samples should be taken away from modern agricultural areas. Wind transport of airborne sea salt in southern Sweden was studied by Gustafsson and Franzén [77] in a 300 km transect. They found that sea salt was transported all over the transect, but with strongly decreasing concentration after ca 20–30 km from the coast, after which concentrations stabilized on a very low level. Tooth enamel is the preferred material for investigating prehistoric human and animal mobility, as it is less susceptible to diagenesis and contamination than bone [49]. Bone undergoes continuous chemical and structural turnover, while enamel stays stable after the formation which in humans takes place in infancy to early adolescence, depending on tooth type [78]. In case a person moves to a new location with a different geology after or during the tooth enamel formation, the Sr isotopic ratio of the enamel will differ from the isotopic signal of the new location, under the premise that local food sources were consumed. Seasonal mobility or movement during the time of tooth formation may result in mixed isotope ratios, as tooth crystallization sometimes can take several years [78]. Teeth with enamel formed at different stages and micro sampling from different levels of the tooth crown (mostly conducted on animal teeth) can give us more detailed information about the movements [61, 79, 80]. Sample material considerations In previous Sr isotope research the terms baseline and isoscape have been used variedly (e.g. [34, 81]). Here, we use these terms as equivalent, referring to the background variation of bioavailable Sr isotope ratios within a region. It is implied that isotopic variation is inherently spatial, and that it is the systematic spatial variation over a region that is of interest, rather than the variation at any specific point, such as a find site. A baseline should not be considered as fixed or stable, as new data accumulate over time, adding more nuances to the picture. The uptake of Sr in humans and animals is mainly controlled by Sr in plants, animals and drinking water coming from a certain area, or from a number of areas visited during a period of time. As discussed above, the Sr isotope ratios characteristic of these different sources can vary and so do the Sr concentrations ([82]: 187). Strontium concentrations are generally higher in plants than in meat, and some researchers argue that most Sr in humans originates from plants while the contribution from animals is of less importance [49]. Frei and Frei [31] also suggest drinking water as a potentially important source of Sr in humans, and recent investigations on modern humans show that local water may be an important source of Sr to human hair [83]. In a controlled feeding study on pigs, Lewis et al [75] found the contribution from drinking water to be less than 5%, however, even with a rather high Sr concentration (2.6 ppm) in the water. The Sr concentration in surface water varies depending on the local PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 5 / 30 A strontium isotope baseline for inland southwestern Sweden geology. Generally, Sr concentrations in surface waters are higher in areas dominated by calcareous rocks (eg. limestones), whereas they are lower in areas dominated by mafic rocks and granitoids. In southwestern Sweden, the Sr concentrations measured by us are very low (Sample preparation and analysis). Therefore, water should be expected to contribute very little to human Sr uptake in our study area. Regardless of the contribution of water to human and animal values, however, such samples are still useful as a proxy for bioavailable Sr isotope ratios [31, 49]. There is at present little consensus on what proxy to use, and the choice of proxy material has been debated [28, 31, 49, 72, 76, 82, 84]. Ongoing methodological development, economical resources, availability and accessibility are some important factors in this choice. Various materials have been suggested including archaeological fauna, modern fauna and flora, human bone, soils and water. Often, isotope studies include several different sample types. Grimstead, Nugent and Whipple [82] advocate a broad sampling strategy of soil, archaeological fauna, vegetation and water to map the Sr isotope system. However, these authors recognize and encourage that current provenance investigations have shifted away from utilizing animals to create baselines [82]. Low mobility faunal species have often been suggested as good reference samples [8, 33, 85]. It is common to use enamel and bone from small mammals and shell from snails, as these animals are unlikely to move over large areas [8, 33, 36, 45, 47, 48]. In some of these studies, both archaeological and modern animals were included. In modern samples, the consumption of non-local foodstuffs cannot be excluded off-hand, and this could also be the case for archaeological domestic animals ([49]: 158). In addition, although the home range of rodents is small, they may sometimes have moved or been transported over large distances both in modern and prehistoric contexts [82, 86]. In a few studies, the Sr isotope signal in snail shells appears to be shifted towards that of rainwater, 0.709 ([28]: 3, [76]: 225). Domestic species, including dogs, cattle and pigs, have also been used for establishing the local Sr isotope signal [36, 47, 66]. However, using these animals as proxies is problematic as we need to consider the possibility of circulation of domestic animals in social networks as well as of seasonal movements of the animals [9, 58, 61, 62, 87]. Human samples are also referred to [36, 45]. In these cases, one needs to be careful not to end up in a circular reasoning, in that the target materials for which the origin is sought is used to define the local baseline. Evans et al. ([28]: 3) used data from diagenetically altered bones, as Sr in groundwater from the burial site was most likely incorporated into these bones. Hence, the Sr signal of the bones would reflect the local Sr signal of the site. Another suggested proxy is modern soil and plant samples [24, 30, 36]. Soil samples capture microvariation, which may not be relevant for humans and other larger animals, who average the Sr intake over their home ranges. When soil is used, the depth of the sample needs to be considered, as the Sr isotope ratio might vary with depth ([31]: 337, [49]: 151, [88]: 316, [89]). Moreover, depending on the leaching technique used during sample preparation, the same soil sample may produce different 87Sr/86Sr ratios, of which not all reflect the bio-available fraction. The topsoil also seems to retain the Sr isotope ratio of rainwater [28]. Likewise, plants from the same location with different root depth can have different isotope signals. Atmospheric factors such as windblown dust may also affect plant samples [49, 82]. Frei and Frei ([31]: 338) conclude that the Sr isotope signal of topsoil and small herbivores in Denmark was 0.15% lower than that defined by lake and stream water, and suggest that this might be due to rainwater and soil leaching/weathering of radiogenic components of Sr in the soil. According to several studies [28– 32, 76], water appears to be a good proxy for identifying the isotope signals relevant to humans. In a study conducted by Maurer et al. [76] Sr isotope ratios in different biological and geological samples were compared, and the authors PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 6 / 30 A strontium isotope baseline for inland southwestern Sweden concluded that the best reference samples with the most accurate estimates for the bioavailable signal were water and tree leaves ([76]: 227). In a study conducted by Wickman and Åberg [90], water samples from rivers in northern Sweden and Finland seemed to correlate rather well with the values from the local bedrock. The published baselines of the British Isles [28] and of Denmark [31, 32] are based on numerous samples from surface water. Also, according to Bentley ([49]: 144), samples from rivers and streams are a good basis for the local isotopic ratio, as the composition of various eroded rocks represents the Sr available for plants and animals. Frei and Frei ([31]: 328) argue in their study that the Sr isotope signal of animal hair (sheep) mirrors the signal of surface water, and proposed that water is a good proxy to delineate the local baseline. To sum up, there are a number of different proxies which are currently used to construct Sr isotope baselines. Considering that this study first and foremost was intended for archaeological research, focusing on humans and domestic animals, water was concluded to be the most suitable proxy and was used as the main source for this investigation. An important factor in this choice was accessibility and availability, as archaeological faunal remains are generally not preserved in the Precambrian area, while numerous springs and small streams are found all over the region. We also wished to avoid Sr from modern and non-local fauna, and we therefore minimized the samples from animals and only use enamel values from small, non-domestic fauna. Further, soil and plant samples were avoided, soil because the relevance for bioavailable Sr ratios is uncertain, and plants because of possible contamination problems from wind-blown dust. The geology of southwestern Sweden The oldest bedrock formation in Europe is the Baltic shield which extends over the Scandinavian Peninsula, Finland and parts of Russia. Within this formation, the oldest, Archaean rocks, which are 3.4 Ga (billion years old), occur in western Russia, eastern Finland and Karelia. As the shield has grown in a westerly direction, the western parts are younger [91, 92]. The Baltic shield is divided into several provinces, including the Svecofennian province (1.9–1.75 Ga) comprising parts of eastern Sweden and Finland, the Sveconorwegian province (1.7–0.9 Ga) covering western Sweden and Norway, and the Transscandinavian granite-porphyry belt (TIB, 1.8–1.6 Ga) which extends between these two provinces (Fig 1). The Sveconorwegian province is usually divided into an eastern and a western segment and was subject to extensive metamorphism [93]. The Baltic shield is made up of Precambrian gneisses and granites [91]. In geological research large numbers of 87Sr/86Sr determinations on whole rock samples from southern Scandinavia have been conducted for dating purposes (http://maps.sgu.se/sguinternetmaps/ alder/viewer.htm). 87Sr/86Sr ratios of Precambrian crystalline rocks are elevated (radiogenic) with 87Sr/86Sr > 0.722 ([8]: 89). Gneisses have more heterogeneous Sr isotope signatures than their unmetamorphosed counterparts, the granites [8]. In the Svecofennian province in eastern and northeastern Sweden, Sr isotope values are generally higher than 0.73 [88, 91, 92, 94, 95]. Most of southwestern Sweden consists of Precambrian crystalline rocks, from the Early and Middle Proterozoic eras ([93, 96] S2). The area, also known as the west Swedish Gneiss Province, is subdivided into an eastern and a western segment. As shown in Fig 2, the bedrock geology has been further divided into subunits, with different mineral and chemical composition, which are described in S2 Appendix. In addition, there are also pockets dominated by younger rocks with different formation histories. There are three areas characterized by Paleozoic sedimentary rocks, Falbygden, Kinnekulle and the Halle/Hunneberg mountains. These rocks once covered large areas, of which PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 7 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 2. Sample locations projected on a geological map of southwestern Sweden, by Malou Blank. Data from the Geological Survey of Sweden (Berggrund 1:50000). Background map created using data from Esri. Data and maps licensed to the University of Gothenburg. For sites numbers see S1 Appendix. 1: Halle and Hunne mountains, 2: Kinnekulle, 3: Falbygden. SNW: Sveconorwegian west, SNE: Sveconorwegian east and TIB: Transscandinavian granite-porphyry belt. A: Lake Vänern, B: Lake Vättern. Colours of the subunits are explained in S2 Appendix. https://doi.org/10.1371/journal.pone.0204649.g002 now only some areas remain due to dolerite/diabase caps protecting them from the glacial ice. Devonian to Permian dolerites, 350 to 250 Ma (million years) in age, intruded at different levels into the sedimentary succession [97]. Covering an area of 50 x 30 km, Falbygden is the largest of these sedimentary terrains (No. 3, Fig 2). Here, a series of sedimentary rocks were deposited in the Lower Paleozoic about 550 to 400 Ma ago. The sedimentary successions include Cambrian, Ordovician and Silurian alum shale, limestone, and slate, on top of sandstone deposited during the lower Cambrian. These rocks are partly covered by dolerite, which now forms the tops of table mountains. A similar remnant of Cambro-Silurian sediments occurs at Kinnekulle (No. 2, Fig 2) near Lake Vänern, 30 km north of Falbygden. At the southern tip of Lake Vänern another very limited area with PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 8 / 30 A strontium isotope baseline for inland southwestern Sweden lower Cambrian sandstone overlaid by Cambro-Silurian sedimentary rocks occurs, the Halle and Hunne mountains. Also here, the sedimentary sequence is covered by Upper Paleozoic dolerite (No. 1, Fig 2, [96]). Finally, local occurrences of intrusive igneous rocks such as gabbro are found within the Precambrian terrain. Normally, these are of limited extent and their influence on Sr isotopic ratios is expected to be marginal or very localised. The geology of western Sweden is complicated by metamorphic overprinting and much younger glacial erosion and deposition of glaciogenic material which today shapes the landscape. The bedrock is in most areas covered by glacial deposits (moraines, glaciofluvial deposits), marine, and by ice lake, and freshwater lake sediments of variable thickness. These deposits add to the complexity of bioavailable Sr and may affect or mask the signatures characteristic of the bedrock. Moraine is by far the most common coverage in the region. Moraine consists of a mixture of materials partly different from the local bedrock and material may be deposited at a distance of several hundred km from where they originally eroded, although the distances are normally in the order of a couple of km [98–100]. In the study area, the ice sheets moved in a southwesterly direction [101, 102]. The effects of glacial processes on the Sr isotope ratios are a smoothening of small-scale local variations and a geographic skewing in the direction of ice movement [100]. Gillberg [101] studied the composition of moraines in the present study region, and showed that limestone was present at levels >1% in the area to the south of Falbygden, at a distance of up to 20 km from the limestone edge. Closer to the edge, the limestone content was gradually higher. A similar gradient has been observed in the CaCO3 content of the moraine ([103]: 65). In depressions, late glacial ice lake and marine as well as postglacial lake sediments have been deposited on top of the moraine. The material in these sediments largely consists of redeposited moraine material, predominantly composed of eroded Precambrian basement rocks. Marine sediments are today found below the marine limit, ca 130 masl in the region of Falbygden. Ice lake sediments are found mainly in basins and depressions to the east of Falbygden, while lake sediments are found in low lying areas of the Väner basin [91, 102]. As Falbygden is situated above the marine limit, no sediments are found here, and the area is covered by moraine. Method and material Sample material The study area is rich in natural springs, small streams and lakes which are suitable for water sampling, and have most probably been important sources for drinking water both for humans and animals in prehistory. The samples in this study consist of ground water from springs and surface water from creeks, streams and lakes (Table 1). The different sources have different uptake areas, thus reflecting different Sr sources. Springs reflect the Sr of various lithological layers and can also be influenced by surface water. Streams and creeks have different and often larger catchment areas and percolate through different lithologies and the Sr isotope ratio is a mix of various water sources with addition of rainwater and soil signals. As the studied area has not been affected by any major environmental changes since prehistory we assume that the bioavailable Sr measured in the modern samples reflects prehistoric conditions. A total of 61 water samples were collected for this study. Two previously published results from Lake Vättern and Hjortmossen were also included [41, 90]. In addition, enamel from five small non-domestic mammals was sampled from archaeological sites in Falbygden (S1 Appendix). Furthermore, 21 enamel and shell samples of low mobility animals (18 small mammals PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 9 / 30 A strontium isotope baseline for inland southwestern Sweden Table 1. Number and type of samples. Sample type Water Fauna This study Previous studies Total Spring 32 0 32 Creek, stream 25 0 25 Lake, pond 4 1 5 Unknown 0 1 1 Enamel 5 17 22 Bone 0 1 1 Shell 0 3 3 66 23 89 Total https://doi.org/10.1371/journal.pone.0204649.t001 and three snails) were included from previous studies [8, 9]. The snail samples were modern, and even though this species tends to show lower 87Sr/86Sr values than the Sr in other species, it still reflects the bioavailable Sr. The mammals were found in archaeological contexts, but a modern/historic origin cannot be ruled out. These values were supplemented by measurements from fishes (three teeth and one throat bone) which were consistent with other local Sr isotope ratios ([8], S1 Appendix). The purpose of the selected animal samples was to complement the water samples and to investigate if the different sample types coincided or not. Some of the samples from the previous study [8] were excluded as they originated from larger mammals with larger home ranges. Table 1 lists a summary of samples used herein. Ethical statements are not relevant to this study. No specific permissions were required for the collection of water samples, according to Swedish legislation. Permissions from the museum of Västergötland and the Swedish History Museum were granted to conduct destructive analysis on the archaeological animal samples. Sample collection Water samples were collected between October 2014 and December 2016. Sampling was avoided in connection with heavy rainfall and ice melting as water Sr isotopic values can change during these periods [51, 90]. This investigation covers a geographical area of 120 x 130 km with the highest concentration of samples in Falbygden (Figs 1 and 2). Our sampling strategy was aimed at achieving a reasonable geographical spread while covering all the different lithologies present in the area. In addition, the proximity to archaeological sites, as well as the accessibility of suitable water was considered. Water samples were collected from 20 localities in the sedimentary area of Falbygden, from 8 localities in the sedimentary area of Kinnekulle, Halle and Hunne mountains, from 32 localities in the Precambrian areas, plus one sample from the Lake Vänern (Fig 2, S1 Appendix). Five small mammals from archaeological sites in Falbygden were sampled with the help of Maria Vretemark at Västergötlands Museum at two different occasions (Fig 2, S1 Appendix). These samples were not 14C dated, however their contexts indicate that they most likely are prehistoric (S1 Appendix). The sampling and preparation process of previous fauna materials is described in Sjögren, Price and Ahlström [8]. Details of the preparation and analysis of the additional two water samples are available in Frei et al. [41] and Wickman and Åberg [90]. In Fig 2 all sample locations are plotted. Sample preparation and analysis Water and animal enamel sample preparation and analysis were carried out at the Curt-Engelhorn-Center for Archaeometry gGmbH, Mannheim, Germany. The samples were centrifuged PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 10 / 30 A strontium isotope baseline for inland southwestern Sweden and filtered in cases of high particle loads. 10–20 ml of water were dried down and the residue taken up with 250 μl of 3 N HNO3. Sr separation was carried out in Teflon columns with Eichrom Sr-Spec resin. The columns were preconditioned with 500 μl of 3 N HNO3, the samples loaded and washed in with 3 x 400 μl of 3 N HNO3. The Sr was eluted with 1.5 ml of 0.4 N HNO3 (0.5 ml + 1 ml steps). In order to provide always sample solutions with the same Sr concentration for the High-Resolution Multi Collector-ICP-MS (HR-MC-ICP MS, Neptune), we determined the Sr concentrations after the column chemistry using a Quadrupole-Inductively Coupled Plasma-Mass Spectrometry (Q-ICP-MS). Strontium concentrations of the water samples ranged between about 5 and 150 ppb. Using 20 ml of water from each sample ensured to elute enough Sr for isotope ratio determination, based on 1 ml with 100 ppb of Sr in three blocks of 30 lines each. Raw data were corrected according to the exponential mass fractionation law to 88Sr/86Sr = 8.375209. Blank values were lower than 10 pg Sr during the whole clean lab procedure. Small mammal teeth were washed in distilled water and enamel samples separated from adhering dentin. Because of the small amounts of samples, no pretreatment with acetic acid was performed. About 3–5 mg of enamel were dissolved in 250 μl of 3 N HNO3 and the Sr separated using Sr-Spec resin as described above for the water samples [63, 64, 104]. During the course of the analyses, the Eimer and Amend (E & A) standards yielded 87Sr/86Sr ratios of 0.70803 ± 0.00004, 2 SD; n = 63 [inter-laboratory mean: 0.70827 ± 0.00004 1 SD [105]; in-house long-term average Jan. 2011-June 2018: 0.70801 ± 0.00008 2 SD (n = 1240], and the NBS 987 standard yielded 0.71025 ± 0.00004, 2 SD; n = 55 [certified value: 0.71034 ± 0.00026 (95% confidence interval)]. Standards and samples were measured in the same concentrations of 100 ppb Sr. Statistical methods Statistical analyses were performed using IBM SPSS version 23. The data evaluation included descriptive statistics and significance tests. As a normal distribution could not be demonstrated in all cases, the non-parametric Mann-Whitney U-test (MWU-test) was used to examine whether the observed differences were statistically significant at the 5% level. This test was preferred because of the sometimes small sample sizes and differences in sample sizes between groups. The Mann-Whitney U-test was used to compare distributions across groups based on sample type, bedrock geology and location. ArcGis 10.1 was used to plot the isotopic values at the sampled locations, using various methods of classifications and numbers of classes depending on scale (Figs 3 and 8). ArcGis was also used to interpolate the Sr isotopic ranges in the studied area. An interpolated surface was produced by a standard circular Empirical Bayesian Kriging method. The Empirical Bayesian Kriging is an interpolation method that accounts for the error in estimating the underlying semivariogram through repeated simulations. This method permits accurate predictions of moderately non-stationary data and is more precise than other kriging methods for small datasets [106]. In this case the method was based on 100 simulations with a searching neighborhood of four sectors and a maximum of 15 and minimum of three neighbors. Results and discussion The Sr isotope ratios of all samples are presented together with lat/long coordinates (WGS 84), context, and geological information in S1 Appendix. For samples collected from archaeological sites, site number in the national Sites and Monuments register are also included. Faunal samples from previous studies, excluded from the present study are coloured red. Initially, different sample types were compared for areas where there is close spatial overlap (S2 Appendix). No statistically significant differences between the two categories of water or PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 11 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 3. Strontium isotope ratios at the sampled locations, grouped at intervals of 0.002 into 9 classes, by Malou Blank. Background map created using data from Esri. Data and maps licensed to the University of Gothenburg. 1: Halle and Hunne mountains, 2: Kinnekulle, 3: Falbygden. A: Lake Vänern, B: Lake Vättern. SNW: Sveconorwegian west, SNE: Sveconorwegian east and TIB: Transscandinavian granite-porphyry belt. https://doi.org/10.1371/journal.pone.0204649.g003 between the water and fauna samples were observed (Tables A, B, C and Fig C in S2 Appendix). Hence, we consider the fauna (low mobility animals) and water (ground and surface water) samples to be compatible and our two data sets were combined to achieve the best resolution possible. Strontium isotope ratios and bedrock geology In Fig 3, the Sr isotope values are plotted for all samples, and a histogram of Sr isotope ratios by bedrock type is given in Fig 4. The general trends are clearly visible: lower isotope values in the Paleozoic areas of Halle and Hunne mountains, Kinnekulle and Falbygden, and higher values in the Precambrian terrain (Figs 3 and 4). This confirms the results from earlier studies. The isotope ratios in the Precambrian regions are internally varied with higher ratios in the east (Fig 3). These trends will be investigated more in detail by statistical testing. First, data from the older (Precambrian) areas are compared with the values from younger (Paleozoic) rocks. Thereafter, we discuss data from the Precambrian areas in more detail. Last, we analyse data from the Paleozoic sedimentary and intrusive regions. Precambrian and Paleozoic bedrock. In Table 2 statistics of Sr isotope values of samples from Precambrian, Lower Paleozoic and Upper Paleozoic areas are summarized. The Precambrian terrain in the study area stretches from the Sveconorwegian west (SNW) and the Sveconorwegian east (SNE) segments to the TIB, while the Paleozoic regions are located in PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 12 / 30 A strontium isotope baseline for inland southwestern Sweden N=19 N=29 N=32 N=7 Fig 4. Histogram of Sr isotope ratios of water and animal samples from the Paleozoic and the Precambrian areas. https://doi.org/10.1371/journal.pone.0204649.g004 Falbygden, in the Kinnekulle and the Halle and Hunne mountains (Fig 2). The mean and median Sr isotope values are highest in the Precambrian regions and lowest on the Upper Paleozoic formations. The small sample size of the Upper Paleozoic group is addressed in the section Paleozoic area. Values in the Precambrian area have rather high standard deviation while the variation in the Lower Paleozoic areas, with sedimentary rocks, is much lower. If the outliers are considered the higher variation in the Precambrian areas is even more pronounced (Fig 5). Table 2. Summary statistics of Sr isotope ratios of water and animal samples by formation age. Formation Era/period Bedrock Age of formation N Mean Std. Dev. Median Min. Max. Upper Paleozoic, DevonianPermian Intrusive: diabase 350–250 Ma 3 0.71383 0.00297 0.71243 0.71181 0.71724 Lower Paleozoic, Cambro- Silurian Sedimentary: slate, limestone, alum shale and sandstone 550–400 Ma 45 0.71463 0.00137 0.71422 0.71190 0.71908 Precambrian, Proterozoic Intrusive: granites and gneisses etc. 1.86–0.9 Ga 39 0.72203 0.00324 0.72185 0.71535 0.72800 https://doi.org/10.1371/journal.pone.0204649.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 13 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 5. Boxplot of Sr isotope ratios in the Paleozoic and Precambrian terrains. Line: median, box: 25th- 75th percentile, whisker: ca 95% of the data. Outliers: Sample no. 21: Hångsdala 82, no. 22: Munkestenskälla, no. 28: Hällekis säteri, no.29: St Brigidakällan, and no. 72: Borgunda. https://doi.org/10.1371/journal.pone.0204649.g005 The difference in Sr isotope values between samples collected in the Precambrian and samples from the Paleozoic areas is highly significant (MWU-test: p < 0.001). Thus, the isotope ratios largely reflect the main divisions in bedrock geology. This is one of the main results of this study, and we can thus confirm the indications from earlier studies. In other Paleozoic sedimentary areas, such as Östergötland, Öland and Gotland (Fig 1), baseline values similar to those in Falbygden have been reported [24, 39, 42, 43, 47, 48, 107]. However, in these regions the variation of the bioavailable Sr seems higher and in the Baltic islands Öland and Gotland Sr isotope ratios as low as 0.708/0.709 have been measured in soil and plants [24, 39, 42, 43, 47, 48, 107]. Variations within the Precambrian terrain. Table 3 shows summary statistics for samples from the different geological provinces within the Precambrian area. The means and medians from the two Sveconorwegian segments are similar, while the average and median from the easternmost province, TIB, are higher (Table 3, Fig 6). As seen in the standard deviations, the variation is considerably higher in the SNE segment than in the other regions, also when the outlier is excluded (Table 3, Fig 6). Statistically, there is a significant difference between the westernmost segment (SNW) and the easternmost province (TIB), with the highest ratios measured in samples from the TIB PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 14 / 30 A strontium isotope baseline for inland southwestern Sweden Table 3. Summary statistics of Sr isotope ratios in the Precambrian area of southwestern Sweden. Geological province/segment Formation Age N Mean Std. Deviation Median Min. Max. SNW Early and Middle Proterozoic (1.7–0.9 Ga) 9 0.72140 0.00104 0.72134 0.72006 0.72356 SNE Early and Middle Proterozoic (1.7–0.9 Ga) 24 0.72174 0.00380 0.72188 0.71535 0.72800 TIB Early Proterozoic (1.8–1.6 Ga) 6 0.72415 0.00230 0.72404 0.72156 0.72751 SNW: Sveconorwegian West, SNE: Sveconorwegian East, TIB: Transscandinavian granite-porphyry belt. https://doi.org/10.1371/journal.pone.0204649.t003 (Tables 3 and 4). The high ratios in samples from the TIB correspond to published reference samples from the Precambrian regions of eastern Sweden [24, 46, 95, 108]. The Motala area, east of the region investigated in this study and east of Lake Vättern, on the edge of a sedimentary plain surrounded by Precambrian bedrock (Fig 1), has recently been subject to Sr isotope analyses [24]. The bioavailable baseline levels in the Cambro-Silurian sedimentary terrain range from 0.714 to 0.728 while values from the Precambrian area of the TIB and the Svecofennian province (Fig 1) range from 0.731 to 0.743, which could be expected from the older bedrock [24]. Even though these values have been measured in other sample material (largely soil Fig 6. Boxplot of Sr isotope ratios from geological provinces in the Precambrian area. SNW: Sveconorwegian West, SNE: Sveconorwegian East, TIB: Transscandinavian granite-porphyry belt. Outlier: Sample 54: Råda Ås. https://doi.org/10.1371/journal.pone.0204649.g006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 15 / 30 A strontium isotope baseline for inland southwestern Sweden Table 4. MWU-test of Sr isotope ratios from the geological provinces of the Precambrian area. Bold: significant at the 5% level. P value TIB-SNE 0.174 TIB-SNW 0.012 SNE-SNW 0.766 TIB- SNE+SNW 0.063 https://doi.org/10.1371/journal.pone.0204649.t004 samples), these results confirm higher isotope ratios of the bioavailable Sr in the more eastern and northern parts of southern Sweden, which have also been verified in other recent papers [44, 46]. Due to high variation the difference between SNE and the two other regions is not statistically significant, even if the outlier (sample 54) is removed. However, the 87Sr/86Sr values lay between the range of values of the two other regions, and thus agree with their geographical position (Fig 1). Hence, the results seem to reflect the general differences in formation ages of the three terrains. Both the lowest and highest ratios in the Precambrian area are found in the SNE segment (Fig 7). The lowest (No. 78: 87Sr/86Sr = 0.7154) derives from a pike collected at a medieval site 30 km south of Falbygden and the highest (No. 37: 87Sr/86Sr = 0.7280) comes from spring water sampled on pasture land 34 km southwest of Falbygden (S1 Appendix). The high variation of 87Sr/86Sr values from the SNE can partly be explained by the occurrence of intrusive rocks in some areas (Figs 2 and 3). Samples from sites south of Falbygden (no. 45, 55, 75, 78) show relatively low Sr isotope ratios, which partly could be explained by the occurrence of gabbros in this area (Fig 2). Another factor is admixture of Sr from ice transported sedimentary rocks from Falbygden as proposed by Gillberg [101], Frei [40] and Sjögren, Price and Ahlström [8]. The presence of gabbro in a narrow band south of Lake Vänern (Fig 2) could explain the lower Sr isotope ratio (0.7157) in sample No. 43 in this area. Unfortunately, this sample is rather isolated and further sampling in this area would be recommended in the future. The high Sr isotope ratio (0.7236) in sample no. 54 collected at the Råda Ås spring (outlier in Fig 6) could possibly be explained by “lake spray” from Lake Vänern which is characterized by a similarly elevated Sr isotope value (0.7244). However, the high ratio could also be a result of admixture of Sr from ice transported Precambrian material stemming from areas to the northeast. The high 87Sr/86Sr ratio of sample no. 37 is more difficult to explain and considering the geological background such elevated signals would be more expected in samples from the TIB, exposed to the east and north of Falbygden. Again, this sample might be influenced by more non-local contributions of Sr from long distance ice transported material from the northeast. Overall, the Sr isotope ratios are consistent with the expected values of the underlying bedrocks of the respective area, but on a more local scale, some deviations can be discerned. As shown in Fig 2, the Precambrian basement rocks have a complex areal distribution with several different geological subunits. These subunits are geologically and chronologically discernible and consist of certain distinctive rock types as categorized by the Swedish Geological Survey (Fig A and Table D in S2 Appendix). A more detailed investigation of the Sr isotope ratios measured in samples from these different subunits is available in S2 Appendix. There is a statistically significant difference only between the Sr isotope values from samples collected in the two subunits 546 and 547 (Fig 2), who dominate the SNE segment. The difference between these two subunits consists in higher 87Sr/86Sr values in subunit 546, mostly present in the northern and eastern part of the SNE segment (S2 Appendix, Table D in S2 Appendix). PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 16 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 7. Boxplot of Sr isotope ratios in the sedimentary areas of southwestern Sweden. Outliers: Sample no. 21: Hångsdala 82, no. 72: Borgunda and no. 64: Falköping stad 3. https://doi.org/10.1371/journal.pone.0204649.g007 Paleozoic areas. In the Paleozoic areas, Cambro-Silurian sedimentary rocks are partly capped by Devonian-Cambrian diabase sills. As shown earlier, the bioavailable Sr isotope signals measured in samples from the Paleozoic areas are significantly lower than the signals from the Precambrian areas. The samples taken from the different sedimentary substrates are quite similar and have low variability, although values on sandstone have a somewhat higher mean and median (Table 5). The sandstone is of a different origin and contains older material than the other sedimentary rocks. Statistical testing shows that none of the differences were significant (S2 Appendix). The covering moraine and other soils have probably evened out the variances in Sr isotope ratios of the underlying geology. The diabase is the youngest bedrock found within the study area and we would expect bioavailable Sr derived from these rocks to show the least radiogenic Sr isotope signals. However, the samples from diabase are too few for statistical evaluation, and are also very divergent. In the Halle and Hunne region, water samples from lakes on top of the diabase returned the lowest 87Sr/86Sr ratios measured in water (Table 5, S1 Appendix). Contrary to this, the single PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 17 / 30 A strontium isotope baseline for inland southwestern Sweden Table 5. Summary statistics of Sr isotope ratios from localities in Paleozoic areas. Area Lithology N Mean Std. Dev. Median Min. Halle and Hunne mountains All 3 0.71413 0.00350 0.71243 0.71181 0.71816 Diabase 2 0.71212 0.00044 0.71212 0.71181 0.71243 Sandstone 1 0.71816 0.00206 0.71570 0.71378 0.71908 0.00272 0.71570 0.71378 0.71762 0.00106 0.71420 0.71190 0.71755 0.00091 0.71405 0.71310 0.71554 Kinnekulle Falbygden All 5 0.71632 Slate 1 0.71570 Limestone 1 0.71542 Alum shale 2 0.71570 Sandstone 1 0.71908 All 40 0.71440 Diabase 1 0.71724 Slate 5 0.71409 Max. Limestone 19 0.71449 0.00098 0.71421 0.71330 0.71755 Alum shale 10 0.71401 0.00117 0.71417 0.71190 0.71558 Sandstone 5 0.71459 0.00046 0.71459 0.71414 0.71527 https://doi.org/10.1371/journal.pone.0204649.t005 water sample collected from a location on diabase in Falbygden (discussed below), revealed the highest Sr ratio (0.7172) measured in this area (Table 5, S1 Appendix). We also compared the three sedimentary areas of Falbygden, Kinnekulle and the Halle and Hunne mountains including the samples on diabase (Fig 7, Tables 5 and 6). There are significantly higher values at Kinnekulle than in Falbygden while other differences are not significant. The higher values at Kinnekulle could be explained by a smaller contribution of bioavailable Sr derived from diabase and a higher contribution from sandstone, but more probably by larger amounts of ice transported Precambrian material. From Halle and Hunne mountains and Kinnekulle only eight samples were collected (Table 5) and even though the areas are rather small, further samples would be desired. In Falbygden, the range of 87Sr/86Sr values is narrower than that of the other two sedimentary areas, despite a few outlier values which are discussed below (Fig 7). The isotope ratios measured in the sandstone areas in Falbygden are slightly lower than the ratios from Halle and Hunne mountains and at Kinnekulle (Table 5). This is probably a result of less radiogenic Sr derived from thicker layers of soils, with primarily Cambro-Silurian material, covering most of the sandstone in Falbygden [97, 102]. Falbygden. In Falbygden, were most of the samples were collected, the spatial resolution is high with 21 water and 19 animal samples (Fig 8). The mean and median 87Sr/86Sr values of different rock types are very similar (values around 0.714 but slightly higher, 0.715 on sandstone) except for the sample on diabase showing a much higher ratio (87Sr/86Sr = 0.7172). The variation of 87Sr/86Sr values is low (Table 5, Fig 7). Statistical significance tests revealed no significant difference between samples from any of the rock types (Tables 5 and 7). The low variation of Sr isotope ratios within Falbygden is probably a result of efficient averaging of various components (Paleozoic sedimentary rocks, Precambrian material and Table 6. MWU-test of Sr isotope ratios in the three sedimentary areas. Bold: p<0.05. Falbygden Halle and Hunne mountains Falbygden P = 0.256 Kinnekulle P = 0.393 P = 0.026 https://doi.org/10.1371/journal.pone.0204649.t006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 18 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 8. Geological map of Falbygden with sample locations and Sr isotope ratios, by Malou Blank (see S1 Appendix). Geological data from the Geological Survey of Sweden. Background map created using data from Esri. The isotopic ranges are classed by geometrical intervals in 9 different groups. A: Mösse- and Ålleberg, B: Gerums- and Varvsberget and C: Billingen. https://doi.org/10.1371/journal.pone.0204649.g008 PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 19 / 30 A strontium isotope baseline for inland southwestern Sweden Table 7. MWU-test of Sr isotope ratios from different sedimentary substrates in Falbygden. Slate Limestone Alum shale Sandstone P = 0.297 P = 0.859 P = 0.222 P = 0.429 P = 0.489 Limestone Alum shale P = 0.371 https://doi.org/10.1371/journal.pone.0204649.t007 Devonian diabase) by glacial processes and the deposition of these thoroughly mixed tills on top of the Cambro-Silurian layers. Local variation is still possible but must then be on a much smaller spatial scale, not coinciding with bedrock type. There are a few exceptions with deviating 87Sr/86Sr values, defined as outliers in Figs 5 and 7 and Fig C in S2 Appendix. Sample no. 21 from a small lake on diabase [40, 41] exhibits a rather high Sr isotope ratio, 0.7172. This was sampled in 2009 and the circumstances of the sample are not clear [41] and should therefore be considered with caution. A plausible explanation for this high ratio is a local deposit of Precambrian material, and this sample is likely not representative for values on diabase. This might also suggest that Sr derived from weathering diabase is not an important component of the overall bioavailable Sr fraction in this area. The lowest ratio measured in Falbygden, 87 Sr/86Sr 0.7119, derives from a snail shell (sample 64) which has been discussed as a species showing 87Sr/86Sr values that are biased towards calcareous components of the soil or rainwater (see above). Sample no. 72 is sampled on a rodent from the eastern part of Falbygden and the high 87Sr/86Sr ratio could indicate that this animal originates from the outskirts of Falbygden or even further away, or it might be a modern rodent affected by non-local sources. However, we cannot exclude the possibility that this animal is affected by isolated pockets of Precambrian glacial deposits. The glacial movements could have affected areas of Falbygden differently, in terms of various types and amount of ice transported material, as suggested by Sjögren, Price and Ahlström [8]. To test this theory, we investigated if potential Sr isotope variations can be correlated with geographical location within Falbygden. For this purpose the samples were divided into three geographical areas (Fig 8): a southwestern part around Mösse- and Ålleberg (A), a southeastern part around Gerums- and Varvsberget (B) and the northern Billingen area (C). As seen in Tables 8 and 9, there are no significant differences in 87Sr/86Sr values at the 5% level between the three areas, although there is a significant difference at the 10% level with higher ratios in the northern area of Falbygden. In Fig 8, a pattern of samples with higher ratios along the eastern and western borders of Falbygden might also be discerned. Surface model When comparing our results from different geological substrates, several areas with distinct baseline ranges appear. Fig 9 clarifies both the differences and the overlaps of the bioavailable Sr isotope values in the study area. The outliers are considered to represent either non-local fauna or very localized anomalies. In any case, these outliers are unlikely to have a significant impact on the overall bioavailable Sr signature of a common human home range. Table 8. Summary statistics of Sr isotope signals in different parts of Falbygden. Area N Mean Std. Dev. Median Min. Max. Southwest (A) 23 0.71415 0.00089 0.71418 0.71190 0.71596 Southeast (B) 9 0.71450 0.00125 0.71418 0.71327 0.71724 North (C) 8 0.71502 0.00116 0.71476 0.71405 0.71755 https://doi.org/10.1371/journal.pone.0204649.t008 PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 20 / 30 A strontium isotope baseline for inland southwestern Sweden Table 9. MWU-test of Sr isotope ratios in different parts of Falbygden. Southeast (B) Southwest (A) P = 0.837 Southeast (B) North (C) P = 0.074 P = 0.321 https://doi.org/10.1371/journal.pone.0204649.t009 The wide range of Sr isotope ratios in the Precambrian terrain, the overlap of Sr isotope ratios between different geological areas as well as the overlay of sediments and moraine is not ideal for modelling the Sr isotope distribution according to bedrock type. Therefore, an Empirical Bayesian kriging function offered by ArcGis was used to produce an interpolated surface of the Sr isotope ratios independently of bedrock or soil type (Fig 10). Fig 9. Overview of Sr isotopes ratios of water and animal samples from different Palaeozoic and Proterozoic provinces presented by boxplots and histograms. https://doi.org/10.1371/journal.pone.0204649.g009 PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 21 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 10. An interpolated surface of the Sr isotope ratios of water and animal samples, by Malou Blank and Karl-Göran Sjögren. Background map created using data from Esri. The ranges are classified by natural breaks in 10 groups. 1: Halle and Hunne mountains, 2: Kinnekulle, 3: Falbygden. A: Lake Vänern, B: Lake Vättern. SNW: Sveconorwegian west, SNE: Sveconorwegian east and TIB: Transscandinavian granite-porphyry belt. https://doi.org/10.1371/journal.pone.0204649.g010 The interpolated surface is a generalisation of the individually measured isotope ratios of the samples, predicting the isotope composition of bioavailable Sr in the intervening areas. The highest 87Sr/86Sr values are, as expected, concentrated in the eastern parts and the lowest values agree with those measured on samples from the younger sedimentary rocks, such as Falbygden (Figs 10 and 11). The model also reveals that low 87Sr/86Sr values are to be found in an area extending towards the south-southwest of Falbygden. A similar pattern can be observed with high limestone and calcium in the moraines south of Falbygden [101, 103]. This trend is compatible with the general direction of movement of the glacial ice in the area [102]. Additional samples from this area are necessary to further substantiate this trend. The interpolation model does not take bedrock composition into consideration, which might lead to an unrealistic smoothing over some bedrock borders in some cases, such as between the younger sedimentary areas and the older intrusive rock terrain. Also, the variable sampling densities are problematic for most interpolation methods, although less so for kriging. The outliers discussed above (Fig 9), do not have a large impact on the model except if they are isolated samples. The model would benefit from additional samples in parts of the Precambrian area. As an example, a critical area between the SNW and SNE segments is less PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 22 / 30 A strontium isotope baseline for inland southwestern Sweden Fig 11. An interpolated surface of the Sr isotope ratios of water and animal samples from southwestern Sweden, by Malou Blank. Sample 43 excluded. Background map created using data from Esri. The ranges are classified by natural breaks in 10 groups. 1: Halle and Hunne mountains, 2: Kinnekulle, 3: Falbygden. A: Lake Vänern, B: Lake Vättern. SNW: Sveconorwegian west, SNE: Sveconorwegian east and TIB: Transscandinavian granite-porphyry belt. https://doi.org/10.1371/journal.pone.0204649.g011 well defined due to very sparse sampling (Fig 10). Here, sample 43, with a very low 87Sr/86Sr ratio discussed earlier, seems to have too much impact on the interpolated surface as the lack of close neighbouring samples causes this sample to appear as an isolated low ratio island (Fig 10). The effect of removing this particular sample from the model is shown in Fig 11. Even though there are some weaknesses, we find that the model is still a good prediction of expected bioavailable Sr isotopic ratios in southwestern Sweden. Conclusion In this study we present the first extensive Sr isotope baseline in Sweden, covering an area of 120 x 130 kilometres of the southwestern inland. Several areas with different baseline ranges can be distinguished, although with overlaps between some areas. Questions considering the construction of baselines were addressed and insights into different proxies highlighted. In our case statistical analyses showed that small non-domestic mammals and water samples were compatible. PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 23 / 30 A strontium isotope baseline for inland southwestern Sweden Our Sr isotope measurements of water and fauna samples largely reflect the underlying bedrock geology of the area. The highest Sr isotope ratios are found in samples located in the oldest geological provinces, while the lowest ratios where measured in samples from the areas comprising the youngest bedrock in the study area. We demonstrate that there is a significant difference between the isotope ratios of the bioavailable Sr of the Paleozoic sedimentary and intrusive rock terrains and the Precambrian granitoid-gneiss areas. Glacially transported till and other glaciogenic materials are a likely cause for deviations from this pattern, increasing the 87Sr/86Sr signals from the younger sedimentary areas and lowering the 87Sr/86Sr isotope composition of samples in parts of the older Precambrian terrains. The isotope ratios in samples from the Precambrian regions are internally varied. The wide range of isotope ratios in the Precambrian terrain can be potentially explained by the complex geology with metamorphic events and glacial deposits, which characterizes the area. Based on our statistical evaluations, there is a significant difference between 87Sr/86Sr values of samples in the most western segment (SNW) and the easternmost province (TIB), with the highest ratios measured in samples from the TIB. In the Precambrian area south of Falbygden, there are sites with lower ratios than expected, probably due to soil deposits by glaciers and the presence of younger intrusive rocks. Samples from the Paleozoic sedimentary areas are characterized by significantly lower and less variable Sr isotope signatures than the Precambrian areas. The lowest variation of Sr isotope ratios was measured in Falbygden, where most of the samples were collected. The narrow Sr isotope range of these sedimentary areas can tentatively be explained by effective mixing and averaging out of various components, such as Paleozoic sedimentary rocks, Precambrian material and Devonian diabase by glaciogenic processes and the deposition of these thoroughly mixed tills on top of the Cambro-Silurian bedrock. The lowest isotope ratios were measured in samples from locations dominated by diabase, the youngest intrusive rock in the area. Furthermore, 87Sr/86Sr values measured in samples from the sedimentary area of Falbygden are significantly lower than in samples from the sedimentary area of Kinnekulle. A possible explanation is differences in the proportions of sedimentary rocks, diabase and Precambrian moraine deposits. We also produced interpolated surface models to predict the isotopic ranges of the bioavailable Sr in the area. This model corresponds well with the other results and shows the influence of the glacial movements in the region, in addition to bedrock substrate. It also shows the narrow Sr isotope range of Falbygden and the subregions with differing Sr isotope ratios within the Precambrian province. Our background data allows for more nuanced and detailed interpretations of human and animal mobility in the region, and we can now identify long distance mobility with greater confidence, eg. from eastern Sweden. It is our hope that this study will be a basis for future research that compiles more samples and achieves a more detailed resolution. Supporting information S1 Appendix. Sample information, Excel file. (XLSX) S2 Appendix. Supplementary information: Geological supplementary information, A comparison of 87Sr/86Sr values in fauna and water samples, Strontium isotope ratios in samples from different Precambrian subunits, Strontium isotope ratios in samples from different sedimentary lithologies. (DOCX) PLOS ONE \| https://doi.org/10.1371/journal.pone.0204649 October 4, 2018 24 / 30 A strontium isotope baseline for inland southwestern Sweden Acknowledgments We are grateful to Melanie Gottschalk, Bernd Höppner, Sigrid Klaus, and Sandra Kraus for sample preparation and Sr isotope analysis at the Curt-Engelhorn Centre for Archaometry gGmbH, Mannheim, Germany. We also want to thank Maria Vretemark at Västergötlands Museum, for helping out with the sampling of animals. We are grateful to the two anonymous reviewers for helpful comments. Author Contributions Conceptualization: Malou Blank, Karl-Göran Sjögren. Formal analysis: Malou Blank, Corina Knipper. Funding acquisition: Jan Storå. 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https://openalex.org/W2946258963	https://akjournals.com/downloadpdf/journals/650/160/22/article-p873.pdf	Hungarian	null	Egy diagnosztikus kihívás: paraduodenalis pancreatitis. <i>Két eset bemutatása</i>	Orvosi hetilap	2,019	cc-by	4,386	Hegedűs Ivett dr.1 ■ Bogner Barna dr.1 ■ Faluhelyi Nándor dr.2 Macygan András dr.3 ■ Illés Anita dr.4 ■ Kelemen Dezső dr.5 1Pécsi Tudományegyetem, Általános Orvostudományi Kar, Pathologiai Intézet, Pécs 2Pécsi Tudományegyetem, Általános Orvostudományi Kar, Radiológiai Klinika, Pécs 3Somogy Megyei Kaposi Mór Oktató Kórház, Dr. Baka József Diagnosztikai, Onkoradiológiai, Kutatási és Oktatási Központ, Kaposvár 4Pécsi Tudományegyetem, Általános Orvostudományi Kar, I. Sz. Belgyógyászati Klinika, Pécs 5Pécsi Tudományegyetem, Általános Orvostudományi Kar, Sebészeti Klinika, Pécs Hegedűs Ivett dr.1 ■ Bogner Barna dr.1 ■ Faluhelyi Nándor dr.2 Macygan András dr.3 ■ Illés Anita dr.4 ■ Kelemen Dezső dr.5 1Pécsi Tudományegyetem, Általános Orvostudományi Kar, Pathologiai Intézet, Pécs 2Pécsi Tudományegyetem, Általános Orvostudományi Kar, Radiológiai Klinika, Pécs 3Somogy Megyei Kaposi Mór Oktató Kórház, Dr. Baka József Diagnosztikai, Onkoradiológiai, Kutatási és Oktatási Központ, Kaposvár 4Pécsi Tudományegyetem, Általános Orvostudományi Kar, I. Sz. Belgyógyászati Klinika, Pécs 5Pécsi Tudományegyetem, Általános Orvostudományi Kar, Sebészeti Klinika, Pécs A paraduodenalis, vagy groove pancreatitis egy kevéssé ismert krónikus hasnyálmirigygyulladás-típus, mely maligni tást utánozva sokszor differenciáldiagnosztikai nehézségeket okoz. Közleményünkben két esetet mutatunk be: mind két esetben műtéti reszekcióra volt szükség a biztos diagnózishoz, az inadekvát preoperatív szövettani mintavétel és a klinikai kép miatt. A képalkotó vizsgálatok az első esetben malignitás gyanúját vetették fel, a második esetben gyo morürülési zavar indikálta a reszekciót. Irodalmi áttekintést adunk az elváltozás klinikopatológiájáról, beleértve annak epidemiológiáját, klinikai megjelenését és az alkalmazható diagnosztikus módszereket, a betegség makroszkópos és mikroszkópos patomorfológiáját. Kialakulásának háttere összetett: az alkohol patogenetikai szerepe mellett a pancre aticus ductalis rendszer anatómiai variációi, a dorsalis pancreas inkomplett involúciójából származó duodenumfali pancreasszigetek vagy az elváltozás részeként gyakran megfigyelt Brunner-mirigy-hyperplasia is szerepet játszhat a minor papilla területén jelentkező pancreasnedv-elfolyási zavarban, mely a jellegzetes lokalizációjú gyulladáshoz ve zet. A legfrissebb kutatások génpolimorfizmusok hajlamosító szerepét is kimutatták a hasnyálmirigy gyulladásos fo lyamataiban. A paraduodenalis pancreatitis differenciáldiagnosztikájának kérdése mellett a terápiás lehetőségekről is szót ejtünk, kiemelve a sikeres elkülönítés esetén jó hatásfokkal alkalmazható konzervatív kezelés lehetőségét. Orv Hetil. 2019; 160(22): 873–879. Kulcsszavak: paraduodenalis pancreatitis, krónikus hasnyálmirigy-gyulladás differenciáldiagnózisa ESETISMERTETÉS ESETISMERTETÉS Rövidítések 1. ábra Hasi CT: duodenumfali részben cystosus elváltozás (nyíl) CEA = carcinoembrionalis antigén; CFTR = (cystic fibrosis transmembrane conductance regulator) cystás fibrosis transz membránkonduktancia-regulátor; CT = (computed tomog raphy) számítógépes tomográfia; CTRC = (chymotrypsin C) kimotripszin C; GIST = gastrointestinalis stromalis tumor; IHC = immunhisztokémia; MRCP = mágneses rezonanciás cholangiopancreatographia; MRI = (magnetic resonance imag ing) mágneses rezonanciás képalkotás; PP = paraduodenalis pancreatitis; SPINK1 = pancreaticus szekretoros tripszininhibi tor; UH = ultrahangvizsgálat A paraduodenalis, vagy groove pancreatitis a krónikus szegmentális hasnyálmirigy-gyulladás megkülönbözte tett formája, mely a minor papilla szomszédságában lévő duodenumfalat, a környező pancreasparenchymát és a köztük lévő teret (groove) érinti. Más krónikus hasnyál mirigy-gyulladások mellett ezen alcsoport mind a klini kusok, mind a patológusok körében kevéssé ismert, vala mint differenciáldiagnosztikai kérdéseket is felvet, gyakran malignitást utánozva [1, 2]. Esetleírásainkkal és a klinikopatológiai jellegzetességek összefoglalásával cé lunk ezen entitás bemutatása. Hasi CT: duodenumfali részben cystosus elváltozás (nyíl) 1. ábra 1. ábra 2. ábra Duodenumfallal és a pancreasszal is összefüggő paraduodenalis fehéres infiltrátum Keywords: paraduodenal pancreatitis, differential diagnosis of chronic pancreatitis Hegedűs I, Bogner B, Faluhelyi N, Macygan A, Illés A, Kelemen D. [A diagnostic challenge: paraduodenal pancrea titis. Two case reports]. Orv Hetil. 2019; 160(22): 873–879. Two case reports The paraduodenal, or groove pancreatitis is a lesser-known type of chronic pancreatitis, often mimicking malignancy, hence resulting in serious differential diagnostic challenges. Herein we report two cases of this entity. Both required analysis of the surgical specimen in order to ensure the diagnosis due to inadequate preoperative histological sam pling and a vague clinical presentation. In the first case, strong suspicion of malignancy following imaging, while in the second, severe gastric outlet stenosis indicated the resection. In our report, we give a clinicopathological sum mary from the literature of this entity, including its epidemiology, clinical presentation and applicable diagnostic methods as well as macroscopic and microscopic pathomorphology. The pathogenesis of this disease is complex. Beside the role of alcohol, anatomic variations of the pancreatic ductal system, pancreatic islets in duodenal wall re sulting from incomplete involution of dorsal pancreas, or Brunner gland hyperplasia (often observed as part of the lesion) can all play a role in the disturbance of pancreatic fluid discharge in the minor papilla area, eventually leading to this specific localised inflammation. In addition, recent investigations revealed a susceptible role of genetic poly morphism in the persistent inflammatory disorders of the pancreas. Besides summarizing the differential diagnostic aspects, we also discuss therapeutic possibilities, underlining the conservative methods, which can be used with good efficacy after a successful identification of this entity. 2019 ■ 160. évfolyam, 22. szám ■ 873–879. 873 DOI: 10.1556/650.2019.31378 ■ © Szerző(k) Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC ESETISMERTETÉS Első eset A 46 éves nő kivizsgálása gyomortáji fájdalom, hányin ger, hányás miatt indult. Régóta dohányzott, korábban rendszeresen fogyasztott alkoholt. Hasi UH-vizsgálattal a gyomorkimenetben írtak le egy 2 × 4 cm-es, a lument szűkítő terimét. Endoszkóppal a duodenum bulbus- postbulbaris szakaszát érintő 1,5 × 12 cm-es leukoplakiát észleltek. A biopsziás minta negatív lett. A hasi CT-vizs gálat során ép pancreas mellett a duodenum falának megvastagodása mutatkozott, a falban 25 mm-es és 12 mm-es cystosus elváltozással (1. ábra). A kép malignitás gyanúját vetette fel. A rebiopsziás minta is negatív ered ményt adott. (A konkrét szöveti képről nincs informáci ónk egyik bioptátum esetében sem.) A jelentős hasi fáj dalom, a duodenumszűkület és elsősorban a nem kizárható malignitás miatt hagyományos Whipple-műtét történt. A reszekátumban a duodenum leszálló szárán 4,5 cm hosszú szakaszt érintően duzzadt redőzet lát szott. Metszlapokon a pancreasfej–duodenum közötti régióban a duodenumfal muscularis rétegét is érintő, 4,5 × 4 × 2 cm-es sárgásfehér, elmosott határú, szolid térfog Duodenumfallal és a pancreasszal is összefüggő paraduodenalis fehéres infiltrátum lalás mutatkozott (2. ábra). Szövettani vizsgálattal a du odenumfal muscularis propriáját is érintve, a pancreasba felületesen terjedve myofibroblastos-myoid, dezmin- és H-kaldezmon-pozitív orsósejtes infiltráció látszott bő fibrosissal, krónikus lobsejtes beszűrődéssel. Helyenként beolvadó akut gyulladás mellett károsodott ductusokból származó eosinophil masszát észleltünk. A duodenum falban Brunner-mirigy-hyperplasia is megfigyelhető volt (3–5. ábra). 874 2019 ■ 160. évfolyam, 22. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC 5. ábra Dezminexpressziót mutató myofibroblastos reakció, elérve a pancreast (dezmin-IHC, 10×) 3. ábra Duodenumfali és paraduodenalis jellegzetes heges, myofibro blastos, gyulladásos reakció, submucosalis Brunner-mirigy- hyperplasiával (HE, 1,1×) Duodenumfali és paraduodenalis jellegzetes heges, myofibro blastos, gyulladásos reakció, submucosalis Brunner-mirigy- hyperplasiával (HE, 1,1×) 3. ábra Dezminexpressziót mutató myofibroblastos reakció, elérve a pancreast (dezmin-IHC, 10×) 4. ábra A pancreast elérő gyulladásos reakció területében károsodott ductusokból származó eosinophil anyag látszott (HE, 10×) 6. ábra Hasi MR: duodenumot szűkítő, cysticus részeket is tartalmazó pancreasfej környéki térfoglalás (folytonos nyíl: duodenum, szaggatott nyíl: pancreasfej) 4. ábra A pancreast elérő gyulladásos reakció területében károsodott ductusokból származó eosinophil anyag látszott (HE, 10×) Hasi MR: duodenumot szűkítő, cysticus részeket is tartalmazó pancreasfej környéki térfoglalás (folytonos nyíl: duodenum, szaggatott nyíl: pancreasfej) Második eset papilla felett 2 cm-rel, a minor papilla területének megfe leltethetően lumenszűkítő duzzadt redőzet látszott egy 2 cm átmérőjű területen. Metszlapon itt a duodenumfal ban és a duodenum és a pancreas között 3 × 1,8 × 2,5 cm-es, elmosott határú, fehéres szövetet azonosítottunk. Szövettani vizsgálattal paraduodenalis hegesedés, myo fibroblastos és gyulladásos reakció, egy-egy bevérzett pseudocysta mellett a duodenum falában hamartomato sus jelleggel pancreas acinaris struktúrák és tágult duc tusszerű képletek is látszottak. A gyulladásos folyamat a pancreast nem érintette (7–10. ábra). A 60 éves férfi anamnézisében biliarisnak tartott akut hasnyálmirigy-gyulladás, köves epehólyag eltávolítása szerepel. Ismételt heveny pancreatitis kapcsán végzett hasi CT-vizsgálat során a duodenum leszálló és alsó víz szintes szárának jelentős falmegvastagodása mutatko zott, a pancreasfej és annak környezetének beszűrtségé vel. Endoszkópos UH-vizsgálattal a duodenumfal megvastagodását látták. A szövettani vizsgálatra vett minta ép duodenum-nyálkahártyát tartalmazott. Az is métlődő gyulladás, illetve a gyomorürülési zavar miatt 4 hónappal később hagyományos Whipple-műtét történt. Megelőző endoszkópos UH-vizsgálattal ödémásan szű kített leszálló duodenum mutatkozott; a hasi MR-vizs gálat a duodenum falát kiterjedten érintő, szűkítő jellegű folyamatot ábrázolt, melyet groove pancreatitisnek véle ményeztek (6. ábra). A műtéti preparátumban a Vater- Kiemelendő, hogy mindkét esetben jó általános álla potban lévő betegek kerültek műtétre, megfelelő műtéti indikációval (fájdalom, duodenalis stenosis, tumorgya nú), multidiszciplináris team mérlegelte a várható ha szon melletti műtéti kockázatot. Mindkét beteg szövőd mény nélkül gyógyult. 875 2019 ■ 160. évfolyam, 22. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC ERTETÉS 9. ábra Duodenumfali és paraduodenalis heges-gyulladásos reakció, be vérzett pseudocysta mellett duodenumfali pancreassziget (HE, 0,7×) ESETISM 7. ábra Vater-papilla (szonda) felett 2 cm-rel lumenszűkítő duzzadt re dőzet ERTETÉS 9. ábra Duodenumfali és paraduodenalis heges-gyulladásos reakció, be vérzett pseudocysta mellett duodenumfali pancreassziget (HE, 0,7×) . ábra Duodenumfali és paraduodenalis heges-gyulladásos reakció, be vérzett pseudocysta mellett duodenumfali pancreassziget (HE, 0,7×) 9. ábra 10. ábra Duodenumfali pancreaticus acinaris és ductalis struktúrák (HE, 3,2×) Vater-papilla (szonda) felett 2 cm-rel lumenszűkítő duzzadt re dőzet 7. ábra 8. ábra Metszlapon elmosott falszerkezet, duodenalis-paraduodenalis fehéres szövet, bevérzett, részben cystosus területekkel 10. ábra Duodenumfali pancreaticus acinaris és ductalis struktúrák (HE, 3,2×) ja, illetve a leggyakrabban groove pancreatitis [2, 4]. A különféle nevek bizonyos megfigyelhető jellemzőkre ref lektálnak, melyek az egyes esetekben változó mértékben prominensek. 8. ábra Metszlapon elmosott falszerkezet, duodenalis-paraduodenalis fehéres szövet, bevérzett, részben cystosus területekkel Először a német szakirodalomban 1973-ban Becker használta a ’Rinnenpankreatitis’ kifejezést, leírva egy szegmentális pancreatitist, mely hegszövetet képez a pancreasfej és a duodenum közti anatómiai területen [5]. Második eset A Becker csoportjához tartozó Stolte groove pancre atitisként fordította le 1982-ben a Becker által alkotott fogalmat, és vezette be az angol szakirodalomba [6]. Emellett megkülönböztetett ún. tiszta (pure) groove pancreatitist, melyben a hegesedés a groove areára korlá tozódik, és szegmentális groove pancreatitist, amely a pancreasfejet is érinti. A groove-ra lokalizálódó forma bizonyítja, hogy e pancreatitistípus külön entitás, és nem az alkoholos krónikus hasnyálmirigy-gyulladás egy meg jelenési formája. Megbeszélés A paraduodenalis pancreatitis (PP) fogalmat mint ún. esernyőterminológiát 2004-ben Adsay és Zamboni alkot ta meg, egyesítve néhány, ugyanazon anatómiai lokalizá cióval és klinikopatológiai jellemzőkkel bíró fibroinflam matoricus folyamatot [3]. A duodenum descendens–mi nor papilla régióban, a pancreaticoduodenalis groove és a csatlakozó pancreas különböző mértékű érintettségé vel létrejött elváltozás számos néven szerepel a szakiro dalomban, mint például a heterotop pancreas cysticus dystrophiája, myoadenomatosis, periampullaris duode nalis fali cysta, a duodenumfal pancreaticus hamartomá 876 2019 ■ 160. évfolyam, 22. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC ESETISMERTETÉS Klinikai megjelenés A PP lefolyása individuális: lehet gyors vagy lassú prog ressziójú, vagy akár klinikailag nem szignifikáns mértékű tünetekkel járó is. Jellemző a súlyos felső abdominalis fájdalom, fogyás, postprandialis hányinger-hányás a duo denumfali hegesedés miatti szűkület és motilitási zavar következtében. Sárgaság a betegség későbbi fázisában fordulhat elő, 6–20%-ban, ha a gyulladásos elváltozás in volválja a ductus choledochust [1, 7, 8]. A képalkotó vizsgálatok közül a mágneses rezonanciás képalkotás, il letve a mágneses rezonanciás cholangiopancreatographia és az endoszkópos ultrahangvizsgálat a legalkalmasabb az elváltozás értékelésére, mindazonáltal hasi ultrahan got és komputertomográfiát is gyakran használnak, bár ezek differenciáldiagnosztikai szempontból kevésbé ha tékony vizsgálati módszerek. A duodenum és a pancreas közti régióban, a duodenumfalat megvastagítóan cysti cus és/vagy szolid elváltozás látszik, gyakran mesze sedéssel, illetve térfoglaló jellegű (pszeudotumor-) formációval. Képalkotó vizsgálatokkal az elváltozás loka lizációja és kiterjedtsége jól meghatározható; a gyulladá sos és a tumoros infiltráció megkülönböztetése egyesek szerint a cysticus formában gyakran sikeres, mások sze rint általánosságban nagyon nehéz – a szolid forma érté kelése valamennyi szerző szerint igen problémás, és gyakran lehetetlen a malignitást nagy valószínűséggel kizárni [14–17]. Szövettani vizsgálattal krónikus gyulladásos folyamat azonosítható a duodenum falában és a környező panc reasszövetben fibrosissal, emellett jellemzően simaizom sejtek és myofibroblastok dens proliferatiója látható, oly kor kis necroticus gócokkal. Az esetek 50–60%-ában mutatkozó, acidophil anyaggal kitöltött cysticus elválto zások pancreaticus ductusszerű kolumnáris jellegű hám mal béleltek, vagy gyakran pseudocysticus jellegűek, gyulladásos granulációs szövet alkotta fallal [11]. A duo denumfalban gyakori a Brunner-mirigy-hyperplasia, il letve szorosan asszociáltan a cystákkal pancreasszövet [1, 3, 7, 17]. Patomechanizmus A PP patomechanizmusa jelenleg még nem tisztázott, csak hipotéziseket állítottak fel [1, 4, 9, 21]. Ezek fő pil léreit a gyulladásos folyamat jellegzetes lokalizációja és a betegek többségében megfigyelhető masszív alkoholfo gyasztás alkotja. Feltételezések szerint a pancreasnedv- elfolyás gátlódik a minor papilla szintjében, aminek hát terében a minor papilla anatómiai és/vagy funkcionális obstrukciója állhat, krónikus lokalizált gyulladást okoz va. Hajlamosító tényezőként a pancreaticus ductalis rendszer egyes anatómiai variációit vetik fel, melyek ér zékenyebbé tehetik a minor papilla régióját az alkohol okozta toxikus károsodásra. Imperforált minor papilla esetében a Santorini-vezetékben ellenirányú áramlás jön létre a Wirsung-vezeték felé, illetve a Santorini-vezeték lehet csökevényes, vagy hiányozhat is. Az elfolyást to vább nehezítheti, ha a Wirsung-vezeték egyébként fizio lógiás megtöretése („Wirsungian knee”) különösen éles [3, 17, 22]. Emellett a PP-ben gyakran megfigyelhető duodenumfali pancreasszövetről is feltételezik, hogy sze repet játszhat az elfolyás akadályozottságában. E panc reasszövetet heterotop jellegűnek tartották (lásd a beteg ség alternatív elnevezéseit), de felvetik, hogy a dorsalis Epidemiológia olykor a malignitás gyanúja is felmerül, amennyiben sej tatípia jelei azonosíthatók, valamint a sejttörmelék össze téveszthető necroticus törmelékkel. Az orsósejtekben gazdag minta lágyrész-daganat, például leiomyoma/lei omyosarcoma, gastrointestinalis stromalis tumor (GIST) lehetőségét vetheti fel [4, 9, 18–20]. A PP predominánsan olyan 40–50 éves férfiakban fordul elő, akiknek az anamnézisében gyakori az alkoholabúzus és a dohányzás [1, 7, 8]. Gyakorisága a fent leírt változa tos nómenklatúra és – részben feltehetően – az ismertség hiányából fakadó aluldiagnosztizáltság miatt sem állapít ható meg biztonsággal. A leggyakrabban krónikus panc reatitis miatt pancreatoduodenectomián átesett betegek anyagait áttekintve határozzák meg, mely esetek 3–24%-a [6, 8–12] felelt meg szövettanilag ezen entitásnak, illetve egy tanulmányban (Casetti, 2009) [13] 70%-os gyakori sággal észleltek a vizsgált mintákban ennek megfelelő képet. A változó gyakoriság oka lehet a tünetek súlyossá ga vagy a malignitás klinikai gyanúja miatti esetenként magasabb reszekciós ráta [9]. A sebészi reszekátum makroszkópos vizsgálatakor jel lemző a minor papillának megfelelő területen a duode numfal heges megvastagodása; a hegesedés gyakran rá terjed a környező pancreasfeji szövetre, komprimálhatja a ductus choledochust. A pancreasfej centrális része azonban szinte sosem érintett. Gyakran észlelhetők cys ticus elváltozások a duodenum falában, a cysticus típus ban 1–10 cm-es cysták figyelhetők meg. A szolid típus ban a tömött, heges szövetben elvétve apró cysták észlelhetők lehetnek. A betegség késői fázisában a hege sedés miatt duodenalis stenosis is létrejöhet, és a heges kompresszió révén a ductus choledochus szűkülete, illet ve elzáródása, valamint a Wirsung-vezeték tágulata is megfigyelhető lehet. Terápia Amennyiben a gondos kivizsgálás után nagy valószínű séggel a paraduodenalis pancreatitis diagnózisa állítható fel, a betegség lefolyásának rendszeres kontrollja mellett lehetséges a konzervatív kezelés, mely magában foglalja a pancreas nyugalomba helyezését, a fájdalom csillapítá sát, szigorú alkoholmegvonás mellett, szükség esetén en doszkóposan elvégezhető beavatkozásokkal, mint a duo denum tágítása, pancreaticus vagy epeúti drenázs, cystenterostomia [8, 9, 13, 27–29]. A szomatosztatin analóg kezelésnek a fájdalomra gyakorolt, noha csak rö vid távon leírt kedvező hatását több szerző is említi. (A pancreasnedv-termelés csökkentése révén az intra pancreaticus nyomást befolyásolja [7, 13, 20, 29–31].) Mindazonáltal a biztonsággal nem kizárható esetleges neoplasticus folyamat miatt gyakran történik primeren részleges pancreatoduodenectomia (hagyományos vagy pylorusmegtartásos Whipple-műtét), mely a betegség progressziója vagy terápiarezisztencia miatt is szükséges sé válhat. Egy 120 beteg anyagát retrospektíven feldol gozó vizsgálatban primeren vagy a kórlefolyás során a betegek 67%-ánál került sor sebészi reszekcióra (nem mutatkozott különbség a cysticus és a szolid variáns kö zött) [7]. Egy szintén friss metaanalízis szerint a feldol gozott 8 tanulmány alapján, mely összesen 335 beteg anyagát reprezentálta, összességében 59%-os volt a sebé szi beavatkozások aránya (80% primer jellegű); a betegek 19%-a részesült elsődlegesen endoszkópos ellátásban, közülük 34%-nak később sebészi kezelésre is szüksége lett [32]. Patomorfológia A gyakran megfigyelhető Brunner-mirigy-hyperplasia szintén szerepet játszhat a minor papilla obstrukciójá ban, illetve diszfunkciójában, valamint összefügghet a krónikus alkoholfogyasztással [6, 23]. Az alkohol ismer ten módosítja a pancreas exokrin funkcióját, növekszik a pancreasnedv viszkozitása a proteinkoncentráció növe kedése miatt, valamint a csökkenő citrátkoncentráció a kalcium : citrát arány eltolódása révén kristályképződésre hajlamosít. E folyamatok kőképződéshez, így ductalis szűkülethez/elzáródáshoz vezetnek, mely nemcsak az alkoholos pancreatitis kialakulásában játszik szerepet, ha nem kedvez a PP létrejöttének is [24]. A betegség prog ressziójában, a duodenalis, majd groove régióról a panc reasra terjedésben a myofibroblastos reakció és a cystaképződés okozta kompresszió is részt vesz, tovább rontva a pancreasnedv-elfolyást, illetve összenyomva a Wirsung-vezetéket. Általánosságban kiemelendő, hogy a krónikus pancreatitis patomechanizmusában napjaink ban az ismert, klasszikus környezeti rizikófaktorok mel lett genetikai tényezők jelenlétét feltételezik, melyek interakciója révén jön létre egy adott egyénben a megbe tegedés. Az elmúlt 20 évben kerültek felfedezésre olyan genetikai mutációk, melyek az egyéb kockázati ténye zőkkel együtt pancreatitis kialakulására hajlamosítanak. E génpolimorfizmusok a SPINK1, CFTR és CTRC gé neket érintik; a biokémiai hátteret a tripszin pancreason belüli fokozott ectopiás aktiválódása, illetve a tripszin inaktiválásért felelős védőmechanizmusok elégtelen mű ködése jelentheti [24, 25]. Patomorfológia Citológiai vizsgálati eredményekről kevés adat áll rendel kezésre, többnyire az esettanulmányok részeként, limi tált leírással. Chute és Stelow közleménye foglalkozik kimondottan a citológiai jellegzetességekkel, 3 eset be mutatása kapcsán. A leggyakrabban stromalis orsósejtek, habos cytoplasmájú sejtek (melyek Brunner-mirigy-hám sejteknek felelhetnek meg) és sejttörmelék azonosítható, gyulladásos sejtek és duodenalis hámsejtek mellett. A lá tott kép általában malignitásra negatív eredményt ad, de 877 2019 ■ 160. évfolyam, 22. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC ESETISMERTETÉS részt az autoimmun pancreatitistől való elkülönítés, mely szteroidterápiával kezelhető, és mint a PP, pszeudotu morjelleget ölthet. (A részletes ismertetés meghaladja a cikk kereteit, ajánljuk Klöppel és Muraki cikkét [1, 21].) pancreas inkomplett involúciójáról lehet inkább szó, a szervfejlődés során a duodenumfalban „csapdába esett” hasnyálmirigyrészletről. E teóriát támogatja az is, hogy a jelen lévő Langerhans-szigetek csak perifériásan és izolál tan tartalmaznak pancreaticus polipeptidsejteket, hason lóképpen az embrionális dorsalis pancreastelephez [17]. A gyakran megfigyelhető Brunner-mirigy-hyperplasia szintén szerepet játszhat a minor papilla obstrukciójá ban, illetve diszfunkciójában, valamint összefügghet a krónikus alkoholfogyasztással [6, 23]. Az alkohol ismer ten módosítja a pancreas exokrin funkcióját, növekszik a pancreasnedv viszkozitása a proteinkoncentráció növe kedése miatt, valamint a csökkenő citrátkoncentráció a kalcium : citrát arány eltolódása révén kristályképződésre hajlamosít. E folyamatok kőképződéshez, így ductalis szűkülethez/elzáródáshoz vezetnek, mely nemcsak az alkoholos pancreatitis kialakulásában játszik szerepet, ha nem kedvez a PP létrejöttének is [24]. A betegség prog ressziójában, a duodenalis, majd groove régióról a panc reasra terjedésben a myofibroblastos reakció és a cystaképződés okozta kompresszió is részt vesz, tovább rontva a pancreasnedv-elfolyást, illetve összenyomva a Wirsung-vezetéket. Általánosságban kiemelendő, hogy a krónikus pancreatitis patomechanizmusában napjaink ban az ismert, klasszikus környezeti rizikófaktorok mel lett genetikai tényezők jelenlétét feltételezik, melyek interakciója révén jön létre egy adott egyénben a megbe tegedés. Az elmúlt 20 évben kerültek felfedezésre olyan genetikai mutációk, melyek az egyéb kockázati ténye zőkkel együtt pancreatitis kialakulására hajlamosítanak. E génpolimorfizmusok a SPINK1, CFTR és CTRC gé neket érintik; a biokémiai hátteret a tripszin pancreason belüli fokozott ectopiás aktiválódása, illetve a tripszin inaktiválásért felelős védőmechanizmusok elégtelen mű ködése jelentheti [24, 25]. pancreas inkomplett involúciójáról lehet inkább szó, a szervfejlődés során a duodenumfalban „csapdába esett” hasnyálmirigyrészletről. E teóriát támogatja az is, hogy a jelen lévő Langerhans-szigetek csak perifériásan és izolál tan tartalmaznak pancreaticus polipeptidsejteket, hason lóképpen az embrionális dorsalis pancreastelephez [17]. 2019 ■ 160. évfolyam, 22. szám Irodalom [1] Klöppel, G. Chronic pancreatitis, pseudotumors and other tu mor-like lesions. Mod Pathol. 2007; 20(Suppl 1): S113–S131. [22] Bang S, Suh JH, Park BK, et al. The relationsphip of anatomic variation of pancreatic ductal system and pancreaticobiliary dis eaeses. Yonsei Med J. 2006; 47: 243–248. [2] Arora A, Bansal K, Sureka B. 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[Bauchspeicheldrüse.] In: Spezielle patholo gische Anatomie, Bd. VI. Springer, Berlin–Heidelbeg–New York, 1973. [German] [26] Patriti A, Castellani D, Partenzi A, et al. Pancreatic adenocarci noma in paraduodenal pancreatitis: a note of caution for con servative treatments. Updates Surg. 2012; 64: 307–309. [6] Stolte M, Weiß W, Volkholz H, et al. A special form of segmental pancreatitis: “groove pancreatitis”. Hepatogastroenterology 1982; 29: 198–208. [27] Chantarojanasiri T, Isayama H, Nakai Y, et al. Groove pancreati tis: endoscopic treatment via the minor papilla and duct of San torini morphology. Gut Liver 2018; 12: 208–213. [7] de Pretis N, Capuano F, Amodio A, et al. Clinical and morpho logical features of paraduodenal pancreatitis. An Italian experi ence with 120 patients. Pancreas 2017; 46: 489–495. [28] Arvanitakis M, Rigaux J, Toussaint E, et al. Endotherapy for paraduodenal pancreatitis: a large retrospective case series. En doscopy 2014; 46: 580–587. [29] Rebours V, Lévy P, Vullierme MP, et al. Clinical and morpho logical features of duodenal cystic dystrophy in heterotopic pan creas. Am J Gastroenterol. 2007; 102: 871–879. [8] Oza VM, Skeans JM, Muscarella P, et al. Groove pancreatitis, a masquerading yet distinct clinicopathological entity. Analysis of risk factors and differentiation. Pancreas 2015; 44: 901–908. [9] DeSouza K, Nodit L. Groove pancreatitis. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. [21] Muraki T, Kim GE, Reid MD, et al. Paraduodenal pancreatitis. Imaging and pathologic correlation of 47 cases elucidates dis tinct subtypes and the factors involved in its etiopathogenesis. Am J Surg Pathol. 2017; 41: 1347–1363. Anyagi támogatás: A közlemény megírása anyagi támo gatásban nem részesült. [17] Zamboni G, Capelli P, Scarpa A, et al. Nonneoplastic mimickers of pancreatic neoplasms. Arch Pathol Lab Med. 2009; 133: 439– 453. Szerzői munkamegosztás: H. I.: A kézirat megírása, a se bészi reszekátumok feldolgozása és a szövettani leletek készítése. B. B.: A szövettani leletek konzulense, a kéz irat lektorálása. F. N.: A második beteg radiológiai leleté nek készítője. M. A.: Az első beteg radiológiai leletének készítője. I. A.: A második beteg belgyógyásza, esetaján lás. K. D.: Mindkét beteg operatőre. A cikk végleges vál tozatát valamennyi szerző elolvasta és jóváhagyta. [18] Chute DJ, Stelow EB. Fine-needle aspiration features of paradu odenal pancreatitis (groove pancreatitis): a report of three cases. Diagn Cytopathol. 2012; 40: 1116–1121. [19] Levenick JM, Gordon SR, Sutton JE, et al. A comprehensive, case-based review of groove pancreatitis. Pancreas 2009; 38: e169–e175. [20] Larjani S, Bruckschwaiger VR, Stephens LA, et al. Paraduodenal pancreatitis as an uncommon case of gastric outlet obstruction: a case report and a review of the literature. Int J Surg Case Rep. 2017; 39: 14–18. A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) Következtetés A legfontosabb a PP elkülönítése a régióban kialakuló daganatoktól, mint a pancreasnak a groove régióban lét rejövő adenocarcinomája, esetlegesen cholangiocarcino ma vagy a duodenumfalból kiinduló adenocarcinoma, valamint regionális cysticus elváltozások és lágyrész-tu morok (például leiomyogen daganatok, GIST). A cysti cus formát és a régió neoplasticus vagy nem neoplasticus cysticus elváltozásait egyes szerzők szerint nagy valószí nűséggel el lehet különíteni preoperatív vizsgálatokkal, azonban a szolid variáns és a periampullaris-paraduode nalis régióban kialakult carcinomák közt igen nehéz, gyakran nem lehetséges a differenciáció, és megbízható diagnózis csak a sebészi reszekátum hisztopatológiai fel dolgozása révén nyerhető [14, 17, 18]. Kiemelendő, hogy a tumormarkerek, mint a CEA és a CA19-9, noha általában nem emelkedettek PP-ben, esetenként leírtak akár magas szérumkoncentrációt is [8, 18, 19, 23]. Megjegyzendő az is, hogy pancreasadenocarcinoma és a PP együttes előfordulása is ismert [14, 26]. Fontos más A paraduodenalis pancreatitis érdekes, ritkának mon dott, bár feltehetően csak kevéssé ismert, így aluldiag nosztizált, diagnosztikus kihívást jelentő megbetegedés, mely gyakran pancreaticus, illetve duodenumfali daganat gyanúját kelti. Összefoglalva, a fent részletezett klinikai képpel jelentkező, majd képalkotó vizsgálatokkal (hasi, illetve lehetőség szerint endoszkópos UH, majd lehető ség szerint MRI, MRCP vagy CT) észlelhető, jellegzetes „groove” lokalizációjú térfoglaló folyamat esetén gon dolnunk kell a paraduodenalis pancreatitis lehetőségére. Az esetleges neoplasticus jelleg kizárása, mint a fentiek ben látható, radiológiailag sokszor nem lehetséges, illet ve akár inkább tumorosnak tűnik a folyamat. A citológiai vagy hisztológiai mintavétel, mint eseteinkben is, gyak ran nem reprezentálja a kérdéses elváltozást. Mindazon által alapos kivizsgálással a radikális sebészi beavatkozás egyes esetekben elkerülhető lehet, noha diagnosztikus kétely vagy konzervatív kezelésre rezisztens tünetek, il 878 ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC ESETISMERTETÉS letve progresszió miatt sokszor az elváltozás nem neo plasticus jellege ellenére gyakran, mint eseteinkben is, szükségessé válik. [14] Malde DJ, Oliveira-Cunha M, Smith AM. Pancreatic carcinoma masquerading as groove pancreatitis: case report and review of literature. J Pancreas (Online) 2011; 12: 598–602. literature. J Pancreas (Online) 2011; 12: 598–602. [15] Arora A, Dev A, Mukund A, et al. Paraduodenal pancreatitis. Clin Radiol. 2014; 69: 299–306. [16] Mittal PK, Harri P, Nandwana S, et al. Paraduodenal pancreatitis: benign and malignant mimics at MRI. Abdom Radiol. 2017; 42: 2652–2674. Anyagi támogatás: A közlemény megírása anyagi támo gatásban nem részesült. Irodalom A brief review of a diagnostic challenge. Arch Pathol Lab Med. 2015; 139: 417– 421. [30] Carvalho D, Loureiro R, Pavão Borges V, et al. Paraduodenal pancreatitis: three cases with different therapeutic approaches. GE Port J Gastroenterol. 2017; 24: 89–94. [31] de Parades V, Roulot D, Palazzo L, et al. Treatment with octreo tide of stenosing cystic dystrophy on heterotopic pancreas of the duodenal wall. Gastroenterol Clin Biol. 1996; 20: 601–604. [Article in French] [10] Manzelli A, Petrou A, Lazzaro A, et al. Groove pancreatitis. A mini-series report and review of the literature. J Pancreas (On line) 2011; 12: 230–233. [11] Becker V, Mischke U. Groove pancreatitis. Int J Pancreatol. 1991; 10: 173–182. [32] Kager LM, Lekkerkerker SJ, Arvanitakis M, et al. Outcomes after conservative, endoscopic, and surgical treatment of groove pan creatitis. A systematic review. J Clin Gastroenterol. 2017; 51: 749–754. [12] Vitali F, Hansen T, Kiesslich R, et al. Frequency and characteriza tion of benign lesions in patients undergoing surgery for the sus picion of solid pancreatic neoplasm. Pancreas 2014; 43: 1329– 1333. (Hegedűs Ivett dr., Pécs, Szigeti út 12., 7624 e-mail: hegedus.ivett@pte.hu) [13] Casetti L, Bassi C, Salvia R, et al. “Paraduodenal” pancreatitis: results of surgery on 58 consecutives patients from a single insti tution. World J Surg. 2019; 33: 2664–2669. 19 ■ 160. évfolyam, 22. szám ORVOSI HETILAP 879 Unauthenticated \| Downloaded 10/24/24 04:43 AM UTC
https://openalex.org/W2778343365	https://ovarianresearch.biomedcentral.com/track/pdf/10.1186/s13048-017-0372-x	English	null	Food restriction during pregnancy and female offspring fertility: adverse effects of reprogrammed reproductive lifespan	Journal of ovarian research	2,017	cc-by	7,493	Abstract Background: Food restriction during pregnancy can influence the health of the offspring during the adulthood. The aim of the present study was to examine the effect of maternal food restriction (MFR) on the reproductive performance in female rat offspring from the first (FR1) and second (FR2) generations. Methods: Adult virgin Wistar female rats were given free access to tap water and were fed ad libitum on standard rodent chow, were mated with virgin adult males, and then were randomly divided into two groups: controls (that was fed ad libitum) and food-restricted group (FR, that was given only 50% of ad libitum food throughout gestation). Their first (FR1) and the second (FR2) generation of offspring were fed ad libitum and sacrificed before puberty and at adulthood. Their ovaries were removed and their histology evaluated by estimating the number of follicles (total and at various stages of folliculogenesis), and the presence of multi-nuclei oocytes and multi-oocyte follicles. Results: Total number of ovarian follicles was lower in FR1 females at week 4 in comparison with controls, while it was not different in FR2 females vs. controls. The number of the primordial follicle was lower in FR1 and FR2 females vs. controls at both week 4 and at week 8. When compared to the controls, the follicles containing multi-nuclei oocytes were more frequent in ovaries from FR1 and FR2 females at week 4, and higher and lower respectively in ovaries form FR1 and FR2 females at week 8. Conclusion: MFR affects ovarian histology by inducing the development of abnormal follicles in the ovaries in first and second generation offspring. This finding could influence the ovarian function resulting in an early pubertal onset and an early decline in reproductive lifespan. Keywords: Follicle, Food restriction, Ovary, Oocyte, Pregnancy Keywords: Follicle, Food restriction, Ovary, Oocyte, Pregnancy of common diseases may be due to the environment that the fetus directly senses via the mother [2, 5–8]. In par- ticular, maternal food restriction (MFR) has been associ- ated with coronary heart disease and increased arterial blood pressure [2], reduced nephron endowment and in- creased renal morbidity in adulthood [9], and may affect physical growth and neurobehavior in newborns [10]. Malnutrition during gestation has been associated with hepatic steatosis, type 2 diabetes, and obesity during adult- hood [5, 11–15]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: hharrath@ksu.edu.sa 1Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia 2Unit of Reproductive and Developmental Biology, Faculty of Science of Tunis University of Tunis El Manar, Tunis, Tunisia Full list of author information is available at the end of the article Abstract During late gestation, MFR is associated with metabolic signaling dysfunction in the liver, and pre- disposes the offspring to insulin resistance [5]. Food restriction during pregnancy and female offspring fertility: adverse effects of reprogrammed reproductive lifespan Abdel Halim Harrath1,2* , Abdulkarem Alrezaki1, Lamjed Mansour1, Saleh H. Alwasel1 and Stefano Palomba3 Harrath et al. Journal of Ovarian Research (2017) 10:77 DOI 10.1186/s13048-017-0372-x Harrath et al. Journal of Ovarian Research (2017) 10:77 DOI 10.1186/s13048-017-0372-x Background Maternal nutritional status during gestation is a key de- terminant for the health and physiology of the offspring at adulthood, and that influence is mainly established during early development, i.e. well before birth [1–5]. Many adulthood diseases can be linked to the environ- ment within which the embryo has developed, including abnormal nutritional, environmental, and hormonal in- sults that may have changed the developmental trajectory of the fetus [1, 2]. According to this hypothesis, the origins Oocyte quality is a critical determinant for the devel- opmental trajectory of the fetus [16, 17]. Many forms of female reproductive disruptions have been linked to the Harrath et al. Journal of Ovarian Research (2017) 10:77 Page 2 of 9 Page 2 of 9 prenatal environment, and it is likely related to early oo- cyte formation, which is vulnerable to numerous envir- onmental effects [4]. In comparison with girls who were born appropriate for the gestational age, girls born small for the gestational age have a reduced reproductive life- span, indicated by a decrease in ovarian size that is asso- ciated with low ovulation rates [18, 19], advanced menarche, and early menopause [20–22]. Polycystic ovarian syndrome (PCOS), one of the most common fe- male endocrine disorders [23], has been suggested to arise through a gene–environment interaction, probably in the developmental milieu within which female gam- etes are formed [24]. Although maternal malnutrition is a major factor that adversely affects fetal growth and is associated with to lifelong consequences, relatively few studies have investigated the effects of MFR on the re- productive outcomes of offspring [4, 25, 26]. In a large epidemiological study women born to mothers exposed to famine were more reproductively successful compared to controls [27]. Moreover, malnutrition during preg- nancy alters reproduction in sheep by inducing poor oo- cyte quality, may cause reproductive disruptions in rats mainly by an early vaginal opening, and induce a decrease in the primordial and antral follicle number [28–30]. During the first trimester of pregnancy in cattle, MFR leads to a reduced ovarian reserve in adulthood, as observed by the increased follicle stimulating hormone (FSH) levels [31]. generation of offspring (FR1) were fed ad libitum. After complete weaning, FR1 and control females were sacri- ficed before puberty (week 4, n. 10) and at adulthood (week 8, n. 10). The ovaries were removed and the fat was discarded. Light microscopy O f Ovaries were fixed in neutral buffer formalin (NBF) or Bouin’s fluid, and were subsequently preserved in 70% alcohol. At least three ovaries from each group have been cut in serial sections to a thickness of 7 μm using a Reichert-Jung microtome. Hematoxylin and eosin (H&E) staining was used to assess ovarian architecture in the group C, FR1, and FR2. The effects of nutrition on folliculogenesis were evalu- ated by counting the number of primordial, primary, secondary, pre-antral, and antral follicles with visible oo- cyte nuclei in some slides for each ovary (see below). Specifically, ovarian follicles were classified according to the scheme of Pedersen and Peters (1968), with modifi- cations. Primordial follicles included oocytes surrounded by a single layer of three to six squamous epithelial cells, whereas primary follicles were composed of oocytes sur- rounded by one layer of numerous cuboidal epithelial cells. Secondary follicles were characterized by oocytes surrounded by more than one layer of granulosa cells with no visible antral spaces. Antral follicles were com- posed of an oocyte surrounded by many layers of cu- boidal granulosa cells, with many visible small antral spaces, or one large antrum. The theca layers and cumu- lus oophorus may be evident. At the moment, the available data regarding the relation- ship between the MFR and the female reproduction in the offspring have many limitations and gaps concerning the potential underlying mechanisms and almost all have evalu- ated the effects of maternal caloric restriction on the first generation only. Based on these considerations, the aim of the present experimental study was to investigate the impact of MFR in rats on ovarian architecture and function in first and second generations of female offspring. Background They were weighed using a digital bal- ance (0.0001 g) and immediately fixed in 10% neutral buffered formalin at room temperature for classical hist- ology. The remainder of FR1 females were allowed to reach sexual maturity, and were treated exactly as their mothers (FR females), i.e. 50% ad libitum food through- out gestation. After birth, a second generation of the doubly food- restricted group females (FR2) was obtained. The FR2 offspring females were humanely sacrificed at 4 and 8 weeks, and their ovaries were collected, weighed and fixed exactly as before detailed for FR1 offspring females. Number of follicles Total follicles Total number of ovarian follicles was significantly (P = 0.0006) lower in FR1 females at week 4 in comparison with controls, while it was not different in FR2 females when compared to the controls (Fig. 2a). At week 8, the total number of follicles in ovaries from both FR1 and FR2 females resulted significantly (P = 0.0485 and P = 0.0013, respectively) lower than in controls. The total number of follicles was significantly (P = 0.0020 and P = 0.0074, re- spectively) higher and lower in FR2 vs. FR1 females at week 4 and 8, respectively. Antral follicles When compared to controls, the number of antral folli- cles was significantly lower (P = 0.0023) in the ovaries of FR1 females at week 4, and significantly higher (P = 0.0084) in the ovaries of FR2 females at week 8 (Fig. 2e). A significant (P = 0.0008 and P < 0.0001, respectively) difference in the number of antral follicles was detected between FR2 and FR1 females at week 4 and 8. Experimental design First- and second-generation offspring [4, 32] of preg- nant rat exposed directly (or indirectly through germline-independent transmission) to MFR were stud- ied. Specifically, adult virgin Wistar female rats weighing 230 ± 20 g obtained from the Animal Unit at King Saud University were given free access to tap water and were fed ad libitum on standard rodent chow (23% protein, 4.5% fat, 3030 kcal/kg; lab diet 5001, Brentwood, MO). After being maintained in separate cages for four days of adaptation, they were mated with virgin adult males, and then were randomly divided into two groups: control group (group C, n. 15) received ad libitum food, and food-restricted group (group FR, n. 20) received only 50% of ad libitum food throughout gestation. The first A particular interest was given to the occurrence of follicles containing multi-nuclei oocytes (MNOFs), key indicator of perturbations during germ cell nest forma- tion [33–38]. The total number of multi-oocyte follicles (MOFs) was also counted in every section of the ovaries from the different groups. To estimate the number of slides to be read for each ovary we used sample size calculations using the following formula [39]: n≅z2 1‐α=2 s2 h2 where: s = sample standard deviation from an initial number (n0) of Harrath et al. Journal of Ovarian Research (2017) 10:77 Page 3 of 9 week 4 (P = 0.0014 and P < 0.0001, respectively), and at week 8 (P = 0.0002 and P = 0.0044, respectively) (Fig. 2b). week 4 (P = 0.0014 and P < 0.0001, respectively), and at week 8 (P = 0.0002 and P = 0.0044, respectively) (Fig. 2b). replications (n0 = 11), Z1 −α/2 = the value retrieved from the normal standard distribution, corresponding to the 1-α/2 probability (we choose α = 0.05) and h: the half width of the confidence interval. Ovarian histopathology MOFs MOFs population was found in all the studied groups and at all follicular stages, from the primordial to the large an- tral stage; these MOFs contained two or more oocytes (Fig. 3a-f). The architecture of the ovaries from FR1 and FR2 females was mainly characterized by more growing follicles when compared to controls. In most of the cases, these were adjacent to each other, suggesting that a fusion has occurred (Fig. 3a). Furthermore, we even reported joining follicles, characterized by the displacement of the oocyte from one follicle into another (Fig. 3b). Primordial follicles The amount of the primordial follicle was significantly lower in FR1 and FR2 females than in controls at both Fig. 1 Effect of MFR on ovarian weight results in a significant reduction in 8-week-old FR2 females when compared to controls (P = 0.0003), and to FR1 females (P = 0.0011); no effect is observed in 4-week-old females When compared to the controls, the MOFs were sig- nificantly (P = 0.0044 and P = 0.006, respectively) more frequent in ovaries from FR1 and FR2 females at week 4 (Fig. 2f). Nevertheless, while this number was signifi- cantly higher (P = 0.0013) in FR1 females compared to control at week 8, it was significantly (P = 0.0075) lower in ovaries from FR2 females. Statistical analysis Statistical analysis For data analysis of follicle number, we used the GraphPad prism version 5. Statistical comparisons were made using a two-tailed t-test. All values are presented as the mean ± standard deviation (SD). Sig- nificance was set at P value less than 0.05. Ovary weight The number of secondary follicles was significantly (P = 0.0002) lower in the ovaries of FR1 females compared to controls only at week 4 (Fig. 2d). Both at week 4 and 8, the number of secondary follicles was significantly (P = 0.0010 and P = 0.0002, respectively) higher in FR2 than in FR1 females. No difference in mean ovary weight was detected be- tween FR1 and FR2 females vs. controls in 4-week-old. Conversely, in 8-week-old animals a significant (P = 0.0003) difference between intervention and control group in ovarian weight for FR2 females only (Fig. 1). A significant (P = 0.0011) difference was also observed in ovary weight in FR2 vs. FR1 females at week 8 (Fig. 1). Primary follicles The number of primary follicles was significantly (P = 0.034 and 0.003, respectively) higher in FR1 and FR2 vs. controls at week 4. That statistical (P = 0.0072) differ- ences were sustained at week 8 only in FR1 females vs. controls (Fig. 2c). A significant (P = 0.0014) difference in the number of primary follicles was observed in FR2 vs. FR1 females at week 8. MNOFs (Heterokaryon) A high frequency of MNOFs was detected in the ovaries of FR1 and FR2 females compared to controls (Fig. 4a- b). A detailed analysis of these MNOFs provided clues on how they were generated. Specifically, we detected Fig. 1 Effect of MFR on ovarian weight results in a significant reduction in 8-week-old FR2 females when compared to controls (P = 0.0003), and to FR1 females (P = 0.0011); no effect is observed in 4-week-old females Harrath et al. Journal of Ovarian Research (2017) 10:77 Page 4 of 9 Fig. 2 Effect of MFR on the number of follicles per section of ovarian tissue. a Total number of follicles: MFR significantly affects the total number of follicles in ovaries from FR1 females at 4 weeks (P = 0.0006) and 8 weeks (P = 0.0485), and from FR2 females at 8 weeks (P = 0.0013). The total number of follicles from FR2 at week 4 is higher vs. FR females (P = 0.0020), whereas it is lower in FR2 females at week 8 compared to FR females (P = 0.0074). b Primordial follicles: significant effect of MFR on the number of primordial follicles in FR1 and FR2 females at week 4 when compared to control (P = 0.0014 and P < 0.0001, respectively) and in FR2 females at week 8 (P = 0.0002). The total number of primary follicles from FR2 at both week 4 and 8 is significantly lower when compared to FR1 females (P = 0.0020 and P = 0.003, respectively). c Primary follicles are significantly higher in number in ovaries of 4-week-old females in both FR1 and FR2 vs. controls (P = 0.034 and P = 0.003). At week 8, FR1 females have significantly higher number of primary follicles vs. control (P = 0.0072), whereas FR2 females have significantly lower number of primary follicles vs. FR1 (P = 0.0014). d Secondary follicles: FR1 females at week 4 have significantly lower number of secondary follicles vs. control (P = 0.0002) and vs. FR2 (P = 0.001), whereas at week 8 FR2 have significantly higher number of secondary follicles vs. FR1 females (P = 0.0002). e Antral follicles: MFR has the same effect as in secondary follicles, except for a significant increase in FR2 females vs. control at week 8 (P = 0.008). f MOFs: The number of MOFs is significantly higher in ovaries from FR and FR2 females vs. MNOFs (Heterokaryon) control at week 4 (P = 0.0044 and P = 0.006, respectively); it is also significantly higher in FR1 females at week 8 (P = 0.0013), and again significantly lower in ovaries from FR2 females vs. control (P = 0.0075). FR2 females at week 8 have significantly lower number of MOFs when compared to FR1 females (P < 0.0001). All results are given as mean ± SD; P < 0.05, FR1 vs. controls (C) and FR2 vs. controls, with error bars: FR1 vs. FR2 Fig. 2 Effect of MFR on the number of follicles per section of ovarian tissue. a Total number of follicles: MFR significantly affects the total number of follicles in ovaries from FR1 females at 4 weeks (P = 0.0006) and 8 weeks (P = 0.0485), and from FR2 females at 8 weeks (P = 0.0013). The total number of follicles from FR2 at week 4 is higher vs. FR females (P = 0.0020), whereas it is lower in FR2 females at week 8 compared to FR females (P = 0.0074). b Primordial follicles: significant effect of MFR on the number of primordial follicles in FR1 and FR2 females at week 4 when compared to control (P = 0.0014 and P < 0.0001, respectively) and in FR2 females at week 8 (P = 0.0002). The total number of primary follicles from FR2 at both week 4 and 8 is significantly lower when compared to FR1 females (P = 0.0020 and P = 0.003, respectively). c Primary follicles are significantly higher in number in ovaries of 4-week-old females in both FR1 and FR2 vs. controls (P = 0.034 and P = 0.003). At week 8, FR1 females have significantly higher number of primary follicles vs. control (P = 0.0072), whereas FR2 females have significantly lower number of primary follicles vs. FR1 (P = 0.0014). d Secondary follicles: FR1 females at week 4 have significantly lower number of secondary follicles vs. control (P = 0.0002) and vs. FR2 (P = 0.001), whereas at week 8 FR2 have significantly higher number of secondary follicles vs. FR1 females (P = 0.0002). e Antral follicles: MFR has the same effect as in secondary follicles, except for a significant increase in FR2 females vs. control at week 8 (P = 0.008). f MOFs: The number of MOFs is significantly higher in ovaries from FR and FR2 females vs. MNOFs (Heterokaryon) control at week 4 (P = 0.0044 and P = 0.006, respectively); it is also significantly higher in FR1 females at week 8 (P = 0.0013), and again significantly lower in ovaries from FR2 females vs. control (P = 0.0075). FR2 females at week 8 have significantly lower number of MOFs when compared to FR1 females (P < 0.0001). All results are given as mean ± SD; P < 0.05, FR1 vs. controls (C) and FR2 vs. controls, with error bars: FR1 vs. FR2 many MOFs in which oocytes were frequently observed very close to each other (Fig. 4d), suggesting they had fused to form a heterokaryon. In some cases, oocytes within the same follicle were apparently undergoing such a joining process (Fig. 4e). follicles that reaches its maximum level around the time of birth, and is gradually depleted during reproductive life [40–42]. At prepubertal age (week 4), we found that the number of primordial follicles was significantly de- creased in FR1 and FR2 rats, suggesting that MFR affects the ovarian reserve of primordial follicles during early fetal life. The significant decrease in the number of primordial follicles at week 4 was associated with an in- crease in the number of primary follicles among the Discussion The fertile reproductive lifespan of female mammals is mainly linked to the initial ovarian reserve of primordial Harrath et al. Journal of Ovarian Research (2017) 10:77 Page 5 of 9 Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Harrath et al. Journal of Ovarian Research (2017) 10:77 Page 6 of 9 (See figure on previous page.) Fig. 3 Hematoxylin and eosin staining of paraffinized ovarian sections from FR1 and FR2 females showing the generation of MOFs. a Ovaries from FR1 and FR2 females are mainly characterized by many growing follicles that are adjacent to each other, indicating their fusion. b-d Follicles in the process of merging (arrowheads); we can see in (d) the displacement of the oocyte from one follicle into the second one. e Primordial follicle with two oocytes. f primordial follicle with three oocytes. g Primary follicle with two oocytes, (h) Primary follicle with three oocytes. i Secondary follicle with two oocytes. (J) Antral follicle with two oocytes; (k) Antral follicle with three oocytes; (l) Antral follicle merging with an early secondary follicle (arrowhead), the large arrow is showing the oocyte position of the antral follicle. Scale bar = 200 μm reproduce successfully [46]. In fact, fetal growth re- striction can be considered as a part of the life his- tory strategy for FR1 and FR2 females that were in utero when their mothers underwent food restriction. Since prenatal undernutrition leads to reduced lon- gevity in mice [47], these females may anticipate a shorter life because of a higher risk of extrinsic mor- tality. It is possible to hypothesize that they may have to adjust their reproductive aptitude by changing the intensity and duration of their lifespan, the timing of the stages of folliculogenesis, as well as the age at which they should reach reproductive maturity. Due to the lack of food sensed through nutritional or endocrine signaling during fetal life [48], and to en- sure reproductive success before death, these females have probably programmed their reproductive lifespan to be very intensive but relatively limited in time, which is consistent with population regulation in the theory of life history [49]. Thus, when they reach different studied groups suggesting that MFR might cause an early menarche by inducing early folliculogen- esis. This hypothesis seems consistent with previous studies [26, 43]. Discussion The rela- tively large number of induced follicles undergoing folliculogenesis at one time makes follicles adjacent to each other and, consequently, highly increases the probability that they will merge to form MOFs [34]. This may have decreased the number of growing oo- cytes (secondary and antral follicles), and may explain the lower total number of follicles, and the higher number of MOFs in 4-week-old FR1 females (vs. con- trols). This strategy is also associated with a faster de- cline in ovarian function with aging, that is clearly supported by the significant decrease in the number of primordial follicles in the ovarian reserve, and also by the significant decrease in the total number of follicles in both FR1 and FR2 females at an early age (8-week), that corresponds in normal females to a high reproductive performance. the assembly of follicles [34] rather than being pro- duced early on during the formation of the ovary. The presence of more than one oocyte in direct con- tact within the same follicle highly increases the pos- sibility of their fusion, and leads to the formation of MNOFs. In fact, any cell brought into contact with another cell and given the right conditions (such as sufficient amounts of fusogen proteins, simultaneously present on each of the two cell surfaces) will fuse with the second cell, even when the latter is foreign [51–53]. This fusion can be beneficial mainly during embryonic development, and for cell-based therapies, and represents a well-known process during reproduction when gametes (spermatozoa and oo- cytes) unite during fertilization to form the zygote. It has also been described in muscle cells, macrophages and nerve cells [54–58]. When cell fusion is blocked during embryonic development, defects in organogen- esis, embryonic lethality, and postembryonic defects can increase [53, 59] suggesting that fused cells are hybrid cells or chimera that function efficiently during embryonic development, driving correct organ forma- tion [54]. In Caenorhabditis elegans, particularly in the proliferative zone of germ cells, two or more crescent shaped nuclei have been observed [60]. MNOFs have also been described during the initial stages of oogenesis in some amphibian species. For example, in Ascaphus truei, oogenesis involves eight nuclei [61], whereas frog oocytes with two nuclei have been described in Leiopelma hochstetteri [62]. Discussion At week 8, the significant decrease in ovarian weight related to the total number of follicles in both first and second generation offspring after MFR is mainly caused by the significant decrease in primordial follicles in the ovarian reserve compared to controls. This could sug- gest an early menopause in FR2, which is less likely in FR1 animals. In fact, previous studies reported that MFR is associated with early menarche and meno- pause [26, 43–45]. To this regard, it is possible to hypothesize that MFR provided first and second gen- eration offspring with a phenotype that is better suited for the lack of food. That new phenotype is consistent with the trade-off theory, i.e. an increase in fertility and a decrease in reproductive lifespan may lead to an increase in the chances of an organism to Fig. 4 Hematoxylin and eosin staining of paraffinized ovarian sections from FR1 and FR2 females showing the generation of MNOFs. a Primordial follicle with two nuclei- oocyte. b Secondary follicle containing one oocyte with two proportional nuclei (c) Secondary follicle containing one merged oocyte with four disproportional nuclei. d Secondary follicle with two oocytes that are very close to each other, suggesting that they will probably fuse soon to form a heterokaryon. e Secondary follicle containing two semi-fused oocytes (arrowhead). f The same secondary follicle in (e) but at another level of section. Scale bar = 200 μm Fig. 4 Hematoxylin and eosin staining of paraffinized ovarian sections from FR1 and FR2 females showing the generation of MNOFs. a Primordial follicle with two nuclei- oocyte. b Secondary follicle containing one oocyte with two proportional nuclei (c) Secondary follicle containing one merged oocyte with four disproportional nuclei. d Secondary follicle with two oocytes that are very close to each other, suggesting that they will probably fuse soon to form a heterokaryon. e Secondary follicle containing two semi-fused oocytes (arrowhead). f The same secondary follicle in (e) but at another level of section. Scale bar = 200 μm Harrath et al. Journal of Ovarian Research (2017) 10:77 Page 7 of 9 Page 7 of 9 prepubertal age, they may upregulate the expression of genes involved in steroidogenesis, which in turn in- duces folliculogenesis for a greater number of primor- dial follicles while concomitantly explaining the significant higher number of in primary follicles in 4- week-old FR1 and FR2 females vs. control. FR1: Female rat offspring from the first generation; FR2: Female rat offspring from the second generation; FSH: Follicle stimulating hormone; H&E: Hematoxylin and eosin; MFR: Maternal food restriction; MNOFs: Follicles containing multi-nuclei oocytes; MOFs: Multi-oocyte follicles; NBF: Neutral buffer formalin; SD: Standard deviation Discussion The most evident example of the necessity for multi- nucleated cells is the syncytiotrophoblast, which is formed when embryonic cytotrophoblast cells fuse with the maternal endometrial epithelium [63, 64]. These cells represent the most important cell type in the placenta, and there is a strong correlation be- tween a successful pregnancy and healthy syncytiotro- phoblasts, likely due to their multi-nuclear state [64]. Of note, a higher number of MOFs in ovaries from 4- week-old first and second generation females was ob- served. Many follicles at these stages may have fused to form MOFs, which explains their significant higher number in FR1 females at four weeks, while this is less likely in FR2 females. While the mechanism of MOFs formation during nest breakdown has been described [33, 50], this is the second study that clearly confirms a new mechanism for the generation of MOFs through the fusion of follicles in the mammalian ovary, and their in- cidence increased sharply at prepubertal age [34]. In- versely, we found that the number of secondary and antral follicles was significantly lower in FR1 and FR2 at prepubertal age when compared to controls. Previous findings have reported that most cases of MOFs repre- sent a fusion between secondary follicles, or a fusion be- tween one secondary and one large antral follicle [34]. Furthermore, the number of secondary/antral follicles observed in 8-week-old females was higher than in con- trols when the number of MOFs was lower. This result is also consistent with the finding of Perez-Sanz and co- workers that showed that the number of MOFs declines significantly in female mice when they become sexually mature [33]. Conclusions Current data suggest that MFR influences ovarian histo- pathology and, in turn, the reproductive health of first and second generation female offspring during fetal de- velopment, and they have been probably programmed to have an early menarche by inducing early folliculogen- esis, and an early decline in ovarian function thereby de- creasing the reproductive lifespan, and leading to an early menopause. The presence of MNOFs is exceptional and repre- sents a challenge for future studies. Two different mechanisms could explain the origin of this phenomenon: the nest breakdown–follicle assembly, and the fusion of more than one oocyte within the same multi-oocyte follicle. Based on our findings, the second mechanism appears the most probable since it is suggested that MOFs are most likely generated by Competing interests 18. Ibanez L, Potau N, Ferrer A, Rodriguez-Hierro F, Marcos MV, De Zegher F. Reduced ovulation rate in adolescent girls born small for gestational age. J Clin Endocr Metab. 2002;87:3391–3. 18. Ibanez L, Potau N, Ferrer A, Rodriguez-Hierro F, Marcos MV, De Zegher F. Reduced ovulation rate in adolescent girls born small for gestational age. J Clin Endocr Metab. 2002;87:3391–3. 19. Ibanez L, Potau N, Enriquez G, De Zegher F. Reduced uterine and ovarian size in adolescent girls born small for gestational age. Pediatr Res. 2000;47:575–7. 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Author details 1 1Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia. 2Unit of Reproductive and Developmental Biology, Faculty of Science of Tunis University of Tunis El Manar, Tunis, Tunisia. 3Unit of Gynecology and Obstetrics, Grande Ospedale Metropolitano “Bianchi – Melacrino – Morelli”, Reggio Calabria, Italy. 22. Ibanez L, de Zegher F. Puberty and prenatal growth. Mol Cell Endocrinol. 2006;254:22–5. 23. Dumesic DA, Oberfield SE, Stener-Victorin E, Marshall JC, Laven JS, Legro RS. Scientific statement on the diagnostic criteria, epidemiology, pathophysiology, and molecular genetics of polycystic ovary syndrome. Endocr Rev. 2015;36:487–525. Received: 11 September 2017 Accepted: 15 December 2017 Received: 11 September 2017 Accepted: 15 December 2017 24. Palomba S, Daolio J, La Sala GB. Oocyte competence in women with polycystic ovary syndrome. Trends Endocrinol Metab. 2017;28:186–98. Authors’ contributions AHH and SA designed the study. 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https://openalex.org/W4205385163	https://www.dora.lib4ri.ch/eawag/islandora/object/eawag%3A24153/datastream/PDF/Chardon-2022-Use_of_iron-coated_sand_for-%28published_version%29.pdf	English	null	Use of iron-coated sand for removing soluble phosphorus from drainage water	Science of the total environment	2,022	cc-by	12,023	H I G H L I G H T S • P removal from drainage water by a pipe drain enveloped with Fe-coated sand was 93%. • Fe in the sand particle coatings was mainly present as siliceous ferrihydrite (Fh). • Reductive release of Fe(II) caused a grad- ual increase in efﬂuent Fe over time. • Fe loss was insigniﬁcant compared to the amount of Fe wrapped around the drain. • Fh stability and its ability to retain P were not affected over time. • Fh stability and its ability to retain P were not affected over time. Contents lists available at ScienceDirect Contents lists available at ScienceDirect ⁎ Corresponding author. E-mail address: gerwin.koopmans@wur.nl (G.F. Koopmans). 1 [ i d] Use of iron-coated sand for removing soluble phosphorus from drainage water Wim J. Chardon a,1, Jan E. Groenenberg b, Jos P.M. Vink c, Andreas Voegelin d, Gerwin F Wageningen Environmental Research, Wageningen University and Research, P.O. Box 47, 6700 AA Wageningen, th Chair Group Soil Chemistry and Chemical Soil Quality, Wageningen University and Research, P.O. Box 47, 6700 A Deltares, Unit Subsurface & Groundwater Systems, P.O. Box 85467, 3508 AL Utrecht, the Netherlands Eawag, Swiss Federal Institute of Aquatic Science & Technology, CH-8600 Duebendorf, Switzerland a Wageningen Environmental Research, Wageningen University and Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands b Chair Group Soil Chemistry and Chemical Soil Quality, Wageningen University and Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands c Deltares, Unit Subsurface & Groundwater Systems, P.O. Box 85467, 3508 AL Utrecht, the Netherlands d Eawag, Swiss Federal Institute of Aquatic Science & Technology, CH-8600 Duebendorf, Switzerland Science of the Total Environment 815 (2022) 152738 Science of the Total Environment 815 (2022) 152738 1 [retired]. ⁎ Corresponding author. E-mail address: gerwin.koopmans@wur.nl (G.F. Koopmans). 1 [retired]. 1. Introduction The aims of our current study are (i) to quantify P retention by the enveloped pipe drain for an extended period of time, i.e., a monitoring period with a total duration of 54 months instead of the ﬁrst 15 months for which data has been reported by Groenenberg et al. (2013), (ii) to test if continuing Mn release from reduction of Mn oxides still prevented reductive dissolution of Fe oxides, (iii) to quantify the effects of (an)oxic conditions on the stability of Fe(III) oxides and P binding inside an undisturbed Fe-coated sand core sampled by using a SOFIE® cell, and (iv) to characterize the Fe oxides in the envelope with Fe-coated sand using X-ray absorption spectroscopy (XAS). Results of this extended ﬁeld experiment will help to support the evaluation of the long-term effective- ness of enveloping pipe drains with Fe-coated sand to prevent P loss to sur- face waters for implementation in practice. Surface water eutrophication, and dense blooms of cyanobacteria in surface waters as a possible consequence, are problems water managers are still facing worldwide (Stroom and Kardinaal, 2016). While point sources of phosphorus (P) have often been reduced effectively, diffuse sources still remain (Macintosh et al., 2018). Due to over-fertilization in the past, many soils became saturated with P and a large part of their ad- sorption capacity is now ﬁlled with P (Schoumans and Chardon, 2015; Kleinman, 2017). Soil P reserves from historic P applications, also called legacy P (Sharpley et al., 2013), may reduce the effectiveness of source- oriented measures taken for preventing dissolved P losses from agricultural land to surface waters. For example, depleting a soil by crop removal with- out replacing P taken up by the crop (phytomining) can be an effective op- tion for reducing the risk of P leaching (Koopmans et al., 2004; van der Salm et al., 2009; Kleinman et al., 2011). However, when the size of the soil legacy P pool is large, the short-term effect of phytomining on surface water quality can be small (McCrackin et al., 2018). Therefore, other op- tions are needed, since a fast improvement of water quality is often neces- sary. For example, the water quality standards of the European Water Framework Directive (EC 2000/60/EC) will have to be met ultimately in 2027. 1. Introduction One option to realize this can be the use of reactive materials for binding P, either for immobilizing P in the topsoil (Koopmans et al., 2020) or for retaining P in buffer strips, vertical barriers, and ﬁlter systems (Klimeski et al., 2012; Groenenberg et al., 2013). These reactive materials often contain Fe or Al oxides (Cucarella and Renman, 2009), which are characterized by a large speciﬁc surface area and a high P adsorption site density (Celi et al., 2003; Hiemstra, 2018; Hiemstra et al., 2019; Hiemstra and Zhao, 2016; Koopmans et al., 2020; Wang et al., 2013). In ﬂat areas, drainage can be more important for discharging excess water from agricul- tural land than surface runoff-mediated transport (King et al., 2015). In such areas, artiﬁcial pipe drainage is usually installed for maintaining a low groundwater level. In The Netherlands, 40% of the agricultural land is drained by pipe drains (Groenenberg et al., 2013). Reactive materials can then be applied around the pipe drains (McDowell et al., 2008; Groenenberg et al., 2013), or in ﬁlters at the end of the pipe drains (Buda et al., 2012; Canga et al., 2016a, 2016b; Penn et al., 2014, 2017; Vandermoere et al., 2018). Groenenberg et al. (2013) described a ﬁeld ex- periment with a pipe drain enveloped with sand, coated with Fe oxides (fur- ther denoted as Fe-coated sand), which is a by-product of the production of drinking water from anoxic groundwater (Sharma et al., 2002; van Beek, 2018). Sharma et al. (2002) characterized Fe-coated sand from 12 drinking water production plants in The Netherlands, where anoxic groundwater is led over a sand ﬁlter in order to remove dissolved Fe(II) by aeration of the raw water. In the coating of the sand particles from these drinking water production locations, they observed amorphous Fe oxides in combi- nation with Ca, silicate, and Mn oxides. Aeration of Fe(II)-containing water in the presence of silicate can lead to the formation of siliceous ferri- hydrite (Fh) at which the adsorption of silicate suppresses the growth of Fh particles (Hiemstra, 2018; Kaegi et al., 2010; Koopmans et al., 2020; Voegelin et al., 2010) while at the same time these particles can densely ag- gregate under the inﬂuence of Ca (Kaegi et al., 2010; Voegelin et al., 2010). The enveloped pipe drain in the ﬁeld experiment of Groenenberg et al. 1. Introduction Fe-coated sand prevented the reductive release of Fe(II) from Fe(III) oxides in the Fe-coated sand under submerged conditions. However, the results of Groenenberg et al. (2013) do not provide insight in the ability of the enveloped pipe drain to retain P from drainage water on the longer term. Consequently, it is difﬁcult to assess whether this measure can be imple- mented in practice. Important research gaps which need further experimen- tal research include the effect of submerged conditions on reductive dissolution of Mn oxides in the Fe-coated sand and how this affects the sta- bility of Fe(III) oxides and their ability to capture P from drainage water on the long term. In the experimental approach of Groenenberg et al. (2013), soil pore water samples were taken in-situ from suction cups either above, 10 cm beside, or 15 cm below the envelope with Fe-coated sand. The chemical composition of these pore water samples indicated anoxic conditions below and beside the enveloped pipe drain. However, this ex- perimental approach did not allow for the determination of the effect of submerged conditions on the stability of Fe(III) oxides and P binding inside the envelope with Fe-coated sand. In our current study, a SOFIE® cell (Sediment Or Fauna Incubation Experiment tool; Vink, 2002, 2009) was used to collect an undisturbed core sample from the envelope of the pipe drain in-situ to enable direct pore water sampling and measurements in the laboratory while preserving redox-discrete ﬁeld conditions in the core sample. Also, in-situ transformation of the Fe oxides in the sand coatings over time into more crystalline Fe phases is an important issue, as this can affect the ability of the enveloped pipe drain to retain P from drainage water. For example, Nielsen et al. (2014) found a half-life of 4 years for the transformation of a siliceous Fh, formed by aerating anoxic groundwater, into goethite after burying it into soil. The maximum P adsorption density for goethite is limited to about 2.5 to 3.5 μmol m−2 (Hiemstra and van Riemsdijk, 1996) whereas the maximum P adsorption density of Fh is usu- ally higher (Celi et al., 2003; Hiemstra and Zhao, 2016; Koopmans et al., 2020; Wang et al., 2013). 1. Introduction (2013) was installed on a site with a calcareous sandy soil located in a seep- age polder (Grifﬁoen, 1994) on which ﬂower bulbs were grown and where concentrations of dissolved reactive phosphorus (DRP) in the water from pipe drains were excessive (2—4 mg L−1). This can at least partly be as- cribed to high concentrations of DRP in seepage water at this location and a net P surplus of 18 kg P ha−1 yr−1 due to compost addition (Groenenberg et al., 2013) in combination with the generally low P binding capacity of calcareous sandy soils (Chardon and Schoumans, 2007; Koopmans et al., 2006; Schoumans, 2014). The results from the ﬁrst 15 months of this ﬁeld experiment were reported by Groenenberg et al. A R T I C L E I N F O Article history: Received 10 November 2021 Received in revised form 23 December 2021 Accepted 23 December 2021 Available online 31 December 2021 Editor: Jan Vymazal Keywords: Phosphorus losses Mitigation measures Enveloped pipe drain Fe-coated sand Siliceous ferrihydrite Mitigation measures are needed for reducing chronic dissolved phosphorus (P) losses from agricultural soils with a leg- acy of excessive P inputs to surface waters. Since pipe drains are an important pathway for P transport from agricultural soils to surface waters in ﬂat areas, removing P from drainage water can be an effective measure. During a 4.5 year- ﬁeld experiment, we tested the performance of a pipe drain enveloped with Fe-coated sand for removing soluble P from drainage water. Iron-coated sand is a by-product of the drinking water industry and has a high ability to bind P. The P concentration in the efﬂuent from the enveloped pipe drain remained at a very low level over the entire mon- itoring period, with a removal percentage amounting to 93% for total P. During the ﬁeld experiment, the enveloped pipe drain was below the groundwater level for a prolonged time. Nevertheless, no reduction of Fe(III) in the Fe- coated sand occurred during the ﬁrst two years, most likely due to preferential reduction of Mn oxides present in the coatings of the sand particles, as reﬂected in elevated efﬂuent Mn concentrations. Thereafter, reductive dissolution of Fe oxides in the coatings caused a gradual increase in the Fe concentration in the enveloped pipe drain efﬂuent over time. Concomitantly, the dissolved Mn concentration decreased, most probably due to the depletion in easily accessi- ble Mn oxides in the Fe-coated sand. The Fe in the Fe-coated sand was identiﬁed as silicate-containing ferrihydrite (Fh). The submerged conditions of the enveloped pipe drain neither affected the stability of Fh in the Fe-coated sand nor the ability of this measure to capture P from drainage water. Enveloping pipe drains with Fe-coated sand is an effective method for reducing dissolved P inputs from agricultural soils to surface waters and holds great promise for implemen- tation in practice. W.J. Chardon et al. Science of the Total Environment 815 (2022) 152738 W.J. Chardon et al. Water samples from the outlet of the enveloped pipe drain and the two reference drains were collected during the winter periods (October to March), when rainfall usually exceeds evaporation in The Netherlands. The outlet of the pipe drains was below the water level of the ditch most of the time, as common in this region. Only when water turbulence in the ditch at the drain outlet indicated water discharge from a drain, the efﬂuent was sampled. To avoid sampling inﬂowing ditch water, efﬂuent water sam- ples were taken via a 2-m-long tube, inserted into the pipe drains via the outlet. During the experimental period October 2010 to April 2015 (54 months), water samples were taken at 32 sampling events from the pipe drain outlets. At all sampling events, also a water sample from the re- ceiving ditch was taken close to the reference pipe drains. ﬁeld sampling was done after 14, 32, and 59 months (denoted as t14, t32, and t59), each time on a new location along the drain. The last sampling of Fe-coated sand at t59 fell outside the period of 54 months when the pipe drain efﬂuent was monitored. For the ﬁeld sampling of the Fe-coated sand from the envelope of the pipe drain, soil (circa 60 cm) above the drain was removed (Groenenberg et al., 2013). Using a piston sampler of 30 cm length, a sample was taken from the entire Fe-coated sand layer. The sample was divided in the ﬁeld in portions originating either from below, beside, or above the enveloped pipe drain, further denoted as the lower layer (L), the middle layer (M), and the upper layer (U). This was done because the Fe-coated sand in the envelope is exposed to different redox conditions at various depths, as concluded from the measurements of the chemical soil pore water composition at different depths near the enveloped pipe drain (Groenenberg et al., 2013). After arrival in the labora- tory, samples taken at t0 and t14 were air-dried at ambient temperature while samples taken at t32 and t59 were freeze-dried, both to avoid the risk of phase transformation of the Fe oxides during drying at elevated tem- perature (Schwertmann and Cornell, 1991; Chardon et al., 2012). 2.5. Total and acid ammonium oxalate-extractable element contents of Fe- coated sand 2.5. Total and acid ammonium oxalate-extractable element contents of Fe- coated sand The Fe-coated sand samples taken at t0, t14, t32, and t59 were ground (50 μm) and subsequently digested with Aqua Regia (Houba et al., 1997) to determine the so-called “pseudo-total” contents of Fe, Al, Mn, and P (Quevauviller, 1998) in the material. For the Aqua Regia digestion, 0.5 g ground material was extracted with a 3:1 (v:v)-mixture of concentrated HCl and HNO3. Furthermore, unground Fe-coated sand samples taken at t32 and t59 were extracted with 0.2 M acid ammonium oxalate (Schwertmann, 1964; McKeague and Day, 1966) to determine the contents of amorphous Fe, Al, and Mn oxides and the content of reversibly adsorbed P. The Fe, Al, Mn, and P concentrations in the Aqua Regia digests and in the acid ammonium oxalate extracts (further denoted as Feox, Alox, Mnox, and Pox) were determined with ICP-AES. During acid ammonium oxalate ex- traction of soil, an SSR of 20 L kg−1 and an extraction time of 2 h are often used (Schoumans, 2000). However, these extraction conditions are not sufﬁcient to extract all amorphous Fe oxides and reversibly adsorbed P from this Fe-rich material (see Section S3 in the Supplementary data). For characterization of the Fe-coated sand, 1.5 g unground material was 2.2. Chemical water analyses Directly after sampling, water samples from the pipe drains and ditch were split in-situ into a ﬁltered (0.45 μm) and an unﬁltered portion. During the ﬁrst 10 months of the ﬁeld experiment (13 sampling events), the pH was measured in-situ in the composite water samples from the suction cups using a HI 9828 Multiparameter Water Quality Portable Meter (Hanna Instruments). All water samples were transported immediately to the laboratory in a cool box and stored at 5 °C until analysis within 1 week of sampling. In the composite water samples from the suction cups and in the 0.45 μm-ﬁltered samples from the pipe drains and ditch, ortho-P was measured as DRP according to Murphy and Riley (1962) using a segmented ﬂow analyzer (SFA; Skalar, SK12) (Houba et al., 2000). Concentrations of metals (Ca, Fe, and Mn), total dissolved P (TDP), and S were measured using an inductively coupled plasma–atomic emission spectrometer (ICP–AES; Iris-3300 DV, Thermo), after acidiﬁcation to pH 1. Chloride was measured using a ﬂow injection analyzer (FIA). Dis- solved unreactive P (DUP) was calculated as the difference between TDP and DRP. Concentrations of NH4–N, (NO3 + NO2)-N, and total dissolved N (TDN) in the composite water samples from the suction cups were mea- sured using the SFA (Houba et al., 2000). Dissolved organic N (DON) was calculated as the difference between TDN and the summed NH4–N and (NO3 + NO2)-N concentrations. Before the SFA analyses of DRP and the dif- ferent N forms, the samples were acidiﬁed by HCl addition to lower the pH to approximately 2 to dissolve Fe(III)-containing colloids which could have been formed in anaerobic water samples due to exposure to air during transport and storage. Dissolved organic C (DOC) in the composite water samples from the suction cups was measured with a Total Organic Carbon analyzer (TC5000, Shimadzu) and calculated as the difference between total and inorganic C. In the unﬁltered part of the pipe drain efﬂuent and ditch water samples, total P was measured after peroxidation digestion (H2SO4, H2O2, and Se). Particulate P (PP) was calculated as the difference between TP and TDP. For further analytical details, see Groenenberg et al. (2013). W.J. Chardon et al. At t32, also an undisturbed Fe-coated sand core was collected in-situ with a SOFIE® cell (Vink, 2002, 2009) while preserving redox-discrete ﬁeld con- ditions when the cell was transported to the laboratory. With this core, the entire Fe-coated sand layer of the enveloped pipe drain was sampled, with material from below, beside, and above the drain. The cell consists of a coring tube which is closed air-tight at both ends during sampling. Both pH and redox potential (Eh) were measured directly in the SOFIE® cell and via 0.1 μm-permeable probes the pore water in the core was sam- pled at the top, in the middle, and near the bottom of the intact core. Pore water samples were analysed on Cl, Br, F, NO3-N, NH4-N, SO4-S, DRP, TDP, Fe, Mn, Na, and Ca. For details on the analytical techniques used for these chemical analyses, see Section S2 in the Supplementary data. For sampling the soil pore water around the enveloped pipe drain, poly- ester acrylate suction cups were installed along the enveloped drain at 5, 15, 25, and 45 m from the ditch. The permeability of these cups is around 0.1 μm (Groenenberg et al., 2013). At each position, one cup was installed 15 cm below, two cups at 10 cm on either side, and one cup straight above the envelope with Fe-coated sand. Soil pore water samples were collected via vacuum bottles (approximately 90 kPa), connected to the cups. Before analysis, the pore water samples from all cups placed at the same height were combined for making a composite sample. From January 2011 to July 2014 (42 months), a total of 23 soil pore water samples were obtained for the suction cups placed above or below the enveloped pipe drain. For the cups placed beside the enveloped pipe drain, no pore water samples could be taken at two sampling events. For these cups, the composite pore water samples of 21 sampling events were analysed. 2.1. Site description and ﬁeld experiment The experiment was done on a ﬁeld used for ﬂower bulb growth, on a calcareous sandy soil in a seepage polder in the Dutch coastal area (Grifﬁoen, 1994) near Egmond aan den Hoef. The ﬁeld site has a net P sur- plus since more P is applied via compost than is removed with harvesting, leading to a build-up of P in the soil proﬁle and leaching of P. The experimental setup of the ﬁeld experiment was extensively described in Groenenberg et al. (2013). In brief, the enveloped pipe drain was installed between two existing pipe drains, which were used as a reference in this ﬁeld experiment. For the installation of the enveloped pipe drain, a 55 m- long trench was dug (depth 90 cm and width 40 cm). In the trench, a coconut ﬁber cloth was placed, then a 10-cm layer of Fe-coated sand with a new 6-cm-i.d. polyvinyl chloride pipe drain placed on the sand. The trench was ﬁlled beside the pipe drain with Fe-coated sand and a 10- to 20-cm layer of coated sand was placed on top of the drain. The co- conut cloth was folded and the trench was ﬁlled with original soil. A total of 0.14 m3 of Fe-coated sand was estimated to be used per m drain, containing 65 kg Fe per m drain (see Section S1 in the Supplementary data). 2 Science of the Total Environment 815 (2022) 152738 W.J. Chardon et al. 2.4. Pore water composition of Fe-coated sand To quantify the chemical pore water composition inside the layer of Fe- coated sand enveloping the pipe drain, samples from the Fe-coated sand taken at t32 and t59 were extracted using 0.01 M CaCl2 at a solution-to- solid ratio (SSR) of 10 L kg−1 (Houba et al., 2000). By shaking during ex- traction, Fe oxide colloids could be released from the coating of the sand particles, which can pass a 0.45 μm-ﬁlter. Therefore, the 0.01 M CaCl2 ex- traction method of Houba et al. (2000) was modiﬁed: shaking was done mildly at 30 rpm whereas the regular shaking time of 2 h was extended to 24 h to compensate for the mild shaking conditions. After 0.45 μm-ﬁltra- tion, the extracts were analysed on pH, Al, Fe, Mg, Mn, TDP, DRP, S, Si, DOC, As, Cd, Cu, Ni, Pb, and Zn, using the same analytical methods as de- scribed above in Section 2.2. 2.6. Structural characterization of Fe in Fe-coated sand by X-ray absorption spectroscopy Four Fe-coated sand samples were characterized by Fe K-edge X-ray ab- sorption spectroscopy (XAS): the grab sample collected at t0 and samples taken at t32 from different layers of the Fe-coated sand envelope. The sam- ples taken at t32 were frozen immediately after sampling in the ﬁeld using liquid N2 for preserving redox-discrete ﬁeld conditions. The samples were ground in a mixer mill and mixed with cellulose for the preparation of a sample pellet. X-ray absorption spectra were recorded at the Swiss- Norwegian Beamline (SNBL) at the European Synchrotron Radiation Facil- ity (ESRF, Grenoble). The measurements were performed in transmission mode at room temperature, using ionization chambers to record the incom- ing and transmitted X-ray photon intensity. For the extraction and interpre- tation of the extended X-ray absorption ﬁne structure (EXAFS) spectra, the software code Athena was used (Ravel and Newville, 2005). For the inter- pretation of the spectra by linear combination ﬁtting, we used four refer- ence spectra: (i) lepidocrocite (Lp), (ii) goethite (Goe), and (iii) 2-line ferrihydrite (2L-Fh) formed by base addition to a concentrated Fe(NO3)3 so- lution (Voegelin et al., 2007) and (iv) silicate-containing Fh (Si-Fh) formed by the oxidation of 0.5 mM Fe(II) in a bicarbonate-buffered solution in the presence of 0.5 mM silicic acid (Senn et al., 2015). Owing to its formation in the presence of Si, Si-Fh features a lower degree of distortion of FeO6 octa- hedra and of corner-sharing linkage of FeO6 octahedra than 2L-Fh (Senn et al., 2015). Fig. 1. Dissolved reactive P (DRP) concentrations in the 0.45 μm-ﬁltered water samples from the reference pipe drains and the enveloped pipe drain and the receiving ditch as a function of time (32 sampling events over 54 months of the whole ﬁeld experiment). chemical speciation of these elements at the transition from oxic to anoxic soil conditions (Kirk, 2004). On average, at all depths, DRP accounts for more than 90% of the TDP concentration over the entire period. There is no indication that the composition of the water from the suc- tion cup below the drain is inﬂuenced by the Fe-coated sand nearby. The high concentrations of Cl, Fe, Mn, DOC, NH4, and P are indicative for brack- ish seepage water, as can be expected in a coastal area with peat layers in the subsoil (Grifﬁoen, 1994). Increased DOC concentrations may well be explained by the mechanisms described by Vink et al. 2.6. Structural characterization of Fe in Fe-coated sand by X-ray absorption spectroscopy (2010): reductive production of Mn2+ increases the metabolic fermentation of organic matter and the production of DOC, which subsequently prolongs the retention of metals and P in solution. Based on the Cl and S concentrations measured in the soil pore water from the cups at the three depths during the ﬁrst sam- pling period, Groenenberg et al. (2013) concluded that downward drainage and upward seepage ﬂuxes contributed equally to the water inﬂux of the enveloped pipe drain. Data in Table 1 show that this remains more or less valid during the whole experiment, since the Cl and S concentrations in the soil pore water samples from the cups placed beside the enveloped pipe drain were close to the average of concentrations in the water from the cups above and below the drain. 3.1. Chemical composition of soil pore water samples During a large part of the ﬁeld experiment, suction cups directly above, beside, and 15 cm below the enveloped pipe drain were sampled. In Table 1, values of pH and the concentrations of major elements, several N species, and P fractions averaged over time are presented. The pattern ob- tained during the ﬁrst 15 months (Groenenberg et al., 2013) was conﬁrmed by the data for the whole experimental period. The soil pore water has a slightly alkaline pH and comparable Ca concentrations at all depths. For other elements, however, the concentrations in the soil pore water samples taken from the cups above the enveloped pipe drain differ strongly from those in the water from the cups below the drain. Above the enveloped pipe drain, the soil pore water is oxic, with 97% of the inorganic N being present as (NO3 + NO2)-N, with relatively low concentrations of Fe and Mn, and a high S concentration compared to the water from the cups below the drain. In contrast, the soil pore water from the cups below the drain is anoxic, with relatively high concentrations of Fe and Mn, a low S concentration, and inorganic N only being present as NH4-N. These obser- vations agree very well with the theoretical concept of redox-sensitive 2.3. Sampling of Fe-coated sand from the enveloped pipe drain Before applying Fe-coated sand for enveloping the pipe drain, a grab sample was taken from the Fe-coated sand in the ﬁeld (denoted as t0) and 3 Science of the Total Environment 815 (2022) 152738 W.J. Chardon et al. Fig. 1. Dissolved reactive P (DRP) concentrations in the 0.45 μm-ﬁltered water samples from the reference pipe drains and the enveloped pipe drain and the receiving ditch as a function of time (32 sampling events over 54 months of the whole ﬁeld experiment). extracted with 300 mL acid ammonium oxalate to realize an SSR of 200 L kg−1 and the extraction time was extended to 4 h. The combination of an SSR of 200 L kg−1 and 4 h was shown by Koopmans et al. (2020) to be sufﬁcient for a complete extraction of both Feox and Pox from iron oxide sludge, which has even a higher Feox content than our Fe-coated sand (Chardon et al., 2012). 2.6. Structural characterization of Fe in Fe-coated sand by X-ray absorption spectroscopy 3.2. Leaching of P from the enveloped and reference pipe drains Fig. 1 shows the trend of the DRP concentration measured in the water samples from the ditch and the efﬂuents of the reference pipe drains and enveloped pipe drain for the entire duration of the ﬁeld experiment. Both reference pipe drains show a large temporal variation. Until the end of the experiment, the DRP concentrations measured in the efﬂuent from the 3.3. Leaching of Fe and Mn from the enveloped and reference pipe drains For the enveloped pipe drain, an average DRP concentration of 0.10 mg L−1 was found, much lower than the average DRP concentration of 3.16 mg L−1 for the two reference pipe drains (Table 2). From these data, a removal percentage of 97% was calculated for DRP. Likewise, the DUP and PP concentrations are lower in the efﬂuent from the enveloped pipe drain than for the reference pipe drains, but the removal percentage of both P forms is lower (81%) than for DRP. However, since the DRP frac- tion of TP exceeds 80% for the reference pipe drains, the removal percent- age of TP is also high: 93% (Table 2). Hence, the enveloped pipe drain is very effective for retaining all these physical-chemical P fractions (Haygarth and Sharpley, 2000). Even after 54 months (4.5 year) of func- tioning, there is no indication of a decrease in the ability of the enveloped pipe drain to capture P from drainage water. Due to the lower removal per- centage, the efﬂuent from the enveloped pipe drain is relatively enriched with P fractions other than DRP: expressed as % of TP, the relative DUP and PP concentrations in the efﬂuent of the enveloped pipe drain are about three times higher than in the efﬂuent of the reference pipe drains (Table 2). However, the concentration of Fe in the enveloped pipe drain ef- ﬂuent is high when compared to TP (Table 2). The Fe, measured in the 0.45 μm-ﬁltered water samples from this drain, will be present as either Fig. 3 shows the average level of dissolved Fe in the 0.45 μm-ﬁltered ef- ﬂuents of the reference pipe drains and the enveloped pipe drain and in the receiving ditch water for the duration of the whole experiment (54 months). Interestingly, the average Fe concentration during the ﬁrst 15 months of the ﬁeld experiment as reported by Groenenberg et al. (2013) completely dif- fers from the period thereafter: during this ﬁrst period, the average Fe con- centration in the efﬂuent from the enveloped pipe drain was much lower than in the efﬂuents from the two reference pipe drains. This is remarkable, since soil pore water with a similarly high Fe concentration was probably transported with upward seepage from the subsoil to all pipe drains (see data from suction cup positioned below the enveloped pipe drain in Table 1). According to Fig. Table 1 Concentrations of major elements, including inorganic C (IC), dissolved organic C (DOC), inorganic N, dissolved organic N (DON), dissolved unreactive P (DUP), and dis- solved reactive P (DRP) in composite soil pore water samples from suction cups above, beside, and below the enveloped pipe drain averaged across the different sampling events (n = 23) of the whole ﬁeld experiment (54 months). Values represent average ± standard deviation. Position pH1 Cl Ca Fe Mn S IC DOC NH4–N (NO3 + NO2)-N DON DUP DRP mg L−1 Above 7.6 ± 0.3 28 ± 11 167 ± 41 1.2 ± 1.8 0.2 ± 0.3 78 ± 40 63 ± 16 26 ± 11 0.19 ± 0.24 6.91 ± 9.60 1.07 ± 0.37 0.05 ± 0.09 0.91 ± 1.16 Beside 7.5 ± 0.2 75 ± 31 154 ± 30 7.6 ± 3.0 0.7 ± 0.2 44 ± 25 96 ± 29 31 ± 12 1.75 ± 0.84 0.46 ± 0.66 1.45 ± 0.41 0.19 ± 0.47 2.49 ± 1.60 Below 7.4 ± 0.3 143 ± 37 179 ± 22 31.1 ± 10.4 3.4 ± 0.4 5.5 ± 6.3 154 ± 46 67 ± 46 8.12 ± 1.43 0.02 ± 0.05 2.52 ± 0.59 0.28 ± 0.82 3.48 ± 1.30 1 pH of pore water in suction cups was measured in the ﬁeld only during the ﬁrst 10 months of the experiment (13 sampling events). 4 Fig. 2. Dissolved reactive P (DRP) in 0.45 μm-ﬁltered water samples (left) and total P (TP) measured after peroxidation digestion of unﬁltered water samples (right) from the receiving ditch, the reference pipe drains, and the enveloped pipe drain. Values represent average ± standard deviation over 54 months of the whole ﬁeld experiment (n = 32). W.J. Chardon et al. Science of the Total Environment 815 (2022) 152738 W.J. Chardon et al. Science of the Total Environment 815 (2022) 152738 Fig. 2. Dissolved reactive P (DRP) in 0.45 μm-ﬁltered water samples (left) and total P (TP) measured after peroxidation digestion of unﬁltered water samples (right) from the receiving ditch, the reference pipe drains, and the enveloped pipe drain. Values represent average ± standard deviation over 54 months of the whole ﬁeld experiment (n = 32). enveloped pipe drain were clearly lower (factor of circa 30) than those for the reference pipe drains. dissolved Fe(II) or in colloidal particles. Baken et al. 3.3. Leaching of Fe and Mn from the enveloped and reference pipe drains 4, the Fe concentration in the efﬂuent from the enveloped pipe drain gradually increased over time to values around 33 mg L−1 at the end of the experiment, which is much higher than the av- erage values found in the efﬂuents from the two reference pipe drains (Fig. 3). Although the Fe concentrations in the efﬂuent from the enveloped pipe drain seem very high, they hardly exceed the average Fe concentration in the soil pore water, sampled via the suction cups placed 15 cm below the enveloped drain, i.e., 31.1 mg L−1 (Table 1). Dilution of the high Fe Table 1 (2015) found that Fe (II), present in anoxic seepage water, was oxidized when the seepage water entered oxic layers of ditch sediments or in the overlying ditch water. The Fe(III) formed was able to immobilize DRP present in the ditch (Baken et al., 2015). Thus, the bioavailability of the P forms in the Fe-enriched enveloped pipe drain efﬂuent and in the ditch water is proba- bly low (Baken et al., 2014). Overall, the average DRP concentration was 2.5 mg L−1 for the refer- ence pipe drain 1 and 3.8 mg L–1 for the reference pipe drain 2 (Fig. 2). For the receiving ditch, an average value of 1.9 mg DRP L−1 was found (Fig. 2). All values were higher than the average DRP concentrations re- ported by Groenenberg et al. (2013) for the ﬁrst 15 months (reference pipe drain 1: 1.4 mg L−1, reference pipe drain 2: 3.1 mg L–1, receiving ditch: 1.7 mg L−1). 3 Average of the two reference pipe drains. 2 Dissolved unreactive P (DUP) was calculated as the difference between total dissolved P and dissolved reactive P (DRP) while p difference between total P (TP) and total dissolved P. 3 Average of the two reference pipe drains. Table 2 Total dissolved Fe (left) and Mn (right) concentrations in 0.45 μm-ﬁltered water samples from the receiving ditch, the reference pipe drains and the enveloped pipe drain. Values represent average ± standard deviation over 54 months of the whole ﬁeld experiment (n = 32). For total dissolved Fe and Mn concentrations from the enveloped pipe drain, the average values for the ﬁrst 15 months are shown as well (Groenenberg et al., 2013). Please note difference in scale of y-axis of both ﬁgures. (3.1 mg kg−1; Groenenberg et al., 2013), according to the reactions (Postma and Appelo, 2000): concentration in the soil pore water from the subsoil with drainage water transported downward from above could explain why the average Fe con- centrations in the efﬂuents from the two reference pipe drains were so much lower. Indeed, the pore water sampled from the cups placed directly above the enveloped drain had an average Fe concentration of 1.2 mg Fe L−1 (Table 1). When using the contributions of the downward drainage and upward seepage ﬂuxes to the water inﬂux into the pipe drains as calcu- lated from the data on Cl (see Section 3.1.), the average Fe concentration in the efﬂuents of the two reference pipe drains would be circa 13 mg L−1, which is still much higher than the measured average Fe concentrations of circa 3 mg L−1 (Fig. 3). Hence, oxidation of Fe2+ and subsequent precip- itation around or in the pipe drain must have contributed to the much lower Fe concentration leaching from the reference pipe drains. Table 2 2Fe2þ þ MnO2 þ H2O↔2FeOOH þ Mn2þ þ 2Hþ ð1Þ Fe2þ þ MnOOH↔FeOOH þ Mn2þ ð2Þ 2Fe2þ þ MnO2 þ H2O↔2FeOOH þ Mn2þ þ 2Hþ ð1Þ 2Fe2þ þ MnO2 þ H2O↔2FeOOH þ Mn2þ þ 2Hþ ð1Þ Fe2þ þ MnOOH↔FeOOH þ Mn2þ ð2Þ Fe2þ þ MnOOH↔FeOOH þ Mn2þ Fe2þ þ MnOOH↔FeOOH þ Mn2þ ð2Þ Alternatively, the mineral Jacobsite (MnFe2O4) can be formed in a sys- tem where both Fe2+ and MnO2 are present, according to the reaction (Villinski et al., 2001, 2003): 2Fe2þ þ MnO2 þ 2H2O↔MnFe2O4 þ 4Hþ ð3Þ ð3Þ According to reaction (1), dissolved Fe(II) from the seepage water is ox- idized in the envelope with Fe-coated sand to insoluble Fe(III) while insol- uble Mn(IV) is reduced to dissolved Mn(II), with two moles of Fe being precipitated by reductive dissolution of one mole of Mn and with the pro- duction of two moles of H+. According to reaction (2), insoluble Mn(III) is reduced to dissolved Mn(II), with one mole of Fe being precipitated by one mole of Mn and with no H+ being produced. Indeed, the Mn concentra- tion in the efﬂuent of the enveloped pipe drain increased during the ﬁrst circa 26 months of the ﬁeld experiment, but then declined quite sharply (Fig. 4). Even more so, this decrease of the Mn concentration coincides with an increase in the Fe concentration in the enveloped pipe drain efﬂu- ent (Fig. 4). However, the decrease of Mn in the drainage water cannot be explained by the exhaustion of the stock of Mn oxides supplied with the Fe-coated sand. Based on the amount of Mn in the Fe-coated sand around the pipe drain, the amount of water passing the drain, and the Mn concentration in the passing water, less than 8% of the ini- tial Mn stock was leached during the whole experimental period of 54 months (see Section S1 in the Supplementary data). However, it is possible that the remaining Mn oxides react more slowly. A potential mechanism behind this could be that the readily accessible Mn oxide pool present on the outer surface of the Fe-coated sand was exhausted over time, or became covered by freshly precipitated Fe oxides (Villinski et al., 2003). Passivated Mn oxides or Mn oxides further inside the Fe coatings may react less readily with Fe(II) in the seepage water. Table 2 Concentrations of different physical-chemical P fractions (DRP, DUP, PP, and TP) and Fe and Mn in efﬂuent water samples from the enveloped pipe drain and from both reference pipe drains. Average values were calculated over the experimental period of 54 months (n = 32). Values represent average ± standard deviation. Drain DRP % sum1 DUP2 % sum1 PP2 % sum1 TP Fe Mn mg L−1 % mg L−1 % mg L−1 % mg L−1 mg L−1 mg L−1 Reference3 3.16 ± 1.60 80 0.16 ± 0.16 4 0.62 ± 1.01 16 3.86 ± 1.45 2.75 ± 1.71 1.01 ± 0.43 With envelope 0.10 ± 0.10 40 0.03 ± 0.03 12 0.12 ± 0.32 48 0.26 ± 0.37 12.6 ± 14.1 4.88 ± 3.20 Removal (%) 97 81 81 93 1 % of (DRP + DUP + PP). 2 Dissolved unreactive P (DUP) was calculated as the difference between total dissolved P and dissolved reactive P (DRP) while particulate P (PP) was calculated as the difference between total P (TP) and total dissolved P. 3 Average of the two reference pipe drains. emical P fractions (DRP, DUP, PP, and TP) and Fe and Mn in efﬂuent water samples from the enveloped pipe drain and from both ere calculated over the experimental period of 54 months (n = 32). Values represent average ± standard deviation. ( ) 2 Dissolved unreactive P (DUP) was calculated as the difference between total dissolved P and dissolved reactive P (DRP) while particulate P (PP) was calculated as the difference between total P (TP) and total dissolved P. 3 Average of the two reference pipe drains. 5 Fig. 3. Total dissolved Fe (left) and Mn (right) concentrations in 0.45 μm-ﬁltered water samples from the receiving ditch, the reference pipe drains and the enveloped pipe drain. Values represent average ± standard deviation over 54 months of the whole ﬁeld experiment (n = 32). For total dissolved Fe and Mn concentrations from the enveloped pipe drain, the average values for the ﬁrst 15 months are shown as well (Groenenberg et al., 2013). Please note difference in scale of y-axis of both ﬁgures. W.J. Chardon et al. Science of the Total Environment 815 (2022) 152738 W.J. Chardon et al. Science of the Total Environment 815 (2022) 152738 Fig. 3. 3.5. Composition of the Fe-coated sand In Table 4, the total contents of Fe, Mn, and P as determined by digesting Fe-coated sand with Aqua Regia are summarized for t0, t14, t32, and t59. Data for Al are not presented as the Al contents in the Fe-coated sand were very small when compared to the Fe contents (< 1 mg kg−1; data not shown). Based on the ratio of the total Fe content in the Fe- coated sand at t59 relative to t0, a large amount of Fe seems to have been lost from the envelope with Fe-coated sand over time, especially from the upper layer. The Fe fraction lost at t59 amounted to 41, 25, and 27% for the upper, middle, and lower layers, respectively. However, leaching as an explanation would not be consistent with the calculated fraction of Fe leached as 0.3% of the initial total Fe content (see Section 3.3. and Section S1 in the Supplementary data). In addition to the Fe content, the total Mn and P contents decreased as well from t0 to t59. The apparent de- crease in the contents of these elements could be due to a heterogeneous composition of the Fe-coated sand. The effect of the latter could have be- come particularly evident because Fe-coated sand samples were collected at different places along the enveloped pipe drain. However, this explana- tion seems unlikely as the total Fe, Mn, and P contents decrease over time in a consistent manner. Alternatively, the coating of the sand particles could have come off, either when the Fe-coated sand was applied during the installation of the enveloped pipe drain or during in-situ sampling in the ﬁeld. The effect of loosening of the coating during Fe-coated sand appli- cation could especially explain the large decrease in the Fe content in the ﬁrst 14 months of the ﬁeld experiment (circa 20%). This large decrease in the Fe content cannot be explained by leaching of Fe. During these ﬁrst 14 months, the Fe concentration in the enveloped pipe drain efﬂuent was still low (Fig. 4), translating into a fraction of Fe leached over this period of only 0.003% of the total amount of Fe initially present around the enveloped pipe drain (see Section S1 in the Supplementary data). Table 2 Consequently, the Fe concentration in the efﬂuent of the enveloped pipe drain increased gradually over time, either due to reductive dissolution of Fe oxides from the Fe-coated sand, or to break- through of Fe2+ from the anoxic groundwater. Still, this did not lead to a concomitant release of DRP to the enveloped pipe drain efﬂuent (Fig. 1). A comparable calculation for Fe as for Mn gave a value of <1% of the total amount of Fe initially present around the enveloped pipe drain Groenenberg et al. (2013) attributed the much lower Fe concentration in the efﬂuent of the enveloped pipe drain during the ﬁrst 15 months of the ﬁeld experiment (Fig. 3) to retardation of Fe2+ by adsorption onto Fh, or to an interaction between Fe(II) dissolved in the seepage water and Mn oxides, present in solid form in the Fe-coated sand used Fig. 4. Total dissolved Fe and Mn concentrations in 0.45 μm-ﬁltered water samples from the enveloped pipe drain as a function of time (32 sampling events over 54 months of the whole ﬁeld experiment). Fig. 4. Total dissolved Fe and Mn concentrations in 0.45 μm-ﬁltered water samples from the enveloped pipe drain as a function of time (32 sampling events over 54 months of the whole ﬁeld experiment). 6 Science of the Total Environment 815 (2022) 152738 W.J. Chardon et al. SO4-S concentration is probably due to sulfate reduction resulting from or- ganic matter mineralization. The concentrations of DRP and TDP are below the detection limit in all layers. So no transfer of P through the Fe-coated sand envelope was observed, which is consistent with the very low levels of DRP leaching from the enveloped pipe drain over the whole ﬁeld exper- iment (Fig. 1). y 2 For t0, the composition of the Fe-coated sand for the three different layers is the same. 3.5. Composition of the Fe-coated sand These Fe contents measured in the Fe-coated sand thus seem unreliable and can- not be used to calculate a mass balance of Fe in the envelope around the pipe drain. For this purpose, the loss of Fe from the envelope could be cal- culated from measurements of Fe in the pipe drain efﬂuent in combination with an estimated efﬂuent ﬂux. Although this seems to be a more reliable approach because the efﬂuent composition was measured at a large number of sampling events, which is the result of Fe release over the total length of the pipe drain, still the estimated efﬂuent ﬂux is uncertain. Unfortunately, Fe concentrations of Fe in the pipe drain efﬂuent were only measured in 0.45 μm-ﬁltered water samples. This could result in underestimating the loss of Fe from the enveloped pipe drain. 1 Measurements were done in pore water collected from PES probes installed at several heights in the sediment core: one probe was installed just under the surface of the Fe-coated sand, denoted as the upper layer (U), another in the middle of the column, denoted as the middle layer (M), and the third one near the bottom, de- noted as the lower layer (L). that was leached during the whole experimental period (see Section S1 in the Supplementary data). In other words, an excess of Fe relative to adsorbed P is still present in the envelope with Fe-coated sand, explaining why the DRP concentration in the enveloped pipe drain efﬂu- ent remained at a very low level over the entire period of the ﬁeld exper- iment. This will be further discussed in Section 3.5. y 2 For t0, the composition of the Fe-coated sand for the three different layers is the same. Table 3 Redox potential (Eh), pH, and chemical composition of the pore water measured within the SOFIE® cell at different layers of an undisturbed Fe-coated sand core taken from the enveloped pipe drain. The Fe-coated sand core was collected after 32 months (t32). Layer Depth Eh pH Fe Mn SO4-S NH4-N NO3-N DRP TDP cm mV mg L−1 U1 0.5 −107 7.9 5.6 1.9 45 0.5 0.23 <0.01 0.61 M1 5 −219 n.d.2 9.4 3.4 36 4.5 <0.02 <0.01 <0.01 L1 10 −233 7.9 15.8 6.1 21 7.4 <0.02 <0.01 <0.01 1 Measurements were done in pore water collected from PES probes installed at several heights in the sediment core: one probe was installed just under the surface of the Fe-coated sand, denoted as the upper layer (U), another in the middle of the column, denoted as the middle layer (M), and the third one near the bottom, de- noted as the lower layer (L). Layer Depth Eh pH Fe Mn SO4-S NH4-N NO3-N DRP TDP cm mV mg L−1 U1 0.5 −107 7.9 5.6 1.9 45 0.5 0.23 <0.01 0.61 M1 5 −219 n.d.2 9.4 3.4 36 4.5 <0.02 <0.01 <0.01 L1 10 −233 7.9 15.8 6.1 21 7.4 <0.02 <0.01 <0.01 3.4. Fe-coated sand: 0.01 M CaCl2 extraction and SOFIE® cell The concentrations of both DRP and TDP in the 0.01 M CaCl2 extracts of the Fe-coated sand from the different layers at t32 and t59 are all very low, as they are all below the detection limit (see Section S4 in the Supplementary data). Also, the levels of heavy metals and arsenic are very low in these ex- tracts. However, these Fe-coated sand samples may have been exposed to oxic conditions during in-situ ﬁeld sampling and subsequent freeze-drying and during the extraction with 0.01 M CaCl2, which may have inﬂuenced the analytical results. Therefore, an undisturbed Fe-coated sand core was sampled using the SOFIE® cell, which gave the opportunity to sample the pore water of the Fe-coated sand in the laboratory at redox-discrete ﬁeld conditions. Table 3 gives a selection of the data measured using the SOFIE® cell while the complete data set is given in Table S2 in Section S2 in the Supplementary data. With increasing depth of the Fe- coated sand layer, the environment is more reduced as follows from the measured Eh. Based on the measured range in Eh, redox reactions including denitriﬁcation and reduction of Mn(III/IV) to Mn(II), Fe(III) to Fe(II), and SO4 2−to S2−could have occurred inside the envelope with Fe-coated sand (Pan et al., 2015). The gradient in Eh corresponds to the differences in the chemical composition of the pore water over the upper, middle, and lower Fe-coated sand layers (Table 3). Nitrate-N was only found in the upper layer; its concentration was below the detection limit in the mid- dle and lower layers, which is probably due to denitriﬁcation. The concen- trations of NH4-N, Fe, and Mn increase with depth and the SO4-S concentration decreased with depth (Table 3), corresponding with the trends seen for the suction cups (Table 1). The decrease with depth of the Due to the binding of P by the Fe-coated sand of the enveloped pipe drain over time, one would expect a gradual increase of the molar P/Fe 1 Samples were taken from three different layers of the Fe-coated sand envelope wrapped around the pipe drain where U denotes the upper layer, M the middle layer, and L the lower layer. 1 Samples were taken from three different layers of the Fe-coated sand envelope wrapped around the pipe drain where U d the lower layer. ition of the Fe-coated sand for the three different layers is the same. sand for the three different layers is the same. Table 4 Based on the estimated P retention by the Fe-coated sand material, the annual re- duction in P loss by enveloping pipe drains with Fe-coated sand at the ﬁeld scale can be extrapolated to 19 kg P ha−1 (see Section S5 in the Supplemen- tary data). Results from the characterization of Fe-coated sand sampled at t0 and t32 by Fe K-edge EXAFS spectroscopy are shown in Fig. S2 in Section S6 in the Supplementary data. The marked oscillations in the k-space EXAFS spectra of lepidocrocite and goethite (Fig. S2a) arise from the edge-sharing (Lp and Goe) and corner-sharing (Goe) coordination of FeO6-octahedra, as can be seen in the corresponding Fourier-transformed spectra (Fig. S2b). The spec- tra of the two nanocrystalline Fh references (2L-Fh and Si-Fh) feature much lower second-shell FeFe signal amplitudes, due to a lower degree and less- ordered second-shell FeFe coordination. The minor differences between the two Fh references arise from a lower distortion of the FeO6 octahedra and a lower degree of corner-sharing FeFe linkage in silicate-ferrihydrite (Si-Fh) than 2-line ferrihydrite (2L-Fh), reﬂecting a lower degree of Fe polymeriza- tion in Si-Fh formed by Fe(II) oxidation in bicarbonate-buffered solution in the presence of silicate than 2L-Fh formed by base addition to an acidic Fe (III) solution. The spectrum of the Fe-coated sand sampled at t0 falls in be- tween the spectra of Si-Fh and 2L-Fh, and can be adequately reproduced by a linear combination of the two Fh reference spectra (0.78 Si-Fh + 0.20 2L- Fh). The Fe-coatings of the sand particles were formed during water treat- ment by the oxidation of Fe(II) in oxygenated groundwater in the presence of silicate (Chardon et al., 2012; Koopmans et al., 2020), in analogy to the synthesis of the Si-Fh reference. The slightly higher degree of Fe polymeri- zation in the coatings than the Si-Fh reference material may indicate that the coatings were either formed at a slightly lower dissolved Si/Fe ratio than the Si-Fh reference or that siliceous Fh in the coatings of sand sampled at t0 had already aged and further polymerized compared to freshly precip- itated Si-Fh. The latter explanation is probable, as sand can be used over several years in ﬁlters used for water treatment in drinking water produc- tion plants before replacement (Sharma et al., 2002), allowing the Fe(III) precipitates to age in contact with aerated Fe(II)-containing water over an extended period of time. Table 4 Rather than transformation of Fh into more crystalline com- pounds, the effect of loosening of the Fe-coating of the sand particles when taking subsamples from the Fe-coated sand samples in the laboratory for the acid ammonium oxalate extraction and Aqua Regia digestion may explain the discrepancy between the contents of Fe, Mn, and P as deter- mined by the two methods. For the Aqua Regia digestion, ground Fe- coated sand (50 μm) was used (Houba et al., 1997), whereas unground ma- terial was used for the acid ammonium oxalate extraction. Since the Fe- coated sand samples may have been non-homogeneous due to the loose Fe oxide coating of the sand particles, it may have been difﬁcult to obtain a representative subsample, causing an underestimation of the Feox, Mnox, and Pox contents if the subsample contained mainly coarse sand particles (Neary and Barnes, 1993). Based on the results of the characterization of Fe-coated sand samples by Fe K-edge EXAFS and the very low DRP concen- trations from the enveloped pipe drain over the whole ﬁeld experiment, the submerged conditions of the enveloped pipe drain did not seem to affect the stability of Fh in the Fe-coated sand and its ability to capture P from drain- age water. ratio in the coated sand. Indeed, this ratio showed a small increase from 0.030 at t0 to 0.033 mol/mol (s.d. 0.0014) at t59 when averaged over the three layers (Table 4). At this low molar P/Fe ratio, a strong adsorbing ac- tion of the Fe-coated sand is expected, even under reduced conditions (Young and Ross, 2001; Loeb et al., 2008). This explains the very low con- centrations of DRP measured in the pore water in the SOFIE® cell (Table 3) and the very low DRP concentrations in the enveloped pipe drain efﬂuent during the whole ﬁeld experiment (Fig. 1). Using the measured TDP con- centrations in the efﬂuents of the pipe drains and estimated water ﬂuxes, we calculated in Section S5 in the Supplementary data the retention of P by the enveloped pipe drain during the whole experimental period, and the resulting increase of the molar P/Fe ratio of the Fe-coated sand. For t59, an increase to 0.031 mol/mol was calculated, which is close to the av- erage P/Fe ratio of 0.033 mol/mol (s.d. 0.0014) found in the ﬁeld. Table 4 Table 4 Composition of Fe-coated sand measured using Aqua Regia digestion. The Fe-coated sand samples were taken after 0, 14, 32, and 59 months (denoted as t0, t14, t32, and t59). Sampling Fe Mn P P/Fe U1 M1 L1 U M L U M L U M L g kg−1 g kg−1 g kg−1 mol/mol t0 2 249 249 249 3.33 3.33 3.33 4.09 4.09 4.09 0.030 0.030 0.030 t14 200 200 195 2.59 2.55 1.96 3.55 3.48 3.01 0.032 0.031 0.028 t32 195 189 168 2.10 1.86 1.49 3.55 3.48 3.01 0.033 0.033 0.032 t59 148 187 181 1.76 1.83 1.82 2.75 3.26 3.43 0.033 0.031 0.034 t59/t0 0.59 0.75 0.73 0.53 0.55 0.55 0.67 0.59 0.75 1 Samples were taken from three different layers of the Fe-coated sand envelope wrapped around the pipe drain where U denotes the upper layer, M the middle layer, and L Science of the Total Environment 815 (2022) 152738 W.J. Chardon et al. Section S6 in the Supplementary data presents data on Feox, Mnox, and Pox in the Fe-coated sand sampled at t32 and t59. The extracted amounts of Alox in these samples were negligible (data not shown). The same was found for iron oxide sludge, produced as a byproduct at the same drinking water production plant (Koopmans et al., 2020). At t32, the measured Feox, Mnox, and Pox contents of the upper and middle layers are only slightly lower than the contents determined by Aqua Regia digestion. For Fe, this is qualitatively in line with the characterization of the solid phase Fe as Fh by EXAFS, as discussed above. For the lower layer, however, the Feox, Mnox, and Pox contents at t32 are clearly lower than for Aqua Regia. Like- wise, the Feox, Mnox, and Pox contents in the samples from all three layers at t59 are much lower than the contents determined by Aqua Regia diges- tion. Since the EXAFS spectrum of the sample from the lower layer taken at t32 was similar to the two spectra from the upper and middle layers (Fig. S2), the discrepancy between the Feox content and the Fe content de- termined by Aqua Regia digestion cannot be explained by substantial Fh transformation into more crystalline compounds. Unfortunately, no EXAFS data are available for the samples collected at t59 to validate this ob- servation. Table 4 Comparison of the spectra of the 32-months ﬁeld-aged Fe-coated sand from the three different layers and the spectrum of the Fe-coated sand at t0 indicated no marked changes in Fe coordination over 32 months, irrespective of the sampled layer. Considering the intense signal amplitudes of the goethite and lepidocrocite reference spectra (Fig. S2), this indicates that at most a very small fraction of the ferrihy- drite had transformed into goethite or lepidocrocite within 32 months. However, because the anoxic state of the Fe-coated sand sample from the lower layer could not be maintained during sample preparation and analysis in the laboratory, some Fe(II) associated with this sample, either as Fe2+ adsorbed onto Fh or as a mixed valence Fe(II/III) phase, may have been overlooked in the ﬁt. Nevertheless, these Fe(II)-phases probably did not account for a major fraction of the total Fe because there is a close match between the three spectra from the three different layers. 4. Conclusions Enveloping a pipe drain with Fe-coated sand resulted in an average DRP re- moval of 93%, over a period of 4.5 years. No indication was found of a de- crease in effectiveness with time. The enveloped pipe drain resulted in efﬂuent concentrations that were below the Dutch water quality criterion of 0.15 mg TP L−1. • While during the ﬁrst 2 years of the experiment Mn oxides present in the Fe-coated sand prevented the reductive release of Fe(II) and P from Fe(III) oxides in the Fe-coated sand, this effect was not found during the latter part of the experiment. This is possibly due to a depletion of easily acces- sible Mn oxides. • The loss of Fe, calculated from the concentrations of Fe in the efﬂuent from the enveloped pipe drain, was insigniﬁcant when compared to the amount of Fe around the drain. • No indications were found for a structural change of the Fe-coatings over the course of the ﬁeld experiment. 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Pepping near Egmond aan den Hoef for making avail- able his land for the ﬁeld experiment with the enveloped pipe drain, the employers of the Flower Bulbs Research Unit of Applied Plant Research (PPO) in Lisse for sampling and supervising the ﬁeld experiment, and the drinking water company Brabant Water Ltd. for providing the Fe-coated sand. The Swiss Norwegian Beamline (SNBL) at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) is acknowledged for the alloca- tion of beamtime for Fe K-edge XAS analyses. This research has been co- ﬁnanced by the Innovation Program Water Framework Directive (Contract KRW08091) and the strategic research program Sustainable spatial devel- opment of ecosystems, landscapes, seas and regions (KB-14) from the Dutch Ministry of Economic Affairs. Additional support was received from the Horticultural Marketing Board (“Productschap Tuinbouw”), the Province Noord-Holland, and the water board Hollands Noorderkwartier. 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Wageningen University, Wageningen, The Netherlands. CRediT authorship contribution statement Wim J. Chardon: Conceptualization, Investigation, Data curation, Writing – original draft, Funding acquisition. Jan E. Groenenberg: Con- ceptualization, Investigation, Data curation, Writing – review & editing, Su- pervision, Funding acquisition. Jos P.M. Vink: Investigation, Writing – review & editing. Andreas Voegelin: Investigation, Writing – review & editing. Gerwin F. Koopmans: Conceptualization, Investigation, Data curation, Writing – review & editing, Supervision, Funding acquisition. The initial Fe-coated sand sampled at t0 and the Fe-coated sand samples aged in-situ for 32 months (t32) in the ﬁeld were further characterized by an acid ammonium oxalate extraction aimed at dissolving amorphous or poorly crystalline Fe oxides like Fh (Schwertmann, 1991). Table S6 in 8 W.J. Chardon et al. Science of the Total Environment 815 (2022) 152738 Hiemstra, T., Zhao, W., 2016. 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https://openalex.org/W2977577238	https://biblio.ugent.be/publication/8634741/file/8634743	English	null	Comparison of Free-Space and Waveguide-Based SERS Platforms	Nanomaterials	2,019	cc-by	7,967	Received: 30 August 2019; Accepted: 28 September 2019; Published: 1 October 2019 Abstract: Surface-Enhanced Raman Spectroscopy (SERS) allows for the highly speciﬁc detection of molecules by enhancing the inherently weak Raman signals near the surface of plasmonic nanostructures. A variety of plasmonic nanostructures have been developed for SERS signal excitation and collection in a conventional free-space microscope, among which the gold nanodomes oﬀer one of the highest SERS enhancements. Nanophotonic waveguides have recently emerged as an alternative to the conventional Raman microscope as they can be used to eﬃciently excite and collect Raman signals. Integration of plasmonic structures on nanophotonic waveguides enables reproducible waveguide-based excitation and collection of SERS spectra, such as in nanoplasmonic slot waveguides. In this paper, we compare the SERS performance of gold nanodomes, in which the signal is excited and collected in free space, and waveguide-based nanoplasmonic slot waveguide. We evaluate the SERS signal enhancement and the SERS background of the diﬀerent SERS platforms using a monolayer of nitrothiophenol. We show that the nanoplasmonic slot waveguide approaches the gold nanodomes in terms of the signal-to-background ratio. We additionally demonstrate the ﬁrst-time detection of a peptide monolayer on a waveguide-based SERS platform, paving the way towards the SERS monitoring of biologically relevant molecules on an integrated lab-on-a-chip platform. Keywords: Raman spectroscopy; SERS; photonic integrated circuit; waveguide-based SERS; nanoplasmonic slot waveguide; gold nanodomes; peptide detection Comparison of Free-Space and Waveguide-Based SERS Platforms Nina Turk 1,2,* , Ali Raza 1,2, Pieter Wuytens 3 , Hans Demol 4,5, Michiel Van Daele 6, Christophe Detavernier 6, Andre Skirtach 2,7, Kris Gevaert 4,5 and Roel Baets 1,2 g y 4 VIB-UGent Center for Medical Biotechnology, 9000 Ghent, Belgium; Hans.Demol@UGent.be (H.D.); Kris.Gevaert@UGent.be (K.G.) 5 Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium 6 CoCooN Research Group, Department of Solid State Sciences, Ghent University, 9000 Ghent, Belgium; Michiel.VanDaele@UGent.be (M.V.D.); Christophe.Detavernier@UGent.be (C.D.) 5 Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium 5 Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium 6 CoCooN Research Group, Department of Solid State Sciences, Ghent University, 9000 Ghent, Belgium; Michiel.VanDaele@UGent.be (M.V.D.); Christophe.Detavernier@UGent.be (C.D.) 7 NanoBioTechnology Laboratory, Department of Biotechnology, Ghent University, 9000 Ghent, Belgium * Correspondence: nina.turk@ugent.be NanoBioTechnology Laboratory, Department of Biotechnology, Ghent University, 9000 Ghent, Belgium * Correspondence: nina.turk@ugent.be * Correspondence: nina.turk@ugent.be nanomaterials nanomaterials nanomaterials www.mdpi.com/journal/nanomaterials 1. Introduction Raman spectroscopy enables for the highly speciﬁc detection of molecules by probing their vibrational states. Such Raman signals are, however, inherently weak. In the last decades, several methods have been developed to enhance Raman signals, such as coherent anti-Stokes scattering that relies on non-linear Raman scattering [1] or Surface-Enhanced Raman Spectroscopy (SERS) [2–15]. Two types of SERS enhancement mechanisms have been proposed to explain the SERS eﬀect—the electromagnetic (EM) mechanism and the chemical enhancement (CE) mechanism [16–19]. The EM enhancement, where the local EM ﬁeld is greatly increased near plasmonic nanostructures by localized surface-plasmon resonances, is thought to be the main contribution to the SERS enhancement [19]. Nanomaterials 2019, 9, 1401; doi:10.3390/nano9101401 www.mdpi.com/journal/nanomaterials 2 of 14 Nanomaterials 2019, 9, 1401 On the other hand, the CE enhancement is characterized by the shifting of the Raman scattering in non-resonance to that in resonance through the formation of charge transfer complexes between adsorbed molecules and metal surfaces, and its contribution is thought to be far smaller than the EM enhancement [19]. In this paper, we will focus solely on the EM mechanism of SERS enhancement. A variety of plasmonic nanostructures providing high SERS enhancements have been developed for SERS signal excitation and collection in a conventional free-space confocal microscope [20]. Colloidal metal nanoparticles enable strong SERS enhancements, yet their fabrication methods oﬀer only limited control of their geometry, size and position, consequently aﬀecting the reproducibility of SERS measurements [20]. Conversely, a variety of SERS substrates has been developed using top-town fabrication techniques such as nanosphere lithography [21–24], and deep-UV [11] and electron beam lithography [25,26]. These techniques enable precise control of the shape and position of the nanostructures, which allows more tunable and reproducible SERS enhancements [20]. Among the top-down fabricated SERS substrates, the gold nanodomes oﬀer one of the highest SERS enhancements [14,15]. Their fabrication is simple and scalable, while also ensuring better control of the hotspot size and enhancement factor as compared to colloidal approaches. p p pp Moreover, novel techniques for the more eﬃcient collection of Raman signals have recently emerged as alternatives to the conventional Raman microscope, such as hollow-core photonic crystal ﬁbers [27] and nanophotonic waveguides on photonic integrated circuits (PICs) [28–31]. 2.1. Fabrication of SERS Substrates Gold nanodomes were fabricated using a nanosphere lithography (NSL) based process, described in detail in [15] and shown in Figure 1. Brieﬂy, we started from a 200 nm thick ﬁlm of silicon nitride (Si3N4) deposited on top of a 4-inch silicon wafer using PECVD deposition (Vision 310-PECVD, AdvancedVacuum, Uppsala, Sweden). We spin-coated a colloidal solution of 450 nm polystyrene beads (microParticles GmbH, Berlin, Germany) on the Si3N4 surface, thereby generating a monolayer of hexagonally-close packed polystyrene beads. The polystyrene beads were then thinned down in an oxygen plasma (GIGA batch 310 M, PVA-TePla, Wettenberg, Germany), and a periodic pattern of nanodomes was etched into the Si3N4 substrate by an anisotropic reactive-ion etch (Vision 320-RIE, AdvancedVacuum, Uppsala, Sweden). These two steps eﬀectively determined the height and the width of the gap between the nanodomes, the two most important parameters for tuning the plasmonic resonance and thus, the SERS enhancement factor [15]. The remains of the polystyrene beads were lifted oﬀin dichloromethane (Sigma-Aldrich, Overijse, Belgium) and the wafers were cleaned in a piranha solution (H2SO4:H2O2, 3:1; purchased from Sigma-Aldrich, Overijse, Belgium). Finally, a 2 nm thick titanium adhesion layer and a 130 nm thick gold layer were sputtered onto the sample (Alcatel SCM600, Bittmann applied technologies, Rotenburg, Germany). The nanodomes were characterized with scanning electron microscopy (SEM; Nova 600 Nanolab, FEI, Hillsboro, OR, USA) and the nanodome gap size was determined to be 12 ± 2 nm. UV-VIS reﬂection and SERS measurements were performed to optimize their localized surface-plasmon resonance wavelength in order to match the 785 nm laser used for the SERS excitation, as published in [14]. 3D Finite-Diﬀerence Time-Domain (FDTD) simulations predict the maximum SERS enhancement factor of the gold nanodomes for a single molecule in the order of 107 [15]. Figure 1. Fabrication process of gold nanodomes. (a) Spincoating of polystyrene beads on a Si3N4 chip. (b) Thinning down of the polystyrene beads by oxygen plasma. (c) Etching of the nanodome pattern in Si3N4. (d) Removal of the remains of the polystyrene beads. (e) Gold deposition. (f) The arrows mark the gap in the gold nanodomes. Figure 1. Fabrication process of gold nanodomes. (a) Spincoating of polystyrene beads on a Si3N4 chip. (b) Thinning down of the polystyrene beads by oxygen plasma. (c) Etching of the nanodome pattern in Si3N4. (d) Removal of the remains of the polystyrene beads. (e) Gold deposition. 1. Introduction Nanophotonic waveguides can be used to eﬃciently excite and collect the Raman signal of the molecules close to the waveguide, opening up the possibility of bringing the selectivity of Raman measurements to an integrated lab-on-a-chip platform. PICs have been also used to collect SERS signals from external colloidal metal nanoparticles [32–34]. However, to enable reproducible waveguide-based excitation and collection of SERS spectra, top-down fabricated plasmonic structures can be integrated on dielectric nanophotonic waveguides. In the ﬁrst demonstration of waveguide-excited and collected SERS on integrated plasmonic nanostructures, gold bowtie antennas were patterned on a silicon nitride waveguide in a two-step electron beam lithography process [35,36]. Electron beam lithography is, however, resource-intensive and time-consuming, so an alternative nanosphere lithography approach was used to fabricate integrated nanotriangles. [37] Besides easier fabrication, the integrated nanotriangles also achieved a signiﬁcant improvement in SERS enhancement compared to the integrated bowties. More recently, new waveguide-based SERS platforms have emerged, such as the nanoplasmonic slot waveguide [38–41] and the nanoporous gold on suspended silicon nitride waveguides [42]. The on-chip SERS platform shows great potential for high-throughput SERS assays on low sampling volumes, which is especially relevant for the detection of biological molecules. However, most biological molecules have low Raman cross-sections, making the SERS enhancements of the integrated bowties and integrated nanotriangle platforms insuﬃcient for their detection. The high SERS enhancement of the nanoplasmonic slot waveguide might, however, prove suﬃcient for the SERS detection of biological molecules [38]. In addition to high SERS enhancement, the nanoplasmonic slot waveguides are fabricated using a combination of atomic layer deposition and deep UV photolithography, enabling mass scale manufacturing. Furthermore, they oﬀer a non-resonant enhancement, making the SERS enhancement independent of excited and scattered wavelengths. In this paper, we evaluate the SERS performance of the two top-performing SERS substrates, one for free-space and other for the waveguide-based excitation and collection of SERS signals. This comparison is highly relevant for future on-chip SERS applications. We compare the gold nanodomes in which the signal is excited and collected in free space, and the waveguide-based nanoplasmonic slot waveguides. We examine the SERS signal enhancement and the SERS background of the diﬀerent SERS platforms using a monolayer of p-nitrothiophenol (NTP). We additionally demonstrate the ﬁrst-time detection of a biomolecule on a waveguide-based SERS platform, paving the way towards SERS detection and the monitoring of biologically relevant molecules on an integrated lab-on-a-chip platform. 1. Introduction 3 of 14 Nanomaterials 2019, 9, 1401 2.2. Acquisition of SERS Spectra of P-Nitrothiophenol Both SERS substrates were functionalized with a self-assembled monolayer of p-nitrothiophenol (NTP; purchased from Sigma-Aldrich, Overijse, Belgium) that selectively binds to the gold via a gold-thiol bond, as described in detail in [38]. In short, the SERS substrates were cleaned with acetone, isopropyl alcohol and deionized (DI) water, and then were dried with a N2 gun. After the oxygen plasma treatment, they were immersed overnight in 1 mM NTP solution in ethanol, and then rinsed with ethanol to remove non-bound NTP. SERS spectra of the two substrates were acquired on a confocal Raman microscope (Alpha 300 R+, WITec, Ulm, Germany) equipped with a −65 ◦C to −70 ◦C cooled CCD camera (iDus 401BR-DD, Andor, Belfast, UK) and a 785 nm diode laser (XTRA II, Toptica, Graefelﬁng, Germany). Stokes scattered light was collected by a 100 µm diameter multimode ﬁber and fed to the spectrometer, which used a 600 l/mm grating to diﬀract the Stokes scattered light on the spectral camera. All lines of the CCD camera were read out (using full vertical binning), using a vertical shift speed of 16.25 µs and a horizontal shift speed of 0.033 MHz. For the gold nanodomes, a 300 µW laser power before the microscope objective and a Nikon PlanFluor 10×/0.3 objective (Nikon, Tokyo, Japan) were used to acquire spectra on a spatially distributed map of 10 × 10 pixels in a 20 × 20 µm area with an integration time of 0.13 s on each point to avoid signal degradation upon laser illumination. Each trace represents a median spectrum of one map. To acquire the SERS spectra of the nanoplasmonic slot waveguide on the confocal microscope, the sample was positioned vertically and end-ﬁre coupled, as shown in Figure 3 and explained in more detail in [38]. The polarization of the 785 nm excitation beam was set to the TE mode of the waveguide and a Zeiss 100×/0.9 EC Epiplan NEOFLUAR:∞/0 objective (Carl Zeiss AG, Oberkochen, Germany) was used to couple the light into the waveguide with the laser power of 350 µW measured before the microscope objective. The integration time was set to 1 s. The SERS signal was collected in back reﬂection using the same objective. The objective and chip were aligned with 100 nm accuracy based on a maximum intensity of the waveguide Raman spectrum. 2.1. Fabrication of SERS Substrates (f) The arrows mark the gap in the gold nanodomes. Nanoplasmonic slot waveguides were fabricated using a combination of atomic layer deposition and deep UV photolithography, as described in detail in [38] and shown in Figure 2. Firstly, 2.3 µm thick SiO2 and 220 nm thick Si3N4 layers were deposited on a 200 nm silicon wafer. The Si3N4 slot waveguides were patterned with 193 nm deep UV optical lithography and subsequently etched by a reactive-ion etch process (fabricated by IMEC, Leuven, Belgium). The resulting average slot width was 150 nm. The minimal width of the slot is limited to 150 nm by the resolution of the deep-UV lithography. To narrow down the slot width, the waveguides were uniformly coated with 58 nm Al2O3 by using atomic layer deposition (ALD; deposited on the home-built ALD setup, Ghent University, Ghent, Belgium) that has a low SERS background [43]. After ALD, gold waveguides were deﬁned using photolithography (MA6 mask aligner, SUSS MicroTec, Garching, Germany), and a 2 nm titanium adhesion layer and a 15 nm thick layer of sputtered gold were deposited. The nanoplasmonic slot waveguides were characterized with scanning electron microscopy and the gap size was determined 4 of 14 Nanomaterials 2019, 9, 1401 to be 15 ± 0.5 nm. Due to the technical limitations of the setup, UV-VIS reﬂection spectra of the waveguide-based SERS substrates could not be measured. To prove the non-resonant enhancement of the nanoplasmonic slot waveguides, we relied on the simulation results [38,44], which are supported by the experimental measurements of the SERS signal at two diﬀerent laser excitation wavelengths (632 and 785 nm), as reported in [38]. Numerical simulations predicted the maximum SERS enhancement factor of the nanoplasmonic slot waveguide for a single molecule to be 1.5 × 107 [38]. Figure 2. Fabrication of a nanoplasmonic slot waveguide. (a) Fabrication of the Si3N4 slot waveguides. (b) Atomic layer deposition of Al2O3. (c) Gold deposition. The arrows mark the gap width of the nanoplasmonic slot waveguide. Figure 2. Fabrication of a nanoplasmonic slot waveguide. (a) Fabrication of the Si3N4 slot waveguides. (b) Atomic layer deposition of Al2O3. (c) Gold deposition. The arrows mark the gap width of the nanoplasmonic slot waveguide. 3.1. Comparison of Free-Space Excited Gold Nanodomes and Waveguide-Based Nanoplasmonic Slot Waveguide In this section, we examine the fabrication processes and the SERS performance of gold nanodomes, where the signal is excited and collected in free space, and waveguide-based nanoplasmonic slot waveguide. 3.1.1. Scalability of the Fabrication Processes 2.3. Acquisition of SERS Spectra of a Peptide 2.3. Acquisition of SERS Spectra of a Peptide Both SERS substrates were functionalized with a self-assembled monolayer of the in-house made peptide NH2-CALNNFCNSFCNGGGGVRGNFSF-COOH, where each letter represents a natural amino acid and FCN represents a non-natural amino acid cyano-phenylalanine. A similar peptide has been previously used to demonstrate the detection of the protease activity on a gold nanodome platform such as in [15], where the fabrication and the labeling are described in detail. Brieﬂy, the in-house synthesized peptide was ﬁrst dissolved in dimethylformamide (Sigma-Aldrich, Overijse, Belgium) and then diluted to the concentration of 100 µM/ml in 10% acetonitrile/water solution. The SERS substrates were cleaned with acetone, isopropyl alcohol and DI water, and then were dried with N2 gun. After the oxygen plasma treatment, the samples were immersed overnight in the peptide solution, and then rinsed with deionized water to remove any peptides that did not covalently bind to the gold. The sample was placed in a Petri dish ﬁlled with 3 ml of 50 mM ammonium bicarbonate buﬀer (pH 7.8; purchased from Sigma-Aldrich, Overijse, Belgium). The SERS spectra of the peptide were acquired with the same confocal microscope that was used for the detection of NTP spectra, as described in the previous paragraph. For gold nanodomes, a 1 mW laser power before the microscope objective and a 40×/0.5 Zeiss water immersion objective (Carl Zeiss AG, Oberkochen, Germany) were used to acquire spectra on a spatially distributed map of 7 × 7 pixels in a 40 × 40 µm area with an integration time of 3 s on each point to avoid signal degradation upon laser illumination. Each trace represents a median spectrum of one map. For the nanoplasmonic slot waveguides, we used 1 mW laser power before the microscope objective and a Zeiss ×/1.0 W-Plan Apochromat ∞/0 objective (Carl Zeiss AG, Oberkochen, Germany) to acquire 10 consecutive spectra with an integration time of 10 s each. 2.2. Acquisition of SERS Spectra of P-Nitrothiophenol Simultaneously, maximum light scattering along the waveguide was observed from a camera imaging the top surface of the chip. 5 of 14 Nanomaterials 2019, 9, 1401 Figure 3. Schematic of an optical setup used to measure the surface-enhanced Raman spectra. Gold nanodomes were measured in a conventional free-space conﬁguration, whereas in the case of the nanoplasmonic slot waveguides, the microscope objective was used to couple the light to the waveguide and then to collect the SERS signal in the back reﬂection. Figure 3. Schematic of an optical setup used to measure the surface-enhanced Raman spectra. Gold nanodomes were measured in a conventional free-space conﬁguration, whereas in the case of the nanoplasmonic slot waveguides, the microscope objective was used to couple the light to the waveguide and then to collect the SERS signal in the back reﬂection. Figure 3. Schematic of an optical setup used to measure the surface-enhanced Raman spectra. Gold nanodomes were measured in a conventional free-space conﬁguration, whereas in the case of the nanoplasmonic slot waveguides, the microscope objective was used to couple the light to the waveguide and then to collect the SERS signal in the back reﬂection. 3. Results and Discussion 3.1. Comparison of Free-Space Excited Gold Nanodomes and Waveguide-Based Nanoplasmonic Slot Waveguide 3.1.1. Scalability of the Fabrication Processes The SEM images of the gold nanodomes are shown in Figure 4. The fabrication of gold nanodomes is simple and scalable. Many SERS substrates are fabricated using electron beam lithography; however, this resource-intensive and time-consuming method is primarily used for prototyping and is not often used in industrial scale fabrication. On the other hand, the fabrication process based on nanosphere 6 of 14 Nanomaterials 2019, 9, 1401 lithography is easily implemented in a mass scale production, making gold nanodomes interesting for industrial applications. Figure 4. Scanning electron microscope images of gold nanodomes. (a) Tilted view. (b) Top-down view. (c) Cross-section of a nanodome-patterned chip with a 12 nm wide gap between nanodomes. Figure 4. Scanning electron microscope images of gold nanodomes. (a) Tilted view. (b) Top-down view. (c) Cross-section of a nanodome-patterned chip with a 12 nm wide gap between nanodomes. Figure 5 shows the schematic and the SEM images of the nanoplasmonic slot waveguide. The nanoplasmonic slot waveguide provides high SERS enhancement that outperforms previously developed waveguide-based SERS platforms, such as integrated bowties [35,36] and integrated nanotriangles [37]. In addition to high SERS enhancement, the nanoplasmonic slot waveguides are fabricated using a combination of atomic layer deposition and deep UV photolithography, enabling mass scale manufacturing. Figure 5. Nanoplasmonic slot waveguide. (a) Schematic showing that the input and Stokes powers are guided by the waveguide. (b) Scanning electron microscope image of the gold-covered slot in top view. (c) Cross-section of a nanoplasmonic slot waveguide with a gap of 15 nm. Figure 5. Nanoplasmonic slot waveguide. (a) Schematic showing that the input and Stokes powers are guided by the waveguide. (b) Scanning electron microscope image of the gold-covered slot in top view. (c) Cross-section of a nanoplasmonic slot waveguide with a gap of 15 nm. Both gold nanodomes and nanoplasmonic slot waveguides are, therefore, fabricated using widely available, mass-scalable fabrication methods that make them interesting for industrial applications. Both gold nanodomes and nanoplasmonic slot waveguides are, therefore, fabricated using widely available, mass-scalable fabrication methods that make them interesting for industrial applications. 3.1.2. SERS Performance Comparison To compare the SERS performance of gold nanodomes and nanoplasmonic slot waveguides, we used a monolayer of p-nitrothiophenol (NTP). NTP shows prominent SERS peaks at the Raman shifts of 1110, 1339 and 1573 cm−1 that correspond to C–S stretching, symmetric nitro stretching and phenyl ring modes of the nitrothiophenol molecule [45]. We used the integrated counts of the 1339 cm−1 NTP mode as a metric of the SERS signal and the SERS background. We additionally assessed the coeﬃcient of variation on the SERS signal across a single chip on the gold nanodomes to be 6–7%, as previously reported in [15]. For the metal slot waveguides, this coeﬃcient cannot be deﬁned since we are exciting the SERS response through a single access waveguide. We acquired the SERS spectra of the gold nanodomes and nanoplasmonic slot waveguides using diﬀerent excitation laser powers and integration times. These parameters were optimized so that no 7 of 14 Nanomaterials 2019, 9, 1401 photoinduced reduction of NTP to dimercaptoazobenzene was observed, since these chemical changes would aﬀect the SERS signal strength of the 1339 cm−1 mode of the NTP [24]. On Figure 6, we show the averaged SERS spectra of the NTP monolayer acquired on gold nanodomes and on the nanoplasmonic slot waveguide. The ﬁrst step to consistent comparison of the SERS signals was to normalize the SERS signal strength on the integration time. Since the signal increases linearly with the integration time, we simply normalized the SERS signal strength to the integration time of 1 s. To include the eﬀect of the diﬀerent excitation laser powers, we calculated the ratio of total collected Stokes power PS over input power Pin as: P η = PS Pin , (1) η = PS Pin , (1) where Pin is the laser power that is used to excite the SERS response of our analyte. In the case of nanodomes, all the free-space laser power is used to excite the SERS response of our analyte, so Pin is the laser power coming out of the microscope objective, whereas in the waveguide-based SERS platforms, we deﬁne Pin as the laser power guided in the waveguide to the plasmonic structure, as shown in Figure 5a. Stokes power PS is the power of the SERS scattered photons, which in our case was at the 1339 cm−1 peak. 3.1.2. SERS Performance Comparison We see that the free-space excited gold nanodomes oﬀer very high SERS enhancements; however, their high background contribution limits their SERS performance. On the other hand, the nanoplasmonic slot waveguides provide lower SERS enhancements, yet their SERS background is reduced compared to that of the nanodomes. Figure 7. Comparison of SERS background power PBG (x-axis) and SERS Stokes power PS (y-axis) of diﬀerent SERS platforms. Both parameters are normalized on the input power and the integration time. FS indicates free-space and WG is the waveguide-based excitation and collection of the SERS signal. Figure 7. Comparison of SERS background power PBG (x-axis) and SERS Stokes power PS (y-axis) of diﬀerent SERS platforms. Both parameters are normalized on the input power and the integration time. FS indicates free-space and WG is the waveguide-based excitation and collection of the SERS signal. SERS background is still not suﬃciently understood, and in the last few years, several new models were proposed to describe its origins [46–49]. Exact identiﬁcation of the origin of the SERS background in gold nanodomes and nanoplasmonic slot waveguides is, therefore, a diﬃcult task that is beyond the scope of this paper. We can, however, try to narrow down the possible origins of the SERS background. The SERS background was experimentally linked to the strength of the localized surface plasmons, the identity of the adsorbate and adsorbate coverage [46]. In our case, we use the same adsorbate (NTP) under identical labeling conditions to characterize both the nanodomes and the nanoplasmonic slot waveguides. Furthermore, we always use newly fabricated SERS substrates in order to avoid any contaminants from previous labelings. We thus argue that the diﬀerences in the SERS background probably do not arise from the adsorbate itself or the contamination of our samples. Conversely, the eﬀects of plasmons on the SERS background are harder to evaluate. We fabricated the structures using the same gold deposition technique, indicating that the diﬀerences in the SERS background did not originate in the diﬀerence of the gold deposition. SERS enhancement was then achieved via localized surface-plasmon resonance in gold nanodomes [15] and via propagating the surface plasmon polariton in the nanoplasmonic slot waveguide [38], which might at least in part cause the diﬀerences in the SERS background. 3.1.2. SERS Performance Comparison To convert the measured CCD counts to PS, we use the formula: PS = Fphh(ν0 −νS)T−1 m . (2) (2) Figure 6. Averaged SERS spectra of the NTP monolayer acquired on gold nanodomes and on the nanoplasmonic slot waveguide. The spectrum on the gold nanodomes was obtained using a laser power of 300 µW and an integration time of 0.13 s. The spectrum on the nanoplasmonic slot waveguide was obtained using a laser power of 350 µW and an integration time of 10 s. The SERS spectrum on the nanodomes was divided by a factor of 40 to allow for better visualization. We subtracted the dark counts, but not the SERS background of the spectra. The spectra are oﬀset on the y-axis for clarity, and the dashed line represents the zero line of each spectrum. Figure 6. Averaged SERS spectra of the NTP monolayer acquired on gold nanodomes and on the nanoplasmonic slot waveguide. The spectrum on the gold nanodomes was obtained using a laser power of 300 µW and an integration time of 0.13 s. The spectrum on the nanoplasmonic slot waveguide was obtained using a laser power of 350 µW and an integration time of 10 s. The SERS spectrum on the nanodomes was divided by a factor of 40 to allow for better visualization. We subtracted the dark counts, but not the SERS background of the spectra. The spectra are oﬀset on the y-axis for clarity, and the dashed line represents the zero line of each spectrum. For the 1339 cm−1 NTP peak excited at 785 nm, the Stokes frequency (ν0 −νS) equals 342 THz and the transmission of the microscope Tm is 0.61 at the 870 nm Stokes shifted wavelength. For the speciﬁc case of our spectrometer with settings as described in Methods and Materials, the photon ﬂux Fph equals 5.9 times the number of counts in the Raman spectrum. The same calculation was also applied to calculate the SERS background signal. In Figure 7, the performance of the gold nanodomes and nanoplasmonic slot waveguides is compared in terms of the SERS signal strength and the SERS background. Additionally, we included the SERS performances of integrated bowties [35] and integrated nanotriangles [37] in the graph to highlight the progress that has been achieved in the 8 of 14 Nanomaterials 2019, 9, 1401 last few years in the ﬁeld of waveguide-based SERS platforms. 3.1.2. SERS Performance Comparison Additional diﬀerences in the SERS background might also have arisen from the two diﬀerent ways of exciting the SERS response, that is via free-space and waveguide-based excitation for the gold nanodomes and nanoplasmonic slot waveguides, respectively. In the case of the nanoplasmonic slot waveguide, the silicon nitride waveguides might have provided some additional contribution to the SERS background. To compare the diﬀerent SERS platforms, we considered both the SERS enhancement and SERS background in one ﬁgure of merit. We propose signal-to-background ratio (SBR) and signal-to-noise ratio (SNR) as relevant ﬁgures for comparison of the SERS performance of diﬀerent platforms. In the case of strong SERS signals compared to the background, the accuracy of the SERS signal analysis was limited by the imperfect background subtraction, since the background needed to be estimated from Nanomaterials 2019, 9, 1401 9 of 14 algorithmic extrapolation or from separate measurements [50]. In this case, we could use SBR as metric to evaluate the SERS performance: P SBR = PS PBG = PS Pin PBG Pin . (3) (3) The SBR of diﬀerent SERS platforms are shown in Table 1. We see that the nanoplasmonic slot waveguide oﬀers a signiﬁcant improvement compared to other waveguide-based SERS platforms. It provided more than 10-times higher SBR than integrated bowties and 60% higher SBR than integrated nanotriangles, respectively. The nanoplasmonic slot waveguide, therefore, approached the SBR of the free-space excited gold nanodomes, providing only 3-times lower SBR than the gold nanodomes. Table 1. The signal-to-background ratio (SBR) and signal-to-noise ratio (SNR) values at Pin = 1 mW and t = 1 s for diﬀerent SERS platforms evaluated based on the 1339 cm−1 mode of a NTP monolayer. If we use another input power or integration time, the absolute value of the SNR will change, but the relative SNR of diﬀerent SERS platforms will remain the same. FS indicates free-space and WG is the waveguide-based excitation and collection of the SERS signal. For the sake of this table, only the shot noise contribution from the background is taken into account (as it will be more relevant when looking for weaker peaks than the 1339 cm−1 mode). The SNR is then deﬁned as: 3.2. SERS Detection of a Peptide on a Waveguide-Based Platform A waveguide-based SERS platform oﬀers several advantages over conventional free-space SERS substrates for biological applications. The waveguide-based SERS platform allows parallel measurements of a large number of SERS analytes, enabling high-throughput assays that are of particular interest for pharmacological drug discovery. Furthermore, the eﬃcient SERS enhancement mechanisms allow for the measurement to be performed on low sampling volumes, additionally minimizing the cost of such assays. To demonstrate the detection of a biomolecule on a waveguide-based SERS platform for the ﬁrst time, we chose the peptide NH2-CALNNFCNSFCNGGGGVRGNFSF-COOH. Here each letter represents a natural amino acid and FCN represents a non-natural amino acid cyano-phenylalanine. A similar peptide containing only natural amino acids has been previously used to demonstrate the detection of protease activity on the gold nanodome platform [15]. Proteases are enzymes that catalyze the hydrolysis of peptide bonds and that therefore play important roles in various human diseases [51]. A real-time, multiplexed method for detection of protease activity is, therefore, important for the development of new drugs that, for instance, could inhibit disease-associated proteases. In the peptide NH2-CALNNFCNSFCNGGGGVRGNFSF-COOH, the SERS signal originates from the aromatic amino acids phenylalanine and cyano-phenylalanine. We observe the SERS peak of phenylalanine at 1003 cm−1 assigned to the trigonal ring breathing of the benzene ring [52], and the peak of cyano-phenylalanine at 1180 cm−1. To additionally increase the Raman cross-section of the peptide, we incorporated doubled aromatic amino acids in the peptide, eﬀectively doubling the Raman cross-section independently of the SERS platform that is used to detect the peptide. We ﬁrst detected the SERS spectra of the peptide on the gold nanodomes. Next, we obtained the SERS spectra of the peptide on the nanoplasmonic slot waveguide, which is, to the best of our knowledge, the ﬁrst-time detection of a biomolecule on a waveguide-based SERS platform. Both spectra are shown in Figure 8. Figure 8. Background-subtracted SERS spectra of the peptide NH2-CALNNFCNSFCNGGGGVR-GNFSF-COOH acquired on gold nanodomes and on the nanoplasmonic slot waveguide. The spectrum on the gold nanodomes was obtained using a laser power of 1 mW and an integration time of 1 s. The spectrum on the nanoplasmonic slot waveguide was obtained using a laser power of 1 mW and an integration time of 30 s, and the SERS intensity was divided by a factor of 10 to allow for better visualization. 3.1.2. SERS Performance Comparison SERS Platform SBR SNR (Pin = 1 mW, t = 1 s) Gold nanodomes (FS) 3.28 3 × 104 Integrated bowties (WG) 0.12 102 Integrated nanotriangles (WG) 0.72 103 Nanoplasmonic slot waveguide (WG) 1.16 2 × 103 On the other hand, when the photon number of the total signal within the integration time was below the critical level, the accuracy of the SERS signal analysis was not limited by the imperfect accuracy of the background subtraction, but rather by shot noise. SNR then provided a more relevant metric for the SERS performance of diﬀerent SERS platforms [50]. The noise N, due to shot noise, was proportional to the square root of the sum of the SERS peak and the SERS background signal. In the case of low SERS signals, the main contribution to the noise N (being the rms value of the signal ﬂuctuation) is, therefore, from the background signal: N = r PBGt hν . (4) (4) The SNR is then deﬁned as: SNR = PSt hν q PBGt hν = PS Pin q PBG Pin r Pint hν . (5) (5) We list the SNR of the diﬀerent SERS platforms in Table 1. Since the SNR also depends on the square root of the input power Pin and integration time t, we set the input power at 1 mW and the integration time to 1 s to be able to do a relative comparison of the SNR of the diﬀerent SERS platforms. If we use another input power, the absolute value of SNR will change, but the relative SNR of diﬀerent SERS platforms will remain the same. We see that the nanoplasmonic slot waveguides provide signiﬁcant SNR improvements compared to the other waveguide-based SERS platforms. Their SNR ratio is 20-times better than that for integrated bowties and 2-times better than in the case of integrated nanotriangles. Nevertheless, free-space excited gold nanodomes still outperform the nanoplasmonic slot waveguide with 15-times higher SNR. Besides the improved SBR, the nanoplasmonic slot waveguide also oﬀers non-resonant SERS enhancement, making the SERS enhancement independent of excited and scattered 10 of 14 Nanomaterials 2019, 9, 1401 wavelengths, which oﬀers an advantage when compared to the highly resonant gold nanodomes. Moreover, the waveguide-based SERS platform shows great potential for high-throughput SERS assays on low sampling volumes, and is especially relevant for the detection of biological molecules. 3.2. SERS Detection of a Peptide on a Waveguide-Based Platform The spectra are oﬀset on the y-axis for clarity, and the dashed line represents the zero line of each spectrum. Background-subtracted SERS spectra of the p Background-subtracted SERS Figure 8. Background subtracted SERS spectra of the peptide NH2-CALNNFCNSFCNGGGGVR-GNFSF-COOH acquired on gold nanodomes and on the nanoplasmonic slot waveguide. The spectrum on the gold nanodomes was obtained using a laser power of 1 mW and an integration time of 1 s. The spectrum on the nanoplasmonic slot waveguide was obtained using a laser power of 1 mW and an integration time of 30 s, and the SERS intensity was divided by a factor of 10 to allow for better visualization. The spectra are oﬀset on the y-axis for clarity, and the dashed line represents the zero line of each spectrum. 11 of 14 Nanomaterials 2019, 9, 1401 Previously developed waveguide-based SERS platforms, such as integrated bowties [35] and integrated nanotriangles [37], have been able to detect organic molecules with relatively high Raman cross-sections, such as NTP. However, their SERS enhancements were not high enough to detect biological molecules with low Raman cross-sections. We have used nanoplasmonic slot waveguide in combination with the peptide with doubled aromatic amino acids that intrinsically increase its Raman cross-section in order to detect a biological molecule for the ﬁrst time on a waveguide-based SERS platform, paving the way towards SERS detection and the monitoring of biologically relevant molecules on an integrated lab-on-a-chip platform. 4. Conclusions In this paper, we evaluated the SERS performance of gold nanodomes in which the signal is excited and collected in free space and in waveguide-based nanoplasmonic slot waveguides. Both SERS platforms are fabricated using simple and scalable fabrication methods, making them interesting candidates for industrial applications. We compared the SERS signal enhancement and the SERS background of the diﬀerent platforms using a monolayer of p-nitrothiophenol. We showed that the SERS enhancement of the gold nanodomes is higher than in the nanoplasmonic slot waveguide; however, their SERS performance is limited by the high SERS background. We demonstrated that the nanoplasmonic slot waveguide approaches the performance of the free-space excited gold nanodomes in terms of the signal-to-background ratio, showing great promise for future waveguide-based SERS applications. Combining the improved SERS signal-to-background ratio of the nanoplasmonic slot waveguide with the possibility of parallel measurements of a large number of SERS analytes on a lab-on-a-chip platform is especially interesting for biological applications. We, therefore, additionally demonstrated the ﬁrst-time detection of a peptide monolayer on a waveguide-based SERS platform, paving the way towards SERS detection and the monitoring of monolayers of biologically relevant molecules on an integrated lab-on-a-chip platform. Author Contributions: R.B. and K.G. directed the research. P.W. and A.R. fabricated the gold nanodomes and nanoplasmonic slot waveguides respectively, and acquired the NTP spectra. M.V.D. performed the ALD deposition of aluminum oxide under guidance of C.D. H.D. fabricated the peptide. N.T performed the peptide experiments on both SERS substrates. A.S. contributed with guidance on the use of Raman microscope. N.T. and A.R. performed the SERS performance analysis. N.T. wrote the original manuscript. All authors discussed the results and contributed to the manuscript. Funding: N.T. acknowledges the Research Foundation Flanders (FWO) for predoctoral fellowship. R.B. acknowledges Methusalem project ‘Smart photonic chips’. We also thank Bijzonder Onderzoeksfonds of Ghent University (BOF UGent) for support. Acknowledgments: The authors thank Dieter Cuypers for performing the gold deposition and Liesbet Van Landschoot for taking the SEM pictures. 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https://openalex.org/W1574330400	http://www.scielo.br/pdf/reben/v66n3/a16v66n3.pdf	Portuguese	null	[Learning in online education: analysis of concept].	null	2,013	cc-by	4,726	Viviane Rolim de HolandaI, Ana Karina Bezerra PinheiroII, Lorita Marlena Freitag PagliucaII I Universidade Federal do Ceará, Programa de Pós-Graduação em Enfermagem (Doutoranda). Fortaleza-CE, Brasil. II Universidade Federal do Ceará, Departamento de Enfermagem. Pesquisadora do Conselho Nacional de Desenvolvimento Científi co e Tecnológico. Fortaleza-CE, Brasil. PESQUISA RESUMO Objetivou-se clarifi car o conceito aprendizagem na educação online, expresso pela literatura da área da saúde. Trata-se de um estudo de análise de conceito, baseado nas etapas do Modelo Evolucionário, destacando-se os atributos, antecedentes, consequentes e termos substitutos. No contexto do ensino online, a aprendizagem é expressa por um processo dinâmico e contínuo de construção ativa do conhecimento e aquisição de habilidades, com separação física entre alunos e professores. Entre os eventos antecedentes abordados sobressaíram: interesse e motivação para aprender; dedicação e autogerenciamento do tempo; interação e ferramentas de comunicação. Quanto aos principais consequentes evidenciados mencionam-se: autonomia do aluno; estudo independente e ativo; e construção do próprio conhecimento. Como termos substitutos predominantes sugiram aprendizagem colaborativa e autoaprendizagem. A compreensão do conceito poderá contribuir para a sua aplicação nas práticas de ensino de enfermagem em ambientes virtuais. Descritores: Aprendizagem; Formação de Conceito; Educação a Distância; Instrução por Computa Descritores: Aprendizagem; Formação de Conceito; Educação a Distância; Instrução por Computador; Enfermagem. ABSTRACT This work aimed at clarifying the concept learning in online education, expressed by the literature in the area of health. It is a study of analysis of concept, based on the Evolutionary Model, being highlighted attributes, background, consequents and substitute terms. Learning in the context of online education is characterized by a dynamic and continuous process of active construction of knowledge and acquisition of skills, with physical separation between students and teachers. Among the background events discussed stood out: interest and motivation in learning; dedication and time self-management; and interaction and communication tools. The main evident consequents were: student’s autonomy; independent and active study; and construction of own knowledge. The prevailing substitute terms were collaborative learning and self-learning. The understanding of the concept can contribute to its implementation in nursing teaching activities in virtual environments. Key words: Learning; Concept Formation; Distance Education; Computer-Assisted Instruction; Nursing. Aprendizagem na educação online: análise de conceito Learning in online education: analysis of concept Aprendizaje en la educación online: análisis de concepto MÉTODO Trata-se de um estudo de análise de conceito baseado nas etapas do Modelo Evolucionário, no qual o destaque é a investigação indutiva e descritiva, com ênfase à natureza do conceito. Compreende o conceito como dinâmico e in- fluenciado pelo contexto, representando uma ideia abstrata expressa pelo grupo de atributos que o constitui, no intuito de clarificá-lo e reduzir problemas conceituais existentes(6). As Diretrizes Curriculares Nacionais dos cursos de gradu- ação da área da saúde recomendam a prática de estudo inde- pendente, com vistas a uma progressiva autonomia intelectu- al a fim de preparar o graduando para enfrentar os desafios das transformações da sociedade e do mercado de trabalho(3). Nesse sentido, acredita-se que as TICs geram estímulos e de- safios para a prática de ensino e aprendizagem. As etapas desse modelo são inter-relacionadas e incluem: identificar o conceito de interesse e expressões associadas; identificar e selecionar campo apropriado para coleta de da- dos; realizar a coleta de dados; analisar os dados distinguindo as características do conceito, seus antecedentes, consequen- tes e termos substitutos; identificar caso modelo do conceito; identificar hipóteses e implicações para outros estudos(6). De acordo com o Decreto 5.622/2005 que regulamenta a Lei de Diretrizes e Bases da educação nacional (LDB), a educação a distância é uma forma de ensino que possibilita a aprendizagem com a mediação de recursos didáticos sistema- ticamente organizados, apresentados em diferentes suportes de informação, utilizados isoladamente ou combinados, e vei- culados pelos diversos meios de comunicação(4). O conceito de interesse selecionado foi aprendizagem no contexto da educação online, considerando sua relevância para a prática de ensino de enfermagem. Em seguida, pro- cedeu-se à busca na Biblioteca Virtual de Saúde (BVS), Base de Dados em Enfermagem (BDENF) e na PubMed (National Library of Medicine and National Institutes of Health), no mês de outubro de 2011, seguindo-se como descritores: aprendizagem (Learning/ Aprendizaje) e educação a distância (Education, Distance/ Educación a Distancia); aprendizagem e instrução por computador (Computer-Assisted Instruction/ Instrucción por Computador). Contudo, a educação a distância não mais se caracteriza pela distância, porquanto a virtualidade permite encontros cada vez mais efetivos que favorecem o processo ensino/ aprendizagem. É, pois, oportuno adotar o termo educação online para o processo de ensino mediado pelas TICs em am- bientes digitais de aprendizagem. As tecnologias de informação propiciam um alto poder de interação entre os participantes rompendo com a ideia de es- paço e tempo. RESUMEN Este trabajo tuvo por objetivo aclarar el concepto aprendizaje en la educación online, expresado por la literatura en el área de salud. Se trata de un estudio de análisis de concepto, basado en el Modelo Evolucionario, destacándose atributos, antecedentes, consecuentes y términos substitutos. El aprendizaje, en el contexto de enseñanza online, se caracteriza por ser un proceso dinámico y continuo de construcción activa del conocimiento y adquisición de habilidades, con separación física entre alumnos y profesores. Entre los eventos antecedentes tratados se destacaron: interés y motivación en aprender; dedicación y administración del tiempo; e interacción y herramientas de comunicación. Los principales consecuentes demostrados fueron: autonomía del alumno; estudio independiente y activo; y construcción del propio conocimiento. Los términos substitutos predominantes fueron aprendizaje colaborativo y autoaprendizaje. La comprensión del concepto podrá contribuir para su aplicación en las prácticas de enseñanza de enfermería en los ambientes virtuales. p p alabras clave: Aprendizaje; Formación de Concepto; Educación a Distancia; Instrucción por Computador; Enfermería. Palabras clave: Aprendizaje; Formación de Concepto; Educación a Distancia; Instrucción por Com Viviane Rolim de Holanda E-mail: vivi_rolim@yahoo.com.br AUTOR CORRESPONDENTE Rev Bras Enferm, Brasília 2013 mai-jun; 66(3): 406-11. 406 Aprendizagem na educação online: análise de conceito INTRODUÇÃO conceito aprendizagem mediada na educação online, enfati- zando os elementos contextuais presentes em suas definições e aplicações na enfermagem, proporcionando ampla compre- ensão desse conceito. Dessa forma, pode-se contribuir para sua correta utilização. Os avanços das tecnologias de informação e comunicação (TICs) e a expansão da internet romperam as barreiras geográ- fico-temporais de acesso à educação. Com o surgimento da web no final dos anos 1990 possibilitou-se uma nova forma de aprendizagem baseada em computador, que se difundiu impulsionada pela disponibilidade de sistemas específicos - softwares para a área acadêmica - conhecidos como ambien- tes virtuais de aprendizagem(1-2). O estudo justifica-se pela necessidade de maior compreen- são conceitual diante das possíveis aplicações no ensino de enfermagem, na tentativa de responder às necessidades emer- gentes de formação seja na graduação, na pós-graduação ou educação permanente. Logo, objetivou-se clarificar o concei- to aprendizagem na educação online, expresso pela literatura da área de saúde. A mediação das TICs na aprendizagem tem propiciado a formação de ambientes educacionais apoiados em teorias so- cioconstrutivistas as quais resultaram em mudanças no pro- cesso de formação dos profissionais e, consequentemente, nas atitudes, percepções e usos dessas tecnologias nos pro- cessos de trabalho. Em acréscimo, a associação das TICs e a flexibilidade da educação a distância apontam um espaço importante para o processo de aprendizagem(1). 407 Rev Bras Enferm, Brasília 2013 mai-jun; 66(3): 406-11. MÉTODO Desta forma, nos ambientes online de apren- dizagem o que era distante pode se tornar perto. A dimensão do tempo e do espaço são instituídas consoantes as necessi- dades, os interesses e a vontade dos aprendizes, ampliando as possibilidades da educação(5). Os critérios de inclusão adotados foram: ser artigo com- pleto e estar disponível eletronicamente, estar publicado nos idiomas português, inglês ou espanhol no período de 2001 a 2011, ter relação com o conceito em foco. Excluíram-se arti- gos repetidos, estudos que não tinham relação com o concei- to em análise ou que não possuíam elementos suficientes para a análise do conceito. Desse modo, há interesse em entender o conceito de aprendizagem no cenário da educação online que facilite compreender os fatores associados à apropriação e uso dos conhecimentos e habilidades desenvolvidos nesses ambien- tes, bem como os elementos que promovem e limitam o uso dessas tecnologias na educação. Neste contexto, o esclareci- mento do significado, do uso e da aplicação de um conceito é um passo importante no processo de desenvolvimento de uma disciplina. Com um conceito definido pode-se caracteri- zar fenômenos de interesse e avaliar suas forças e seus limites além de mantê-lo útil e aplicável a um determinado contexto. Procedeu-se à leitura do título e do resumo para verificar se atendiam aos critérios de inclusão estabelecidos. Em seguida, fez-se a leitura crítica dos artigos selecionados, buscando a identificação dos elementos constituintes do conceito: atribu- tos, antecedentes e consequentes. A análise dos atributos essenciais que expressam a natureza do conceito foi guiada pelas questões: Como o autor define o conceito? Quais as características/atributos apontados por ele? Que ideias o autor discute sobre o conceito aprendizagem? Em face do exposto, ressalta-se a relevância de analisar o Para a identificação dos eventos antecedentes, ou seja, 407 Rev Bras Enferm, Brasília 2013 mai-jun; 66(3): 406-11. 407 Holanda VR, Pinheiro AKB, Pagliuca LMF. Holanda VR, Pinheiro AKB, Pagliuca LMF. situações que precedem o conceito de interesse usou-se a se- guinte pergunta norteadora: Que eventos contribuem para a iminência do conceito aprendizagem nos ambientes virtuais? E por fim, para evidenciar os eventos consequentes resultan- tes da aprendizagem, utilizou-se a questão: O que aconteceu como resultado da aprendizagem? Inicialmente encontrou-se 153 artigos, mas com a aplica- ção dos critérios de inclusão foram eliminados 121. Desse modo, a amostra do estudo compôs-se de 32 artigos. Desses, a maioria datava do período de 2007 a 2011. RESULTADOS Em seguida, apresenta-se o caso modelo com a presença dos atributos críticos para o conceito aprendizagem em am- biente de ensino online. Quadro 1 – Síntese da análise do conceito aprendizagem no contexto da educação online, segundo Método Evolucionário, 2012. Análise do conceito aprendizagem no contexto do ensino online Termos substitutos Aprendizagem colaborativa Autoaprendizagem Aprendizagem a distância Aprendizagem online Aprendizagem em rede Atributos essenciais Construção ativa do conhecimento Processo dinâmico e contínuo Aquisição de habilidades motoras, cognitivas e intelectuais Experiência assíncrona Mudança de comportamento Desenvolvimento de sentido crítico (gerar reflexões sobre o assunto) Desenvolvimento de significados e conhecimento compartilhado Estímulo às capacidades investigativas do aluno Ressignificação de saberes Capacidade de aprender a aprender Antecedentes Interação e estímulo Interesse e motivação para aprender Dedicação, disciplina e gerenciamento de tempo (autogestão) Ferramentas de comunicação (assíncronas e síncronas) Materiais didáticos organizados e informações adequadas (método ativo) Mudança no papel do professor e do aluno Colaboração (trabalho colaborativo) Mediação Ambiente de afetividade e dialógico Consequentes Estudo mais independente e ativo Protagonismo e autonomia do aluno Construção do próprio conhecimento Atualização e produção de conhecimentos Aprendizagem significativa e contextualizada Liberdade para construir seu próprio percurso e ritmo de estudo Formação de sujeitos críticos Desenvolvimento de habilidades Conhecimento cooperativo Quadro 1 – Síntese da análise do conceito aprendizagem no contexto da educação online, segundo Método Evolucionário, 2012. CASO MODELO Graduando em enfermagem matricula-se no ambiente vir- tual da universidade para curso online de uma determinada disciplina. No decorrer das aulas, passa a construir ativamente o seu conhecimento utilizando-se das ferramentas assíncro- nas, da interação e colaboração entre professor e outros alu- nos do curso. O estudante é incentivado a organizar melhor o seu tempo e priorizar os conteúdos a serem estudados. Ao final do semestre, o aluno adquire novas habilidades e refere que se sentiu motivado para o aprender. MÉTODO Quanto ao idio- ma, analisaram-se 23 artigos em português, cinco em inglês e quatro em espanhol. Após leitura exaustiva do material, os dados foram revisa- dos e organizados de maneira indutiva e temática. A apre- sentação dos resultados está organizada em quadro e tabela com frequência absoluta. Para esclarecer e revelar o estado atual do conhecimento relacionado ao conceito em interesse, desenvolveu-se a análise dos dados. O quadro 1 apresenta a síntese da análise do conceito, constam os atributos essenciais e os termos substitutos que estão frequentemente associados ao conceito aprendizagem, nos mais diversos contextos da educação online. E apontam- -se os eventos antecedentes prevalentes para expressar a ocor- rência do conceito em análise e os eventos consequentes que ocorrem como resultado do uso do conceito. DISCUSSÃO Após leitura crítica do material analisado, percebeu-se que aprendizagem é um conceito presente em diversas disciplinas acadêmicas da área da saúde com semelhanças nas definições assumidas no uso do conceito na perspectiva do ensino online. É É importante inferir que os termos substitutos são meios alternativos de expressar o mesmo conceito, diferentemente da palavra ou expressão selecionada pelo pesquisador para focar o estudo(6). Eles devem ser identificados, em alguma di- mensão, durante a coleta de dados mediante permuta da ter- minologia, e compartilham dos mesmos atributos do conceito em análise. Antecedentes Os atributos essenciais expressam a natureza do conceito e permitiram perceber como os autores definem o conceito, as características a ele atribuídas e as ideias que discutiam sobre a aprendizagem e a aplicação e uso no contexto da educação online. A análise dos artigos resultou na definição do conceito de aprendizagem como processo dinâmico e contínuo de cons- trução ativa do conhecimento e aquisição de habilidades. Evi- dencia-se que os atributos – processo dinâmico e construção ativa do conhecimento - foram consistentes no ambiente de ensino online. Consequentes A utilização de ferramentas assíncronas aumenta o nível de motivação dos participantes e a realização das atividades pro- postas. Assim, as informações publicadas ficam disponíveis para o acesso do grupo, socializando as discussões e gerando reflexões sobre o assunto nas comunidades virtuais. Estas são concebidas com o propósito de favorecer a interação dialógi- ca entre seus participantes no ciberespaço onde todos devem se expressar(9,17). No contexto da educação online, a aprendizagem é realiza- da por meio da separação física entre alunos e professores. O aprendizado e a comunicação acontecem via recursos tecno- lógicos que ultrapassam a exposição oral e permitem ao aluno navegar de forma não-linear, ou seja, de acordo com sua neces- sidade de estudo(8). Isto porque as ferramentas assíncronas não exigem a presença simultânea dos participantes e os acessos podem ser feitos nos horários disponíveis de cada um(9). Além disso, essas ferramentas de comunicação promovem a interação e a colaboração porquanto facilitam a intercone- xão de uma série de pessoas com a finalidade de propiciar o fluxo de informação entre elas, e também a realização de trabalhos conjuntos. Estas ferramentas de comunidade virtual em rede incluem: correio eletrônico, chat, mensagens instan- tâneas, blogs, wikis e fóruns(13). Nesta concepção, os níveis de participação e interação hu- mana são elementos críticos para o sucesso das experiências de aprendizagem, pois contribuem significativamente para a efetividade das ações educacionais apoiados em modelos mentais reflexíveis da realidade, capazes de evoluir em suces- sivas e crescentes formalizações(1,10). Percebe-se que a interação social propiciada pelas ferra- mentas síncronas e assíncronas modela o tipo de relações sur- gidas nesses contextos de ensino. Os níveis de participação e interação humana são elementos críticos no sucesso das experiências de aprendizagem, pois possibilitam a presença social que contribui significativamente para a efetividade das ações educacionais(1). Como experiência assíncrona, a aprendizagem permite o acesso independente de tempo ou lugar, atendendo às neces- sidades individuais dos estudantes e possibilitando ao apren- diz a decisão sobre os assuntos a serem explorados, escolha de métodos e estratégias. Desse modo, resulta em uma expe- riência genuína, com integração de novos conhecimentos e desenvolvimento de habilidades mediante conexão de alunos com professores e outros participantes(11,2). Outro evento percebido foi a mudança nos papéis desem- penhados pelos professores e pelos alunos, além de modi- ficações na elaboração dos materiais didáticos direcionados aos ambientes digitais de ensino. Consequentes Também, como se destaca, a aprendizagem nesse ambien- te é uma experiência assíncrona que promove mudança de comportamento, reflexões críticas sobre o assunto e estimula a capacidade investigativa do aluno. 8 Rev Bras Enferm, Brasília 2013 mai-jun; 66(3): 406-11. 408 Aprendizagem na educação online: análise de conceito interação, a motivação e a autogestão do tempo como supor- tes fundamentais para o alcance de seus propósitos(1). Para fornecer uma demonstração prática do conceito e delinear as suas características essenciais buscou-se construir um caso modelo. O caso ideal deve ser universal para ilustrar claramente o conceito em interesse e possibilitar a sua apli- cação efetiva(6). No processo de aprendizagem deve-se considerar a moti- vação para aprender; o aluno precisa ter interesse no que está fazendo; é fundamental ter significado pessoal. Vários autores apontaram a motivação de aprender algo e a autogestão do tem- po como condições propícias ao desenvolvimento da apren- dizagem(14). Determinado estudo revelou que a aprendizagem depende mais do esforço do próprio indivíduo (autogerencia- mento da aprendizagem) do que dos recursos instrucionais(15). No caso modelo apresentado verificam-se os atributos es- senciais. Por ser um fenômeno individual que promove mu- dança de comportamento e desenvolvimento de significados, tal percepção é identificada no aluno ao ter maior autonomia na construção do conhecimento mediante um trabalho coo- perativo, organização do seu próprio ritmo de estudo e referir motivação no processo de aprendizagem. Entretanto, como se ressalta, na educação online é mister po- tencializar a vontade dos aprendizes. O docente deve cativar para construir um ambiente de afetividade e estimular o diálogo por meio de ferramentas que facilitarão o aprendizado(5). Neste contexto, a aprendizagem requer atividades didáticas enrique- cedoras que incluam informação e comunicação por meio de ferramentas e recursos que auxiliem no trabalho colaborativo(16). Como observado, os avanços nas tecnologias de informa- ção e comunicação e a expansão da internet romperam as barreiras geográfico-temporais de acesso à educação. Os au- tores discutiram a aprendizagem como um processo contínuo uma vez que o conhecimento não pode ser transmitido como algo acabado, por ser fruto de uma relação/interação do su- jeito com o meio. Como processo dinâmico, a aprendizagem deixa de ter caráter linear e passa a ser percebida dentro de um processo de simultaneidade expandido pelas práticas de cooperação entre os sujeitos, construídas continuamente por meio da motivação e interação(7). 409 Rev Bras Enferm, Brasília 2013 mai-jun; 66(3): 406-11. CONSIDERAÇÕES FINAIS E IMPLICAÇÕES PARA A ENFERMAGEM por gerar novas propostas de pesquisa e possibilitar o enten- dimento ampliado do conceito de interesse. Com base nos artigos analisados, os consequentes apontam que a aprendiza- gem no contexto da educação online é construída buscando a informação de forma ativa, no ritmo próprio do aluno, des- pertando maior motivação ao estudante. A análise do conceito pelo Método Evolucionário clarificou a compreensão do fenômeno, possibilitando o uso adequado da terminologia aprendizagem no contexto online, expressa pela literatura da área de saúde. A partir dessa análise, segundo evidenciado, essas experiên- cias estimulam a autonomia do aluno uma vez que a separação física induz os alunos a desenvolverem comportamento de ge- renciamento do seu aprendizado, portanto precisam planejar os períodos e o tempo de estudo e estabelecer a prioridade dos con- teúdos a serem aprofundados e revisados por eles próprios(21). Foram associados, como atributos essenciais ao conceito aprendizagem na educação online, o processo dinâmico e a construção ativa do conhecimento. Os eventos antecedentes identificados foram: interação, estímulo, motivação, dedica- ção e autogestão do tempo. Como consequentes destacaram- -se: estudo independente, autonomia do aluno e construção própria do conhecimento. Prevaleceram como termos subs- titutos a aprendizagem colaborativa e a autoaprendizagem. O aluno é considerado como centro do processo de apren- dizagem e tem o poder de tomada de decisões, gerenciamen- to de sua própria aprendizagem. Para tal, precisa interagir com outros alunos e com o professor(22). Como apontam os artigos analisados, em um mundo co- nectado em redes a educação online está se expandindo, e, desse modo, afeta o processo de ensino/aprendizagem, prin- cipalmente no ensino superior. O ensino de enfermagem acompanha essa tendência e possibilita ao aluno buscar co- nhecimentos e habilidades por meios mediados e na troca com o outro em ambientes digitais de aprendizagem. Conforme alguns estudos evidenciam os estudantes assu- mem maior responsabilidade por sua aprendizagem nos cur- sos via internet, pensam criticamente e participam bem mais que na modalidade presencial, e são constantemente incenti- vados a aprender a aprender. A ambiência dos encontros de ensino em rede favorece o acesso aos objetos de informações de maneira personalizada propiciando aos estudantes defini- rem a dinâmica de estudo, a aquisição de habilidades e a atu- alização do conhecimento(13,16). Dessa forma, levante–se a hipótese que a utilização de ambientes online pode responder as necessidades de ensino garantindo uma aprendizagem eficaz para os alunos. Consequentes Por não haver contato direto entre aluno e professor, se requer os conteúdos tratados de modo especial, ou seja, terem a estrutura ou organização que os torne passíveis de aprendizado a distância(16,18). Aprender, neste sentido, é estimular as capacidades investi- gadoras dos alunos, ajudando-os a desenvolver competências e habilidades mentais, internalizar conceitos para lidar com a realidade, resolver problemas, tomar decisões e formular es- tratégias de ação(12-13). Como consta em estudos, os materiais de ensino devem conter atividades sistematizadas que desafiem os alunos, fa- voreçam o diálogo e promovam a busca intuitiva. As ações do professor/tutor devem motivar o aluno a fortalecer sua autoa- prendizagem e a colaborar com os outros alunos, de forma a ampliar o compromisso pedagógico(8,19). Segundo se observa, antecedentes são aqueles eventos ou incidentes que devem ocorrer previamente à ocorrência do conceito e que favorecem o entendimento do contexto no qual ele está sendo usado. Nesta óptica, o professor passa a ser agente e mediador fun- damental da construção do estudante, facilitador dos novos mé- todos de aprendizagem, encorajando os aprendizes a fazerem escolhas sensatas e promover mudança de comportamentos(20). Consoante os artigos analisados revelaram a aprendiza- gem na educação online baseia-se no desenvolvimento de significados compartilhados entre os participantes, tendo a Em relação aos consequentes, a sua identificação é útil 409 Rev Bras Enferm, Brasília 2013 mai-jun; 66(3): 406-11. 409 Holanda VR, Pinheiro AKB, Pagliuca LMF. CONSIDERAÇÕES FINAIS E IMPLICAÇÕES PARA A ENFERMAGEM É pos- sível a educação tradicional combinar a experiência desses ambientes focando a participação do aluno e construindo um modelo de aprendizagem colaborativo, ativo e transformador. Dessa forma, a autonomia concedida ao aluno lhe atribui um papel mais ativo na construção de um conhecimento compartilhado, significativo e contextualizado. A participa- ção em espaços virtuais coletivos significa assumir a responsa- bilidade na construção do próprio conhecimento, permitindo a liberdade para organizar seus estudos e mobilizando com isso a vontade de aprender(1,21). De tal modo, as tecnologias de informação e comunicação auxiliam como ferramentas de ensino de maneira atraente e a aquisição do conhecimento dependerá menos do professor. Portanto, os resultados desse estudo fornecem implicações para a educação em enfermagem, a fim de fortalecer seu pró- prio saber por meio da compreensão do desenvolvimento de conceitos e sua correta utilização. Contudo, recomendam-se o delineamento de outras pesquisas para aprofundar a apli- cação efetiva do conceito aprendizagem relacionada aos am- bientes virtuais que empregam as tecnologias da informação e da comunicação com vistas a refinar elaborações teóricas para seu uso e aplicação na enfermagem. No entanto, o ensino interativo requer do professor sensibi- lidade para promover modificações no pensamento do aluno à medida que os expõem a novas ideias, valorizando suas ex- periências e fortalecendo-os para se tornarem aprendizes crí- ticos para aquisição de conhecimentos novos e relevantes(2). Pode-se afirmar, portanto, que as propostas para práticas de ensino em ambientes online devem observar os atributos essenciais e os antecedentes aqui identificados com vistas a promover uma aprendizagem significativa e mais indepen- dente, e a autonomia do aluno, como sujeito crítico, para a construção e atualização do seu próprio conhecimento. No entanto, o ensino interativo requer do professor sensibi- lidade para promover modificações no pensamento do aluno à medida que os expõem a novas ideias, valorizando suas ex- periências e fortalecendo-os para se tornarem aprendizes crí- ticos para aquisição de conhecimentos novos e relevantes(2). Por fim, destaca-se como limite do estudo o fato de terem sidos avaliados artigos publicados eletronicamente, o que pode refletir em uma revisão incompleta do conceito em análise. Considera-se, então, importante suscitar indagações sobre o conceito de interesse para responder às necessidades emergentes das práticas de ensino da enfermagem no cenário das novas mídias e tecnologias na educação e os desdobra- mentos que essa experiência pode possibilitar aos alunos e aos professores. CONSIDERAÇÕES FINAIS E IMPLICAÇÕES PARA A ENFERMAGEM Pode-se afirmar, portanto, que as propostas para práticas de ensino em ambientes online devem observar os atributos essenciais e os antecedentes aqui identificados com vistas a promover uma aprendizagem significativa e mais indepen- dente, e a autonomia do aluno, como sujeito crítico, para a construção e atualização do seu próprio conhecimento. 3. Brasil. Conselho Nacional de Educação. Câmara de Edu- cação Superior. Parecer nº 1113, de 7 de agosto de 2001. Diretrizes Curriculares nacionais dos cursos de gradua- ção em enfermagem, medicina e nutrição [parecer na in- ternet]. 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https://openalex.org/W4206162091	https://www.meddocsonline.org/annals-of-anesthesia-and-pain-medicine/anaesthetic-management-of-hemiparesis-patient.pdf	English	null	Anaesthetic Management of Hemiparesis Patient with Severe Thoracolumbar Scoliosis for Orthopaedic Surgery by Taylor's Approach of Lumbar Subarachnoid Block in the era of COVID 19 Pandemic	Annals of anesthesia and pain medicine	2,021	cc-by	1,947	Anaesthetic Management of Hemiparesis Patient with Severe Thoracolumbar Scoliosis for Ortho paedic Surgery by Taylor ‘s Approach of Lumbar Subarachnoid Block in the era of COVID 19 Pandemic P Veena1; Mohankumar2; Geethakumari P2; Arun R3 1Assistant professor, Department of Anaesthesiology, SUT Academy of Medical Sciences, Thiruvananthapuram, kerala, India. 2Professor and Head of the Department of Anaesthesiology, SUT Academy of Medical Sciences, Thiruvananthapuram, kerala, India. 3Assistant Professor, Department of Anaesthesiology, SUT Academy of Medical Sciences, Thiruvananthapuram, kerala, India. 4Senior Resident, Department of Anaesthesiology, SUT Academy of Medical Sciences, Thiruvananthapuram, kerala, India. Corresponding Author(s): P Veena Assistant professor, Department of Anaesthesiology, SUT Academy of Medical Sciences, Thiruvananthapuram, kerala, India. *Corresponding Author(s): P Veena Assistant professor, Department of Anaesthesiology, SUT Academy of Medical Sciences, Thiruvananthapuram, kerala, India. Open Access \| Case Report Anaesthetic Management of Hemiparesis Patient with Severe Thoracolumbar Scoliosis for Ortho paedic Surgery by Taylor ‘s Approach of Lumbar Subarachnoid Block in the era of COVID 19 Pandemic MedDocs Publishers MedDocs Publishers Annals of Anesthesia and Pain Medicine Open Access \| Case Report Cite this article: Veena P, Mohankumar, Geethakumari P, Arun R. Anaesthetic Management of Hemiparesis Patient with Severe Thoracolumbar Scoliosis for Orthopaedic Surgery by Taylor ‘s Approach of Lumbar Subarachnoid Block in the era of COVID 19 Pandemic. Ann Anesth Pain Med. 2021; 4(1): 1021. Discussion We report a case of 80year old female patient anaestheti cally managed with Taylor’s approach of lumbar subarachnoid block for right Proximal Femur Nailing (PFN). Known case of diabetes, hypertension, History of cerebrovascular accident 1 year back with residual hemiparesis of right side on dual anti platelet drugs of which clopidogrel was stopped 5 days prior to surgery. No history of other major illness and surgery. On ex amination the patient had adequate mouth opening and airway examination revealed an anticipated difficult airway with par tially edentulous, Mallampatti class 3,short neck and with nor mal temperomandibular joint movement. Intravenous access as well as regional anaesthesia access difficultly was anticipated. Examination of spine revealed severe thoracolumbar scoliosis. Airentry was equal on both sides. Blood investigations after stopping clopidogrel were normal. Chest Xray revealed cardio megaly. Neurological assessment revealed right side residual hemiparesis. Central neuraxial block is controversial with dual antiplatets in these patients and poses an anesthetic challenge, in view of difficulties in palpating anatomical landmarks, per forming dura puncture, and difficulty in predicting the extent of block. We followed the updated universal gold standard ASRA (American society of Regional Anaesthesiology) guidelines re garding antiplatet medication. Geriatric patients undergoing major surgery have a signifi cantly higher incidence of morbidity and morality compared with younger age group because of their reduced cardio respi ratory reserve and concomitant diseases .Spinal anaesthesia is widely used in orthopaedic surgery. Scoliosis (lateral spinal curvature) and in majority, it is usually an idiopathic disorder. Secondary kyphoscoliosis occurs as a result of various neuro muscular, vertebral, or connective tissue disorders. The level of derangements in cardiac and pulmonary function of kyphosco liosis patient is related to the amount of Cobb’s angle in tho racolumbar X-ray. Cardiopulmonary function starts decreasing if this angle is larger than 40° and it becomes very significant with angle >100. In our patient, post cerebrovascular accident induced hemiplegia 1 year back was the cause of progressive thoracolumbar scoliosis, but he had no significant respiratory or cardiovascular instability. Spinal anesthesia in this type of patient is technically challenging to the anesthesiologist. Due to angulation and rotation of vertebral body, epidural space is deviated toward the convexity of angulation. One method of administering spinal or epidural block is by directing the needle toward the convexity of the curve with significant angulation of the needle. Abstract Background: Subarachnoid block in patients with scolio sis always present a unique challenge to the anesthesiolo gist owing to the deformity of spine. Difficulty in perform ing neuraxial anaesthesia may result in neural injury, spinal haematoma, post-dural puncture headache In addition, it may decrease procedure efficiency and increase patient discomfort and dissatisfaction. The most commonly prac ticed technique is the midline approach. This approach is technically difficult in the geriatric patients because of de generative changes in the spine. Calcification of supraspi nous and interspinous ligaments in the geriatric age group makes midline approach difficult. Paramedian approach and its modification Taylor’s approach is not routinely practiced and is definitely a game changer when midline approach of lumbar subarachnoid block has failed or is not possible due to anatomical variations like ankylosing spondylitis, thoraco lumbar kyphoscoliosis. Email: veenap743@gmail.com Received: May 24, 2021 Accepted: Jun 17, 2021 Published Online: Mar 08, 2021 Journal: Annals of Anesthesia and Pain Medicine Publisher: MedDocs Publishers LLC Online edition: http://meddocsonline.org/ Copyright: © Veena P (2021). This Article is distributed under the terms of Creative Commons Attribution 4.0 International License Received: May 24, 2021 Accepted: Jun 17, 2021 Published Online: Mar 08, 2021 Journal: Annals of Anesthesia and Pain Medicine Publisher: MedDocs Publishers LLC Online edition: http://meddocsonline.org/ Copyright: © Veena P (2021). This Article is distributed under the terms of Creative Commons Attribution 4.0 International License We report a case of unilateral hemiparesis patient with severe thoracolumbar scoliosis anaesthetically managed by Taylor’s approach of lumbar Subarachnoid block for Proxi mal Femur Nailing (PFN) of intertrochanteric fracture thus avoiding general anaesthesia and associated aerosol gen eration in the era of COVID19 pandemic. Keywords: Taylor’s approach; Lumbar subarachnoid block; Paramedian; Proximal femur nailing; thoracolumbar scoliosis; Hemiparesis. Cite this article: Veena P, Mohankumar, Geethakumari P, Arun R. Anaesthetic Management of Hemiparesis Patient with Severe Thoracolumbar Scoliosis for Orthopaedic Surgery by Taylor ‘s Approach of Lumbar Subarachnoid Block in the era of COVID 19 Pandemic. Ann Anesth Pain Med. 2021; 4(1): 1021. Cite this article: Veena P, Mohankumar, Geethakumari P, Arun R. Anaesthetic Management of Hemiparesis Patient with Severe Thoracolumbar Scoliosis for Orthopaedic Surgery by Taylor ‘s Approach of Lumbar Subarachnoid Block in the era of COVID 19 Pandemic. Ann Anesth Pain Med. 2021; 4(1): 1021. Conclusion Your hands will do only what the mind thinks of so be up dated in knowledge as well as in skill regarding how to tackle such challenging regional anaesthesia cases in prevailing CO VID19 pandemic condition. Taylor ‘s approach is the best modi fied paramedian approach in difficult spine access for lumbar subarachnoid block though only a few is aware of such an ap proach. After preparation of OT, checking anaesthesia machine with emergency resuscitation drugs, intravenous fluids, infusion pump, difficult regional anaesthesia equipments and equip ment backup for general anaesthesia patient was shifted to OT. Discussion Because of the distortion of the spinous processes in patients with scoliosis, the interspinous space is not felt. Modi fied paramedian approach “Taylor ‘s approach” is definitely a game changer in such challenging spine deformity cases. Abstract 1 MedDocs Publishers MedDocs Publishers Under strict asepsis, Inspite of adequate interspinous space, negotitating a 23G Quincke ‘s spinal needle was difficult and needle was hitting bone in midline as well as in paramedian ap proach. Hence considering the prevailing covid pandemic and to avoid general anaesthesia for the patient we reassured the patient and reverted the patient to sitting position for a modi fied paramedian approach “Taylor’s Approach” of subarach noid blockade. Taylor’s approach attempted under strict asepsis with 23G Quincke spinal needle. Posterior Superior Iliac Spine (PSIS) palpated, 1cm medial and 1cm caudal to PSIS needle inserted and directed towards cephalo-medial direction. After advancing needle bony obstruction was felt. Walking over the bone “Loss of Resistance” was elicited and stylet removed. Posi tion of needle tip in subarachnoid space confirmed by clear and free backflow of cerebrospinal fluid. 2.8ml of 0.5% heavy bu pivacaine with 30 microgram buprenorphine was given. Block adequacy was confirmed in supine position. vitals stable and intraoperatively one pint whole blood was transfused. Surgery completed uneventful. postop period was uneventful . Introduction Subarachnoid blockade is widely used due to its procedural simplicity, low cost and better physiological benefits and thus reduced complications than that of general anaesthesia. Sub arachnoid block can be safely administered via Taylor’s ap proach for anticipated as well as unanticipated difficult spine cases when general anaesthesia has to be avoided as far as possible to prevent aerosol generation during era of COVID19 Pandemic. Severe thoracolumbar scoliosis with hemiparesis fol lowing a cerebrovascular accident in a geriatric age group holds array of challenges for both general anaesthesia and regional anaesthesia. Considering the prevailing COVID19 Pandemic and thoracolumbar scoliosis with hemiparesis its always safe to pro ceed with regional anaesthesia as far as possible. Subarachnoid block is preferred by many anesthesiologists in view of cost ef fectiveness, low risk of cognitive dysfunction, thrombo-embolic events, post-operative respiratory morbidity, renal failure and prolonged post-operative hospital stay. Anaesthetic management After arranging adequate blood products, a detailed writ ten informed high risk consent was obtained for the planned anaesthesia procedure along with a backup plan for general anaesthesia in view of anticipated difficult regional anaesthe sia access. Sedative premedications were avoided, Patient was reassured to ally anxiety and was given T. Pantoprazole 40mg, along with patient ‘s own medication , N.P.O was maintained. Annals of Anesthesia and Pain Medicine Annals of Anesthesia and Pain Medicine 4. Chin KJ, Macfarlane AJR, Chan V, Brull R. The use of ul trasound to facilitate spinal anesthesia in a patient with previous lumbar laminectomy and fusion: A case report J Clin Ultrasound. 2009378482510.1002/jcu.20588 Acknowledgement Department of Anaesthesiology, Department of orthopae dics, OT & RICU staffs SUT Academy of Medical Sciences, Thiru vananthapuram, kerala, India. On receiving inside OT – Monitors for ECG, Non-invasive Blood pressure, pulse oximetry, temperature probe were con nected. Two wide bore cannulae were established under strict asepsis under local anaesthesia and was preloaded judiciously with balanced salt solution. 2 5. https://www.academia.edu/47848195/The_Role_of_ Perioperative_Physicians... 6. Taylor%E2%80%99s-Approach (ijmsir.com) 7. (PDF) Taylor’s Approach/Modified paramedian approach a game changer in unanticipated difficult spine during the era of Covid pandemic - A Case Report (researchgate.net) 8. Guidelines for Neuraxial Anesthesia and Anticoagulation (ihc.com). References 1. Brull R, Macfarlane AJ, Chan VW, Miller RD, Cohen NH, et al. Young Spinal, Epidural, and Caudal Anesthesia Miller’s Anesthesia Eighth Edition Elsevier Philadelphia. 20151692 2. Center NCG. The Management of Hip Fracturein Adult. Regional vs General Anaesthesia. London: Royal College of Physician. 2011. 3. Asra Guidelines Anticoagulation Pdf (helpforchildren. info). 4. Chin KJ, Macfarlane AJR, Chan V, Brull R. The use of ul trasound to facilitate spinal anesthesia in a patient with previous lumbar laminectomy and fusion: A case report J Clin Ultrasound. 2009378482510.1002/jcu.20588 5. https://www.academia.edu/47848195/The_Role_of_ Perioperative_Physicians... 6. Taylor%E2%80%99s-Approach (ijmsir.com) 7. (PDF) Taylor’s Approach/Modified paramedian approach a game changer in unanticipated difficult spine during the era of Covid pandemic - A Case Report (researchgate.net) 8. Guidelines for Neuraxial Anesthesia and Anticoagulation (ihc.com). References 1. Brull R, Macfarlane AJ, Chan VW, Miller RD, Cohen NH, et al. Young Spinal, Epidural, and Caudal Anesthesia Miller’s Anesthesia Eighth Edition Elsevier Philadelphia. 20151692 2. Center NCG. The Management of Hip Fracturein Adult. Regional vs General Anaesthesia. London: Royal College of Physician. 2011. 3. Asra Guidelines Anticoagulation Pdf (helpforchildren. info). 4. Chin KJ, Macfarlane AJR, Chan V, Brull R. The use of ul trasound to facilitate spinal anesthesia in a patient with previous lumbar laminectomy and fusion: A case report J Clin Ultrasound. 2009378482510.1002/jcu.20588 5. https://www.academia.edu/47848195/The_Role_of_ Perioperative_Physicians... 6. Taylor%E2%80%99s-Approach (ijmsir.com) 7. (PDF) Taylor’s Approach/Modified paramedian approach a game changer in unanticipated difficult spine during the era of Covid pandemic - A Case Report (researchgate.net) 8. Guidelines for Neuraxial Anesthesia and Anticoagulation (ihc.com). References 3. Asra Guidelines Anticoagulation Pdf (helpforchildren. info). 3. Asra Guidelines Anticoagulation Pdf (helpforchildren. info). 4. Chin KJ, Macfarlane AJR, Chan V, Brull R. The use of ul trasound to facilitate spinal anesthesia in a patient with previous lumbar laminectomy and fusion: A case report J Clin Ultrasound. 2009378482510.1002/jcu.20588 3
https://openalex.org/W4390712427	https://jurnal.stisummulayman.ac.id/positajhki/article/download/128/51	Indonesian	null	Keharmonisan dalam Rumah Tangga Pengaruh Terhadap Material di Mesjid Tuha	Posita	2,023	cc-by-sa	2,491	Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 Abstract In this study, the problem is how harmony in the household affects the material at the Tuah Mosque. The purpose of this research is to find out and explain the effect of harmony on household materials. The research used by the author is field research with qualitative methods. The data source used in this research is primary data about the economic crisis and secondary data is family documents in Tringgadeng. The data set strategy used in this research is interview. The information evaluation method makes use of ideas, especially techniques of systematic effort to find and organize information obtained from interviews, subject notes, and other substances so that it can be easily understood and findings can be known. for everything else. From the results of research on material harmony in the household at the Tuha Mosque, it has a very large influence in meeting material needs such as inadequate housing and children's education which ends in the middle of the road so that chaos becomes the end of family relationships that are far from harmony. Keywords: Harmony, Household, Material Keywords: Harmony, Household, Material Abstrak Dalam penelitian ini yang menjadi permasalahan bagaimana keharmonisan dalam rumah tangga berpengaruh terhadap material di Mesjid Tuah. Tujuan penelitian ini untuk mengetahui dan menjelaskan pengaruh keharmonisa terhadap material dalam rumah tangga. Penelitian yang digunakan oleh penulis adalah penelitian lapangan dengan metode kualitatif. Sumber data yang digunakan dalam penelitian ini yaitu data primer tentang krisis ekonomi dan data sekunder adalah dokumen keluarga di tringgadeng. Strategi rangkaian data yang digunakan dalam penelitian ini yaitu wawancara. Metode evaluasi informasi memanfaatkan gagasan, khususnya teknik upaya sistematis untuk mencari dan menyusun informasi yang diperoleh dari wawancara, catatan subjek, dan substansi lain agar dapat dengan mudah dipahami dan temuan dapat diketahui. untuk yang lainnya. Dari hasil penelitian keharmonisan terhadap material dalam rumah tangga di Mesjid Tuha memberikan pengaruh yang sangat besar dalam memenuhi kebutuhan material seperti kurang memadai tempat tinggal dan pendidikan anak yang berakhir ditengah jalan sehingga kericuhan menjadi akhir dari hubungan keluarga yang jauh dari keharmonisan. Kata Kunci: Keharmonisan, Material, Rumah Tangga Keharmonisan dalam Rumah Tangga Pengaruh Terhadap Material di Mesjid Tuha Munawarsyah1 1STIS Ummul Ayman, Pidie Jaya Email Korespondensi: cutputroemunawar@gmail.com PENDAHULUAN Keharmonisan merupakan situasi dan kondisi dalam keluarga dimana di dalamnya tercipta kehidupan beragama yang kuat, suasana yang hangat, saling menghargai, saling pengertian, saling terbuka, saling menjaga dan diwarnai kasihsayang dan rasa saling percaya sehingga memungkinkan anak untuk tumbuh dan berkembang secara seimbang dan sedangkan keluarga yang harmonis dapat dilihat dari rasa serba kecukupan dalam hal material baik fisik dan non-fisik. Keharmonisan dalam keluarga juga merupakan suatu kondisi yang didasarkan pada kesempurnaan kasih dan cinta keluaraga dalam memenuhi kebutuhan material dan kepuasan lahir dan batin serta menjalani semua peranan dalam rumah tangga dalam melengkapi kebutuhan yan sempurna (Awaliyah & Rostanti, 2020). 10 DOI: doi.org/10.52029/pjhki.v1i1.128 Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 Kepuasan batin juga banyak orang turut mendukung agar tercapainya keharmonisan dalam rumah tangga. Maka dalam membangun rumah tangga yang harmonis salah satunya pengaruh terhadap material. Untuk mencapai kondisi ekonomi yang memadai, maka suatu rumah tangga harus memiliki pendapatan yang diperoleh melalui ikhtiar dan usaha. Yang dimaksud iktiar di sini yaitu berupa kegiatan yang dilakukan baik secara langsung maupun tidak langsung untuk mendapatkan penghasilan dalam bentuk uang, barang, dan jasa dalam memenuhi kebutuhan (Amruddin, 2020). Material adalah suatu kebutuhan benda yang berwujud baik perlengkapan rumah dan sebagainya dan material memiliki dua katagori diataranya; fisik dan no-fisik, kebutuhan fisik juga berupa kelengkapan kebutuhan sehari-hari (makan –minum) dan non-fisik tergolong dalam pendidikan anak, keamanan arumah tangga, kesehatan rumah tangga dan lainnya (Nadhiroh, 2020). Dalam menyempurnakan kebutuhan material bahwa kebutuhan material sebuah tantangan yang sangat besar dalam mempertahankan keharmonisan keluarga dalam melengkapi kebutuhan keluarga (Sumarni dkk, 2022). Karena kebutuhan material dalam rumah tangga memiliki kekuatan dan pondasi awal dari pembentukan rumah tangga, hal tersebut suatu kewajiban yang waji terpenuhi dalam rumah tangga (Imasyah, 2009). ) Berdasarkan penjelasan diatas dipahami bahwa masalah keharmonisan dalam keluarga/rumah tangga sering terjadi dari masalah kebutuhan material, sebab kurangnya keuangan dalam rumah tangga sehingga sulitnya tercapai dalam kebutuhan material dan membawa dampak dalam keluarga, misalnya masalah kurangnya keharmonisan dalam keluarga, kericuhan dan berbagai macam persoalan lainnya. g g p y Dari hasil pengamatan dilapangan menjelaskan bahwa masih banyak rumah yang belum layak untuk dihuni dan apalagi kebutuhan lainnya, dan masih banyak juga penganguran dikalangan SMA dengan melihat biaya pendidikan yang tidak dapat terjangkau oleh kalangan masyarakat rendah sehingga lahirlah berbagai kericuhan antara suami-istri dalam menghadapi keadaan ekonomi yang tidak stabil sehingga hilangnya keharmonisan dalam rumah tangga. g y gg Maka dari penjelasan diatas dapat dijelaskan bahwa keharmonisan pengaruh terhadap material di Mesjid Tuha sudah tidak asing lagi di terdenger dan justrus menjadi suatu hal yang harus dikaji dan diteliti dalam karya ilmiah ini. METODE Metode penelitian ini menggunakan kualitatif yang menggambarkan kejadian, fonomena yang terjadi di lapangan sesuai dengan kenyataan mengenai keharmonisan dalam rumah tangga pengaruh terhadap material di Mesjid Tuha (Arikunto, 2020). 1. Rumah Tangga Rumah tangga merupakan sekumpulan anggota yang berkumpul dalam satu rumah, bangunan, gudung dan memiliki satu dapur dalam mempertahankan kehidupan sehari-hari. Sedangkan dalam pendapat yang lain rumah tangga merupakan sejumlah 11 DOI: doi.org/10.52029/pjhki.v1i1.128 Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 orang yang tinggal bersama dalam satu rumah dan masing-masing anggota merasakan adanya pertautan batin sehingga terjadi saling mempengaruhi, saling memperhatikan, dan saling menyerahkan diri, sementara secara paedagogis, oleh karena itu persekutuan hidup yang dijalin oleh kasih sayang antara pasangan dua jenis manusia yang dikukuhkan dengan pernikahan, yang bermaksud untuk saling menyempurnakan, yang mana tiap-tiap pribadi memiliki kedudukan di dalamnya, tugas dan tanggung jawab, hak dan kewajiban yang harus dipenuhi dan dilaksanakan Rumah tangga suatu tempat dimana semua di tanggung oleh satu kepala keluarga (ayah) dan apabila kurangnya kebutuhan dalam rumah tanggga maka biasanya berbagai macam perselisihan terjadi baik cara pandang istri dan lainnya sehingga ujungnya berekhir dengan kekerasan dari tingkat penghasilan yang berbeda seringkali menjadi kendala dalam sebuah rumah tangga (Muta’li, 2019). Rumah tangga kan tentram berawal dari; a. Diawali dengan pernikahan yang sah b. Saling menjaga dalam hal ibadah c. kewajiban dan tanggung jawab terpenuhi d. kebutuhan keluarga yang cukup e. hubunganmesra Oleh karena itu tujuan dalan dalam rumah tangga agar tercapai dalam kesejahteraan yang senpurna dengan kondisi ekonomi keluarga yang dapat mencukupi kebutuhan rumah tangga. Sebagai tugas utama dalam rumah tangga bahwa suami yang bekerja dan mencari nafkah untuk keluarga, sedangkan fokus dan tugas utama dari istri adalah mendukung suami dan mengurus segala urusan rumah tangga termasuk pengasuhan anak Dari penjelasan tersesebut bahwa rumah tangga merupakan sekumpulan anggota yang terdiri dari ayah dan ibu serta anak dalam memenuhi untuk mencapai ridha Allah, dan rumah yang dihiasi dengan kedamaian, ketenangan, kasih sayang, keturunan, pengorbanan, saling melengkapi, sempurna, tolong menolong. dan kerjasama untuk mencapai keharmonisan dalam rumah tangga.sekumpulan orang yang memiliki hubungan darah yang hidup bersama, yang memiliki peran dan tanggungjawab masing- masing yang harus dilaksanakan demi mencapai ridhoIlahi, dan dalam rumah tangga yang dihiasi dengan ketenangan, ketentraman, kasih sayang, keturunan, pengorbanan, saling melengkapi, menyempurnakan, saling membantu, dan bekerja sama agar tercapainya keharmonisan dalam rumah tangga. 2. Keharmonisan h Keharmonisan asal dari kata harmonis yang berarti hal (keadaan) serasi, selaras. Keharmonisan suatu situasi yang selaras dan cocok dalam menggapai tujuan dalam rumah tangga dari sikap saling menjaga serta keharmonisan harmonis hanya akan tercipta apabila salah satu anggota berkaitan dengan kebahagiaan anggota-anggota keluarga lainnya. Secara umum keharmonisan akan tercapai diantaranya: Pertama; keinginan tercapai, cita2 dan harapan rumah tangga dan Kedua; minimnya terjadi kericuhan antar suami-istri. 12 DOI: doi.org/10.52029/pjhki.v1i1.128 3. Pengaruh Keharmonisan Terhadap Material dalam Rumah Tangga 3. Pengaruh Keharmonisan Terhadap Material dalam Rumah Tangga Pengaruh keharmonisan terhadap material dalam rumah tangga dapat dilihat dari Kebutuhan keluarga yang bersifat material serta finansial dalam keuangan, kebutuhan tersebut terdiri dari kebutuhan fiisik / non fisik; kebutuhan keseharian/kebutuhan yang berhubungan dengan hiburan, pendidikan dan kesehatan. g g p Persyaratan material fisik meliputi; keluarga mengutamakan pemenuhan kebutuhan. Ini karena, misalnya, pria dan wanita baru memulai kariernya, namun bisa saja kebutuhan akan meja (akomodasi) didahulukan, padahal keduanya sudah memiliki tabungan yang cukup. Begitu juga dengan jumlah fisik yang dibutuhkan pria dan wanita untuk merencanakan dan memprioritaskan kebutuhan mereka. Misalnya, biaya persalinan menjadi prioritas jika ternyata seorang wanita akan hamil dalam beberapa bulan setelah pernikahan. Biaya pendidikan menjadi prioritas ketika anak sudah cukup umur untuk bersekolah. Kebutuhan material dalam rumah tangga suatu penopang utama dan kebutuhan hidup keluarga, sedangkan kelangkaan merupakan awal dari kehancuran keluarga. Oleh karena itu, pertemuan ini perlu menjadi perhatian utama bagi seluruh anggota keluarga. Sebagaimana hasil wawancara dari Ibu Aminnah bahwa kebanyakan suami sering menyia-nyiakan waktu dalam berkerja dan membawa pulang hasil kerjanya hanya sedikit dari istrinya. Sedangkan Ibu Siti juga menjelaskan bahwa kurangnya kebutuhan dalam rumah tangga dan banyaknya tanggungan dalam keluarga. Dari Pengaruh keharmonisan suatu keharusan dalam mencukupi kebutuhan material keluarganya serta istri sering menjadi pembatu dalam penghasilan suaminya yang tidak mencukupi kebutuhan anak-anaknya sehingga istri aktiif mencari nafkah siang dan malam dan hal tersebut menjadi suatu kondisi dimana anak kurang mendapat perhatian dan bimbingan khusus dari orang tuanya dan inilah suatu permasalahan yang sangat dratis dalam keluarga yang menimbulkan kurang tentram dan harmonis (Arfiani, 2009). ) Berdasarkan uraian diatas menjelaskan bahwa keharmonisan terhadap material dalam rumah tangga sangat berpengaruh. Hal tersebut dapat dilihat dari kebutuhan marerial yang belum terpenuhi. Sebagaimana hasil wawancara dari Irwan di bahwa sulit mencari kerja yang cocok untuk penghasilan kesehariannya dan mampu menutupi kebutuhan keluarga. Sedangkan Ramli menjelaskan bahwa dari pada sia-sia dalam bekerja lebih baik duduk dan santai menunggu pekerjaan yang sesuai dengan upah didapat. p Dengan demikian dalam membangun suatu bahtera rumah tangga yang harmonis dengan mencukupi semua kebutuhan keluaraga karena suami sebagai kepala keluaraga. Sebagaimana mawar menjelaskan bahwa kebanyakan laki-laki bekerja sebagai buruh kasar tapi tidak bias mencukupi kebutuhan keluarganya dan bahwa istri juga harus ikut bekerja. Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 Dari hal inilah keharmonisan ini dapat menciptakan keseimbangan dan kenyamanan dalam pikiran, kesukaan dan tindakan masing-masing individu anggota keluarga sehingga tidak terjadi hal-hal yang terlalu menekan. Keharmonisan juga ditentukan oleh sikap kedewasaan jiwa laki-laki dan perempuan, rasa tanggung jawab terhadap diri sendiri, masyarakat dan keluarga, sikap terbuka dengan pikiran terbuka, perasaan sosial yang tidak mudah putus asa, keseimbangan kreatif antara luar. kepuasan dan kepuasan batin. p Keharmonisan tersebut dapat terwujud keseimbangan dan kesesuaian alam pikiran, persamaan dan perbuatan masing-masing individu anggota keluarga sehingga tidak terjadi hal-hal yang menegangkan secara berlebih-lebihan. Keharmonisan juga ditentukan dalam sikap kedewasaan jiwa suami istri dengan rasa tanggung jawab terhadap diri sendiri, terhadap masyarakat dan terhadap keluarganya, memiliki sikap terbuka tanpa prasangka, memiliki rasa sosial, tidak lekas putus asa, sanggup menciptakan keseimbangan antara kepuasan lahiriah dan kepuasan batiniah. Keharmonisan merupakan suatu bentuk interaksai dalam keluarga dengan utuh dan sesuai tampa ada sikap permusuhan dan agresif serta keutuhan keluarga, kecocokan hubungan antara suami dan istri serta adanya ketenangan dan keharmosian ini ditandai dengan suasana rumah yang teratur, tidak cenderung pada konflik dan peka terhadap kebutuhan rumah tangga. gg Dari pengertian di atas dapat disimpulkan bahwa, keluarga harmonis merupakan keluarga yang memiliki suasana yang nyaman dan tentrem, anggota keluarga yang saling menyayangi, dan minim akan pertengkaran-pertengkaran karena mampu menangani perseilsihan. Oleh kerena itu keharmonisan dalam rumah tangga akan tercapai dengan tercapainya kebutuhan material dala rumah tangga karena hubungan keharmonisan dalam memenuhi kebutuhan material keluarga menjadi hambatan terbesar dalam rumah tangga disebabkan beberapa kendala diantaranya : keluarga tidak mampu memenuhi kebutuhan keuangan, seringkali menjadi masalah besar dalam keluarga, sehingga keuangan menjadi suatu kendala dalam memulai sebuah rumah tangga suatu kendala yang sangat mempengaruhi fungsi rumah tangga terhadap kebutuhan material baik fisik maupun non fisik (Bong dkk, 2019). Kebutuhan material suatu kebutuhan keseharian sepeti makanan/minuman serta pakaian, tempat tinggal, kebutuhan tersebut memberikan masukan bagi vitiman bagi tubuh manusia dalam melakukan sesuatu dan yang termasuk kebutuhan yang mengandung vitamin biasa sayur dan buahan, inilah kebutuhan fisik manusia yang harus ada, kebutuhan makan dan minum juga sebagai bahan /alat yang dijadikan sebagai pelindung utama yang sangat dibutuhkan. p g y g g Berdasarkan penjelasan diatas dapat dijelaskan bahwa keharmonisan dalam rumah tangga mudah tercapai dengan meberikan kebutuhann material yang sepadan dan cukup agar keluarga dapat hidup layak. Namun dari kenyataan yang terjadi menjadi pengaruh keharmonisan rumah tangga terhadap material di masjid tuha suatu permasalahan utama yang harus diperhatikan. 13 DOI: doi.org/10.52029/pjhki.v1i1.128 Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 3. Pengaruh Keharmonisan Terhadap Material dalam Rumah Tangga SIMPULAN Dari penjelasan diatas dapat dijelaskan bahwa keharmonisan terhadap material dalam rumah tangga di Mesjid Tuha memberikan pengaruh yang sangat besar dalam memenuhi kebutuhan material seperti kurang memadai tempat tinggal dan pendidikan anak yang berakhir ditengah jalan sehingga kericuhan menjadi akhir dari hubungan keluarga yang jauh dari keharmonisan. Posita: Jurnal Hukum Keluarga Islam, Vol. 01 No. 01, Juni 2023 Bedasarkan penjelasan diatas dapat dijelaskan bahwa keharmonisan terhadap material dalam rumah tangga di Mesjid Tuha masih sangat berpengaruh seperti masih kurangnya kebutuhan materian yang terselesaika seperti tempat penginapan dll. 3. Pengaruh Keharmonisan Terhadap Material dalam Rumah Tangga j Dari situasi inilah membuat keharmonisan terhadap material dalam rumah tangga sangat berdampak pada keharmonisan rumah tangga dalam menggapai kebutuhan material karena masih banyak kebutuhan material lainnya yang belum tercapai seperti salah satunya tempat tinggal. 14 DOI: doi.org/10.52029/pjhki.v1i1.128 DAFTAR RUJUKAN Amruddin, Strategi Nafkah Petani di Desa Perbatasan Perkotaan, Bandung: CV Media, 2020. Amruddin, Strategi Nafkah Petani di Desa Perbatasan Perkotaan, Bandung: CV Media, 2020. Anis Nur Nadhiroh Pemberian Upah Pekerja/Buruh YnagAdil dan LayakPerpektif HukumPositif Amruddin, Strategi Nafkah Petani di Desa Perbatasan Perkotaan, Bandung: CV Media, 2020. Anis Nur Nadhiroh, Pemberian Upah Pekerja/Buruh Ynag Adil dan Layak Perpektif Hukum Positif dan Islam Indonesia: Guepedia 2020 Anis Nur Nadhiroh, Pemberian Upah Pekerja/Buruh Ynag Adil dan Layak Perpektif Hukum Positif dan Islam, Indonesia: Guepedia, 2020. Arikunto, Prosedur Penelitian suatu Pendekatan Praktek, Jakarta: Rineka Cipta. 2020. Awaliyah, G, & Rostanti, Keharmonisan Rumah Tangga, Jakarta: Arjasa Pratama, 2020 Badan Pusat Statistik, Profil Kemiskinan di Indonesia, 2020. Bong, Soeseno dkk, Manajemen Resiko, Keluarga Harmonis, Jakarta: T. Gremedia, 2019. Devi Arfiani, Brantas Kemiskinan, Semarang: ALRINT, 2009. Bong, Soeseno dkk, Manajemen Resiko, Keluarga Harmonis, Jakarta: T. Gremedia, 2019. D i A fi i B t K i ki S ALRINT 2009 Hakim, L. N. Ulasan, Metodologi Kualitatif, Jakarta: PT. Bumi Aksara, 2015. Imas Novita Juaningsih, Analisis Kebijakan PHK bagi Para Pekerja pada Masa Pandemi Covid-19 di Indonesia, Pusat Studi Konstitusi dan Legislasi Nasional (Poskolegnas), Universitas Islam Negeri, 2020. Lufi Muta’li, Dinamika Peran Rumah Tangga Indonesia, Yokyakarta; IKAPI, 2019. M. Handry Imasyah, Menyempurnakan Kebutuhan Keluarga, Jakarta: Kompas Gramedia, 2009. Maryunani & Axellina Muara Setyanti, Ekonomi Pendesaan, Jakarta: UB Press, 2020. oeseno Bong dkk, Manajemen Resiko, Krisis dan Bencana Industri Pariwisata yang Berkelanjutan, Jakarta: T. Gremedia, 2019. Sumarni dkk, Nafkah Petani, Jakarta; IKAPI. 2022. Sumarni dkk, Nafkah Petani, Jakarta; IKAPI. 2022. Yusuf, Konsep Penentuan Upah dalam Ekonomi Islam, Jurnal Al-Ulum, 2010. Yusuf, Konsep Penentuan Upah dalam Ekonomi Islam, Jurnal Al-Ulum, 2010. 15 DOI: doi.org/10.52029/pjhki.v1i1.128
https://openalex.org/W4387001284	https://zenodo.org/records/8377018/files/269-278.pdf	Indonesian	null	Pengaruh Konsep Diri Terhadap Tingkat Kepercayaan Diri Pada Mahasiswa	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	5,013	Abstract This study aims to determine whether there is an influence between self-concept and the level of self- confidence in student at University X in Bekasi, as well as the extent of the influence of self-concept on the level of self-confidence . The research method used quantitative research methods involving 263 students, using convenience sampling. Data was collected using two questionnaires, namely self- concept and and selef-confidence, which was tested its reliability and validity. Data was processed using simple linear regression. The results showed that self concept had influenced 76,8% to seld- confidence. f Keywords: self-confidence, self-concept, undergraduate student. 2023 Madani : Jurnal Ilmiah Multidisipline 2023 Madani : Jurnal Ilmiah Multidisipline Madani: Jurnal Ilmiah Multidisiplin Volume 1, Nomor 8, September 2023, Halaman 269-278 Licenced by CC BY-SA 4.0 E-ISSN: 2986-6340 DOI: https://doi.org/10.5281/zenodo.8377018 Abstrak Penelitian ini bertujuan untuk mengetahui apakah terdapat pengaruh dari konsep diri dengan tingkat kepercayaan diri pada mahasiswa di Universitas X di Bekasi, dan sejauh mana pengaruh tersebut. Metode penelitian yang digunakan adalah metode penelitian kuantitatif dengan melibatkan 263 responden mahasiswa, yang diambil dengan cara convenience sampling, sesuai dengan kriteria yang telah ditentukan. Pengumpulan data dilakukan dengan menggunakan kala konsep diri dan kepercayaan yang telah diuji validitas dan reliabilitasnya. Pengolahan data dilakukan dengan menggunakan uji regresi linear sederhana. Pada uji regresi terlihat bahwa konsep diri memiliki pengaruh terhadap kepercayaan diri., dan memberikan peran sumbangan sebanyak 76,8% terhadap kepercayaan diri. p y Kata kunci: kepercayaan diri, konsep diri, mahasiswa. Article Info Received date: 15 August 2023 Revised date: 25 August.2023 Accepted date: 21 Sept. 2023 Article Info Received date: 15 August 2023 Revised date: 25 August.2023 PENDAHULUAN Kepercayaan diri sangat penting dalam kehidupan manusia karena dengannya seseorang dapat melakukan berbagai hal tanpa merasa malu atau terhambat. Rasa percaya diri adalah keyakinan seseorang terhadap semua kelebihan yang dimilikinya, yang memungkinkannya mencapai berbagai tujuan dalam hidupnya (Thursan, 2005). Kepercayaan diri juga dapat didefinisikan sebagai keyakinan yang dimiliki setiap orang terhadap dirinya sendiri dan cara mereka melihat diri mereka sendiri melalui konsep diri (Davies, 2006). Kepercayaan diri adalah bagian penting dari kepribadian seseorang; jika seseorang tidak memiliki kepercayaan diri, hal itu dapat menyebabkan masalah. Kepercayaan diri adalah sifat kepribadian yang mencakup keyakinan yang kuat terhadap kemampuan diri sendiri (Lauster, 1992). Lebih lanjut, dinyatakan bahwa Kepercayaan diri adalah sikap dan perasaan yang percaya pada kemampuan diri sendiri sehingga membuat seseorang merasa lebih tenang dan tidak terlalu cemas tentang apa yang mereka lakukan. Mereka juga akan merasa lebih bebas Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) 269 \| Vol. 1 No. 8 2023 Madani : Jurnal Ilmiah Multidisipline untuk melakukan apa pun yang mereka suka, dan mereka akan merasa tanggung jawab atas apa yang mereka lakukan. Mereka juga sopan dan ramah dengan orang lain. (Lauster, 1992). untuk melakukan apa pun yang mereka suka, dan mereka akan merasa tanggung jawab atas apa yang mereka lakukan. Mereka juga sopan dan ramah dengan orang lain. (Lauster, 1992). y g j g g g Hasil penelitian terdahulu juga menunjukkan bahwa salah satu sifat yang paling penting dari seseorang adalah kepercayaan diri; jika seseorang memiliki kepercayaan diri, mereka dapat lebih mudah menghadapi masyarakat dan menyalurkan potensi mereka. Jika seseorang tidak memiliki kepercayaan diri, mereka akan menghadapi masalah bagi diri mereka sendiri (Ghufron & Rini, 2010). Kepercayaan diri merupakan hal penting dari kehidupan dikarenakan dapat mempengaruhi seseorang dalam berpendapat di depan orang lain (Indriawati, 2018). Terdapat banyak hal yang dapat mempengaruhi kepercayaan diri, salah satunya adalah konsep diri seseorang. Studi sebelumnya menunjukkan korelasi yang positif dan signifikan antara konsep diri siswa dan kepercayaan diri mereka. Hasilnya menunjukkan bahwa siswa memiliki kepercayaan diri yang lebih besar ketika mereka memiliki konsep diri yang lebih tinggi, dan sebaliknya, siswa memiliki kepercayaan diri yang lebih rendah ketika mereka memiliki konsep diri yang lebih rendah (Eliza, 2019). Selain itu, Azizi (2014) mengungkapkan bahwa terdapat hubungan yang positif dari konsep diri yang positif terhadap peningkatan rasa percaya diri seseorang. Lebih lanjut, berdasarkan penelitian Sari dan Khoirunnisa (2021) terlihat terdapat hubungan positif antara konsep diri dan kepercayaan diri. 270 \| Vol. 1 No. 8 PENDAHULUAN Dalam hal ini, siswa dengan konsep diri yang tinggi akan memiliki kepercayaan diri yang tinggi juga. Sebaliknya, siswa dengan konsep diri yang rendah akan memiliki kepercayaan diri yang rendah juga, menunjukkan hubungan antara konsep diri dan kepercayaan diri. p y Sementara itu, Mahasiswa adalah generasi muda yang kelak akan meneruskan cita- cita bangsa, jadi mereka diharapkan dapat mencapai potensi terbaik mereka dan menguasai ilmu pengetahuan agar mereka dapat berpartisipasi secara aktif dalam pembangunan bangsa dan menjadi sumber daya manusia yang berkualitas di masa depan. Mahasiswa, menurut Widiastuti dan Gumulya (2013), adalah kelompok remaja muda yang memasuki masa dewasa awal. Dengan demikian, siswa harus dapat mengisi waktu mereka dengan berbagai kegiatan bermanfaat agar mereka menjadi orang yang bermanfaat bagi masyarakat dan negara di masa depan. Kehidupan kampus telah mengubah gaya hidup siswa dan menuntut adanya penyesuaian dari mahasiswa untuk beradaptasi pada nilai-nilai dan norma-norma baru yang terdapat di perguruan tinggi yang berbeda dibandingkan dengan di sekolah menengah atas. Perubahan tersebut, menuntut mahasiswa untuk merespon terhadap kurikulum yang diberikan, yang dapat menjadikannya memiliki wawasan yang cukup luas, dan cara berfikir yang lebih maju. Sementara itu, setiap individu adalah unik, hal ini juga memengaruhi perbedaan pemikiran di antara para mahasiswa, khususnya bila mahasiswa berasal dari daerah yang beragam yang memiliki kultur budaya dari masing-masing daerah. Hal ini juga akan memengaruhi keberhasilan mahasiswa, dalam arti bila mahasiswa memiliki kepercayaan diri maka dia tidak akan terlalu terpengaruh terhadap tuntutan di kampus yang berbeda pada waktu mereka di sekolah menengah atas. Berdasarkan penelitian Sumiarsih dan Novita (2021) terlihat bahwa kepercayaan diri merupakan modal dasar seorang mahasiswa dalam memenuhi berbagai kebutuhannya. Rasa percaya diri yang dimiliki mampu membuat siswa dapat bertumbuh dalam pengalaman dan kemampuan sehingga menjadikan individu yang sehat dan mandiri. Jika mahasiswa tidak mempunyai rasa percaya diri, kemungkinan mahasiswa akan sulit dalam bergaul serta kemampuan yang dimiliki tidak akan tertampilkan karena kurangnya keberanian. Kurangnya rasa percaya diri juga dapat menimbulkan masalah bagi mahasiswa dalam mengungkapkan pendapat. Hal ini disebabkan karena mahasiswa merasa malu, belum terbiasa, takut salah, Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) 270 \| Vol. 1 No. 8 2023 Madani : Jurnal Ilmiah Multidisipline dan takut diejek dengan teman apabila jawaban yang merekan jawab salah, meskipun mungkin mahasiswa tersebut sebenarnya mampu. Penelitian ini menggunakan teori humanistik dari Carl Rogers (1959) yang mendefinisikan konsep diri sebagai kecenderungan individu untuk bertindak dengan cara mengaktualisasikan dirinya, yang mengarah pada diferensiasinya dari sekelompok pengalaman. PENDAHULUAN karenanya adalah dibedakan dan dilambangkan dalam kesadaran-kesadaran sebagai pengalaman diri yang jumlahnya membentuk konsep diri individu. Lebih lanjut, Rogers (1959) menyampaikan bahwa pengalaman masa kecil itu penting yang akan memberikan dampak positif pada konsep diri seseorang. Konsep diri tidak langsung dimiliki ketika seseorang lahir di dunia, melainkan suatu rangkaian proses yang terus berkembang dan membedakan individu satu dengan lainnya (Tarwoto, 2003). Hal ini di dukung pula oleh pernyataan McLeod (2007) yang menyatakan bahwa lingkungan luar memiliki pengaruh positif pada pembentukan konsep diri. Dalam hal ini, konsep diri yang telah terbangun seringkali sulit untuk berubah dan perubahan terjadi ketika adanya penerimaan dari orang lain, yang membantu seseorang untuk mengurangi kecemasan dan ancaman serta mengakui dan menerima pengalaman-pengalaman yang sebelumnya ditolak (Feist & Feist, 2010). Pengalaman masa lalu dapat memengaruhi konsep diri seseorang sehingga akan membentuk kepercayaan diri seseorang. Lebih lanjut, Afif (2015) menyatakan konsep sebagai bentuk gambaran diri yang terbentuk dari identitas sosial dan identitas personal yang dalam pemunculannya dapat bergantian ataupun bersamaan. Konsep diri mencakup apa yang dirasakan serta dipikirkan oleh individu tentang dirinya sendiri, penilaian seorang individu untuk dirinya sendiri, sehingga dengan begitu berarti konsep diri akan menjadi salah satu aspek yang penting bagi seorang individu dalam perilaku (Widiarti, 2017). Konsep diri dan tingkat kepercayaan diri pada setiap mahasiswa berbeda-beda, semua itu terbentuk atau tidak terbentuk di dasari dari latar belakang kehidupan di sekitarnya, baik keluarga, perekonomian maupun dilingkungan social atau yang lainnya. Menurut Arnett (2015) mahasiswa adalah Emerging Adulthood, yang merupakan periode transisi dari remaja ke dewasa, terjadi dari usia 18-25 tahun. Peneliti bertujuan untuk mengetahui seberapa besar pengaruh konsep diri terhadap kepercayaan diri pada mahasiswa di Universitas X Bekasi. Pentingnya topik ini untuk di teliti karena, mahasiswa diharapkan dapat memiliki kepercayaan diri sebagai modal dasar berinteraksi di lingkungan kampus dan mengikuti berbagai kegiatan yang ada serta untuk menjalin komunikasi dengan para mahasiswa maupun dosen pengajar. Penelitian ini bertujuan untuk mengetahui apakah terdapat pengaruh konsep diri terhadap tingkat kepercayaan diri pada mahasiswa dan sejauh mana pengaruh tersebut. 271 \| Vol. 1 No. 8 TINJAUAN TEORITIS Kepercayaan diri adalah sikap atau keyakinan bahwa seseorang dapat melakukan apa yang mereka inginkan, sehingga mereka tidak cemas dalam bertindak, bebas melakukan apa yang mereka suka, bertanggung jawab atas apa yang mereka lakukan, dan mampu mengenal kelebihan dan kekurangan mereka sendiri (Lauster, 2012). Konsep yang digunakan untuk menerangkan kepercayaan diri menggunakan konsep dari Lauster (2012), yang membahas pengalaman mengenai kemampuan diri, kelebihan dan kekurangan diri. Terdapat empat dimensi menurut Lautser (2012) yang menjadi pembentuk rasa percaya diri, yaitu:1. Percaya pada kemampuan sendiri. kemampuan seseorang untuk berkembang sehingga mereka dapat mengenal kemampuan mereka sendiri dan tidak bergantung pada orang lain. 2. Bertindak mandiri dalam mengambil keputusan. Yang berarti, seseorang dapat mengambil keputusan secara mandiri atau tanpa bantuan orang lain. 3. Memiliki rasa positif terhadap diri sendiri. Memiliki perasaan positif terhadap diri sendiri berarti memiliki penilaian yang positif terhadap diri sendiri, termasuk pandangan dan tindakan yang diambil, yang menghasilkan 271 \| Vol. 1 No. 8 Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) 2023 Madani : Jurnal Ilmiah Multidisipline perasaan positif terhadap diri sendiri dan masa depan. 4. Berani mengungkapkan pendapat. Sikap individu untuk memiliki kemampuan dalam mengungkapkan perasaan dalam diri yang ingin disampaikan kepada orang lain tanpa merasa terhalang atau terhalang oleh hal-hal yang menghambat pengungkapan perasaan tersebut. Lebih lanjut Lauster (2012) menyatakan bahwa salah satu faktor yang dapat memengaruhi kepercayaan diri seseorang adalah pengalaman hidup. Dalam hal ini faktor yang paling sering menyebabkan rasa rendah diri adalah kepercayaan diri yang rendah yang diperoleh dari pengalaman yang mengecewakan.. Terutama bila individu pada dasarnya memiliki rasa tidak aman, kurang kasih sayang dan kurang perhatian. perasaan positif terhadap diri sendiri dan masa depan. 4. Berani mengungkapkan pendapat. Sikap individu untuk memiliki kemampuan dalam mengungkapkan perasaan dalam diri yang ingin disampaikan kepada orang lain tanpa merasa terhalang atau terhalang oleh hal-hal yang menghambat pengungkapan perasaan tersebut. Lebih lanjut Lauster (2012) menyatakan bahwa salah satu faktor yang dapat memengaruhi kepercayaan diri seseorang adalah pengalaman hidup. Dalam hal ini faktor yang paling sering menyebabkan rasa rendah diri adalah kepercayaan diri yang rendah yang diperoleh dari pengalaman yang mengecewakan.. Terutama bila individu pada dasarnya memiliki rasa tidak aman, kurang kasih sayang dan kurang perhatian. Sementara itu, konsep diri menggunakan teori dari Carl Rogers (1959) karena ia adalah salah satu tokoh psikologi ternama mengenai konsep diri, yang menyatakan bahwa konsep diri adalah kecenderungan seseorang untuk bertindak dengan cara yang mengaktualisasikan dirinya sendiri. TINJAUAN TEORITIS Menurut Rogers (1959), konsep diri memiliki tiga dimensi, yaitu diri ideal, citra diri dan harga diri. a) Diri ideal (ideal self) adalah seseorang yang diinginkan, yaitu atribut atau kualitas yang ingin di capai, serta diri seperti yang di inginkan. b) Citra diri (self image). mengacu pada bagaimana seseorang melihat diri sendiri pada saat ini. Atribut seperti karakteristik fisik, ciri kepribadian, dan peran sosial semuanya berperan dalam citra diri tersebut. c) Harga diri (self esteem), adalah seberapa besar seseorang menyukai, menerima, dan menghargai diri sendiri. Konsep diri dapat dipengaruhi oleh sejumlah faktor termasuk bagaimana orang lain melihat dirinya, bagaimana seseorang berpikir dan membandingkan dirinya dengan orang lain. Konsep diri mempunyai hubungan yang positif dalam meningkatkan rasa percaya diri individu, dalam arti ketika seseorang memiliki konsep diri yang positif, mereka lebih percaya diri. (Azizi, 2014). Begitu juga berbagai penelitian terdahulu yang menunjukkan bahwa terdapat keterkaitan antara konsep diri dengan kepercayaan diri, dimana semakin positif konsep diri seseorang akan diikuti dengan semakin tingginya tingkat kepercayaan diri, begitu juga sebaliknya (Savira & Suhardhani, 2017; Sari & Khoirunnisa, 2021).Hasil yang sama juga didapat oleh Eliza (2019), serta penelitian dari Sumiarsih dan Novita (2021) yang dilakukan pada murid SD kelas V dan VI, juga mengungkapkan bahwa terdapat pengaruh positif dari konsep diri dengan kepercayaan diri. Bila dilihat dari penelitian terdahulu tersebut, tampak bahwa yang diteliti adalah mengenai hubungan antara konsep diri dan kepercayaan diri, dan hal ini telah terbukti termasuk juga pada mahasiswa. Untuk mengetahui kedalaman dari hubungan tersebut, pada penelitian ini peneliti akan melihat pengaruh dari konsep diri terhadap kepercayaan diri mahasiswa, dengan hipotesa sebagai berikut: Hipotesa: Konsep diri memiliki pengaruh positif dan signifikan terhadap kepercayaan diri mahasiswa. 272 \| Vol. 1 No. 8 METODE PENELITIAN Penelitian ini menggunakan pendekatan kuantitatif karena memiliki karakteristik yang dapat di klasifikasikan, konkrit, teramati dan terukur berdasarkan aksioma dasar penelitian kuantitatif dari sifat reabilitas (Sugiyono, 2018). Metode pada penelitian ini akan menggunakan metode penelitian regresi untuk melihat pengaruh konsep diri (IV) terhadap kepercayaan diri (DV) pada mahasiswa. Sesuai apa yang disampaikan oleh Sugiyono (2018) mengenai populasi yang merupakan generalisasi terdiri atas obyek/subyek yang memiliki kualitas dan karakteristik tertentu. Populasi dalam penelitian ini adalah seluruh mahasiswa pada perguruan tinggi X dengan jumlah populasi secara keseluruhan adalah 8611 orang. Lebih lanjut, Sugiyono (2018) menyatakan sampel adalah bagian dari jumlah dan harus memiliki karakteristik dari populasi tersebut. Pada penelitian ini karakteristik responden yang digunakan sebagai responden adalah sebagai berikut: a) Responden merupakan mahasiswa di Perguruan Tinggi Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) 2023 Madani : Jurnal Ilmiah Multidisipline X di Bekasi, dan b)Responden yang berusia 18-25 tahun. Untuk menentukan jumlah sampel, menggunakan tabel sampel menurut Isaac dan Michael, dengan tingkat kesalahan sebesar 10% . Hal ini sejalan dengan yang dinyataka Sugiyono (2018) bahwa peneliti dapat menggunakan taraf kesalahan 10% jika populasi yang digunakan cukup besar. Pada penelitian ini yang menjadi responden adalah mahasiswa Universitas X di Bekasi, yang terdiri dari 7 (tujuh) fakultas yaitu, hukum, ekonomi, psikologi, ilmu teknik, ilmu pendidikan, ilmu komunikasi, dan ilmu komputer. Pada penelitian ini responden adalah 263 mahasiswa aktif dengan karakteristik usia 18-25 tahun. X di Bekasi, dan b)Responden yang berusia 18-25 tahun. Untuk menentukan jumlah sampel, menggunakan tabel sampel menurut Isaac dan Michael, dengan tingkat kesalahan sebesar 10% . Hal ini sejalan dengan yang dinyataka Sugiyono (2018) bahwa peneliti dapat menggunakan taraf kesalahan 10% jika populasi yang digunakan cukup besar. Pada penelitian ini yang menjadi responden adalah mahasiswa Universitas X di Bekasi, yang terdiri dari 7 (tujuh) fakultas yaitu, hukum, ekonomi, psikologi, ilmu teknik, ilmu pendidikan, ilmu komunikasi, dan ilmu komputer. Pada penelitian ini responden adalah 263 mahasiswa aktif dengan karakteristik usia 18-25 tahun. Tabel 1. METODE PENELITIAN Profil Responden Penelitian Keterangan Profil N Persentasi Gender Pria Wanita 134 129 51.0% 49.0% Usia 18 – 21 thn >21 – 25 thn 136 127 51.7% 48.3% Fakultas Hukum Ekonomi Psikologi Ilmu Teknik Ilmu Pendidikan Ilmu komunikasi Ilmu komputer 23 46 67 35 41 40 11 8.7% 17.5% 25.5% 13.3% 15.6% 15.2% 4.2% Status lokasi tempat tinggal Penduduk Bekasi Penduduk Jakarta Perantau / pendatang 154 76 33 58.6% 28.9% 12.5% Semester Semester 2 Semester 4 Semester 6 Semester 8 23 43 76 121 8.7% 16.3% 28.9% 46.0% Total 263 100.0 % Tabel 1. Profil Responden Penelitian Keterangan Profil N P 273 \| Vol. 1 No. 8 Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) Total Dari tabel di atas, dapat dilihat bahwa profil responden pria dan wanita nyaris seimbang dengan pria 51.0% dan wanita 49,3%. Mayoritas rentang usia adalah 18-21 tahun (51.7%). Mayoritas mahasiswa adalah berasal dari fakultas psikologi (25.5%) dan paling sedikit dari Fakultas ilmu Komputer (4.2%). Mayoritas mahasiswa adalah penduduk Bekasi (58.6%), dan terdiri dari semester delapan (46.0%). Data dikumpulkan dengan menggunakan skala konsep diri Lauster (2012), dan kepercayaan diri (Rogers, 1959). Jenis skala pengukuran adalah menggunakan skala Likert. Menurut Sugiyono (2018), skala Likert digunakan untuk mengukur pendapat, sikap dan persepsi seseorang atau sekelompok orang terhadap fenomena sosial. Terkini peneliti melakukan modifikasi terhadap dua skala tersebut. Modifikasi skala dilakukan untuk mengubah beberapa bagian yang kurang sesuai dengan sampel penelitian dengan cara menambah atau mengurangi aitem (Saifuddin, 2020). Dalam pernyataan skala Likert, terdapat dua bentuk pernyataan yaitu pernyataan positif (favorable) untuk mengukur sikap positif, dan bentuk pernyataan negatif (unfavorable) untuk mengukur sikap negatif. Skala likert memiliki 4 pilihan yaitu, STS (sangat tidak setuju, TS (tidak setuju), S (setuju), dan SS (sangat setuju). Lebih lanjut, untuk mengetahui apakah alat ukur yang digunakan adalah sahih dan dapat dipercaya maka peneliti melakukan uji validitas, sesuai dengan apa yang dinyatakan oleh Gronlund (2009) validitas didefinisikan sebagai interpretasi yang diperoleh dari hasil penilaian. Dalam penelitian ini validitas instrumen menggunakan corrected item Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) 273 \| Vol. 1 No. 8 2023 Madani : Jurnal Ilmiah Multidisipline total correlation. Uji coba validitas instrumen pada penelitian ini menggunakan internal consistency. y Alat ukur variabel konsep diri disusun berdasarkan dimensi dari teori Carl Rogers (1959) yaitu, harga diri, diri ideal, dan citra diri. Total aitem terdiri dari 18 butir pernyataan, dinyatakan valid 18 aitem. Proses uji validitas aitem dilakukan dua kali dengan rentang skor uji beda sebesar 0,317-0,663. Sementara itu, menurut Lauster (2012), ada empat dimensi yang membentuk alat ukur kepercayaan diri; percaya pada kemampuan sendiri, bertindak mandiri dalam mengambil keputusan, memiliki rasa positif terhadap diri sendiri, dan berani menyatakan pendapat. y p p .Total aitem terdiri dari 32 aitem, aitem yang dinyatakan valid sebanyak 16 butir aitem. Proses uji validitas aitem dilakukan dua kali, dengan rentang skor uji beda sebesar 0,303-0,472. Tabel 2. Uji Validitas Variabel Skor Validitas Keterangan Konsep diri 0,317-0,663 Valid Kepercayaan diri 0,303 – 0,472 Valid Dalam hal ini aitem di nyatakan valid dengan nilai minimum 0,30, sehingga kedua variabel konsep diri dan kepercayaan diri dinyatakan valid. Total Peneliti juga melakukan pengujian reliabilitas dengan menggunakan Alpha Cronbach. Hasil dari uji reliabilitas seperti terlihat pada tabel dibawah ini. p p Tabel 3. Reliabilitas skala penelitian Variabel Skor reliabilitas Keterangan Konsep diri 0,822 Reliabel Kepercayaan diri 0,777 Reliabel Alat ukur dinyatakan reliabel apabila p>0,6, jika nilai Alpha Cronbach >0,6 maka kuesioner dianggap reliabel atau konsisten, tetapi jika nilai Alpha Cronbach <0,6 maka kuesioner dianggap tidak reliabel atau tidak konsisten (Sujarweni, 2014). Berdasarkan dari tabel diatas pada nilai skor alat ukur variabel kepercayaan diri 0,777 dan pada variabel konsep diri memiliki nilai 0,822. Untuk itu, dapat dikatakan alat ukur pada penelitian ini reliabel karena skor p>0,6. p Data kemudian diolah dengan menggunakan analisis statistik, menggunakan metode regresi sederhana (rsimple regression) agar dapat menentukan apakah konsep diri mempengaruhi kepercayaan diri dengan menggunakan program SPSS for Windows. Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) 2023 Madani : Jurnal Ilmiah Multidisipline Profil Demografis Tabel 5. Karakteristik Responden Karakteristik Responden Konsep diri Kepercayaan diri Mean SD Sign. Mea n SD Sig n. Gender .952 .061 Pria 2.75 0.33 2.73 0.29 Wanita 2.75 0.35 2.80 0.33 Fakultas .443 .087 Hukum 2.82 0.32 2.76 0.31 Ekonomi 2.84 0.43 2.85 0.37 Psikologi 2.71 0.33 2.60 0.30 Ilmu teknik 2.71 0.29 2.81 0.28 Ilmu pendidikan 2.74 0.28 2.72 0.29 Ilmu komunikasi 2.78 0.34 2.82 0.29 Ilmu komputer 2.61 0.25 2.81 0.25 Status lokasi tempat tinggal .344 .527 Penduduk Bekasi 2.73 0.31 2.77 0.29 Penduduk Jakarta 2.76 0.38 2.78 0.36 Perantau/pendatang 2.83 0.32 2.71 0.28 Usia .454 .943 18 – 21 tahun 2.76 0.35 2.76 0.32 22 – 25 tahun 2.74 0.32 2.78 0.30 Semester . 277 .198 Semester 2 2.84 0.40 2.66 0.42 Semester 4 2.69 0.35 2.75 0.26 Semester 6 2.78 0.30 2.82 0.28 Semester 8 2.74 0.34 2.76 0.32 l.o.s p< 0,05 Berdasarkan tabel di atas, terlihat bahwa dari 2 (dua) variabel konsep diri dan kepercayaan diri pada berbagai karakteristik, baik karakteristik gender, fakultas, lokasi tempat tinggal, usia dan semester tidak memiliki perbedaan yang signifikan. Dengan perkataan lain, meskipun terlihat terdapat perbedaan skor pada ke dua variabel tersebut, tetapi tidak dapat diambil kesimpulan adanya perbedaan karena tidak signifikan. Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) HASIL DAN PEMBAHASAN Analisis Deskriptif Pada penelitian ini menggunakan skala linier dengan nilai skor 1 sampai 4, secara umum hasil menunjukkan bahwa konsep diri memiliki mean 2,75, median 2,72 kepercayaan diri memiliki mean 2,77, median 2,81 Tabel 4. Analisis Deskriptif Variabel Mean Median S.D Konsep diri 2.75 2.72 0.34 Kepercayaan diri 2,77 2,81 0,31 274 \| Vol. 1 No. 8 2023 Madani : Jurnal Ilmiah Multidisipline 2023 Madani : Jurnal Ilmiah Multidisipline l.o.s (p<0,01), p KonDi: konsep diri, KepDi: kepercayaan diri, Smtr:semester. Hasil analisis interkorelasi menunjukkan bahwa kepercayaan diri dan konsep diri berkorelasi. Sementara itu, tidak terdapat korelasi antara konsepd diri dengan usia dan semester maupun antara kepercayaan diri dengan usia semester. Hasil Analisis Kategorisasi Dari hasil kategorisasi yang telah dilakukan, ditemukan bahwa mayoritas mahasiswa di Universitas perguruan tinggi X di Bekasi memiliki tingkat kepercayaan diri yang sedang sebesar 81.7%. Artinya mahasiswa sebagian besar dari Universitas perguruan tinggi X di Bekasi memiliki rasa kepercayaan diri yang sedang. pada konsep diri mahasiswa di Universitas X perguruan tinggi di Bekasi berada di kategori sedang sebesar 74.9%. Artinya mahasiswa sebagian besar mampu menilai positif terhadap keadaan dirinya dan merasa yakin dengan kemampuan yang dimiliki individu sehingga mampu menyesuaikan perilakunya pada lingkungan sekitarnya. Analisis Interkorelasi Untuk mengetahui apakah terdapat korelasi antar variabel, maka dilakukan analisis interkorelasi dengan menggunakan analisis Pearson Correlation. Untuk mengetahui apakah terdapat korelasi antar variabel, maka dilakukan analisis elasi dengan menggunakan analisis Pearson Correlation. Tabel 6. Analisis interkorelasi M SD KonDi KepDi Usia Smtr KonDi 2.75 0.34 1 .000* -.030 -.041 KepDi 2.77 0.31 .000 1 .033 .056 Usia - - -.030 .033 1 .408 Smtr - - -,041 .056 .408 1 275 \| Vol. 1 No. 8 2023 2023 Madani : Jurnal Ilmiah Multidisipline l.o.s (p<0,01), 276 \| Vol. 1 No. 8 Azwar, S. (2017). Reliabilitas dan Validitas. Yogyakarta: Pustaka Belajar Afif, A. (2015). Teori Identitas Sosial.Yogyakarta: UII Press. Agustiani, Hendrianti. (2006). Psikologi perkembangan: Pendekatan Ekologi Kaitannya dengan Konsep Diri dan Penyesuaian Diri pada Remaja Bandung: Refika Aditama PEMBAHASAN Penelitian ini menunjukkan bahwa konsep diri memiliki efek positif dan signifikan terhadap kepercayaan diri mahasiswa.. Penelitian ini selaras dengan peneltiian yang dilakukan oleh Sumiarsih dan Novita (2021) pada murid SD kelas v dan VI, yang menunjukkan keterkaitan positif antara kepercayaan diri dan konsep diri. Selain itu, studi ini juga mendukung penelitian sebelumnya yang telah dilakukan oleh peneliti terdahulu (Suhardhani & Siti ,2017; Eliza, 2019; Sari, & Khoirunnisa, 2021) yang menunjukkan bahwa kepercayaan diri dan konsep diri berkorelasi positif dan signifikan.. p y p p g Penelitian ini memiliki beberapa keterbatasan antaral lain 1) penelitian ini hanya dilakukan pada universitas X di Bekasi, sehingga tidak dapat digeneralisasikan. Untuk itu, penelitian selanjutnya dapat dilakukan di Universitas lain baik universitas negeri maupun universitas swasta di lokasi lain untuk dapat memeroleh data yang lebih akurat. 2) Keterbatasan kedua adalah berhubungan dengan variabel peneltian. Pada penelitian ini hanya menggunakan 2 (dua) variabel, yaitu konsep diri dan kepercayaan diri, dan hasil menunjukkan bahwa terdapat variabel lain yang dapat memengaruuhi kepercayaan diri. Untuk itu, penelitian selanjutnya disarankan dengan menggunakan variabel yang berbeda. Pada penelitian ini terlihat tidak terdapat perbedaan yang sigifikan dari nilai rerata pada konsep diri dan kepercayaan diri baik dari aspek gender, usia, fakultas, semester maupun asal lokasi mahasiswa. Meskipun demikian, bisa saja bila penelitian dilakukan di tempat lain akan memeroleh hasil yang berbeda. Analisis Uji Hipotesis Peneliti menggunakan analisis statistik dengan menggunakan metode simple regression sesuai dengan penelitian ini, agar dapat mengetahui apakah terdapat pengaruh dari konsep diri terhadap kepercayaan diri. Uji Analisis Regresi Tabel 7. Hasil uji regresi ANOVAa Model Sum of squares df Mean Square F Sig. 1 Regression 1539.338 1 1539.338 78.919 .000b Residual 5090.882 261 19.505 Total 6630.221 262 a. Dependent Variable: Kepercayaan diri b. Predictors: (Constant), Konsep diri Uji Analisis Regresi Berdasarkan tabel diatas, dari output tersebut di ketahui bahwa nilai F hitung = 78.919 dengan tingkat signifikansi sebesar 0,000 p<0,1 maka dapat diartikan bahwa variabel konsep diri (X) memiliki pengaruh terhadap variabel kepercayaan diri (Y). Tabel 8: Koefisien Model Unstandardized Coefficients Standardized Coefficients B Std. Error Beta t Sig. 1 (Constant) 24.753 2.221 11.147 .000 Konsep diri .395 .044 .482 8.884 .000 Model r R square Adjusted R square Std. Error estimate 1 .482a .232 .229 4.41648 Berdasarkan hasil pengujian, menunjukkan bahwa konsep diri memberi pengaruh sumbangan sebanyak 23,2% terhadap kepercayaan diri. Berdasarkan tabel koefisiensi yang Berdasarkan hasil pengujian, menunjukkan bahwa konsep diri memberi pengaruh gan sebanyak 23,2% terhadap kepercayaan diri. Berdasarkan tabel koefisiensi yang Pengaruh Konsep Diri terhadap (Syifa Asha Umarta, dkk.) 276 \| Vol. 1 No. 8 2023 Madani : Jurnal Ilmiah Multidisipline telah di lampirkan diatas, diperoleh persamaan regresi sederhana yaitu Y= 24,753 + 0,395 X. Pada hasil nilai F hitung= 78.919 dengan tingkat signifikansi sebesar 0,000 p<0,1 maka dapat diartikan bahwa variabel konsep diri (X) memiliki pengaruh terhadap variabel kepercayaan diri (Y). Uji hipotesis yang telah dilakukan pada penelitian ini adalah dengan melakukan uji regresi linear sederhana.. Pada uji regresi F hitung sebesar 78,919 dengan signifikansi sebesar 0,000 (p< 0,05) yaitu dapat diartikan bahwa konsep diri memiliki pengaruh terhadap kepercayaan diri. Nilai pada tabel R yaitu 0,482 artinya nilai koefisien regresi bernilai positif (+) sehingga dapat disebutkan arah pengaruh konsep diri terhadap kepercayaan diri adalah positif, dan hipotesis diterima. Kesimpulannya adalah penelitian ini menunjukkan bahwa konsep diri memengaruhi kepercayaan diri pada mahasiswa Universitas Perguruan Tinggi X di Bekasi. Untuk itu, dalam upaya meningkatkan kepercayaan diri dapat dilakukan dengan cara meningkatkan konsep diri. KESIMPULAN Dengan mempertimbangkan hasil penelitian, dapat disimpulkan bahwa konsep diri mempengaruhi kepercayaan diri siswa. Untuk itu, bila ingin meningkatkan kepercayaan diri seseorang, dalam hal ini mahasiswa maka konsep diri perlu ditingkatkan. Workshop dan pelatihan adalah dua metode untuk meningkatkan konsep diri. Untuk dapat memeroleh hasil yang lebih lengkap, pengulangan penelitian dengan berbagai variabel serta pada populasi yang berbeda diharapkan akan dapat memperkaya hasil mengenai pengaruh dari konsep diri terhadap kepercayaan diri yang tidak hanya pada kalangan mahasiswa saja. 2023 Madani : Jurnal Ilmiah Multidisipline Arnett, J. J. (2015). The Winding Road from The Late Teens through The Twenties: Emerging Adulthood 2nd Edition. New York: Oxford University Press. g g y Azizi, K. (2014). Hubungan Konsep Diri Dengan Rasa Percaya Diri. Agus Hasan Burns, R. (1993). Konsep Kepercayaan Diri: Teori, Pengukuran, Perkembangan & Perilaku (penerjemah: Eddy). Jakarta: Arcan Da vies, P. (2006). Meningkatkan Rasa Peraya Diri. Jogjakarta: Torrent Book Eliza, M. (2019). Hubungan Antara Konsep Diri Dengan Kepercayaan Diri Pada Mahasiswa Dalam Menghadapi Sidang Skripsi (Doctoral dissertation, UIN Raden Intan Lampung). Feist dan Feist. (2010). Teori Kepribadian. Jakarta : Salemba Humanika. Firdaus, I. C. 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https://openalex.org/W2888500444	https://pure.rug.nl/ws/files/64528547/Versluis2018_Article_L1CAMExpressionInUterineCarcin.pdf	English	null	L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial–mesenchymal transition	Virchows Archiv	2,018	cc-by	5,989	Abstract Uterine carcinosarcoma (UCS) has been proposed as a model for epithelial–mesenchymal transition (EMT), a process charac- terized by a functional change facilitating migration and metastasis in many types of cancer. L1CAM is an adhesion molecule that has been involved in EMT as a marker for mesenchymal phenotype. We examined expression of L1CAM in UCS in a cohort of 90 cases from four different centers. Slides were immunohistochemically stained for L1CAM and scored in four categories (0%, < 10%, 10–50%, and > 50%). A score of more than 10% was considered positive for L1CAM. The median age at presentation was 68.6 years, and half of the patients (53.3%) presented with FIGO stage 1 disease. Membranous L1CAM expression was positive in the epithelial component in 65.4% of cases. Remarkably, expression was negative in the mesenchymal component. In cases where both components were intermingled, expression limited to the epithelial component was confirmed by a double stain for L1CAM and keratin. Expression of L1CAM did not relate to overall or disease-free survival. Our findings suggest L1CAM is either not a marker for the mesenchymal phenotype in EMT, or UCS is not a good model for EMT. Keywords Endometrial neoplasm . Neural cell adhesion molecule L1 . L1CAM . Epithelial–mesenchymal transition . Histology Keywords Endometrial neoplasm . Neural cell adhesion molecule L1 . L1CAM . Epithelial–mesenchymal transition . Histology Keywords Endometrial neoplasm . Neural cell adhesion molecule L1 . L1CAM . Epithelial–mesenchym L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial–mesenchymal transition Received: 19 July 2018 /Revised: 14 August 2018 /Accepted: 15 August 2018 # The Author(s) 2018 Received: 19 July 2018 /Revised: 14 August 2018 /Accepted: 15 August 2018 # The Author(s) 2018 L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial-mesenchymal transition Versluis, Mac; Plat, A; de Bruyn, M; Matias-Guiu, X; Trovic, J; Krakstad, C; Nijman, H W; Bosse, T; de Bock, G H; Hollema, H Published in: Virchows Archiv : an International Journal of Pathology L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial-mesenchymal transition Versluis, Mac; Plat, A; de Bruyn, M; Matias-Guiu, X; Trovic, J; Krakstad, C; Nijman, H W; Bosse, T; de Bock, G H; Hollema, H Published in: Virchows Archiv : an International Journal of Pathology DOI: 10.1007/s00428-018-2444-8 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2018 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Versluis, M., Plat, A., de Bruyn, M., Matias-Guiu, X., Trovic, J., Krakstad, C., Nijman, H. W., Bosse, T., de Bock, G. H., & Hollema, H. (2018). L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial-mesenchymal transition. Virchows Archiv : an International Journal of Pathology, 473(5), 591-598. https://doi.org/10.1007/s00428-018-2444-8 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). 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Download date: 24-10-2024 Download date: 24-10-2024 Virchows Archiv https://doi.org/10.1007/s00428-018-2444-8 ORIGINAL ARTICLE L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial–mesenchymal transition MAC Versluis1 & A Plat1 & M de Bruyn1 & X Matias-Guiu2,3,4 & J Trovic5,6 & C Krakstad5,6 & HW Nijman1 & T Bosse7 & GH de Bock8 & H Hollema9 ORIGINAL ARTICLE AL ARTICLE Introduction as well as a mesenchymal component [1, 5]. Various models have been proposed to explain this feature ranging from collision of two separate and original components to conversion of a common monoclonal neoplasm of epithe- lial (carcinomatous) origin [5–8]. Currently, most studies support the theory of conversion of an endometrial carci- noma into a cancer consisting of an epithelial and a mes- enchymal component. Uterine carcinosarcoma (UCS) is a rare subtype of endo- metrial cancer (EC) with a poor prognosis. Approximately 60% of cases present with advanced disease, and recur- rence occurs in approximately 50% of patients [1–4]. Accordingly, 5-year survival ranges from 33 to 39%. Histology is peculiar because it contains both an epithelial 5 Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway 6 Department of Clinical Science, Center for Cancer Biomarkers, University of Bergen, Bergen, Norway 7 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands 8 Department of Epidemiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 9 Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands * MAC Versluis m.a.c.versluis@umcg.nl 1 Department of Gynecology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands 2 Department of Pathology and Molecular Genetics/Oncologic Pathology Group, Arnau de Vilanova University Hospital, IRBLleida, University of Lleida, Lleida, Spain 3 Centro de Investigación Biomédica en Red de Oncología (CIBERONC), Madrid, Spain 4 Department of Pathology, University Hospital of Bellvitge, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Catalonia, Spain 5 Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway 6 Department of Clinical Science, Center for Cancer Biomarkers, University of Bergen, Bergen, Norway 7 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands 8 Department of Epidemiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 9 Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 5 Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway 6 Department of Clinical Science, Center for Cancer Biomarkers, University of Bergen, Bergen, Norway 7 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands 8 Department of Epidemiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 9 Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 5 Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway Study population Four-micrometer formalin-fixed slides were assembled from four separate centers (Haukeland University hospital Bergen, Norway; University hospital Lleida, Spain; Leiden University Medical Center, The Netherlands; and University Medical Center Groningen, the Netherlands). Sufficient material from 88 primary tumors and 10 metastases was available. With an overlap of 8 cases, this added up to a total of 90 cases with UCS diagnosed between 1980 and 2012 to be included in this study. Data concerning age at diagnosis, International Federation of Gynecology and Obstetrics (FIGO) stage as well as patient and tumor characteristics concerning age at diagnosis, histology of both components (homologue or heterologue for the epithelial component), differentiation grade, LVSI, were delivered by each participating center. All material was revised at the University Medical Center Groningen by HH and MV blinded from findings at the pri- mary center. For some cases, clinicopathological information was missing and could not be retrieved from the slides, for example, when information on lymphovascular space in- volvement was missing. Follow-up data was provided by each center in an anonymized dataset and completed until October 2014. Datasets were combined into a final password-protected database. Patient identity was protected by study-specific pa- tient numbers. Informed consent was obtained according to local protocol in each participating center. A possible marker for a mesenchymal phenotype is L1 cell adhesion molecule (L1CAM), a transmembrane adhe- sion molecule important for embryonic development [17]. The extracellular domain interacts with other binding mol- ecules and integrins expressed on either the same or other cells. The cytoplasmic tail mainly interacts with cytoskel- etal proteins. Several studies support involvement of L1CAM in EMT [18–21]. Exposure of both endometrial cancer (EC) and breast cancer cell lines to transforming growth factor B1 (TGF-B1), a known inducer of EMT, results in increased expression of L1CAM [19, 21]. Huszar et al. found that increased expression of L1CAM relates to reduced membranous expression of E-cadherin. L1CAM could be a marker for a mesenchymal phenotype of EMT in UCS. Expression of L1CAM previously described as immu- nohistochemical staining of more than 10% is absent in normal endometrium [22]. In addition, expression of L1CAM is related to metastasis and poor prognosis in many cancers, including EC [17, 22–28]. In early-stage EC with endometrioid histology, expression of L1CAM is related to unfavorable pathological findings, distant recurrence and poor survival [22, 23]. Study population Correspondingly, in high grade EC and non-endometrioid EC (NEEC), L1CAM expression is related to unfavorable pathological findings such as advanced stage and lymphovascular space invasion (LVSI) as well as distant recurrence but not survival [26–28]. * MAC Versluis m.a.c.versluis@umcg.nl * MAC Versluis m.a.c.versluis@umcg.nl 6 Department of Clinical Science, Center for Cancer Biomarkers, University of Bergen, Bergen, Norway 6 Department of Clinical Science, Center for Cancer Biomarkers, University of Bergen, Bergen, Norway 1 Department of Gynecology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands 2 Department of Pathology and Molecular Genetics/Oncologic Pathology Group, Arnau de Vilanova University Hospital, IRBLleida, University of Lleida, Lleida, Spain 3 Centro de Investigación Biomédica en Red de Oncología (CIBERONC), Madrid, Spain 4 Department of Pathology, University Hospital of Bellvitge, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Catalonia, Spain 1 Department of Gynecology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands 2 Department of Pathology and Molecular Genetics/Oncologic Pathology Group, Arnau de Vilanova University Hospital, IRBLleida, University of Lleida, Lleida, Spain 3 Centro de Investigación Biomédica en Red de Oncología (CIBERONC), Madrid, Spain 4 Department of Pathology, University Hospital of Bellvitge, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Catalonia, Spain 1 Department of Gynecology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands 7 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Pathology and Molecular Genetics/Oncologic Pathology Group, Arnau de Vilanova University Hospital, IRBLleida, University of Lleida, Lleida, Spain 8 Department of Epidemiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Virchows Arch Methods Because UCS contains both an epithelial and mesenchy- mal component, UCS has been suggested as a model for epithelial–mesenchymal transition (EMT) [5, 9–14]. EMT is a process that is characterized by a functional change in tumor cells that facilitates migration and metastasis [11, 15]. By loss of cell polarity and cell–cell junctions, and by reorganization of their cytoskeleton, tumor cells acquire the ability to migrate. This transformation is thought to be essential to metastasis in gynecologic cancers as well as other tumors [13, 16]. In a study on 76 cases of UCS, Castilla et al. found a different expression of epithelial and mesenchymal markers between the two components. Expression of E-cadherin for example was limited to the epithelial component. Histology Slides, 4-μm formalin-fixed paraffin-embedded sections were immunohistochemically stained as described by Bosse et al. [23]. BBriefly, formalin fixed paraffin-embedded (FFPE) tis- sue blocks were cut into 4 μm slides and mounted on Starfrost slides. Antigen retrieval was achieved by microwave oven procedure in 10 mmol/L citrate buffer, pH 6. Sections were incubated overnight with primary monoclonal antibodies against L1CAM (CD171, clone 14.10, SIG-3911, Convance Inc., lot number D13KF03087, Biolegend, San Diego, USA) in a 1:500 dilution. Sections were incubated and stained for 30 min using a secondary antibody (Poly-HRP-GAM/R/R; DPV0110HRP; ImmunoLogic, Duiven, The Netherlands). The slides were counterstained with Mayer’s Haematoxylin, dehydrated and mounted. Omission of the primary antibody was used as a negative control and a highly L1CAM- expressing serous ovarian cancer as an external positive control.^ i h i diffi l diff i b We hypothesize that L1CAM could be involved in EMT in UCS as a marker for the mesenchymal phenotype and may relate to metastasis. Therefore, we aimed to evaluate L1CAM expression in a large cohort of 90 UCS patients assembled in 4 oncologic centers participating in the European Network for Individualized Treatment of Endometrial Cancer (ENITEC) collaboration. Expression was evaluated separately in the epithelial and the mesen- chymal component and related to patient and tumor char- acteristics and survival. In eight cases, it was difficult to differentiate between L1CAM expression by the epithelial or mesenchymal compo- nents as both components were intermingled. Therefore, these selected cases were double stained for both L1CAM and Virchows Arch Table 1 Patient and tumor characteristics in 90 cases of uterine carcinosarcoma (UCS). FIGO = International Federation of Gynecology and Obstetrics #345779) diluted 1:5 for 1 h at room temperature. Sections were then incubated with secondary antibody (RAMbio; Dako) and tertiary antibody (streptavidin-AP; Dako), both diluted 1:300, for 30 min each at room temperature. Cytokeratin staining was visualized using Fast Blue BB/ Napthhol-AS-MX-phosphate (Sigma, F3378) for 30 min at room temperature. Sections were rinsed with demineralized water and mounted with Kaiser’s glycerol gelatine (109242; Merck). Omission of the primary antibody was used as a neg- ative control and tonsil tissue as a positive control. carcinosarcoma (UCS). Histology FIGO = International Federation of Gynecology and Obstetrics Number % or IQR Median age (years) 68.6 62.0–75.5 FIGO stage (2009) - I and II 50 55.36 - III and IV 40 44.4 Myometrial invasion - Less than 50% 43 53.1 - More than 50% 38 46.9 - Unknown 9 Lymph node status - Negative 36 78.3 - Positive 10 21.7 - Unknown 44 Lymphovascular space invasion (LVSI) - No 24 36.4 - Yes 42 63.6 - Missing 24 Differentiation grade carcinoma component - Grade 2 6 8.3 - Grade 3 63 87.5 - Undifferentiated 3 4.2 - Unknown 18 Histologic type carcinoma component - Endometrioid 27 39.1 - Non-endometrioid 42 60.9 - Unknown 21 Histologic type sarcoma component - Homologue 39 53.4 - Heterologue 34 46.6 - Unknown 17 Evaluation of immunohistochemistry Evaluation of immunohistochemistry was performed by two independent investigators (MV and AP), blinded for clinical outcome data. Expression of L1CAM in either component was scored as the percentage of positive membranous staining and categorized into four groups: 0%, 0–10%, 10–50%, or > 50% as described by Meier et al. [29]. In conformity with previous publications, tumors with more than 10% expression of L1CAM were considered positive [23, 29]. There was no disagreement in evaluation of the mesenchymal component. There was disagreement in evaluation of 21 cases of the car- cinomatous component mainly on a score of either 3 or 4. Disagreement was resolved by consensus. Location of L1CAM staining was scored as expression at the tumor center, tumor margin, or diffusely over the tumor. - Grade 2 Statistical analysis All statistical analyses were done by using SPSS (version 23, IBM Statistics, Chicago USA). For analysis, age was dichot- omized into 65 and below versus above 65 years as this is an important cutoff in treatment of endometrial cancer. Recurrence was classified as local, pelvic, or distant. Disease-free survival (DFS) was defined as time until any recurrence or death of disease and disease-specific survival (DSS) as time until death of disease. Associations between patient and tumor characteristics were compared using logistic regression analyses. For survival analysis, a relation between L1CAM expression and DFS, DSS, or distant recurrence was examined using Cox regression analyses. Patients without ev- idence of disease were censored at last date of follow-up. - Unknown cytokeratin-8 as expression of cytokeratin is limited to the epithelial component. Tissue sections were dewaxed in xylene and hydrated in a graded series of alcohol to tap water. Antigen retrieval was performed in 10 mM EDTA pH 8.0 in a microwave at 400 W for 15 min. Endogenous peroxidase was blocked by incubating sections for 30 min in 1% H2O2. Primary antibody against L1CAM (anti-CD171, Biolegend, Sig-3911-1000, San Diego, USA) was applied in a 1:500 di- lution and left to incubate overnight at 4 °C. Subsequently, tissue sections were incubated with secondary antibody (RAM-HRP; Dako, Glostrop, Denmark) and tertiary antibody (GAR-HRP; Dako, Glostrop, Denmark), both diluted at 1:100, for 30 min each at room temperature. L1CAM staining was visualized with 3,3′-diaminobenzidine. After washing the sections in demineralized water, they were incubated with the antibody against cytokeratin-8 (CAM 5.2, BD Biosciences, cytokeratin-8 as expression of cytokeratin is limited to the epithelial component. Tissue sections were dewaxed in xylene and hydrated in a graded series of alcohol to tap water. Antigen retrieval was performed in 10 mM EDTA pH 8.0 in a microwave at 400 W for 15 min. Endogenous peroxidase was blocked by incubating sections for 30 min in 1% H2O2. Primary antibody against L1CAM (anti-CD171, Biolegend, Sig-3911-1000, San Diego, USA) was applied in a 1:500 di- lution and left to incubate overnight at 4 °C. Subsequently, tissue sections were incubated with secondary antibody (RAM-HRP; Dako, Glostrop, Denmark) and tertiary antibody (GAR-HRP; Dako, Glostrop, Denmark), both diluted at 1:100, for 30 min each at room temperature. L1CAM staining was visualized with 3,3′-diaminobenzidine. After washing the sections in demineralized water, they were incubated with the antibody against cytokeratin-8 (CAM 5.2, BD Biosciences, Fig. 1 L1CCAM staining in carcinosarcoma. a L1CAM expression scored as 0%. b L1CAM expression scored as less than 10%. c L1CAM expression scored as 10–50%,. d L1CAM expression scored as more than 50%. All figures are presented at × 10 magnification Population Tissue from the primary tumor was available in 88 cases. In 10 cases, metastatic tissue was available. With an overlap of 8 cases (primary as well as metastatic tissue), this added up to a Virchows Arch Fig. 1 L1CCAM staining in carcinosarcoma. a L1CAM expression scored as 0%. b L1CAM expression scored as less than 10%. c L1CAM expression scored as 10–50%,. d L1CAM expression scored as more than 50%. All figures are presented at × 10 magnification Immunohistochemistry All figures are presented at × 10 magnification Double stain for L1CAM and keratin in case 9. e HE stain for case 14. This case was originally scored as L1CAM expression > 50% in the epithelial component and < 10% in the mesenchymal component. f Double stain for L1CAM and keratin in case 14. All figures are presented at × 10 magnification Double stain for L1CAM and keratin in case 9. e HE stain for case 14. This case was originally scored as L1CAM expression > 50% in the epithelial component and < 10% in the mesenchymal component. f Double stain for L1CAM and keratin in case 14. All figures are presented at × 10 magnification Fig. 2 Examples of double stain for L1CAM (brown) and keratin (blue). a HE stain of case 8. This case was originally scores as L1CAM expres- sion > 50% in the epithelial component and 0% in the mesenchymal component. b Double stain for L1CAM and keratin in case 8. c HE stain for case 9. This case was originally scores as L1CAM expression > 50% in the epithelial component and 0% in the mesenchymal component. d 95%CI 0.96–3.34), and differentiation grade (HR 3.81 95%CI 1.13–12.80). LVSI was not a significant predictor for DFS. Findings were similar for DSS. Expression of L1CAM did not relate to either DFS (HR 1.48 95%CI 0.74–3.00) or DSS (HR 1.48 95%CI 0.74–3.00). There was also no relation between L1CAM expression and distant recurrence (HR1.02 95%CI 0.45–2.30). expression was scored less than 10%. In some of these cases, both components were intermingled and therefore difficult to distinguish. To differentiate between L1CAM expression by either component, a double stain for L1CAM and keratin was performed in eight cases. In all eight cases, L1CAM expres- sion was limited to cells expressing keratin. Figure 2 shows examples of double staining for L1CAM. Immunohistochemistry total of 90 cases to be evaluated. Not all tissue contained both components. The epithelial component was available in 81 cases and the mesenchymal component in 83 cases. Patient and tumor characteristics are shown in Table 1. The median age at diagnosis was 68.6 years (IQR 62.0–75.5). A small majority of cases presented with FIGO stage 1 disease (53.3%), most other cases presented with stage 3a or 3c disease (14.4 vs 12.2%). LVSI was observed in 53.8%, and non-endometrioid histology of the epithelial component was 56.5%. Figure 1 shows examples of L1CAM expression in both com- ponents. Expression was not limited to either the tumor center or margin. Table 2 shows the results of L1CAM staining. The epithe- lial component was L1CAM positive in a majority of cases (65.4%). Remarkably, the mesenchymal component was de- termined as negative (< 10%) for L1cam in all cases. In 86.7%, no expression was found. In 13.3%, L1CAM Table 2 Expression of L1CAM in both components in 88 primary UCS tumors and 7 metastatic tumors Epithelial component, primary tumor, n (%) Mesenchymal component, primary tumor, n (%) Epithelial component, metastasis, n (%) Mesenchymal component, metastasis, n (%) 0% expression 16 (20.2) 72 (86.7) 1 (12.5) 4 (80) < 10% expression 11 (15.2) 11 (13.3) 3 (37.5) 1 (20) 10–50% expression 22 (27.8) 4 (50.0) > 50% expression 30 (38.0) Component not available 9 5 2 5 Table 2 Expression of L1CAM in both components in 88 primary UCS tumors and 7 metastatic tumors of L1CAM in both components in 88 primary UCS tumors and 7 metastatic tumors Virchows Arch Fig. 2 Examples of double stain for L1CAM (brown) and keratin (blue). a HE stain of case 8. This case was originally scores as L1CAM expres- sion > 50% in the epithelial component and 0% in the mesenchymal component. b Double stain for L1CAM and keratin in case 8. c HE stain for case 9. This case was originally scores as L1CAM expression > 50% in the epithelial component and 0% in the mesenchymal component. d Double stain for L1CAM and keratin in case 9. e HE stain for case 14. This case was originally scored as L1CAM expression > 50% in the epithelial component and < 10% in the mesenchymal component. f Double stain for L1CAM and keratin in case 14. Discussion An explanation for the ab- sence of a relation could be the already poor survival in co- horts of NEEC and the high expression of L1CAM in NEEC as compared to EEC as suggested by van der Putten et al. [27]. Remarkably, L1CAM expression is not expressed in the mes- enchymal component. Our working hypothesis was that L1CAM expression could act as a marker for a mesenchymal component in UCS. Several other studies describe increased expression of L1CAM after induction of EMT. In endometrial cancer cell lines, TGF-B1 induces expression of L1CAM and EMT [19, 21]. Exposure to TGF-B1 results in increased ex- pression of L1CAM through upregulation of the EMT tran- scription factor Slug. In turn, expression of L1Cam relates to cell migration and invasion [21]. TGF-B1-induced L1CAM expression is related to decreased expression of E-cadherin, and E-cadherin expression is limited to the epithelial compo- nent in UCS [10, 19]. Considering our findings, L1CAM is of L1Cam positive tumors and no relation between L1CAM expression and survival. A strong relation between expression and survival is therefore unlikely. An explanation for the ab- sence of a relation could be the already poor survival in co- horts of NEEC and the high expression of L1CAM in NEEC as compared to EEC as suggested by van der Putten et al. [27]. Remarkably, L1CAM expression is not expressed in the mes- enchymal component. Our working hypothesis was that L1CAM expression could act as a marker for a mesenchymal component in UCS. Several other studies describe increased expression of L1CAM after induction of EMT. In endometrial cancer cell lines, TGF-B1 induces expression of L1CAM and EMT [19, 21]. Exposure to TGF-B1 results in increased ex- pression of L1CAM through upregulation of the EMT tran- scription factor Slug. In turn, expression of L1Cam relates to cell migration and invasion [21]. TGF-B1-induced L1CAM expression is related to decreased expression of E-cadherin, and E-cadherin expression is limited to the epithelial compo- nent in UCS [10, 19]. Considering our findings, L1CAM is either not a marker for the mesenchymal phenotype in EMT, or UCS is not a good model for EMT. However, this does not exclude a relevant contribution of L1CAM expression in an early stage of EMT where cancer cells gain motility and invasive properties. This could be by direct function as an adhesion molecule or indirect by acting as a signaling molecule [17, 20]. Table 3 Patient and tumor characteristics and L1CAM expression. Data exclude missing values. FIGO = International Federation of Gynecology and Obstetrics Discussion L1CAM is frequently expressed in UCS, but expression in UCS is limited to the epithelial component. Expression does not relate to patient characteristics, recurrence, or survival. Table 3 shows patient and tumor characteristics stratified for L1CAM expression. Logistic regression showed no relation between patient or tumor characteristics and L1CAM expres- sion. L1CAM expression survival in the overall cohort was poor with a median DFS of 2.83 years (95%CI 0.80–4.87) and DSS of 3.30 (1.10–5.50). Known prognostic variables related to DFS in univariate Cox regression analysis: FIGO stage (HR 1.67 95%CI 1.30–2.14), myometrial invasion (HR 1.80 Expression of L1CAM in UCS is in accordance with other studies on L1CAM expression in aggressive histologic sub- types of EC such as non-endometrioid EC. These studies de- scribe an L1CAM expression of more than 55% and no rela- tion with survival [22, 23, 27]. We also find a high percentage Virchows Arch Table 3 Patient and tumor characteristics and L1CAM expression. Data exclude missing values. FIGO = International Federation of Gynecology and Obstetrics Epithelial component (n = 79) L1CAM negative (%) L1cAM positive (%) Odds ratio (95% CI) Age below/above 65 - 65 and below 12 (44.4) 19 (37.3) Reference 0.74 - Above 65 15 (55.6) 32 (62.7) (0.29–1.19) FIGO stage (2009) - I and II 18 (66.7) 28 (53.8) Reference - III and IV 9 (33.3) 24 (46.2) 0.58 (0.22–1.54) Myometrial invasion - Less than 50% 14 (53.8) 26 (54.2) Reference - More than 50% 12 (46.2) 22 (45.8) 1.01 (0.39–2.64) Lymph node status - Negative 14 (87.5) 21 (72.4) Reference - Positive 2 (12.5) 8 (27.6) 0.38 (0.07–2.03) Lymphovascular space invasion (LVSI) - No 13 (58.1) 29 (67.4) Reference - Yes 9 (40.9) 14 (32.6) 1.43 (0.50–4.15) Differentiation grade carcinoma component - Grade 2 3 (13.0) 3 (7.0) Reference - Grade 3 18 (18.3) 39 (90.7) 2.17 (0.40–11.80) - Undifferentiated 2 (8.7) 1 (2.3) 0.50 (0.03–8.92) Histologic type carcinoma component - Endometrioid 11 (52.4) 15 (35.7) Reference - Non-endometrioid 10 (47.6) 27 (64.3) 0.5 (0.17–1.46) Histologic type sarcoma component - Homologue 12 (50.0) 24 (57.1) Reference - Heterologue 12 (50.0) 18 (42.9) 1.33 (0.49–3.65) of L1Cam positive tumors and no relation between L1CAM expression and survival. A strong relation between expression and survival is therefore unlikely. References 1. Cantrell LA, Blank SV, Duska LR (2015) Uterine carcinosarcoma: a review of the literature. Gynecol Oncol 137:581–588 19. Huszar M, Pfeifer M, Schirmer U, Kiefel H, Konecny GE, Ben- Arie A, Edler L, Munch M, Muller-Holzner E, Jerabek-Klestil S, Abdel-Azim S, Marth C, Zeimet AG, Altevogt P, Fogel M (2010) Up-regulation of L1CAM is linked to loss of hormone receptors and E-cadherin in aggressive subtypes of endometrial carcinomas. J Pathol 220:551–561 2. Vorgias G, Fotiou S (2010) The role of lymphadenectomy in uterine carcinosarcomas (malignant mixed mullerian tumours): a critical literature review. Arch Gynecol Obstet 282:659–664 3. Versluis MAC, Pielsticker C, van der Aa MA, de Bruyn M, Hollema H, Nijman HW (2018) Lymphadenectomy and adjuvant therapy improve survival with uterine carcinosarcoma: a large ret- rospective cohort study. Oncology:1–9 20. Kiefel H, Bondong S, Hazin J, Ridinger J, Schirmer U, Riedle S, Altevogt P (2012) L1CAM: a major driver for tumor cell invasion and motility. Cell Adhes Migr 6:374–384 4. Boll D, Verhoeven RH, van der Aa MA, Pauwels P, Karim-Kos HE, Coebergh JW, van Doorn HC (2012) Incidence and survival trends of uncommon corpus uteri malignancies in the Netherlands, 1989- 2008. Int J Gynecol Cancer 22:599–606 21. Kiefel H, Bondong S, Pfeifer M, Schirmer U, Erbe-Hoffmann N, Schafer H, Sebens S, Altevogt P (2012) EMT-associated up- regulation of L1CAM provides insights into L1CAM-mediated integrin signalling and NF-kappaB activation. Carcinogenesis 33:1919–1929 5. Matias-Guiu X, Prat J (2013) Molecular pathology of endometrial carcinoma. Histopathology 62:111–123 22. Zeimet AG, Reimer D, Huszar M, Winterhoff B, Puistola U, Azim SA, Muller-Holzner E, Ben-Arie A, van Kempen LC, Petru E, Jahn S, Geels YP, Massuger LF, Amant F, Polterauer S, Lappi-Blanco E, Bulten J, Meuter A, Tanouye S, Oppelt P, Stroh-Weigert M, Reinthaller A, Mariani A, Hackl W, Netzer M, Schirmer U, Vergote I, Altevogt P, Marth C, Fogel M (2013) L1CAM in early- stage type I endometrial cancer: results of a large multicenter eval- uation. J Natl Cancer Inst 105:1142–1150 6. de Jong RA, Nijman HW, Wijbrandi TF, Reyners AK, Boezen HM, Hollema H (2011) Molecular markers and clinical behavior of uter- ine carcinosarcomas: focus on the epithelial tumor component. Mod Pathol 7. McCluggage WGRS (2009) Mesenchymal uterine tumors and adenomyosis. In: Robboy SJ, Mutter GL, Prat J, Bentley RC, Russell P, Anderson MC (eds) Pathology of the female reproductive tract, 2nd edn. Churchill Livingstone Elsevier, Amsterdam, pp 427–456 23. Discussion At a later stage, motility and invasive properties may become less relevant when tumor cells form a secondary tumor. A strength of this study is a large cohort of 90 UCS cases made possible through an international collaboration. The resulting population covers well the diversity of this type of cancer as can be seen in Table 1. Another strength is the stepwise approach with a double stain for cases where histol- ogy proved difficult. A limitation is the retrospective nature of the study. In conclusion, we describe a large cohort of 90 cases with UCS where the majority of tumors stained positive for L1CAM but where expression is limited to the epithelial Virchows Arch 8. McCluggage WG (2002) Malignant biphasic uterine tumours: car- cinosarcomas or metaplastic carcinomas? J Clin Pathol 55:321–325 component. L1CAM could be involved in development of UCS and EMT at an early stage but is not a marker for the mesenchymal phenotype. Our study provides further insight into the possible mechanism of EMT and metastasis. Expression of L1CAM did not relate to unfavorable patient and tumor characteristics, recurrence, or survival. 9. Guarino M, Rubino B, Ballabio G (2007) The role of epithelial- mesenchymal transition in cancer pathology. Pathology 39:305–318 10. Castilla MA, Moreno-Bueno G, Romero-Perez L, Van De Vijver K, Biscuola M, Lopez-Garcia MA, Prat J, Matias-Guiu X, Cano A, Oliva E, Palacios J (2011) Micro-RNA signature of the epithelial- mesenchymal transition in endometrial carcinosarcoma. J Pathol 223:72–80 Acknowledgements The authors thank all participating centers. Acknowledgements The authors thank all participating centers. 11. Chaffer CL, Weinberg RA (2011) A perspective on cancer cell metastasis. Science 331:1559–1564 Author contributions All authors have made a substantial contribution to conception and execution of the study including acquisitions of data, processing of material, analysis and interpretation of data, as well as writing of the manuscript. All authors approve of publication. 12. Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams ED, Thompson EW (2007) Epithelial-mesenchymal and mesenchymal-epithelial transitions in carcinoma progression. J Cell Physiol 213:374–383 13. Stewart CJ, McCluggage WG (2013) Epithelial-mesenchymal tran- sition in carcinomas of the female genital tract. Histopathology 62: 31–43 Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. 16. Colas E, Pedrola N, Devis L, Ertekin T, Campoy I, Martinez E, Llaurado M, Rigau M, Olivan M, Garcia M, Cabrera S, Gil- Moreno A, Xercavins J, Castellvi J, Garcia A, Ramon y Cajal S, Moreno-Bueno G, Dolcet X, Alameda F, Palacios J, Prat J, Doll A, Matias-Guiu X, Abal M, Reventos J (2012) The EMT signaling pathways in endometrial carcinoma. Clin Transl Oncol 14:715–720 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 17. Altevogt P, Doberstein K, Fogel M (2016) L1CAM in human can- cer. Int J Cancer 138:1565–1576 18. Doberstein K, Harter PN, Haberkorn U, Bretz NP, Arnold B, Carretero R, Moldenhauer G, Mittelbronn M, Altevogt P (2015) Antibody therapy to human L1CAM in a transgenic mouse model blocks local tumor growth but induces EMT. Int J Cancer 136: E326–E339 Compliance with ethical standards 14. Piulats JM, Guerra E, Gil-Martin M, Roman-Canal B, Gatius S, Sanz-Pamplona R, Velasco A, Vidal A, Matias-Guiu X (2017) Molecular approaches for classifying endometrial carcinoma. Gynecol Oncol 145:200–207 The experiments reported here were carried out in agreements with the Declaration of Helsinki principles and in agreement with local legislation at each participating center. 15. Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial- mesenchymal transitions in development and disease. Cell 139: 871–890 Garcia A, Mancebo G, Alameda F, Trovik J, Kopperud RK, Huvila J, Schrauwen S, Koskas M, Walker F, Weinberger V, Minar L, Jandakova E, Snijders MP, van den Berg-van Erp S, Matias-Guiu X, Salvesen HB, Amant F, Massuger LF, Pijnenborg JM (2016) L1CAM expression in endometrial carcinomas: an ENITEC collaboration study. Br J Cancer 115:716–724 References Bosse T, Nout RA, Stelloo E, Dreef E, Nijman HW, Jurgenliemk- Schulz IM, Jobsen JJ, Creutzberg CL, Smit VT (2014) L1 cell Virchows Arch Garcia A, Mancebo G, Alameda F, Trovik J, Kopperud RK, Huvila J, Schrauwen S, Koskas M, Walker F, Weinberger V, Minar L, Jandakova E, Snijders MP, van den Berg-van Erp S, Matias-Guiu X, Salvesen HB, Amant F, Massuger LF, Pijnenborg JM (2016) L1CAM expression in endometrial carcinomas: an ENITEC collaboration study. Br J Cancer 115:716–724 adhesion molecule is a strong predictor for distant recurrence and overall survival in early stage endometrial cancer: pooled PORTEC trial results. Eur J Cancer 50:2602–2610 24. Dellinger TH, Smith DD, Ouyang C, Warden CD, Williams JC, Han ES (2016) L1CAM is an independent predictor of poor surviv- al in endometrial cancer—an analysis of The Cancer Genome Atlas (TCGA). Gynecol Oncol 141:336–340 28. 28. van Gool IC, Eggink FA, Freeman-Mills L, Stelloo E, Marchi E, de Bruyn M, Palles C, Nout RA, de Kroon CD, Osse EM, Klenerman P, Creutzberg CL, Tomlinson IP, Smit VT, Nijman HW, Bosse T, Church DN (2015) POLE proofreading mutations elicit an antitu- mor immune response in endometrial cancer. Clin Cancer Res 21: 3347–3355 25. Fogel M, Gutwein P, Mechtersheimer S, Riedle S, Stoeck A, Smirnov A, Edler L, Ben-Arie A, Huszar M, Altevogt P (2003) L1 expression as a predictor of progression and survival in patients with uterine and ovarian carcinomas. Lancet 362:869–875 26. Fadare O, Roma AA, Desouki MM, Gwin K, Hanley KZ, Jarboe EA, Liang SX, Quick CM, Zheng W, Hecht JL, Parkash V, Wang XJ (2018) The significance of L1CAM expression in clear cell carcinoma of the endometrium. Histopathology 72:532–538 29. Meier F, Busch S, Gast D, Goppert A, Altevogt P, Maczey E, Riedle S, Garbe C, Schittek B (2006) The adhesion molecule L1 (CD171) promotes melanoma progression. Int J Cancer 119:549–555 27. van der Putten LJ, Visser NC, van de Vijver K, Santacana M, Bronsert P, Bulten J, Hirschfeld M, Colas E, Gil-Moreno A,
https://openalex.org/W4394594090	https://pubs.rsc.org/en/content/articlepdf/2024/tb/d3tb02631k	English	null	The effect of charge and albumin on cellular uptake of supramolecular polymer nanostructures	Journal of materials chemistry. B	2,024	cc-by	11,596	a Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600 MB, The Netherlands. E-mail: P.Y.W.Dankers@tue.nl b Department of Biomedical Engineering, Laboratory for Cell and Tissue Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB, The Netherlands c Department of Biomedical Engineering, Laboratory of Chemical Biology, Eindhoven University of Technology, PO Box 513, 5600 MB, The Netherlands † Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d3tb02631k The eﬀect of charge and albumin on cellular uptake of supramolecular polymer nanostructures† Jiankang Song,abc Peter-Paul K.H. Fransen,ac Maarten H. Bakker, ac Sjors P.W. Wijnands,ac Jingyi Huang,ac Shuaiqi Guoa and Patricia Y. W. Dankers abc Open Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42:52 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: J. Mater. Chem. B, 2024, 12, 4854 Received 6th November 2023, Accepted 30th March 2024 DOI: 10.1039/d3tb02631k rsc.li/materials-b n Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42:52 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: J. Mater. Chem. B, 2024, 12, 4854 Cite this: J. Mater. Chem. B, 2024, 12, 4854 Jiankang Song,abc Peter-Paul K.H. Fransen,ac Maarten H. Bakker, ac Sjors P.W. Wijnands,ac Jingyi Huang,ac Shuaiqi Guoa and Patricia Y. W. Dankers abc Intracellular delivery of functional biomolecules by using supramolecular polymer nanostructures has gained significant interest. Here, various charged supramolecular ureido-pyrimidinone (UPy)-aggregates were designed and formulated via a simple ‘‘mix-and-match’’ method. The cellular internalization of these UPy-aggregates in the presence or absence of serum proteins by phagocytic and non- phagocytic cells, i.e., THP-1 derived macrophages and immortalized human kidney cells (HK-2 cells), was systematically investigated. In the presence of serum proteins the UPy-aggregates were taken up by both types of cells irrespective of the charge properties of the UPy-aggregates, and the UPy-aggregates co-localized with mitochondria of the cells. In the absence of serum proteins only cationic UPy-aggregates could be eﬀectively internalized by THP-1 derived macrophages, and the internalized UPy-aggregates either co-localized with mitochondria or displayed as vesicular structures. While the cationic UPy-aggregates were hardly internalized by HK-2 cells and could only bind to the membrane of HK-2 cells. With adding and increasing the amount of serum albumin in the cell culture medium, the cationic UPy-aggregates were gradually taken up by HK-2 cells without anchoring on the cell membranes. It is proposed that the serum albumin regulates the cellular internalization of UPy- aggregates. These results provide fundamental insights for the fabrication of supramolecular polymer nanostructures for intracellular delivery of therapeutics. Received 6th November 2023, Accepted 30th March 2024 DOI: 10.1039/d3tb02631k rsc.li/materials-b This journal is © The Royal Society of Chemistry 2024 Journal of Materials Chemistry B View Article Online View Journal \| View Issue The eﬀect of charge and albumin on cellular uptake of supramolecular polymer nanostructures† Jiankang Song,abc Peter-Paul K.H. Fransen,ac Maarten H. Bakker, ac Sjors P.W. Wijnands,ac Jingyi Huang,ac Shuaiqi Guoa and Patricia Y. W. Dankers abc Paper It was hypothesized that serum proteins rule over charge on cellular internalization, and that serum albumin dominates the uptake process. To this end, an amphiphilic molecule based on Although the influence of serum proteins on the cellular uptake of conventional nanocarriers has been indicated from a large number of studies,26,29,32,34–37 this influence on supramo- lecular nanostructures has not yet been investigated in detail. Besides serum proteins, the surface charge of the nanocarriers is a key parameter known to influence their cellular internaliza- tion even though the internalization procedure is not fully understood.35,37–41 Moreover, the influence of the charge on the cellular uptake of nanocarriers is cell-type dependent, e.g., phagocytic vs. non-phagocytic cells.38,39,42 It is reported that the internalization of a self-assembled supramolecular scaffold based on 1,3,5-benzenetricarboxamide amphiphiles was either charge or receptor dominated.15 However, a possible difference of the influence of serum proteins on cellular internalization by either phagocytic or non-phagocytic cells was not investigated. Fig. 1 Chemical structures of the ureido-pyrimidinone (UPy)-monomers and characterizations of the various UPy-aggregates. (a) Chemical structures of the UPy-monomers with diﬀerent functional groups. (b) Composition and illustration of the UPy-aggregates with diﬀerent charge properties. (c) Nile red (NR) measurement for the confirmation of the assemblies of UPy-aggregates in PBS with the formation of lateral hydrophobic pockets, where the NR could be encapsulated and emitted intensive fluorescent signals. (d) Fluorescence resonance energy transfer (FRET) measurement with NR/Cy5 pair to confirm the encapsulation of fluorescent reporter UPy–Cy5 into the UPy-aggregates in PBS. (e) Total internal reflection fluorescence microscope (TIRF) of the various UPy-aggregates in PBS, the morphology of these aggregates were examined with Cy5 fluorophore. Scale bars represent 10 mm. (f) Zeta-potential measurements of the various UPy-aggregates (50 mM) in 5 mM HEPES buffer at pH 7.4, the transition of the charge properties of the UPy-aggregates from anionic to cationic has been confirmed. Fig. 1 Chemical structures of the ureido-pyrimidinone (UPy)-monomers and characterizations of the various UPy-aggregates. (a) Chemical structures of the UPy-monomers with diﬀerent functional groups. (b) Composition and illustration of the UPy-aggregates with diﬀerent charge properties. (c) Nile red (NR) measurement for the confirmation of the assemblies of UPy-aggregates in PBS with the formation of lateral hydrophobic pockets, where the NR could be encapsulated and emitted intensive fluorescent signals. (d) Fluorescence resonance energy transfer (FRET) measurement with NR/Cy5 pair to confirm the encapsulation of fluorescent reporter UPy–Cy5 into the UPy-aggregates in PBS. Paper Paper Journal of Materials Chemistry B quadruple hydrogen-bonding ureido-pyrimidinone (UPy) supra- molecular moiety,43–45 connected to an alkyl spacer containing an urea functionality, linked via an urethane group to hydrophilic oligo(ethylene glycol) (OEG) was selected as the base monomer (Fig. 1(a)). The chemical structure of the UPy-monomers i.e., length of alkyl spacer and PEG densities, was optimized to achieve robust UPy-aggregates formation (unpublished data). This monomer has been shown to assemble into stable fibrous aggregates owing to the stacking of hydrogen-bonded UPy- dimers in the lateral direction, assisted by additional hydrogen- bonding of the urea functionalities and possibly the urethane groups between the alkyl and OEG part. The modularity of this supramolecular polymer system enables for diverse functiona- lization of monomers to achieve multifunctional UPy- aggregates in a ‘‘mix-and-match’’ fashion.9,10,25 Previously, our group has shown to be able to successfully complex RNAi therapeutics with such cationic UPy-aggregates for cellular internalization.9,46 In the current study, the UPy-monomers were functionalized with different end groups to obtain a supramolecular toolbox for the fabrication of four types of UPy-aggregates with various charge properties, i.e., neutral, anionic, neutral (+/) and cationic (Fig. 1(a) and (b)). To systematically investigate the influence of serum proteins and charge properties on cellular internalization, the uptake of Although the influence of serum proteins on the cellular uptake of conventional nanocarriers has been indicated from a large number of studies,26,29,32,34–37 this influence on supramo- lecular nanostructures has not yet been investigated in detail. Besides serum proteins, the surface charge of the nanocarriers is a key parameter known to influence their cellular internaliza- tion even though the internalization procedure is not fully understood.35,37–41 Moreover, the influence of the charge on the cellular uptake of nanocarriers is cell-type dependent, e.g., phagocytic vs. non-phagocytic cells.38,39,42 It is reported that the internalization of a self-assembled supramolecular scaffold based on 1,3,5-benzenetricarboxamide amphiphiles was either charge or receptor dominated.15 However, a possible difference of the influence of serum proteins on cellular internalization by either phagocytic or non-phagocytic cells was not investigated. Therefore, the aim of the current study was to systematically investigate cellular uptake of various charged supramolecular polymer nanostructures (1) with the presence of serum proteins to mimic the complex in vivo administration environment and (2) for both phagocytic and non-phagocytic cells to explore the dominate factor that determines cellular uptake. Introduction composed of tunable molecular recognition motifs which can self-assemble through dynamic and reversible non-covalent interactions.23,24 These inherent characteristics allow for deli- cate control of the composition and physicochemical properties of the supramolecular nanostructures on a molecular level to achieve efficient delivery outcomes.25 Eﬃcient and eﬀective intracellular delivery of biomolecules and exogenous compounds for advanced cell-based therapies, regenerative medicine, gene editing and fundamental biology remain to be challenged.1–4 To tackle this challenge, numerous synthetic nanocarriers have been developed based on inorganic nanomaterials, polymers and lipids.2,5–7 Additionally, targeting ligands, cell penetrating peptides and other functional molecules have been incorporated to achieve selectivity.2,5–8 Among these nanocarriers, various types of supramolecular nanostructures9–22 have gained significant interest. These nanostructures are Despite the promising potential of using nanocarriers for intracellular delivery, these platforms confront a complex bio- logical barrier which severely limits their therapeutic outcomes upon systemic or local administration.1,7 This barrier begins with non-specific adherence of a mixture of plasma proteins including serum albumin onto their surfaces,7,26–30 followed by clearance of the opsonized nanocarriers by the mononuclear phagocyte system which mainly consists of resident macro- phages.31 In addition, this protein binding alters the overall functional and physicochemical properties of the nanocarriers and thus dramatically influences their entry into targeting cells.26,32–38 Therefore, it is crucial to evaluate the internalization of the nanocarriers by both phagocytic and targeting cells in the presence or absence of serum proteins to provide fundamental information for developing new intracellular delivery therapies. This journal is © The Royal Society of Chemistry 2024 4854 \| J. Mater. Chem. B, 2024, 12, 4854–4866 View Article Online Journal of Materials Chemistry B This journal is © The Royal Society of Chemistry 2024 Characterization of the UPy-aggregates Supramolecular monomers were designed and synthesized to assemble in aqueous medium in a modular manner. To obtain functional UPy-aggregates with diﬀerent charge properties, the UPy-monomers were end-functionalized with various charged groups, i.e., non-charged methoxy, cationic amine and anionic carboxylate groups (Fig. 1(a)). As reporter molecules, fluoro- phore Cy5 or fluorescein was grafted onto the UPy-molecule to help monitor the cellular uptake of the UPy-aggregates (Fig. 1(a)). The UPy-aggregates were prepared via a ‘‘mix-and- match’’ method. With this method, the critical aggregation concentration of the UPy-monomers was determined at approximately 1 mM (unpublished data), while characterization of the UPy-aggregates in PBS was performed with optimized concentration of 50 mM. The UPy-monomers dissolved in methanol solutions were thoroughly mixed at required ratios (Table S1, ESI†), followed by adding phosphate buﬀered saline (PBS) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (5 mM, pH 7.4) buﬀer to reach an UPy-monomer concentration of 50 mM. The UPy-suspension was then equili- brated overnight with shaking at 200 rpm to form the UPy- aggregates. Zeta-potential of the UPy-aggregates were measured in 5 mM HEPES buﬀer at pH 7.4. The shift of the zeta-potential values of the four types of UPy-aggregates from negative to positive was observed, i.e., 42.40 8.57, 18.8 5.76, 5.53 3.70 and 33.6 8.60 mV for anionic, neutral (+/), neutral and cationic UPy-aggregates, respectively (Fig. 1(f)). These results demon- strate that UPy-aggregates were successfully prepared with distinctive charge properties that correspond to the functional groups. The assembly of UPy-monomers in PBS was characterized by examining the hydrophobic pocket formation at the lateral direction of the aggregates using Nile Red (NR) as a probe.9 The NR produces a hyperchromic eﬀect upon encapsulation into a hydrophobic pocket.47 For all of the four types of systems (Fig. 1(b)), strong intensity of fluorescent NR signals were observed, which suggested the formation of hydrophobic domains in all cases (Fig. 1(c)). Besides, the wavelength of the emission spectra peaks for the systems of neutral (+/), catio- nic, anionic and neutral were 640, 637, 635 and 633 nm, respectively (Fig. 1(c)), which showed a blue shift. The shorter wavelength of the emission peaks indicated a more hydropho- bic domain48 that represented a better fibrous assemblies formation. This blue shift suggests that the fibrous assemblies formation capability of the systems were in the order of neutral 4 anionic 4 cationic 4 neutral (+/). Paper (e) Total internal reflection fluorescence microscope (TIRF) of the various UPy-aggregates in PBS, the morphology of these aggregates were examined with Cy5 fluorophore. Scale bars represent 10 mm. (f) Zeta-potential measurements of the various UPy-aggregates (50 mM) in 5 mM HEPES buffer at pH 7.4, the transition of the charge properties of the UPy-aggregates from anionic to cationic has been confirmed. This journal is © The Royal Society of Chemistry 2024 This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4855 View Article Online Journal of Materials Chemistry B Paper these UPy-aggregates by THP-1 derived macrophages and immortalized human kidney cells (HK-2 cells) in the presence or absence of fetal bovine serum (FBS) were performed. Secondly, the location of the UPy-aggregates inside the cells after being incubated with cells for a certain period was probed. Finally, to test the regulatory role of bovine serum albumin (BSA) on cellular uptake, BSA was added into the cell culture medium containing UPy-monomers and subsequent internali- zation of the formed UPy-aggregates was monitored. needed for monitoring intracellular uptake of the UPy-aggregates by fluorescence microscopy. Strong fluorescent signal of Cy5 was observed upon excitation of the encapsulated NR, which indicates the two fluorophores are in close proximity and the incorporation of UPy–Cy5 was successfully achieved (Fig. 1(d)). The morphology of the UPy-aggregates containing 1 mol% UPy–Cy5 was then examined using the Total Internal Reflection Fluorescence (TIRF) microscope. Distinct UPy-aggregates were observed for all of the four systems (Fig. 1(e)). The neutral and anionic aggregates show long fibrous structures while their counterparts of neutral (+/) and cationic aggregates are more rod-like particles (Fig. 1(e)). These results are in accordance with the NR measurements that also showed more blue shift of the wavelengths of the emission peaks for the neutral and anionic aggregates. Previously, it was reported that the incor- poration of positively charged supramolecular monomers in the system impeded their growth and final polymer size.49 The rod-like structure formation of the cationic UPy-aggregates is proposed to be caused by the electrostatic repulsion of the protonated amine groups within a single supramolecular poly- mer. Although the micrograph shows fibrous structure for the anionic UPy-aggregates, its NR emission peak wavelength at 635 nm was longer than that of the neutral UPy-aggregates at 633 nm (Fig. 1(c) and (e)). Characterization of the UPy-aggregates Dynamic light scattering (DLS) measurement showed that these UPy-aggregates had a hydrodynamic diameters between 50 and 300 nm (Fig. S1, ESI†). Fluorescence resonance energy transfer (FRET) experi- ments were performed by using the NR/Cy5 pair to confirm the incorporation of UPy–Cy5 into the UPy-aggregates, which is This journal is © The Royal Society of Chemistry 2024 Paper This result also suggests that the charge repulsion between the deprotonated carboxylates impeded the supramolecular polymerization, which was also reported previously,50 although it was not as strong as their amine counterparts. Open Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42 This article is licensed under a Creative Commons Attribution 3.0 Un These results are in consistence with the results of live confocal imaging. charge property of nanocarriers are beneficial for their cellular uptake, this charge property may induce cytotoxicity.42 The UPy-aggregates used in the present study, however, did not show cytotoxicity even at a concentration of 20 mM. In the absence of FBS in the medium, live confocal imaging results showed that both the cationic and neutral (+/) UPy- aggregates were internalized by THP-1 derived macrophages and HK-2 cells, with significantly more of the cationic UPy- aggregates being internalized compared to the neutral (+/) aggregates (Fig. 4(a)). The anionic and neutral UPy-aggregates, however, were not taken up by both types of cells since no visible signal could be observed (Fig. 4(a)). Identical results were obtained from the flow cytometry experiments, with significant stronger fluorescence intensity and relative mean fluorescence intensity per cell for the cationic and neutral (+/) UPy-aggregates compared to their anionic and neutral counterparts (Fig. 4(b) and (c)). Besides, the cationic UPy-aggregates with a higher positive charge were significantly more internalized than the neutral (+/) UPy-aggregates (Fig. 4(b) and (c)). These results show that the internalization of UPy-aggregates by both THP-1 cells and HK-2 cells are charge dominated in the absence of serum proteins. Importantly, in this condition the cellular internalization of positively charged UPy-aggregates was enhanced. Influence of serum proteins and charge on the internalization of the UPy-aggregates It is hypothesized that serum proteins strongly aﬀect the charge influence of UPy-aggregates on cellular uptake. To test this hypothesis, the internalization of the UPy-aggregates by both THP-1 derived macrophages and HK-2 cells was qualitatively and quantitatively examined by live confocal imaging and flow cytometry, respectively. With FBS in the medium, the confocal live imaging results showed that UPy-aggregates were interna- lized by THP-1 derived macrophages in 5 min (Fig. S2a, ESI†). More aggregates were internalized with increasing incubation time until 120 min (Fig. 3(a) and Fig. S2a, ESI†). No significant diﬀerences, however, could be observed among the UPy- aggregates with diﬀerent charge properties (Fig. 3(a) and Fig. S2a, ESI†). In contrast, the UPy-aggregates were interna- lized by HK-2 cells in a slower manner with visible cellular uptake after 30 min (Fig. S3a, ESI†). No significant diﬀerences could be observed among diﬀerent charged UPy-aggregates at 120 min (Fig. 3(a)). In conclusion, the presence of serum proteins stimulated the cellular uptake of supramolecular UPy-aggregates for both phagocytic and non-phagocytic cells irrespective their charge properties (Fig. 3(a)). These results supported our hypothesis that the serum proteins overrule the influence of charge on cellular internalization of supramolecular nanostructures, although it is well recognized that the cationic nanocarriers are more favourable for cellular internalization.39 Moreover, these results indicate that the serum proteins may strongly interact with the UPy-aggregates, thus influence their cellular uptake and possibly their cellular locations. Flow cytometry results showed that both pure UPy–Cy5 and Cy5–NH2 were internalized by HK-2 cells (Fig. 3(b), (c) and Fig. S3b, c, ESI†). In contrast, only UPy–Cy5 was taken up by THP-1 derived macrophages even up to 120 min (Fig. 3(b), (c) and Fig. S2b, c, ESI†). Although pure UPy–Cy5 could be internalized by cells, the amount of internalized UPy–Cy5 as quantified from the fluorescent signal was much less compared to the fluor- escent signal of the UPy-aggregates containing the same amount of UPy–Cy5 (Fig. 3(b), (c) and Fig. S2b, c, S3b, c, ESI†). These results suggest that the existence of UPy-aggregates stimulated their cellular internalization by both types of cells. Moreover, no significant difference on cellular internalization was observed among the UPy-aggregates with different charge properties upon 120 min incubation with cells (Fig. 3(b) and (c)). This journal is © The Royal Society of Chemistry 2024 Cytocompatibility of the UPy-aggregates The cationic UPy-aggregates may bind to the membrane of the cells and disrupt the plasma membrane to induce cytotoxicity due to their positive charged property. With the disruption of the plasma membrane, the Lactate Dehydrogenase (LDH) in the cytoplasm is released and hence the cytotoxicity of the UPy-aggregates can be correlated to the damage of cell membranes.51 Therefore, the cytocompatibility of the UPy- aggregates was determined with an LDH test. To prepare the UPy-aggregates, the required amount of UPy-monomers in methanol solution were thoroughly mixed, followed by addition of cell culture medium containing 10% FBS and 1% antibiotics. The UPy-suspensions were then overnight equilibrated on a shaking bed at 200 rpm to form the UPy-aggregates with diﬀerent concentrations. The LDH results showed that all four types of UPy-aggregates had good cytocompatibility for both THP-1 derived macrophages and HK-2 cells from 5 to 20 mM (Fig. 2). Although previous studies suggested that the positive This journal is © The Royal Society of Chemistry 2024 4856 \| J. Mater. Chem. B, 2024, 12, 4854–4866 View Article Online Paper Paper Fig. 2 Cytocompatibility of the UPy-aggregates. The various UPy-aggregates all have good cytocompatibility with examined concentrations for both (a) THP-1 derived macrophages and (b) Human kidney cells (HK-2 cells). The tests were performed with an LDH method with the presence of fetal bovine serum, the cells without the addition of any UPy-aggregates were used as the control group. Error bars are the standard deviation of the mean value averaged over triplicates. Paper Journal of Materials Chemistry B Fig. 2 Cytocompatibility of the UPy-aggregates. The various UPy-aggregates all have good cytocompatibility with examined concentrations for both (a) THP-1 derived macrophages and (b) Human kidney cells (HK-2 cells). The tests were performed with an LDH method with the presence of fetal bovine serum, the cells without the addition of any UPy-aggregates were used as the control group. Error bars are the standard deviation of the mean value averaged over triplicates. Location of the UPy-aggregates in cells Live confocal images showed no overlap of fluorescent signals between Cy5 and membrane staining for THP-1 derived macro- phages in all of these internalization studies (Fig. 3(a), 4(a) and Fig. S2a, ESI†). However, overlap of the fluorescent signals from This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4857 4857 View Article Online Journal of Materials Chemistry B Journal of Materials Chemistry B Fig. 3 Internalization of UPy-aggregates (10 mM) by THP-1 derived macrophages and human kidney cells (HK-2) in the presence of fetal bovine s (FBS). (a) Confocal laser scanning micrographs of the internalization of the UPy-aggregates at 120 min. The UPy-aggregates, nuclei and membran the cells were labeled red, blue and green, respectively. All of the UPy-aggregates with diﬀerent charge properties were internalized by both THP- HK-2 cells. Scale bars represent 30 and 50 mm for THP-1 and HK-2 cells, respectively. (b) Flow cytometry analysis of the internalization of the aggregates by THP-1 derived macrophages and HK-2 cells. The results are illustrated as the signal distribution of the 10 000 gated cells. The experim group without addition of UPy-aggregates was used as the control. Both the internalizations of pure UPy–Cy5 and Cy5–NH2 were compared the other four types of UPy-aggregates containing the same amount of Cy5, and much stronger fluorescence intensity for the cells of the four typ UPy-aggregates can be observed. (c) Illustration of the average fluorescence intensity of the 10 000 gated cells incubated with different materials. bars are the standard deviation of the mean cell fluorescence intensity averaged over triplicates. p o 0.0001 (*). Journal of Materials Chemistry B Pa Paper Paper Open Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42:52 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 Internalization of UPy-aggregates (10 mM) by THP-1 derived macrophages and human kidney cells (HK-2) in the presence of fetal bovine serum (FBS). (a) Confocal laser scanning micrographs of the internalization of the UPy-aggregates at 120 min. The UPy-aggregates, nuclei and membranes of the cells were labeled red, blue and green, respectively. All of the UPy-aggregates with diﬀerent charge properties were internalized by both THP-1 and HK-2 cells. Scale bars represent 30 and 50 mm for THP-1 and HK-2 cells, respectively. This journal is © The Royal Society of Chemistry 2024 Location of the UPy-aggregates in cells (b) Flow cytometry analysis of the internalization of the UPy- aggregates by THP-1 derived macrophages and HK-2 cells. The results are illustrated as the signal distribution of the 10 000 gated cells. The experimental group without addition of UPy-aggregates was used as the control. Both the internalizations of pure UPy–Cy5 and Cy5–NH2 were compared with the other four types of UPy-aggregates containing the same amount of Cy5, and much stronger fluorescence intensity for the cells of the four types of UPy-aggregates can be observed. (c) Illustration of the average fluorescence intensity of the 10 000 gated cells incubated with different materials. Error bars are the standard deviation of the mean cell fluorescence intensity averaged over triplicates. p o 0.0001 (). Previous studies reported that Cy5-labeled oligonucleotides could colocalize with mitochondria after cellular internalization.52,53 The high magnification confocal micrographs of the interna- lized UPy-aggregates with diﬀerent charge properties, which were prepared in the presence of serum proteins, showed Previous studies reported that Cy5-labeled oligonucleotides could colocalize with mitochondria after cellular internalization.52,53 Cy5 and membrane staining of HK-2 cells on the internalization of cationic UPy-aggregates in the absence of serum proteins was observed (Fig. 4(a)). This overlap of signals indicates the binding of positively charged UPy-aggregates onto the membranes of HK-2 cells. The high magnification confocal micrographs of the interna- lized UPy-aggregates with diﬀerent charge properties, which were prepared in the presence of serum proteins, showed 4858 \| J. Mater. Chem. B, 2024, 12, 4854–4866 This journal is © The Royal Society of Chemistry 2024 View Article Online Paper Journal of Materials Chemistry B Internalization of the UPy-aggregates (10 mM) by THP-1 derived macrophages and human kidney cells (HK-2) in the absence of fetal bo (FBS). (a) Confocal laser scanning micrographs of the internalization of UPy-aggregates at 120 min. The UPy-aggregates, nuclei and membrane s were labeled red, blue and green, respectively. Both neutral and anionic UPy-aggregates were much less internalized compared to cationic l (+/) counterparts, which indicates the internalization of UPy-aggregates without the presence of FBS is charge dominated. Scale bars repre 50 mm for THP-1 derived macrophages and HK-2 cells, respectively. (b) Flow cytometry analysis of the internalization of the UPy-aggregate and HK-2 cells. The results are illustrated as the signal distribution of the 10 000 gated cells. The experimental group without addition of U ates was used as the control. Location of the UPy-aggregates in cells Both the internalizations of pure UPy–Cy5 and Cy5–NH2 were compared with the other four types of U ates containing the same amount of Cy5, and much stronger fluorescence intensity for the cells of the cationic and neutral (+/) groups of U ates can be observed. (c) Illustration of the average fluorescence intensity of the 10 000 gated cells incubated with different materials. Error standard deviation of the mean cell fluorescence intensity averaged over triplicates. p o 0.0001 (). r Journal of Materials Chemistr Open Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42:52 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Internalization of the UPy-aggregates (10 mM) by THP-1 derived macrophages and human kidney cells (HK-2) in the absence of fetal bovine serum (FBS). (a) Confocal laser scanning micrographs of the internalization of UPy-aggregates at 120 min. The UPy-aggregates, nuclei and membranes of the cells were labeled red, blue and green, respectively. Both neutral and anionic UPy-aggregates were much less internalized compared to cationic and neutral (+/) counterparts, which indicates the internalization of UPy-aggregates without the presence of FBS is charge dominated. Scale bars represent 30 and 50 mm for THP-1 derived macrophages and HK-2 cells, respectively. (b) Flow cytometry analysis of the internalization of the UPy-aggregates by THP-1 and HK-2 cells. The results are illustrated as the signal distribution of the 10 000 gated cells. The experimental group without addition of UPy- aggregates was used as the control. Both the internalizations of pure UPy–Cy5 and Cy5–NH2 were compared with the other four types of UPy- aggregates containing the same amount of Cy5, and much stronger fluorescence intensity for the cells of the cationic and neutral (+/) groups of UPy- aggregates can be observed. (c) Illustration of the average fluorescence intensity of the 10 000 gated cells incubated with different materials. Error bars are the standard deviation of the mean cell fluorescence intensity averaged over triplicates. p o 0.0001 (**). rod-like morphology around the nuclei of cells from the fluorescent signal of Cy5 (Fig. S4, ESI†). This rod-like mor- phology is identical to the structure of mitochondria. There- fore, the mitochondria of the cells were labelled with green fluorescent proteins (GFP), which did not interfere with the surface properties of mitochondria.54 The locations of the UPy-aggregates in the cells were determined by using live confocal imaging. This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4859 Location of the UPy-aggregates in cells After 120 min incubation, the UPy-aggregates wer washed away and the Dulbecco’s Modified Eagle Medium (DMEM) containing full FBS was added to incubate the cell for another 120 min. Live confocal imaging results showed tha part of the UPy-aggregates were internalized and co-localize with mitochondria, while part of aggregates anchored at th membranes of the cells (Fig. S6, ESI†). This result suggests tha FBS can stimulate the internalization of cationic UPy-aggregate In the presence of FBS, the internalized cationic UPy- aggregates were co-localized with mitochondria for both THP- 1 derived macrophages (Fig. 5(a)) and HK-2 cells (Fig. 5(b)), since the fluorescent signal of Cy5 overlapped with the GFP of mitochondria. Lacroix et al. also reported identical results for Cy5- and Cy3-labeled DNA nanostructures or oligonucleotides55,56 with both a cervical cancer cell line HeLa cells and a human liver cancer cell line HepG2 cells. In the absence of serum proteins, however, the location of the internalized cationic UPy-aggregates were significantly diﬀerent for THP-1 derived macrophages and HK-2 cells. After internalization by THP-1 derived macrophages, the fluorescent Cy5 signal of the UPy-aggregates partly overlapped with the fluorescent signal of mitochondria while other parts of the Cy5 signal were displayed as vesicular structures (white arrows in Fig. 5(a)). These vesicular structures are proposed to be the phagosomes or endocytic vesicles containing internalized UPy-aggregates that were formed through phagocytosis (UPy- aggregates size 4 0.5 mm) or endocytosis (UPy-aggregates size o 0.5 mm), respectively.57,58 For HK-2 cells, however, most of the UPy-aggregates were present at the surface of the cell membrane with minor internalization (Fig. 5(b)). These results indicate that the uptake and biodistribution of the UPy-aggregates by both THP-1 derived macrophages and HK-2 cells were dictated by serum proteins. To further determine if these interactions between cationic UPy-aggregates and HK-2 cells were influenced by the mole- cular structure of the fluorescent dye, UPy-Fluorescein piper- azine (UPy-F) was prepared and used as a fluorescent reporter to repeat the cellular internalization experiments. Identically, the cationic UPy-aggregates containing UPy-F prepared in the absence of serum protein presented mainly at the membranes of HK-2 cells, while they were mostly internalized by cells other than binding to membranes in the presence of serum protein (Fig. S5, ESI†). These results suggest that the interna- lization behaviour of UPy-aggregates into HK-2 cells was not likely influenced by the molecular nature of the fluorescent probes. Location of the UPy-aggregates in cells This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4859 View Article Online Fig. 5 Locations of the cationic UPy-aggregates (10 mM) after being incubated with mammalian cells for 120 min. The cationic UPy-aggregates, GFP- modified mitochondria and nuclei of the cells were labelled red, green and blue, respectively. (a) The cationic UPy-aggregates can be internalized by THP-1 derived macrophages under the conditions of presence or absence of fetal bovine serum (FBS), and the Cy5 signal overlapped with mitochondria. Scale bars represent 10 mm. (b) The cationic UPy-aggregates can be internalized by human kidney cells (HK-2 cells) under the condition of presence of FBS, and the Cy5 signal overlapped with mitochondria. Under the condition of absence of FBS, the cationic UPy-aggregates anchored on the membranes of the HK-2 cells with subtle internalization. Scale bars represent 20 mm. Journal of Materials Chemistry B Paper View Article Online g. 5 Locations of the cationic UPy-aggregates (10 mM) after being incubated with mammalian cells for 120 min. The cationic UPy-aggregates, modified mitochondria and nuclei of the cells were labelled red, green and blue, respectively. (a) The cationic UPy-aggregates can be internalize HP-1 derived macrophages under the conditions of presence or absence of fetal bovine serum (FBS), and the Cy5 signal overlapped with mitocho cale bars represent 10 mm. (b) The cationic UPy-aggregates can be internalized by human kidney cells (HK-2 cells) under the condition of presen ournal of Materials Chemistry B Pa View Article O Journal of Materials Chemistry B Paper y Open Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42:52 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 Locations of the cationic UPy-aggregates (10 mM) after being incubated with mammalian cells for 120 min. The cationic UPy-aggregates, GFP- modified mitochondria and nuclei of the cells were labelled red, green and blue, respectively. (a) The cationic UPy-aggregates can be internalized by THP-1 derived macrophages under the conditions of presence or absence of fetal bovine serum (FBS), and the Cy5 signal overlapped with mitochondria. Scale bars represent 10 mm. (b) The cationic UPy-aggregates can be internalized by human kidney cells (HK-2 cells) under the condition of presence of FBS, and the Cy5 signal overlapped with mitochondria. This journal is © The Royal Society of Chemistry 2024 4860 \| J. Mater. Chem. B, 2024, 12, 4854–4866 Location of the UPy-aggregates in cells Under the condition of absence of FBS, the cationic UPy-aggregates anchored on the membranes of the HK-2 cells with subtle internalization. Scale bars represent 20 mm. In the presence of FBS, the internalized cationic UPy- aggregates were co-localized with mitochondria for both THP- 1 derived macrophages (Fig. 5(a)) and HK-2 cells (Fig. 5(b)), since the fluorescent signal of Cy5 overlapped with the GFP of mitochondria. Lacroix et al. also reported identical results for Cy5- and Cy3-labeled DNA nanostructures or oligonucleotides55,56 with both a cervical cancer cell line HeLa cells and a human liver cancer cell line HepG2 cells. In the absence of serum proteins, however, the location of the internalized cationic UPy-aggregates were significantly diﬀerent for THP-1 derived macrophages and HK-2 cells. After internalization by THP-1 derived macrophages, the fluorescent Cy5 signal of the UPy-aggregates partly overlapped with the fluorescent signal of mitochondria while other parts of the Cy5 signal were displayed as vesicular structures (white arrows in Fig. 5(a)). These vesicular structures are proposed to be the phagosomes or endocytic vesicles containing internalized UPy-aggregates that were formed through phagocytosis (UPy- aggregates size 4 0.5 mm) or endocytosis (UPy-aggregates size o 0.5 mm), respectively.57,58 For HK-2 cells, however, most of the UPy-aggregates were present at the surface of the cell membrane with minor internalization (Fig. 5(b)). These results indicate that the uptake and biodistribution of the UPy-aggregates by both THP-1 derived macrophages and HK-2 cells were dictated by serum proteins. To further determine if these interactions between cationi UPy-aggregates and HK-2 cells were influenced by the mole cular structure of the fluorescent dye, UPy-Fluorescein pipe azine (UPy-F) was prepared and used as a fluorescent reporte to repeat the cellular internalization experiments. Identically the cationic UPy-aggregates containing UPy-F prepared in th absence of serum protein presented mainly at the membrane of HK-2 cells, while they were mostly internalized by cell other than binding to membranes in the presence of serum protein (Fig. S5, ESI†). These results suggest that the interna lization behaviour of UPy-aggregates into HK-2 cells was no likely influenced by the molecular nature of the fluorescen probes. To further explore the influence of FBS on the location o UPy-aggregates after interacting with HK-2 cells, the HK-2 cell were incubated with the cationic UPy-aggregates in the absenc of FBS. Serum albumin regulates the cellular internalization of the UPy-aggregates Serum albumin is the most abundant proteins in FBS, as well as in the bloodstream. It can bind to lipophilic molecules and assist their transport into the cells.59,60 In the present study, it was hypothesized that bovine serum albumin (BSA) presents in the FBS dominates the internalization of UPy-aggregates into HK-2 cells. The formation of UPy-aggregates in the DMEM medium without BSA was examined. The results of the NR assay showed that all of the four types of UPy-aggregates could be formed in the DMEM medium since the samples containing UPy-monomers showed an increased NR intensity compared to the pure DMEM Finally, the internalization of the UPy-aggregates in DMEM medium containing BSA with UPy : BSA at diﬀerent molar ratios was monitored to confirm the function of BSA during cellular Fig. 6 Bovine serum albumin (BSA) determined the internalization of cationic UPy-aggregates (10 mM) by human kidney cells. (a) Nile red (NR) test for the confirmation of the formation of UPy-aggregates in the DMEM medium with the formation of lateral hydrophobic pockets, where the NR could be encapsulated and emitted intensive fluorescent signals. The DMEM medium without addition of UPy-monomers was used as the control. (b) Zeta- potential of the cationic UPy-aggregates (50 mM) incubated with diﬀerent amount of BSA in the medium of 5 mM HEPES buﬀer at pH 7.4. BSA strongly interacted with the cationic UPy-aggregates as shown by the zeta-potential shifting from positive to negative with diﬀerent amount of BSA. (c) Confocal fluorescent micrographs of the internalization of the cationic UPy-aggregates with diﬀerent amount of BSA. The cationic UPy-aggregates, GFP- modified mitochondria and nuclei of the cells were labeled red, green and blue, respectively. Scale bars represent 20 mm. With the addition of BSA into the cationic UPy-aggregates, the UPy-aggregates were gradually internalized by the cells and co-localized with mitochondria instead of anchoring on the membrane of the cells. Fig. 6 Bovine serum albumin (BSA) determined the internalization of cationic UPy-aggregates (10 mM) by human kidney cells. (a) Nile red (NR) test for the confirmation of the formation of UPy-aggregates in the DMEM medium with the formation of lateral hydrophobic pockets, where the NR could be encapsulated and emitted intensive fluorescent signals. The DMEM medium without addition of UPy-monomers was used as the control. Location of the UPy-aggregates in cells To further explore the influence of FBS on the location of UPy-aggregates after interacting with HK-2 cells, the HK-2 cells were incubated with the cationic UPy-aggregates in the absence of FBS. After 120 min incubation, the UPy-aggregates were washed away and the Dulbecco’s Modified Eagle Medium (DMEM) containing full FBS was added to incubate the cells for another 120 min. Live confocal imaging results showed that part of the UPy-aggregates were internalized and co-localized with mitochondria, while part of aggregates anchored at the membranes of the cells (Fig. S6, ESI†). This result suggests that FBS can stimulate the internalization of cationic UPy-aggregates 4860 \| J. Mater. Chem. B, 2024, 12, 4854–4866 This journal is © The Royal Society of Chemistry 2024 4860 View Article Online Journal of Materials Chemistry B Paper Paper Journal of Materials Chemistry B by HK-2 cells. Conclusively, the internalization process of amphi- philic cationic UPy-aggregates by HK-2 cells and the location of the aggregates were strongly influenced by serum proteins. medium (Fig. 6(a)). Besides, the NR signal intensity of these UPy- aggregates was identical to those formed in the PBS (Fig. 1(c)), which suggests that the medium composition did not have significant influence on the formation of these UPy-aggregates. The zeta-potential of UPy-aggregates formed in 5 mM HEPES buﬀer (pH 7.4) with BSA at diﬀerent UPy:BSA molar ratios was examined. The cationic UPy-aggregates became negatively charged with the addition of BSA into HEPES buﬀer containing UPy-monomers (Fig. 6(b)). These results show that BSA interacted strongly with these UPy-aggregates. Moreover, identical zeta- potential values of UPy-aggregates formed at diﬀerent molar ratios of UPy:BSA were observed (Fig. 6(b)). Similar interactions between UPy-aggregates and BSA also occurred for the other UPy-aggregates with diﬀerent charge properties (Fig. S7, ESI†). This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4861 Serum albumin regulates the cellular internalization of the UPy-aggregates (b) Zeta- potential of the cationic UPy-aggregates (50 mM) incubated with diﬀerent amount of BSA in the medium of 5 mM HEPES buﬀer at pH 7.4. BSA strongly interacted with the cationic UPy-aggregates as shown by the zeta-potential shifting from positive to negative with diﬀerent amount of BSA. (c) Confocal fluorescent micrographs of the internalization of the cationic UPy-aggregates with diﬀerent amount of BSA. The cationic UPy-aggregates, GFP- modified mitochondria and nuclei of the cells were labeled red, green and blue, respectively. Scale bars represent 20 mm. With the addition of BSA into the cationic UPy-aggregates, the UPy-aggregates were gradually internalized by the cells and co-localized with mitochondria instead of anchoring on the membrane of the cells. This journal is © The Royal Society of Chemistry 2024 J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4861 View Article Online View Article Online Journal of Materials Chemistry B Paper aggregates with different charge properties (Table S1, ESI†), a certain amount of monomers were thoroughly mixed, fol- lowed by addition of PBS or HEPES to reach a UPy-monomer concentration of 50 mM, the UPy-suspensions were then equili- brated overnight under gentle shaking at 200 rpm. For cell internalization purpose, the UPy-aggregates (10 mM) were pre- pared by mixing required amount of monomer solutions, followed by addition of the culture medium containing 1% penicillin/streptomycin, with or without 10% FBS and equili- brated overnight or three hours on a shaking bed at 200 rpm, respectively. internalization process. With molar ratio of UPy : BSA = 10 : 1, only small amount the formed cationic UPy-aggregates were internalized by HK-2 cells and they co-localized with mitochon- dria (Fig. 6(c)). Most of the aggregates, however, remain adhered onto the membrane of the cells. With increasing the amount of BSA, gradually more of the cationic UPy-aggregates were internalized and less of the aggregates bounded onto the membrane (Fig. 6(c)). At the molar ratio of UPy : BSA = 1 : 1, the cationic UPy-aggregates were internalized by HK-2 cells and no membrane binding of these aggregates was observed (Fig. 6(c)). These results demonstrate that BSA played a strong regulatory role during the internalization of the cationic UPy-aggregates by HK-2 cells. Synthesis UPy–OCH3 was synthesized as described previously,63 with LC–MS [M] calculated m/z 1035.67, found 518.92 [M + 2H]2+ and 1036.33 [M + H]+. UPy–NH2 and UPy–COOH were synthe- sized as described previously,64 for UPy–NH2 with LC–MS [M] calculated m/z 1064.69, found 533.50 [M + 2H]2+ and 1065.58 [M + H]+, for UPy–COOH with [M] calculated m/z 1137.70, found 570 [M + 2H]2+ and 1138.67 [M + H]+. UPy–Cy5 and UPy-F were synthesized as described in ESI,† with LC–MS [M] of UPy– Cy5 calculated m/z 1701.11, found 567.75 [M + 3H]3+, 851.06 [M + 2H]2+ and 1701.33 [M + H]+; with LC–MS [M] of UPy-F calculated m/z 1519.82, found 1519.42 [M + H]+. Due to the polydisperse properties of the UPy-aggregates, size and size distribution of the UPy-aggregates were measured with multi-angle Dynamic Light Scattering (DLS) with cumulant analysis.65 The UPy-aggregates were prepared at 50 mM in PBS and measured with a range of angles between 451 and 1501 at an interval of 151. Each angle was measured for 60 s with triplets at 20 1C. The hydrodynamic diameters of the UPy- aggregates were analyzed via cumulant analysis to obtain the z-average size by considering the UPy-aggregates as particles. Zeta-potential of the UPy-aggregates with a concentration of 50 mM in HEPES buﬀer (5 mM, pH 7.4) was measured by Laser Doppler Electrophoresis with a Malvern Zetasizer Nano-Z Characterizations of UPy-aggregates Formation of the UPy-aggregates in PBS or DMEM medium was examined by a NR encapsulation test. In detail, NR was added into the 50 mM UPy-aggregates suspension to reach a concen- tration of 5 mM, with the UPy-monomer : NR molar ratio of 10 : 1. The suspension was then equilibrated by shaking at 150 rpm for 5 min. The emission spectrum was recorded from 565 nm to 800 nm using an excitation wavelength of 550 nm on a Varian Cary Eclipse fluorescence spectrometer (Agilent Tech- nologies) using Quartz cuvettes. Encapsulation of Cy5 into the UPy-aggregates was examined by FRET measurement using the NR/Cy5 pair. The UPy–Cy5 was mixed with UPy-monomers at a molar ratio of 1 : 100 to reach the final concentrations of 0.5 mM and 50 mM, respectively. After formation of the UPy-aggregates, NR was then added to reach a concentration of 5 mM. After equilibrating, the emission spectrum was recorded from 540 to 800 nm using an excitation wavelength of 520 nm on the Varian Cary Eclipse fluorescence spectrometer. For all of these mea- surements, data of five scans were collected and averaged. Although the morphology change of the UPy-aggregates influences their cellular internalization in the presence of BSA, the contribution of BSA on cellular internalization cannot be ruled out. Besides, other factors such as the density of PEG on the UPy-aggregates also have an impact on their interactions with BSA61,62 to influence cellular internalization. Therefore, further systematical investigation on the interaction between various types of UPy-aggregates and BSA, and the mechanism of BSA-regulated internalization are beneficial for the develop- ment of UPy-based supramolecular nanostructures for intracel- lular delivery. Materials Total Internal Reflection Fluorescence (TIRF) images of the UPy-aggregates were acquired with a Nikon N-STORM system. The UPy-aggregates were prepared at 50 mM containing 1% UPy–Cy5 in PBS and diluted to 2 mM with PBS, followed by flowing in a chamber between a glass slide and microscope coverslip (Menzel-Gla¨ser, #1, 24 24 mm) which were sepa- rated by a piece of double-sided tape. The sample was annealed for 2 min and subsequently washed twice with PBS. The Cy5 was excited at 647 nm and the fluorescence was filtered through a Nikon 97335 quad-band pass dichroic filter. The fluorescence was observed with a Nikon 100 objective (1.4 NA oil immersion) and the images were recorded with an EMCCD camera (ixon3, Andor with pixel size 0.165 mm). All solvents were purchased from commercial sources and used as received unless stated otherwise. Hoechst 33342, Invitro- gent Live Cell Imaging Solution, CellMaskt Green plasma membrane stain, CellLightt Mitochondria-GFP with BacMam 2.0 were purchased from Thermo Fisher Scientific. Phorbol- 12-myristate-13-acetate (PMA) was ordered from Sigma-Aldrich. Serum albumin regulates the cellular internalization of the UPy-aggregates With suﬃcient amount of BSA, which was at the molar ratio of UPy : BSA = 1 : 1, the aggregates can strongly interact with BSA to assist their intracellular uptake other than bind onto the membrane of the cells. Open Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42:52 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This journal is © The Royal Society of Chemistry 2024 ¼ 100% Aggregates LDH Medium control LDH Maximum LDH Medium control LDH 100% ¼ 100% Aggregates LDH Medium control LDH Maximum LDH Medium control LDH 100% ¼ 100% Aggregates LDH Medium control LDH Maximum LDH Medium control LDH 100% Flow cytometry experiment The amount of internalized UPy-aggregates by THP-1 derived macrophages and HK-2 cells were quantified by flow cytometry on a FACS Aria III (BD Biosciences) machine equipped with a 70 mm nozzle. The UPy-aggregates (Fig. 1(b) and Table S1, ESI† 10 mM) containing one percent of UPy–Cy5 as reporter were prepared in DMEM and RPMI media. THP-1 monocytes were seeded at a density of 2 105 cells per cm2 in the 24-well plates (n = 3) while HK-2 cells were seeded onto the 6-well plates with a density of 2 104 cells per cm2 to avoid the overlap of the cells in the middle of each well (n = 3). After attachment, the cells were washed three times with PBS and incubated with UPy- aggregates for a certain period. The cells were then gently washed three times with PBS and trypsinized with trypsin for 2 min. Trypsin was deactivated by the addition of full medium and the cells were collected by centrifugation for 5 min at 150 and 130 g for THP-1 derived macrophages and HK-2 cells, respectively. The collected cell pellet was re-suspended in 500 mL of PBS containing 1 mg mL1 BSA and loaded into the flow cytometry. A total amount of 10 000 single cells were counted and gated based on their forward vs. side scatter signals. The fluorescence intensity of per cell was measured by exciting the Cy5 in the UPy-aggregates with a 633 laser and detect through a 650/670 bandpass filter. All of the samples were measured with triplicate at a flow rate of 1 mL min1. Cytocompatibility of the UPy-aggregates Cytocompatibility of the various charged UPy-aggregates for both THP-1 derived macrophages and HK-2 cells was investi- gated with a LDH assay following the standard protocol (Thermo Fisher Scientific). Briefly, the cells were seeded onto a 48-well plate with the above mentioned density (n = 3). After attachment of the cells, the cell culture medium containing UPy-aggregates at various concentrations was then added into each well. The wells containing only culture medium were used as control. After 24 hours of incubation, lysis buﬀer was added into wells without UPy-aggregates and incubated for 45 min at 37 1C in 95% air/5% CO2 atmosphere, these wells were used as Maximum LDH. After this, a 50 mL aliquot of metabolic medium of each well was transferred into a 96-well flat- bottom plate with duplicates, followed by the addition of 50 mL of reaction mixture. The plate was then incubated at room temperature for 30 min protected from light. After incubation, 50 mL of stop solution was added into each well and the absorbance at 490 nm and 680 nm was measured at a plate reader. The LDH absorbance value was obtained by subtracting the 680 nm absorbance value (background) from the 490 nm absorbance value. The cytocompatibility of the UPy- aggregates was calculated with the follow equation: Preparation of UPy aggregates UPy-monomers were dissolved in methanol with a concen- tration of 2 mM, except for UPy–Cy5 and UPy-F at the concentration of 0.1 and 0.4 mM, respectively. To prepare 4862 \| J. Mater. Chem. B, 2024, 12, 4854–4866 This journal is © The Royal Society of Chemistry 2024 View Article Online Paper Journal of Materials Chemistry B instrument (Worcestershire, UK) using a folded capillary cell (DTS 1060). instrument (Worcestershire, UK) using a folded capillary cell (DTS 1060). instrument (Worcestershire, UK) using a folded capillary cell (DTS 1060). For the experiments in the presence of FBS, the UPy-aggregates were incubated with cells for 5, 30 and 120 min. For the studies in the absence of FBS, the UPy-aggregates were incubated with cells for 120 min. At each time point, the UPy-aggregates were washed away with PBS, followed by sequential staining with Hoechst 33342 and CellMaskt Green plasma membrane stain for nuclei and membranes, respectively. After staining, the cells were washed three times with PBS and Invitrogent Live Cell Imaging Solution was added into each well for live imaging. The live imaging was performed under a Leica SP5 CLSM at 37 1C. For the experiments in the presence of FBS, the UPy-aggregates were incubated with cells for 5, 30 and 120 min. For the studies in the absence of FBS, the UPy-aggregates were incubated with cells for 120 min. At each time point, the UPy-aggregates were washed away with PBS, followed by sequential staining with Hoechst 33342 and CellMaskt Green plasma membrane stain for nuclei and membranes, respectively. After staining, the cells were washed three times with PBS and Invitrogent Live Cell Imaging Solution was added into each well for live imaging. The live imaging was performed under a Leica SP5 CLSM at 37 1C. Determination of the location of internalized UPy-aggregates CLSM live imaging with the mitochondria staining was per- formed to explore the location of the UPy-aggregates after incubation with cells. Cationic UPy-aggregates (Fig. 1(b) and Table S1, ESI† 10 mM) containing one percent of UPy–Cy5 as a reporter were prepared in DMEM medium in the presence or absence FBS. THP-1 and HK-2 cells were seeded in an 8-well Thermo Fisher Scientifict Nunct Lab-Tekt Chamber with #1 borosilicate glass bottom (n = 4). After attachment of the cells, 20 and 8 mL of CellLights Mitochondria-GFP BacMam 2.0 Reagent were added into each well for THP-1 derived macro- phages and HK-2 cells, respectively, to induce the mitochondria GFP secretion. The cells were then washed three times with PBS after overnight culture. The cationic UPy-aggregates (0.4 mL) in media with or without FBS were added and the cells were incubated for 120 min. After that, the cells were gently washed three times with PBS and stained by Hoechst 33324. Location of Cytocompatibility This journal is © The Royal Society of Chemistry 2024 Cell culture THP-1 human monocytic cells and HK-2 cells were purchased from ATCC and cultured at 37 1C in 95% air/5% CO2 atmo- sphere with DMEM and Gibcot Roswell Park Memorial Insti- tute (RPMI) 1640 medium, respectively, supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). THP-1 cells in suspension culture were passed every other day while the HK-2 cells were passed twice per week. For confocal fluorescence live imaging experiments and cytocompatibility measurement, the THP-1 derived macrophages and HK-2 cells were seeded at the density of 2.5 105 and 2.5 104 cells per cm2, respec- tively. To induce the diﬀerentiation of THP-1 monocytes into macrophages, PMA was added into the culture medium (50 ng mL1) and incubated for 48 h. Open Access Article. Published on 09 April 2024. Downloaded on 10/24/2024 5:42:52 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4863 Confocal fluorescence microscope Diﬀerent types of UPy-aggregates (Table S1, ESI† 10 mM) con- taining one percent of UPy–Cy5 as a reporter were prepared in DMEM and RPMI medium accordingly. THP-1 and HK-2 cells were seeded in an 8-well Thermo Fisher Scientifict Nunct Lab-Tekt Chamber with #1 borosilicate glass (n = 4). After attachment, the cells were washed three times with PBS and 0.4 mL of UPy-aggregates suspension was added into each well. This journal is © The Royal Society of Chemistry 2024 4863 J. Mater. Chem. B, 2024, 12, 4854–4866 \| 4863 View Article Online Journal of Materials Chemistry B Paper Paper the cationic UPy-aggregates was then observed under CLSM live imaging at 37 1C. the cationic UPy-aggregates was then observed under CLSM live imaging at 37 1C. conceptualization, visualization, supervision, writing – review & editing. All authors have given approval to the final version of the manuscript. References 1 E. Blanco, H. Shen and M. Ferrari, Nat. Biotechnol., 2015, 33, 941. 1 E. Blanco, H. Shen and M. Ferrari, Nat. Biotechnol., 2015, 33, 941. Conclusions Zhou and W. J. Parak, ACS Nano, 2017, 11, 2313–2381. 3 B. Pelaz, C. Alexiou, R. A. Alvarez-Puebla, F. Alves, A. M. Andrews, S. Ashraf, L. P. Balogh, L. Ballerini, A. Bestetti, C. Brendel, S. Bosi, M. Carril, W. C. W. Chan, C. 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Therefore, we propose that BSA regulates the cellular internalization of UPy-aggregates. This study opens the door for the investigation of serum albumin on the internalization of supramolecular polymer assemblies and provides fundamental insights for the fabrication of supramolecular polymer nanostruc- tures for intracellular delivery purposes. J , , , , 3 B. Pelaz, C. Alexiou, R. A. Alvarez-Puebla, F. Alves, A. M. Andrews, S. Ashraf, L. P. Balogh, L. Ballerini, A. Bestetti, C. Brendel, S. Bosi, M. Carril, W. C. W. Chan, C. Chen, X. Chen, X. Chen, Z. Cheng, D. Cui, J. Du, C. Dullin, A. Escudero, N. Feliu, M. Gao, M. George, Y. Gogotsi, A. Gru¨nweller, Z. Gu, N. J. Halas, N. Hampp, R. K. Hartmann, M. C. Hersam, P. Hunziker, J. Jian, X. Jiang, P. Jungebluth, P. Kadhiresan, K. Kataoka, A. Khademhosseini, J. Kopecˇek, N. A. Kotov, H. F. Krug, D. S. Lee, C.-M. Lehr, K. W. Leong, X.-J. Liang, M. Ling Lim, L. M. Liz-Marza´n, X. Ma, P. Macchiarini, H. Meng, H. Mo¨hwald, P. Mulvaney, A. E. Nel, S. Nie, P. Nordlander, T. Okano, J. Oliveira, T. H. Park, R. M. Penner, M. Prato, V. Puntes, V. M. Rotello, A. Samarakoon, R. E. Schaak, Y. Shen, S. Sjo¨qvist, A. G. Skirtach, M. G. Soliman, M. M. Stevens, H.-W. Sung, B. Z. Tang, R. Tietze, B. N. Udugama, J. S. VanEpps, T. Weil, P. S. Weiss, I. Willner, Y. Wu, L. Yang, Z. Yue, Q. Zhang, Q. Zhang, X.- E. Zhang, Y. Zhao, X. This journal is © The Royal Society of Chemistry 2024 4864 \| J. Mater. Chem. B, 2024, 12, 4854–4866 Acknowledgements We acknowledge the support from the Netherlands Cardiovas- cular Research Initiative (CVON 2012-01), the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development and the Royal Netherlands Academy of Sciences. Additionally, this work was financially supported by the Eur- opean Research Council (FP7/2007–2013) ERC Grant Agree- ment 308045, the Ministry of Education, Culture and Science (Gravity programs 024.003.013 and 024.005.020). Conflicts of interest The cellular internalization of cationic UPy-aggregates with the presence of diﬀerent amount of BSA was examined to determine the function of BSA on the cellular uptake of UPy- aggregates by HK-2 cells. The cationic UPy-aggregates (Fig. 1(b) and Table S1, ESI† 10 mM) containing one percent of UPy–Cy5 as a reporter were prepared in DMEM media containing differ- ent amounts of BSA. The HK-2 cells were seeded in an 8-well Thermo Fisher Scientifict Nunct Lab-Tekt Chamber with #1 borosilicate glass bottom (n = 4). After cell attachment, 8 mL of CellLights Mitochondria-GFP BacMam 2.0 Reagent was added into each well with overnight incubation to induce the mito- chondria GFP secretion. 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https://openalex.org/W2989240412	https://europepmc.org/articles/pmc6833254?pdf=render	English	null	Bioelectrical impedance phase angle in sport: a systematic review	Journal of the International Society of Sports Nutrition	2,019	cc-by	9,072	Abstract Background: Phase angle (PhA) is a raw BIA variable that has been gaining attention in recent years because it is supposed to be an index of the ratio between extracellular and intracellular water, body cell mass, and cellular integrity. The aim of this systematic review was to evaluate the variability of PhA between different sports and its relationships with sport performance. Additionally, we investigated whether PhA depends on gender or age, and analyzed the differences between athletes and controls. Methods: A systematic research using PubMed, Scopus and Web of Science up to June 2019 was performed. Selection criteria included studies on subjects who practice sports in planned and continuous modality at competitive or elite level. Results: Thirty-five papers met the inclusion criteria (twenty-one cross-sectional data, fourteen longitudinal data). A few but convincing studies have shown that mean PhA is higher in athletes vs. controls. PhA increases with age and is likely to be higher in male than female athletes. A large variability in PhA is observed for the same sport, while it is still uncertain to what extent PhA differs between various sports. There are no clear relationships of PhA with sport performance or training/untraining. Conclusion: It is still to be defined to what extent PhA varies between different sports and changes with training/ untraining. It can be argued that for a given sport much more data should be collected in a systematic way and for a period of time appropriate in order to determine changes and trends. This is even more crucial in the case of intervention studies. Keywords: Phase angle, Bioimpedance analysis, Athletes, Sport, Physical activity Keywords: Phase angle, Bioimpedance analysis, Athletes, Sport, Physical activity Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 https://doi.org/10.1186/s12970-019-0319-2 https://doi.org/10.1186/s12970-019-0319-2 Bioelectrical impedance phase angle in sport: a systematic review Olivia Di Vincenzo1, Maurizio Marra1* and Luca Scalfi2 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background which is also stated as the arctangent of the Xc to R ratio, describes the angular shift (phase difference) between voltage and current sinusoidal waveforms; in humans the current reaches at regular intervals its maximum/minimum peaks after the voltage (positive PhA values) and this lag is most likely due to cell mem- branes and tissue interfaces [1, 2]. Bioelectrical impedance analysis (BIA) is a widely used, non-invasive field method for assessing body compos- ition, which measures the electrical characteristics of human body either at 50 kHz (single-frequency BIA) or at several frequencies in the range 1–1000 kHz (multifre- quency BIA and BIS = bioimpedance spectroscopy). Impedance (Z) is the opposition of the body to an alter- nating current, resulting from resistance (R) to the current that flows through tissue containing water plus electrolytes, and reactance (Xc), which is associated with the capacitive component of tissues (e.g. cell membranes and tissue interfaces) [1]. In addition, phase angle (PhA), Using BIA, total body water (TBW) and fat-free mass (FFM) can be estimated by means of predictive equations, which include BIA variables and almost always variables such as age, stature and weight. Alternatively, directly- measured raw BIA variables, such as PhA at 50 kHz or impedance ratio (IR = the ratio between Z at higher frequencies and Z al lower frequencies), have been gaining attention because they are considered indexes of water distribution (ratio between extracellular water-ECW and * Correspondence: maurizio.marra@unina.it 1Department of Clinical Medicine and Surgery, Federico II University Hospital, Via S. Pansini 5, 80138 Naples, Italy Full list of author information is available at the end of the article * Correspondence: maurizio.marra@unina.it 1Department of Clinical Medicine and Surgery, Federico II University Hospital, Via S. Pansini 5, 80138 Naples, Italy Full list of author information is available at the end of the article Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Page 2 of 11 Page 2 of 11 Page 2 of 11 intracellular water-ICW), body cell mass (BCM), and cel- lular integrity [2]. PhA and IR have been shown to be sig- nificantly associated with muscle strength and physical activity [3, 4] and to vary between gender and with aging [5, 6] in line with that is known about physiological changes in BCM and ECW/ICW. physical activity, “O” (outcome) corresponded to PhA, and “S” (study design) indicated cross-sectional or longi- tudinal studies. The following eligibility criteria were applied: a) stud- ies on athletes following exercise programmes with or without a control group; b) papers published from in- ception to June 2019; c) full papers published in peer- reviewed journals or in relevant congress proceedings; d) studies evaluating body composition using BIA phase- sensitive devices and yielding overt data on PhA; e) stud- ies written in English. No restriction was applied to age of participants and sample size. g In sport science the assessment of body composition has different applications such as identifying individual’s characteristics critical to performance, evaluating the ef- fects of training programs, managing weight strategies in weight-category sports, etc. In this regard, BIA has been used in athletes as a field technique for estimating TBW and FFM. Indeed, there is still limited research and it is uncertain to what accuracy BIA may be used in athletes for single measurements or for tracking body compos- ition changes [7]. Even less attention has been paid to raw BIA data. A recent review has shown that Bioelec- trical Impedance Vector Analysis (BIVA) of both R and Xc has yielded some conflicting results on the use if BIA for identifying dehydration [8, 9]. On the other hand, at least in theory, the use of PhA or IR may be crucial in evaluating athletes’ body composition because it can provide useful data on the percentage of BCM in FFM (structural muscle quality) in both cross-sectional and longitudinal studies. A recent paper [10] supported this view showing in 202 athletes that PhA significantly cor- related with ICW and the ICW/ECW ratio. In this context, the purpose of this systematic review was to evaluate the variability of PhA among athletes and its relationship with sports performance. Search strategy Methodological quality was assessed using [1] the Quality Assessment Tool for Observational Cohort and Cross- Sectional Studies in observational studies [2]; the Quality Assessment Tool for Before-After (Pre-Post) Studies With No Control Group in before-after (pre-post) studies. Both tools are recommended by the National Institute of Health, U.S. Department of Health and Human Services [12], which were based on Evidence-based Practice Cen- ters (AHRQ) criteria (Additional file 1: Table S1). The [1] tool consists of 14 criteria and the [2] tool of 12 criteria that are used to assess quality, including whether the population studied was clearly specified and defined, whether the outcome assessors were blinded, and an assessment of the participation rate. The criteria were classified as “yes”, “cannot be determined”, “not reported”, or “not applicable”. Two authors (ODV and MM) independently performed a literature search up to June 2019 of the electronic da- tabases PubMed, Scopus, and Web of Science. The following terms were used as search strategy string: (“bioelectrical impedance” OR “bioimpedance” OR BIA) AND “phase angle” AND (spor* OR athlet* OR “physical activity” OR fitness OR train*). The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [11] were followed for performing the present review. Due to the study type (systematic review), ethical approval was not necessary according to local registration. Study selection and data extraction Titles and abstracts from the electronic searches were screened independently by two authors (ODV and MM). The full texts of selected articles were checked by the same two authors to consider the fit with eligibility cri- teria. A third reviewer (LS) revised any differences in opinion to make a final decision. An electronic database was designed to store all relevant data. Data were extracted separately by two investigators (ODV and MM), and in the event of disagreement LS cross-examined doubtful data. The following data were extracted: first author, year of publication, country of ori- gin, study type (cross-sectional or longitudinal), study population (sample size, age, gender, period of data collec- tion, and country of residence), type of sport/exercise, presence of a control groups, assessment method and when they were studied. Additionally, we wanted to investigate whether PhA differs between ath- letes and controls or between different sports. Studies with the following criteria were excluded: a) non-healthy athletes; b) articles without full-text avail- ability, opinion pieces, review articles and editorials. Study selection The literature search revealed a total of 196 studies. After exclusion of duplicates (n = 99), by screening ti- tles and abstracts 59/97 studies were excluded be- cause included ill subjects or subjects not practicing a sport or because they were not otherwise appropri- ated. Five reviews were also excluded. The full text of 38 studies was independently examined by two re- viewers. Thirty-five studies (21 cross-sectional and 14 longitudinal studies, of which 12 giving also cross- sectional data) meeting the inclusion criteria and be- ing suitable for the systematic review (Fig. 1). Of the 35 papers analysed, 32 (91.3%) used the classic BIA, one used Tanita (2.9%), one used Inbody720 (2.9%) and one used mBCA Seca (2.9%). Piccoli et al. [13] and Matias et al. [14] measured PhA with bioelectrical im- pedance spectroscopy (BIS) analyser. In 29 studies phase Eligibility criteria Eleven studies evaluated soccer players (34.4%), eight cy- clists (22.9%), six judo players (17.1%), six swimmers (17.1%), six volleyball players (17.1%), five triathlon ath- letes (14.3%), four water polo athletes (11.4%), four hand- ball (11.4%) and four basketball players (11.4%). Other 31 sport specialties were considered in only one study. Eligibility criteria The PICOS strategy was defined as follows: “P” (pa- tients) corresponded to participants of any age, sex or ethnicity, “I” (intervention) designated regular physical exercise at amateur, elite and professional level, “C” (comparison) indicated no physical exercise or low Quality rates were good, fair, or poor as judged by two independent observers (ODV and MM) following the in- structions given by the National Institute of Health and Page 3 of 11 Page 3 of 11 Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 from 1992 to 2019 but most of them (85.7%) appeared in the last 10 years. Overall, 3703 athletes (3172 in cross- sectional and 531 in longitudinal studies) were taken into consideration in this systematic review, with more males (n = 2699) than females (n = 1264), and including children, adolescents and adults. Most of the cross-sectional studies were carried out in Europe (n = 14), especially in Italy (n = 9), six in the United States, Central or South-America and only one in Asia. All the longitudinal studies were performed in Europe (n = 7 in Italy, n = 2 Spain and Portugal, and n = 1 in France, UK and Czech Republic). Eleven studies evaluated soccer players (34.4%), eight cy- clists (22.9%), six judo players (17.1%), six swimmers (17.1%), six volleyball players (17.1%), five triathlon ath- letes (14.3%), four water polo athletes (11.4%), four hand- ball (11.4%) and four basketball players (11.4%). Other 31 sport specialties were considered in only one study. taking into consideration the number of positive re- sponses. High risk of bias translates to a rating of poor quality. Low risk of bias translates to a rating of good quality. from 1992 to 2019 but most of them (85.7%) appeared in the last 10 years. Overall, 3703 athletes (3172 in cross- sectional and 531 in longitudinal studies) were taken into consideration in this systematic review, with more males (n = 2699) than females (n = 1264), and including children, adolescents and adults. Most of the cross-sectional studies were carried out in Europe (n = 14), especially in Italy (n = 9), six in the United States, Central or South-America and only one in Asia. All the longitudinal studies were performed in Europe (n = 7 in Italy, n = 2 Spain and Portugal, and n = 1 in France, UK and Czech Republic). Differences between athletes and controls Six studies have compared PhA in athletes and controls. In the paper by Piccoli et al. [13], professional male bodybuilders (n = 30, 31.2 ± 5.7 yrs) had a higher PhA (+ 17.8% at 50 kHz) than control subjects. This finding suggested more cell membranes per fluid volume unit, i.e. increased intracellular water and BCM. Study characteristics The main characteristics of the selected studies are sum- marized in Tables 1 and 2. The articles were published Fig. 1 Flowchart on the search and selection of articles included in the review Fig. 1 Flowchart on the search and selection of articles included in the review Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Page 4 of 11 Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Page 4 of 11 ble 1 Descriptive characteristics of cross-sectional included studies (n = 21) Table 1 Descriptive characteristics of cross-sectional included studies (n = 21) Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Page 5 of 11 Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Page 5 of 11 Table 2 Descriptive characteristics of longitudinal included studies (n = 14) Table 2 Descriptive characteristics of longitudinal included studies (n = 14) Table 2 Descriptive characteristics of longitudinal included studies (n = 14) rating in terms of quality, eight were rated as fair and four as poor (Additional file 1: Table S1). rating in terms of quality, eight were rated as fair and four as poor (Additional file 1: Table S1). angle was measured at 50 kHz. Piccoli et al. [13], with BIS methodology, measure PhA at 5 and 50 kHz assum- ing that the current path is only extracellular at the low- est frequencies and that is both extra- and intracellular at the highest frequencies. Authors in fourteen papers performed BIA and BIVA. Other information is available in in Table 1 and Table 2. Risk of bias Sample size was small especially in longitudinal studies (Table 2). Measurement conditions of BIA were some- times not completely described. Furthermore, the time period in which patients were included in the studies was not always clearly described. In the same year, D’Alessandro et al. [15] found that female rhythmic gymnasts (n = 55, 15.2 ± 2.2 yrs) had PhA values within the normal range for age and sex. No direct comparison with a control group was reported. The risk of overall bias was moderate to high. Three of the observational studies had an overall good rating in terms of quality, while sixteen were rated as fair and two as poor. Only two of the before-after (pre-post) studies had an overall good Later, Marra et al. [16] showed that female ballet dancers (n = 15, 18.9 ± 1.7 yrs) had significantly higher PhA compared to controls, not only for the whole body Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Page 6 of 11 Page 6 of 11 Page 6 of 11 (+ 9.6%) but also for upper limbs (+ 22.2%) and lower limbs (+ 10.0%). (+ 9.6%) but also for upper limbs (+ 22.2%) and lower limbs (+ 10.0%). Torres et al. [22] studied 158 elite athletes (13–48 yrs) practicing adventure running, cycling, judo, long- distance running, short-distance running, soccer, swim- ming, triathlon and water polo. PhA was lower in adolescents (10–19 years) than in young adults (20–29 years), and increased with age within the same specialty (soccer). The highest mean value was observed in the third decade of life. In the athletes aged 10–19 years, 57% of PhA values were lower than the 5th reference percentile (6) whereas in the other three age groups the corresponding values were 2%, 0% and 0% respectively. Meleleo et al. [17] studied two groups of children: competitive individuals attending swimming and gym- nastics sports clubs (n = 29, 8.0–10.5 yrs) vs. a control group of age-matched healthy children (n = 190, 8.2– 10.5 yrs). At study entry PhA was significantly lower in competitive vs. non-competitive children and this differ- ence was maintained even after 6 months. After one-year follow-up, PhA decreased in competitive children but no statistically significant differences were obtained. y g Recently, Marra et al. Risk of bias [18] evaluated 27 young males: 9 cyclists (28.8 ± 3.5 yrs), 9 dancers (19.2 ± 1.3 yrs) and 9 young control normal-weight men (18.9 ± 2.8 yrs). Data of cyclists were collected during a three-week stage race, while dancers were studied during the ballet season. Whole-body PhA was similar between cyclists and dancers being significantly higher vs. controls (+ 11.4% and + 12.0%). The highest upper limb PhA was observed in dancers with non-significantly differences between cy- clists and controls. Lower-limb PhA is similar in cyclists and dancers but lower in the control group (−15.4%). Mala et al. [23] evaluated PhA in three teams of female national volleyball players: a senior national team (SNT, n = 12, 24.0 ± 1.1 yrs), a junior national team (under 19, n = 12, 18.0 ± 0.6 yrs), and a youth national team (under 17, n = 14, 16.6 ± 0.5 yrs). SNT and U17 players were eval- uated 3 weeks before the European Championship 2008 whereas U19 players were studied 2 weeks before qualifi- cation at European Championship 2008. The highest PhA values were recorded in the SNT group, with a significant difference between SNT and Under 19 players. p y In the study by Koury et al. [24] on male adolescent (n = 105, 15.1 ± 2.1 yrs) and adult (n = 90, 28.9 ± 7.3 yrs) athletes, considering several sport groups (athletics, foot- ball, swimming, water polo, triathlon, basketball, adven- ture running, cycling, marathon and judo), adolescent athletes showed lower PhA than adult athletes (−15.9%). PhA in the adolescents remained lower when sport type was used as a covariate in a multivariate general linear model (p < 0.001). A positive correlation between PhA and age was observed in adolescents, whereas adult ath- letes exhibited a negative correlation. The influence of age on PhA persisted when controlled for sport type. In another paper the same authors [19] studied 28 male marathon runners (personal best in the last year < 195 min; 39.4 ± 9.5 yrs) and 29 male control subjects with aerobic physical activity < 60 min/week. A signifi- cant difference between groups emerged (PhA + 9.7% in marathon runners). Comparisons between different sport disciplines Comparisons between different sport disciplines Five studies compared PhA between athletes practicing different sports. Comparisons between different sport disciplines Five studies compared PhA between athletes practicing different sports. Kim et al. [27], in a conference paper, showed that PhA was higher in 6 female gymnasts (20.8 ± 0.8 yrs., PhA 5.9 ± 0.5 degrees) than 10 female dancers (20.7 ± 0.7 yrs., PhA 5.0 ± 0.3 degrees). Differences between genders Differences in PhA between genders were consistently evaluated in three studies. Veitia et al. [20] performed BIA in 943 Cuban athletes (620 males, 22.8 ± 4.1 yrs., and 323 females 22.4 ± 3.5 yrs) specialized in 26 different sports. Mean PhA value was significantly higher (+ 15.5%) in males than females, with a difference for most of the sports considered. More recently, Carrasco-Marginet et al. [25] evaluated young female elite synchronized swimmers of two age categories (34 comen, 13.9 ± 0.9 yrs., and 15 junior, 16.3 ± 0.6 yrs) performing a single long, high-intensity training session. They found that PhA was significantly higher in junior (+ 7.1%) than comen, with a positive correlation between PhA and age. The same year, Mala et al. [21] assessing whole-body BIA variables in adolescent judo athletes (39 males, 12.1 ± 1.5 yrs., and 20 females, 12.4 ± 1.4 yrs) members of the Czech cadet and junior teams, observed that gender did not have a significant effect on PhA and that there was no difference between the dominant or non- dominant body sides. Finally, Giorgi et al. [26] reported that in 525 male road cyclists (30.1 ± 11.3 yrs) PhA values were higher (not significantly) in youth elite compared to adult elite athletes or adult amateurs. Lastly, in the recent study by Marini et al. [10] on 202 athletes involved in 11 different sports mean PhA was definitely higher in males than females (+ 13.2%). No data were available for males and females practicing the same sport. Differences due to age In the paper by Koury et al. (see above) [24], differ- ences in PhA between various sports were evaluated. PhA in athletes of various age were determined in five studies. Page 7 of 11 Page 7 of 11 Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Page 7 of 11 Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Adolescent football players had a lower mean value than track and field athletes (−31.7%) or basketball players (− 15.3%), An overall significant difference was observed between adult athletes practicing athletics, swimming, tri- athlon, water polo, adventure running, cycling, marathon and judo but no pairwise comparisons were performed. Of note, sample size was small in most experimental groups (even < 10 subjects). Differences due to racial and genetic profile Differences due to racial and genetic profile In the only study reporting data on racial profile, Horto- bagyi et al. [32] showed that mean PhA was higher in 55 black (19.4 ± 1.2 yrs) compared to 35 white (19.7 ± 1.5 yrs) Division I American Football players. y p y Levi Micheli et al. [33] determined the genetic profile in a group of young adolescent Italian medium to high- level soccer players (< 17 yrs) assessing the distribution of ACE genotypes (DD, ID, II) and VDR gene (FF, Ff and ff) polymorphisms, because of their association with performance-related functions. They assessed body com- position with BIA and studied the athletic performance by standard functional performance field tests (squat jump, countermovement jump, 10- and 20-m sprint time). Concerning ACE genotypes, PhA was higher in athletes harboring the D allele. Furthermore, regarding VDR gene, the FF genotype was associated with a mean PhA higher than that observed with FF and ff genotypes. Galanti et al. [28] in male adolescents observed that the average value of PhA was slightly but significantly higher (7.3 ± 0.6 vs. 7.1 ± 0.5 degrees) in cyclists (n = 17, 14–16 yrs) than soccer players (n = 30, 15–16 yrs). Mala et al. [29] studied 80 elite female players (24.9 ± 4.4 yrs) of five team sports (volleyball, softball, basketball, soccer and handball). They observed significant differ- ences in body composition between groups (for instance, with respect to FFM), but did not detect any significant differences in PhA. Differences due to age The variability of PhA was high in all groups, as indicated by the large standard deviation values. Correlation with other variables In their large study, Veitia et al. (see above) [20] stud- ied 943 subjects which made up the Cuban adult na- tional selection in 26 sports. In males, athletes practicing triathlon, weight lifting, boating, artistic gymnastics and wrestling had average values of PhA ≥7 degrees that were higher compared to those of other athletes. In fe- males, athletes from boating, artistic gymnastics and weight lifting had higher average values of PhA (≥6.5 de- grees) than athletes from other sports. Seven studies have evaluated the relationships between PhA and other variables. In the study by Torres et al. [22] (see above) PhA was positively correlated with BMI (r = 0.66; p < .001). Simi- larly, Koury et al. (see above) [24] observed a positive association with both weight and BMI (r = 0.498 and 0.583, respectively, p < 0.01). Ney et al. [34] studied 20 male short-distance swim- mers (18.1 ± 4.1 yrs., 50 and 100 m freestyle) and found significant correlations of PhA with fatty acid and toc- opherol composition in plasma and erythrocytes mem- branes. PhA was positively related (r = 0.51, p = 0.024) with erythrocyte 22:5 n-3 (an index of DHA deficiency). On the contrary, PhA was neither associated with other erythrocyte PUFAs, nor with indices of PUFA and DHA status, or erythrocyte tocopherols. Short-term studies and longitudinal studies A study of Pollastri [42] on 8 elite cyclists (28.8 ± 4.7 yrs) investigated whether body water changes during a multiple-stage bicycle race affected the average maximal mean power (MMP) of different time durations. PhA at baseline was associated with the best MMP over 15 s as observed during competition (20 measurements). Only three papers evaluated changes in PhA immedi- ately before and after a training session. In two of the three short-term studies [36, 37] there were no details regarding intensity and/or volume of the exercise session probably due to the study type (conference papers). Moreno et al. [36] showed that in 12 male cyclists (45.0 ± 8.8 yrs) there was a non-significant difference be- tween PhA during 30 min of exercise in standing pos- ition and on bicycle position. Hard training cyclists exhibited significant PhA changes at exercise peak, but this was not the case for the low training cyclists. Peaks correspond to maximal heart rate. Matias et al. [14] in 20 male judo athletes (22.9 ± 2.9 yrs) observed that PhA did not differ from a period of weight stability to prior competition; mean change in weight was −0.8 ± 2.2 kg. There was a positive associ- ation between changes in PhA and those in serum and RBC Mg levels. Meleleo et al. [17] studied two groups of children: competitive subjects attending swimming and gymnas- tics sports clubs (n = 29, 8.0–10.5 yrs) and ‘control’ age- matched healthy children (n = 190, 8.2–10.5 yrs). At baseline PhA was significantly lower in competitive sub- jects and this difference was maintained even after 6 months. After one-year follow-up, PhA decreased, but not significantly, in competitive children. In another conference paper, junior (n = 18, 16.7 ± 0.9 yrs) and pre-junior (n = 41, 13.9 ± 0.9 yrs) female syn- chronized swimmers were studied by Irurtia et al. [37]. All BIA parameters, except PhA, in both groups varied after training session. More recently, Carrasco-Marginet et al. [25] (see above) observed a significantly increased PhA between pre and post-training (p < 0.05) in both junior (208.4 ± 10.3 min of training with 6.8 ± 0.6 rating of per- ceived exertion, following the RPE scale) and comen (149.6 ± 3.3 min of training with 6.4 ± 0.5 of RPE) elite synchronized swimmers. PhA was negatively related to BIA-derived ECW/TBW ratio. Comparisons within the same sport discipline Journal of the International Society of Sports Nutrition Page 8 of 11 Page 8 of 11 zinc concentration (p = 0.047) were both positive predic- tors of PhA. through (Δ = −0.51 ± 0.45, p < 0.001) and at the end of the race (Δ = −1.00 ± 0.27, p < 0.001). The same authors [41] assessed segmental BIA in a group of professional cyclists (n. 9, 28.8 ± 3.5 yrs) participating in a multiple- stage bicycle race. Whole-body PhA did not significantly change after the first half of the race but significantly de- creased at the end (p < 0.05). Upper-limb PhA did not significantly change whereas a significant reduction was reported for lower-limb PhA. In a relevant cross-sectional study in 202 athletes, Ma- rini et al. [10] showed that in both males and females PhA was negatively correlated with the ECW/ICW ratio (males: r = −0.493, p < 0.001; females: r = −0.408, p < 0.001), while there was a positive association with ICW (males: r = 0.327, p < 0.001; females: r = 0.243, p = 0.080). Comparisons within the same sport discipline Three studies evaluated the possible variation of PhA due to different performance levels. Maly et al. [30] stud- ied two volleyball teams (n = 12, 24.3 ± 2.7 yrs., and n = 9, 20.8 ± 2.1 yrs), participating in the CEV Champion League 2008–2009. The first team did not pass beyond the basic round, whereas the second one participated in the quarterfinal round. There was no significant differ- ence in mean PhA between the two teams. Levi Micheli et al. (see above) [33] claimed that in a well-trained population, PhA and anthropometric values were not correlated with athletic performance. In the abovementioned study by Mala et al. [21] in judo adolescent athletes a significant correlation emerged be- tween PhA and handgrip strength (boys: r = 0.64, p < 0.01, girls: r = 0.61, p < 0.01) for the dominant limb. In the study by Levi Micheli et al. [31] 893 male soccer players (24.1 ± 5.1 yrs) were subdivided in five groups according to performance level (i.e. the div- ision in which the team plays). An increased PhA was observed in the elite-level group compared to the other groups (high-level, medium-level, medium-low level and low-level). In a recent study Koury et al. [35] evaluated the rela- tion between minerals and PhA. It was found that in 40 adolescent male soccer athletes (13.4 ± 0.6 yrs), PhA tended (p = 0.010) to be higher in adolescents classified by bone age as “Early” compared to “Late”. PhA also cor- related (p < 0.05) with bone age (r = 0.562), BMI (r = 0.382), FFM (r = 0.468) and erythrocyte zinc concentra- tion (r = 0.379). PhA was higher in adolescents with erythrocyte zinc concentration above the median than those below the median. Multiple linear regression ana- lysis revealed that bone age (p = 0.001) and erythrocyte Finally, Giorgi et al. (see above) [26] reported that PhA of elite (n = 79, 21.1 ± 2.9 yrs) and amateur cyclists (n = 232, 39.0 ± 10.5 yrs) (but not that of youth elite cyclists, n = 59, 16.8 ± 1.1 yrs) was lower (p < 0.05) in comparison to professionals (n = 155, 26.3 ± 4.7 yrs). Among these latter PhA was lower for climbers compared to sprinters and all-rounders (p < 0.05). Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 Di Vincenzo et al. Discussion BIA is applied in athletes as a field technique to estimate body composition, being useful in sport science for sin- gle measurements or for tracking body composition changes [7]. On the other hand, raw BIA variables, such as PhA or IR, are commonly related to ECW/ICW ratio, BCM, and cellular integrity [2]. In addition, an associ- ation between muscle strength and PhA has been ob- served in various pathophysiological conditions (for instance,1–3), suggesting that raw BIA may be useful in assessing muscle quality. The papers selected for gender diversity are in line with the aforementioned findings, with no difference in young adolescent judo athletes [21] and significant higher values in adolescent/adult male compared to female athletes [20]. Similarly, four out of five selected papers reported an age trend in various sports [22–25], whereas a single paper found the opposite, with higher PhA in adolescent male than adult male road cyclists [26]. It should be noted that differences in years of practice and training programmes may influence changes with time. In this context, only a few papers have so far evaluated raw BIA variables in athletes. A recent systematic review examined the applications of BIVA in sports and exercise, a methodology giving information on hydration status by analyzing the length of bioimpedance vector and its inclin- ation [9]. The authors concluded that the current tech- nique, called “classical BIVA”, is not fully reliable to identify dehydration in individual athletes. The review by Custodio Martins et al. [47] explored the use of different BIA-derived estimates of body composition in athletes, adding a concise, preliminary view on PhA, a raw BIA variable that has been considered in recent years for assessing body composition in various pathophysiological conditions [1–3]. A key point of the present review was to evaluate whether and to what extent PhA differs between different sports and performance levels. Overall, the selected papers have provided inconsistent and puzzling findings, possibly because of inappropriate study design (for instance, in selecting subjects) or small sample sizes. The variability of PhA was high, as indicated by large standard deviation values [27–29]. Variations between sports emerge but no definite conclusions could be drawn on endurance vs. resistance training or recreational vs. competitive sports, although some results suggest indirectly that PhA increase with muscle-strengthening activities [20]. Short-term studies and longitudinal studies Journal of the International Society of Sports Nutrition (2019) 16:49 Page 9 of 11 Page 9 of 11 (2019) 16:49 topic for future research on sport nutrition. The authors studied the effects of a resistance training programme (at least 3 h/week) in 14 resistance-trained individuals (8 males 30 ± 6 yrs.; 6 females 33 ± 6 yrs). They underwent two 10-day isocaloric dietary regimes with a protein con- tent of 1.8 g × kg−1 × d−1 (PROMOD) or 2.9 g × kg−1 × d−1 (PROHIGH). On days 8–10 (T1-T3), participants undertook resistance exercise under controlled condi- tions, performing 3 sets of squats, bench press and bent- over rows at 80% 1 repetition maximum until volitional exhaustion. In PROHIGH group PhA increased at T3 compared to T1 and T2, while it tended to decrease in the PROHIGH group, although not significantly. PhA was slightly higher at T3 for PROHIGH (+ 2.2%) com- pared with PROMOD (p = 0.012). also BCM and muscle cell mass. The first question in this study sought to determine whether PhA differs between athletes and control subjects. Surprisingly, only few papers have so far addressed this issue, sometimes in small groups of athletes. A very marked increase in PhA was observed in bodybuilders [13] (+ 17.8% on the average), female dancers [16] (+ 9.6%), male dancers [18] (+ 12.0%), cyclists [18] (+ 11.4%) and marathon runners [19] (+ 9.7%). Thus, these findings suggest that muscle strengthening causes a greater increase in PhA compared to endurance training. Indeed, contrary to expectations, Meleleo et al. [17] reported that PhA was significantly lower in com- petitive vs. non-competitive children, suggesting that the effects of training on PhA may be different in childhood. As far as main individual’s characteristics are con- cerned, in the general population PhA increases with age in both genders until late adulthood and then decrease in the elderly [22–26], with a between-gender difference that becomes greater through adolescence [48, 49] and with mean values in adult age consistently higher in males than females [5, 6]. Thus, these findings suggest that muscle strengthening causes a greater increase in PhA compared to endurance training. Indeed, contrary to expectations, Meleleo et al. [17] reported that PhA was significantly lower in com- petitive vs. non-competitive children, suggesting that the effects of training on PhA may be different in childhood. Short-term studies and longitudinal studies No correlations were ob- served between bioelectrical pre to post changes in rela- tion to BM. Mala et al. [43] studied 10 elite youth judo athletes (22.1 ± 2.8 yrs) before and after pre-competitive weight loss (6 day on average, using dehydration). BIA was per- formed before and after the weight reduction period, 6 days apart. Mean PhA significantly decreased after weight loss (−4.1%, p < 0.01). In the study of Melchiorri et al. [44], 21 male elite water polo athletes intensively trained based on a detailed pro- gram for 3 months before the Olympic Games. Only 13 athletes (OA, 29.7 ± 3.4 yrs) participated to the Olympics Games, 8 were excluded (NOA, 27.4 ± 5.5 yrs). PhA was evaluated after the first (T0), second (T1) and third (T2) month of training. There was no statistical difference among the three measurements for PhA in the OA group. Furthermore, data showed no statistically significant dif- ferences of PhA between the OA and NOA groups. Eleven papers have evaluated changes in PhA with time due to training programmes and/or other planned interventions. Mascherini et al. [38] reported data on 11 professional male soccer players (22.4 ± 1.8 yrs) measuring their PhA eight times during regular season. Mean PhA was signifi- cantly lower than baseline 3 weeks and 9 weeks after starting training. Later, in 18 professional italian soccer players (21.8 ± 3.0 yrs) the same authors [39] found that PhA increased significantly at mid-season compared to pre-season (p < 0.05). Campa et al. [45] performed BIA in 58 athletes at baseline and after 6 months during the competitive sea- son. PhA variations were positively associated with TBW and ICW and negatively associated with the ECW/ICW ratio. Marra et al. [40] evaluated whole-body PhA in profes- sional cyclists (n = 9, 26.7 ± 2.5 yrs) participating in a 3- week stage race. They collected data at the beginning (the day before the race), halfway through (rest day, after the 9th lap) and at the end of the race (the last day, after the 20th lap). PhA was significantly decreased halfway Finally, although not concerning athletes, we consid- ered Roberts’s study [46] because it gave some informa- tion on the effects of protein supplementation plus physical activity on phase angle. This is an interesting Di Vincenzo et al. Consent for publication Not applicable. Consent for publication Not applicable. Funding Not applicable. Authors’ contributions LS, MM, and ODV conceived the review. ODV and LS undertook the literature search, data extraction, and drafted the report. MM assisted in interpretation of results and was involved in the critical revision of report. All authors read and approved the final manuscript. Finally, longitudinal evaluation of body composition may offer, at least in theory, relevant information on the changes in body composition and hydration due to training or untraining, which might be associated with physical performance. Unfortunately, the papers se- lected for the present review [14, 17, 25, 36–46] have given inconsistent results. A comprehensive view of the issue cannot be formed because they considered differ- ent athletic disciplines and had very different experi- mental protocols (sometimes with small experimental groups). Received: 18 July 2019 Accepted: 16 October 2019 Received: 18 July 2019 Accepted: 16 October 2019 Discussion In this systematic review, we aimed to extend previous information on PhA values as measured in athletes by focusing in depth on different issues of interest. Thirty- five papers were selected according to inclusion and ex- clusion criteria. In almost all cases single-frequency BIA has been performed (on the whole body). Although it is well known the standardization of measurement condi- tions is essential for obtaining accurate and reproducible BIA data, most of selected studies did not give enough details in this respect, in particular on the length of time since the last training session (a critical aspect especially in the case of strenuous exercise). Turning to athletes of the same sport, two studies [26, 31] demonstrated that PhA was higher in soccer players and cyclists with a better performance level, whereas another one did not find differences between a stronger and a weaker volleyball teams [30]. Thus, it could be argued (but not definitely demonstrated), that the rela- tionships between PhA and performance level may vary in different sports and are possibly influenced by the criteria used to assess performance level. Interestingly, changes emerge also for the same sport when athletes differ depending on their physical characteristics. For One might expect that training, especially muscle strengthening, should affect not only muscle function but Page 10 of 11 Di Vincenzo et al. Journal of the International Society of Sports Nutrition (2019) 16:49 instance, among cyclists PhA was lower for climbers compared to sprinters and all-rounders [26]. Author details 1 1Department of Clinical Medicine and Surgery, Federico II University Hospital, Via S. Pansini 5, 80138 Naples, Italy. 2Department of Public Health, School of Medicine, Federico II University, Naples, Italy. 1Department of Clinical Medicine and Surgery, Federico II University Hospital, Via S. Pansini 5, 80138 Naples, Italy. 2Department of Public Health, School of Medicine, Federico II University, Naples, Italy. Competing interests Competing interests The authors declare that they have no competing interests. Conclusions This systematic review aimed to summarise the current knowledge on the evaluation of BIA-derived PhA in ath- letes. Of note, two recent studies strongly support the idea that PhA is an index of ECW/ICW ratio or BCM [10, 45]. PhA increases with age and is likely to be higher in males. Unfortunately, it is still uncertain to what extent PhA varies between different sports and changes with training/untraining. It can be argued that for a given sport much more data should be collected in a systematic way and for a period of time appropriate in order to determine changes and trends. This is even more crucial in the case of intervention studies. References 1. Norman K, Stobäus N, Pirlich M, Bosy-Westphal A. Bioelectrical phase angle and impedance vector analysis - clinical relevance and applicability of impedance parameters. Clin Nutr. 2012;31(6):854–61. 1. Norman K, Stobäus N, Pirlich M, Bosy-Westphal A. Bioelectrical phase angle and impedance vector analysis - clinical relevance and applicability of impedance parameters. Clin Nutr. 2012;31(6):854–61. 2. Lukaski HC, Kyle UG, Kondrup J. 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Abbreviations BCM: Body Cell Mass; BIA: Bioelectrical Impedance Analysis; BIS: Bioelectrical Impedance Spectroscopy; BIVA: Bioelectrical Impedance Vector Analysis; ECW: Extracellular Water; FFM: Fat-Free Mass; FM: Fat Mass; ICW: Intracellular Water; IR: Impedance Ratio; PhA: Phase Angle; R: Resistance; TBW: Total Body Water; Xc: Reactance; Z: Impedance Overall, in order to interpret variability of PhA, a sin- gle study [33] indicated that PhA is influenced by ACE or VDR gene polymorphisms, in line with their involve- ment in a variety of performance-related functions. In addition, another study has shown that mean PhA was higher in white than black football players [32], which may not surprising given that differences in body com- position due to ethnicity are well known [50]. Availability of data and materials ll d h l All data pertaining to the conclusions of the study are found within the article. The corresponding data set used is available under reasonable requests. 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https://openalex.org/W4320511388	https://zenodo.org/records/6558047/files/%D0%9C%D1%83%D1%81%D0%B0%D0%B5%D0%B2%20%D0%97%D0%B8%D0%BA%D0%B8%D1%80%20%D0%97%D0%BE%D0%BA%D0%B8%D1%80%D0%BE%D0%B2%D0%B8%D1%87.pdf	Russian	null	ИНФЛЯЦИЯНИ ЮЗАГА КЕЛТИРУВЧИ ОМИЛЛАР ВА УНГА ҚАРШИ СИЁСАТНИНГ ЎЗИГА ХОС ХУСУСИЯТЛАРИ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,643	ИНФЛЯЦИЯНИ ЮЗАГА КЕЛТИРУВЧИ ОМИЛЛАР ВА УНГА ҚАРШИ СИЁСАТНИНГ ЎЗИГА ХОС ХУСУСИЯТЛАРИ https://doi.org/10.5281/zenodo.6558047 Мусаев Зикир Зокирович магистрант, Мирзо Улуғбек номидаги Ўзбекистон Миллий университети, Тошкент, Ўзбекистон и.ф.д. проф. Вахабов А. В. Мирзо Улуғбек номидаги Ўзбекистон Миллий университети, Тошкент, Ўзбекистон Аннотация: Инфляция даражасини янада пасайтириш борасидаги чора-тадбирларнинг амалга оширилиши аҳоли турмуш даражасини яхшилаш ва барқарор иқтисодий ўсиш учун шароитлар яратишга қаратилган иқтисодий ислоҳотлар самарадорлигини таъминлашнинг муҳим омили ҳисобланади. Мақола мазмуни, инфляциянинг иқтисодиётимизга таъсирини очиб беради. Калит сўзлар: инфляция; ЯИМ; ЯИМ дифлятори; экспорт; импорт. Инфляция - бу товарлар ва хизматлар баҳоларининг ўсиши натижасида муомаладаги пул массасининг кўпайиши ва шунинг натижасида пулнинг қадрсизланишидир. П.Грауд ва М.Полан томонидан амалга оширилган ва инфляцияга бағишланган жиддий илмий тадқиқотлардан бири бўлиб, ушбу илмий иш 165 мамлакатни ва 1969- 1999 йилларни қамраб олади [1]. Мазкур тадқиқот муаллифлари инфляциянинг миқдорийлик назарияси қоидаларидан бири бўлган - пулларнинг инг ръати ва ия си а тўғри иблик деган ўғри ва ик отларда анади, хулосага р. пуллар экзоген 1-Расм. Инфляцияга таъсир этувчи омиллар ҳақида маълумот 1-Расм. Инфляцияга таъсир этувчи омиллар ҳақида маълумот ўсиш суръати ва инфляция даражаси ўртасида тўғри мутаносиблик мавжуд, деган қоида тўғри ва статистик маълумотларда тасдиқланади, деган хулосага келдилар. Демак, пуллар таклифи экзоген омиллар туфайли юзага келган пулларга бўлган талабнинг ўзгаришига мослашади. Инфляциянинг оқилона даражасига эга бўлган мамлакатларда корреляция коэфффициэнти 0,24 га тенг бўлган бўлса, инфляция даражаси юқори бўлган мамлакатларда ушбу коэффициэнт 1 га яқинлашди. Инфляция – пул муомаласи қонунининг бузилиши, иқтисодиётда товар массаси ҳамда пул массаси ўртасидаги номутаносиблик жараёнида вужудга келади. Инфляцияни вужудга келишига қатор омиллар таъсир қилиб, уларни ички ва ташқи омилларга ажратиш мумкин [1]. 1-расмда қайд этилгандек, ички омиллар таркибига ортиқча пул массаси вужудга келиши, миллий валютага нисбатан ишончнинг пасайиши, мамлакатнинг номутаносиб тўлов баланси кабиларни киритиш мумкин. Ташқи омилларга мамлакат ташқи сиёсатининг беқарорлиги, иқтисодий инқирозлар, молия ва фонд бозорларида индексларнинг ўзгариши кабиларни келтириш мумкин. Инфляциянинг вужудга келишига муомала ва ишлаб чиқариш соҳаси, шунингдек, мамлакатдаги иқтисодий – сиёсий омиллар ҳам сабаб бўлади. Инфляциянинг вужудга келишида пул муомаласи омилларига бюджет тақчиллигини қоплаш мақсадида муомалага қўшимча тарзда чиқарилган пуллар, иқтисодиётда асоссиз равишда берилган кредитлар ҳажмининг ортиб бориши, мамлакат пул – кредит сиёсатига аҳоли ишончининг пасайиши ва бошқалар сабаб бўлади. Ишлаб чиқариш ва иқтисодий сиёсий омилларга мамлакатда импорт ҳажмининг экспорт ҳажмидан ортиб кетиши, товарлар ва кўрсатилаётган хизматлар ҳажми ва сифатининг пастлиги, бюджет – солиқ сиёсати, мамлакатнинг ташқи ва ички иқтисодий муносабатлар доирасида амалга ошираётган сиёсати ва бошқалар сабаб бўлади. Инфляциянинг вужудга келишининг асосий сабабларидан бири иқтисодиётдаги ялпи талаб ва ялпи таклиф ўртасидаги мувозанатнинг бузилишидир. Инфляция шароитида капитал ишлаб чиқариш доирасидан муомала доирасига оқиб ўта бошлайди, чунки ишлаб чиқариш жараёни иқтисодий жиҳатдан самарасиз соҳага айланиб боради. Муомала жараёни кўп вақтни талаб этмаганлиги боис, дастлабки босқичларда унинг иштирокчиларига маълум миқдорда иқтисодий фойда келтиради, лекин инфляция жараёнларини тезлаштиришга хизмат қилади. Инфляция механизми ўз – ўзидан ривожланиб, барча соҳа ва тармоқларни қамраб олади, унинг таъсирида жамғармалар ҳажми қисқаради, кредит, инвестиция ва товарлар таклифи камаяди [2]. 2-расмда қайд этилгандек, инфляция миллий даромадга ва ишсизликка кескин таъсир кўрсатади. 2 Р И ф й Яъни, юқоридагидек, уларнинг салмоғи ўсиш ёки пасайиш тенденциясига эга бўлади. Инфляцияни келтириб чиқарувчи биринчи сабаб – мамлакатда бозор механизмларининг тўлиқ жорий этилмаслиги, давлатнинг бюджет даромадлари ва харажатлари устидан монопол тартибини жорий этиши ҳисобланади. Бунда давлат бюджетининг даромадларидан ортиқча бўлган харажатлар, яъни бюджет тақчиллиги муомалага қўшимча пуллар чиқариш йўли билан қопланади. Бюджет тақчиллигини қоплаш учун муомалага чиқарилган пуллар товар моддий қийматликлари билан таъминланмаганлиги оқибатида мамлакатда инфляция даражасининг ортишига олиб келади. Иккинчи сабаби – асоссиз равишда иш ҳақи ва бошқа тўловларнинг оширилиши, ушбу тўловлар давлат бюджети харажатлари таркибида қўзда тутилмаган бўлса, муомалага қўшимча эмиссия ҳисобига амалга оширилади. Бунинг натижасида иқтисодиётда товарлар ва хизматларнинг баҳоси ошади ва инфляция даражаси ортиб боради. Учинчи сабаби – мамлакатда ишлаб чиқарилаётган товарларнинг экспорт қилиш даражасининг пастлиги ва импорт даражасининг юқорилиги билан ифодаланади [2]. Мамлакатга кириб келаётган импорт товарлар ҳисобига хорижий валюталарнинг четга оқиб кетиши юз беради, натижада мамлакатда ишлаб чиқарилаётган товарлар нафақат ташқи эҳтиёжларни, балки ички эҳтиёжларни қондиришга ҳам хизмат қилмай қўяди. Бунинг оқибатида миллий валютанинг сотиб олиш қобилияти таборо салбийлашиб, инфляция даражаси ортиб боради. Ҳозирги иқтисодиётни модернизациялаш шароитида аҳоли ва хўжалик юрутувчи субъэктлар жорий ҳисобрақамларидаги валюта маблағларини тижорат банкларининг муддатли ва жамғарма депозит ҳисобрақамларига жалб этиш ва уларни иқтисодиётнинг устувор йўналишларига кредит сифатида йўналтириш иқтисодиётни ривожлантиришнинг устувор вазифаларидан бири ҳисобланади. Ушбу вазифани ҳал этиш, ўз навбатида, хорижий валюталарда банкларга жалб этилган муддатли ва жамғарма депозитларига тўланадиган фоиз ставкаларини ошириш йўли билан мазкур депозитларнинг аҳоли ва корхоналар учун жозибадорлигини оширишни тақозо этади [3]. Инфляцияни юзага келтирувчи асосий омиллар сифатида қуйидагиларни кўрсатиш мумкин: 1. Ўзбекистон Республикасида бюджет ташкилотлари ходимлари иш ҳақининг ўсиши билан истеъмол товарлари баҳоларининг ўсиши ўртасидаги бевосита боғлиқлик мавжуд. 2. Республикамизда талаб инфляциясининг кучайиши натижасида ЯИМ дефляторининг юқори даражаси сақланиб қолмоқда. 3. Табиий монополиялар маҳсулотлари ва хизматлари баҳоларининг ўсиш суръатларига эга эканлиги инфляцион жараёнларнинг юзага келишида муҳим ўрин тутмоқда. 3. Табиий монополиялар маҳсулотлари ва хизматлари баҳоларининг ўсиш суръатларига эга эканлиги инфляцион жараёнларнинг юзага келишида муҳим ўрин тутмоқда. 4. Нақд пулларга бўлган талабнинг нисбатан юқори эканлиги. 5. Монетар сиёсатни такомиллаштириш билан боғлиқ бўлган муаммоларнинг мавжудлиги. Ўзбекистон Республикаси Марказий банки қайта молиялаш сиёсатини такомиллаштириш борасидаги муаммоларни ҳал қилиш Марказий банкнинг кредитларнинг фоиз ставкаларига таъсир этиш орқали муомаладаги пул массасини тартибга солиш имкониятини оширади. Бу эса, пировард натижада, хўжалик юритувчи субъэктлар ва аҳолининг тўлов қобилиятини рағбатлантириш ва миллий валютанинг барқарорлигини таъминлашга хизмат қилади [4]. Ўзбекистон Республикаси Марказий банки қайта молиялаш сиёсатини такомиллаштириш борасидаги муаммоларни ҳал қилиш Марказий банкнинг кредитларнинг фоиз ставкаларига таъсир этиш орқали муомаладаги пул массасини тартибга солиш имкониятини оширади. Бу эса, пировард натижада, хўжалик юритувчи субъэктлар ва аҳолининг тўлов қобилиятини рағбатлантириш ва миллий валютанинг барқарорлигини таъминлашга хизмат қилади [4]. Ўзбекистон Республикаси Марказий банкининг амалдаги мажбурий захира сиёсатининг икки салбий жиҳати мавжуд: Ўзбекистон Республикаси Марказий банкининг амалдаги мажбурий захира сиёсатининг икки салбий жиҳати мавжуд: 1. Мажбурий захира ставкаларини сўмдаги депозитларнинг ҳар бир тури бўйича уларнинг суммасига боғлиқ равишда табақалаштирилмаганлиги натижасида трансакцион депозитларнинг сўмнинг алмашув курсига нисбатан салбий таъсири юзага келмоқда. 2. Тижорат банкларининг хорижий валютадаги депозитларига нисбатан мажбурий захира талабномаларининг жорий этилганлиги уларнинг хорижий валютадаги маблағларни муддатли ва жамғарма депозит ҳисобрақамларига жалб этиш имкониятини янада пасайтиради. Инфляциянинг нисбатан умумий ҳамда анъанавий таърифи қуйидагича: Инфляция — муомалада керагидан ортиқча пул пайдо бўлиб, нарх-наво ўсиб, пул қадр-қиймати яни харид қобилиятининг пасайшни, пулнинг обрўсизланишидир. Инфляция - бу барча товар ва хизматларга бўлган нарх индексининг кўтарилишидир.Инфляция нархларнинг ўсиш даражасини ўлчаш билан аниқланади. Кўпинча бунинг учун нархларнинг ўсиш динамикасини ифодаловчи кўрсаткичлардан фойдаланилади. Асосий кўрсаткич бўлиб нарх индекси ҳисобланади. У жорий йилдаги ўртача нархларни аввалги йилдаги ўртача нархларга нисбати тарзида ҳисобланади. Жаҳон тажрибасида улгуржи баҳолар индекси, чакана нархлар (истеъмол) индекси, ЯИМ, ЯИМ дифлятори — индекси, экспорт ва импорт нархлари ва бошқалар ҳисобланади. Аммо инфляцияни пулнинг қадрини пасайиши, муомалада қоғоз пулларнинг кўпайиб кетиши тарзида кўрсатиш тўлиқ эмас, у нархларнинг ўсиши тарзида намоён бўлса ҳам уни фақат пул билан боғлаш ярамайди. У мураккаб иқтисодий воқелик, у бутун такрор ишлаб чиқаришнинг диспропорцияси, мутаносибликни бузилишини ифодалайди. Пул муомаласи ҳолатига боғлиқ бўлмаган ҳолда ҳам товар нархи кўтарилиши мумкин. Меҳнат унумдорлигини ўзгариши, циклик ва сезонли тебранишлар, таркибий силжишлар, иқтисодиётни давлат томонидан тартибга солиниши, янги солиқ ставкаларини белгилаш, бозор конюнктурасининг ўзгариши, ташқи иқтисодий алоқаларнинг таъсири, стихияли фалокатлар ҳам нархларга таъсир қилади. Хуллас нархнинг ўсишига турли сабаблар бўлиши мумкин. Лекин ҳар қандай нархнинг ўсиши ҳам инфляция бўлавермайди. Юқорида кўрсатиб ўтган сабаблардан ҳақиқий инфляцияни олиб келувчиларни ажратиш зарур [5]. Конюнктуранинг циклик тебранишлар, табиий офатлари натижасида Конюнктуранинг циклик тебранишлар, табиий офатлари натижасида нархларнинг ўсишини инфляция деб айта олмаймиз. Муомаладаги тўлов воситаларини мўллиги товарлар таклифи чекланганлиги билан тўқнашиб, у нархлар умумий даражасининг ўсишига олиб келади. Таклиф ёки харажатлар инфляцияси – (cост-пуш инфлатион) нархларнинг ишлаб чиқариш харажатларини ўсиши билан характерланади. Харажатларни ўсишига нархни монополистик бозор томонидан шаклланиши, давлатнинг молия сиёсати, хом-ашё нархларининг кўтарилиши, касаба уюшмаларининг талабига кўра иш ҳақининг ўсиши ва бошқалар сабаб бўлади. Аввалги миқдордаги қилинган харажатга аввалгидан кам миқдорда маҳсулот ишлаб чиқарилади. Натижада ўсган харажатларни қоплаш учун нарх оширилади. Амалиётда талаб инфляцияси билан таклиф инфляциясини ажратиш қийин. Улар бир-бири билан чамбарчас боғланган. Масалан иш ҳақининг ўсиши ҳам талаб, ҳам таклиф инфляцияси тарзида ифодаланиши мумкин. Шунга аҳамият бериши керакки, ХХ асрнинг иккинчи ярмида узоқ вақт ҳеч қайси ривожланган мамлакатларда бир вақтнинг ўзида тўла бандлик, очиқ бозор ва нархлар барқарорлиги кузатилмади. Нархлар доимий ўсиб бораяпти, уни иқтисодий ҳамкорлик ва ривожланиш ташкилоти маълумотларидан кўриш мумкин. Инфляция суръатлари нисбатан ривожланаётган мамлакатларда, айниқса лотин Америкаси мамлакатларида юқори. Бунга сабаб давлат бюджетининг катта тақчиллиги ва ташқи қарзларнинг кўплигидир. 60 йилларнинг охирларидан бошлаб нархлар хатто иқтисодий тушкунлик, тургунлик даврларида ҳам ХИХ аср охири ва ХХ асрнинг биринчи ярмида ишониб бўлмайдиган ҳолда ўсган. Инфляция юз бериш миқёсига кўра: локал — бир мамлакат миқёсидаги, ҳамда жаҳон миқёсида юз берадиган инфляция тарзида ҳам изоҳланади. Бундан ташқари иқтисодий адабиётларда баланслашган ҳамда баланслашмаган инфляция тушунчалари ҳам мавжуд. Баланслашган инфляция деганда бир вақтнинг ўзида кўпгина товар ва хизматлар нархларининг унча кўп бўлмаган миқдорда ўсиши тушунилади. Нархларнинг йиллик ўсиш бўйича, % ставкаси ҳам кўтарилади, буни стабил нархлар билан тенглаштириш мумкин. Баланслашмаган инфляция эса ҳар хил товарлар нархининг ўсиш суръати турлича бўлишини ифодалайди. Кутилаёттан инфляция билан кутилмаган инфляцияни ҳам фарқлаш зарур. Кутилаётган инфляция олдиндан айтиш мумкин ёки уни ҳукумат томонидан режалаштирилади. Кутилмаган инфляция тўсатдан нархларнинг кўтарилиб кетиши билан характерланади. У пул муомаласи ва солиқ тизимига ёмон таъсир қилади. Бундан аҳоли иложи борича пулидан тезроқ қутилишга ҳаракат қилади. Бу харакат оқибати қандай бўлишини биламиз. Тўсатдан бўлган инфляция иқтисодиётда кутилаётган, лекин ҳали бошланмаган инфляция жараёнида юз берса, аҳолининг ўзини тутиши тамомила бошқача бўлиши мумкин. Нархларнинг ўсиши қисқа фурсатда юз берадиган ҳолат деб истеъмолчилар нархларни пасайишини пойлаб, кам пул сарфлайдилар. Бу ўз навбатида талабни пасайтиради ва натижада нархларни пасайиши кузатилади [6]. Дунё тажрибаси кўрсатишича, бир тизимдан иккинчи тизимга ўтиш шароитида, одатда кучли инфляцион жараёнлар билан содир бўлади. Ушбу холат собиқ иттифоқ таркибига кирган барча республикалар, шу жумладан Ўзбекистон Республикасида ҳам кузатилди. Мутақилликнинг дастлабки йилларида мамлакатимизда кучли инфляция кузатилган бўлса, охирги тўққиз йилда уни жиловлашга муваффақ бўлинди. Мустақилликнинг 20 йили давомида инфляциянинг турли шакллари намоён бўлди. Инфляциянинг оқибатлари жуда салбий холатларга олиб келади: аҳоли жамғармалари ва пулнинг қадрсизланиши ва ҳ.к. Замонавий ўзбек иқтисодиётига нисбатан икки хил қараш мавжуд. Биринчи қарашга биноан, ўзбек иқтисодиёти бошқалар каби иқтисодиёти, фақат бошланғич шартлари билан фарқ қилади. Бошқа қарашга кўра эса Ўзбекистонда ўзига хос хусусиятлар шунчалик кўпки, унинг муаммоларини ҳал қилиш учун янги иқтисодий назария бўлмасада, ҳеч бўлмаганда, мавжуд стандарт қарашларни сезиларли даражада модификация қилиш талаб қилинади. Мамлакат миллий валютасининг харид қобилиятини мустаҳкамлаш, валюта курси барқарорлигини сақлаш, иқтисодиётнинг пулга бўлган талаб ва таклифини мувозанатли равишда қондириш, миллий ва халқаро ҳисоб-китоблар тизимининг узлуксиз ҳамда самарали ишлашини таъминлаш бошқа омиллар билан бир қаторда, монетар сиёсатда қўлланиладиган таргетлаш воситаларига ҳам боғлиқдир. Иқтисодий категориялар хилма – хил бўлиши билан бирга ҳар турли иқтисодий тизимларда амал қилиш хусусияти ва шартлари ҳам турлича бўлади. Талаб, таклиф, рақобат каби терминлар режали иқтисодиёт учун ёд тушунчалар бўлгани ҳолда инфляция атамаси барча иқтисодий тизимлар учун хос бўлган категория ҳисобланади. Инфляцион жараёнлар бошқарилиши муаммоси барча иқтисодий тизимларда мавжуд. Инфляция бошқарилиши бир вақтнинг ўзида барча макроиқтисодий кўрсаткичларга, иқтисодий субъэктлар фаолиятига, аҳоли даромадларига ва инвестицион фаолликка таъсир кўрсатади [7]. Ҳозирги даврда инфляция муаммоси иқтисодчи олимлар қашшоқ ёки ривожланаётган мамлакатлар ҳамда иқтисодиёти тараққий топган мамлакатлар ҳукуматларининг ҳам асосий диққат эътиборида турган умумиқтисодий ҳолат ҳисобланади. Айниқса, ушбу муаммо ҳамон давом этаётган жаҳон молиявийиқтисодий инқирози оқибатларини бартараф этишда жиддий тус олмоқда. Ҳар бир алоҳида олинган мамлакатда инфляция суръатларини бошқариш масаласи жуда муҳим аҳамият касб этади. Инфляцияга қарши кураш сиёсатининг асосий мақсади бўлиб, инфляция устидан назорат ўрнатиш ва инфляцион жараёнларнинг чуқурлашишига йўл қўймаслик ҳисобланади. Инфляцияга қарши кураш сиёсатининг муҳим йўналишлари сифатида қуйидагиларни кўрсатиш мумкин: Инфляцияга қарши кураш сиёсатининг муҳим йўналишлари сифатида қуйидагиларни кўрсатиш мумкин: 1. Инфляцияни юзага келтирувчи монетар ва номонетар омилларнинг ҳақиқатдаги даражасига баҳо бериш. 2. Табиий монополиялар маҳсулотлари ва хизматлари баҳоларини тартибга солиш механизмини такомиллаштириш. 3. Хўжалик юритувчи субъэктлар ва аҳолининг инфляцион кутиш психологиясига барҳам бериш. 4. Молия бозорларини ривожлантириш ва давлат томонидан тартибга солиш механизмини такомиллаштириш. 5. Пул массасининг ўсиш суръатларининг барқарорлигини таъминлаш. Иқтисодиётни модернизациялаш босқичида аҳоли ва корхоналар жорий ҳисобрақамларидаги валюта маблағларини тижорат банкларининг муддатли ва жамғарма депозит ҳисобрақамларига жалб этиш ва уларни иқтисодиётнинг устувор йўналишларига кредит сифатида йўналтириш миллий иқтисодиётни ривожлантиришнинг устувор вазифаларидан бири ҳисобланади. Ушбу вазифани ҳал этиш, ўз навбатида, хорижий валюталарда банкларга жалб этилган муддатли ва жамғарма депозитларига тўланадиган фоиз ставкаларини ошириш йўли билан мазкур депозитларнинг аҳоли ва корхоналар учун жозибадорлигини оширишни тақозо этади [8]. Инфляцияни юзага келтирувчи асосий омиллар сифатида қуйидагиларни кўрсатиш мумкин: Инфляцияни юзага келтирувчи асосий омиллар сифатида қуйидагиларни кўрсатиш мумкин: 1. Ўзбекистон Республикасида бюджет ташкилотлари ходимлари иш ҳақининг ўсиши билан истеъмол товарлари баҳоларининг ўсиши ўртасидаги бевосита боғлиқлик мавжуд. Ушбу боғлиқликнинг юзага келиши қуйидаги омилларга асосланади: – бюджет ташкилотларида иш ҳақининг ўсиши истеъмол товарларига бўлган талабнинг ошишига олиб келади, чунки истеъмол товарларининг сезиларли қисми аҳоли томонидан нақд пулларга харид қилинади; – чакана савдо шохобчалари орқали истеъмол товарларини сотаётган савдо ходимларининг онгига иш ҳақларининг ўсиши кучли психологик таъсир кўрсатмоқда. 2. Республикамизда талаб инфляциясининг кучайиши натижасида ЯИМ дефляторининг юқори даражаси сақланиб қолмоқда. Инфляциянинг классик талқинида, баҳоларнинг ўсиши, пулнинг айланиш тезлиги ўзгармасдан қолган шароитда, пул таклифининг ошишига боғлиқ. Пуллар таклифи ва пулнинг айланиш тезлиги ўзгармаган шароитда алоҳида товарларнинг баҳоси ўзгарганда ҳам мамлакатда баҳоларнинг умумий даражаси ўзгармасдан қолади. Шу сабабли, баҳолар умумий даражасининг ўсиши билан уларнинг алоҳида олинган товар позициялари бўйича ўсишини жиддий фарқлаш лозим. 3. Фуқароларимизнинг хорижий давлатлардан жўнатаётган пул ўтказмалари инфляцияни юзага келтираётган омиллар ичида асосий ўринни эгалламоқда. 4. Табиий монополиялар маҳсулотлари ва хизматлари баҳоларининг ўсиш суръатларига эга эканлиги инфляцион жараёнларнинг юзага келишида муҳим ўрин тутмоқда. 5. Нақд пулларга бўлган талабнинг нисбатан юқори эканлиги. Аҳоли пуллик тўловларининг асосий қисмини нақд пулларда амалга оширилаётганлиги, пластик карточкалар ва пуллик чеклар воситасидаги тўловларнинг ривожланмаганлиги нақд пулларга бўлган талабнинг юқори даражада қолишига сабаб бўлмоқда. Хулоса қилиб айтиш мумкинки, нархлар барқарорлиги нол даражадаги инфляция орали изоҳланади. Инфляция ўзи билан бирга катта иқтисодий ва ижтимоий харажатларни олиб келади. Натижада ресурслар нотўғри жойлаштирилади ва бу ўз навбатида, иқтисодий самарадорликнинг пасайишига олиб келади. Шунингдек, инфляция иқтисодий вакилларга ўз фаолиятини келгусида режалаштиришга халақитберади, ҳамда уларнинг олиб борилаётган иқтисодий сиёсатга бўлган ишонч даражасини пасайтиради. Бундан ташқари, у миллий даромадни аҳолининг бир қисми фойдасига ва иккинчи қисми зарарига қайтатақсимлайди. Шу боисдан инфляцияга карши курашбутун дунёда ҳукуматнинг асосийвазифаларидан бири ҳисобланади.Ўтиш даври иқтисодиётини бошиданкечираётган мамлакатлар тажрибаси шуни кўрсатадики, юқори суръатдаги инфляция кузатилган ҳаммажойларда у аҳолининг турмуш даражасига ёмон таъсиркўрсатган. Бундай мамлакатларда барқарорлаштиришгадоир барча дастурларнинг диққат марказидаинфляцияга қарши кураш масаласи туриши бежиземас. Ўзбекистон Республикаси Марказий банкининг монетар сиёсатини асосий вазифалрдан бири ҳам инфляция суръатларини пасайтиришдан иборат.Чунки Марказийй банкнинг монетар сиёсатин тўғри олиб борилишига таъсир қилувчи омиллардан бири бўлиб инфляция ҳисобланади. АДАБИЁТЛАР: 1. Graude P., Polan M. Is Inflation Always and Everywhere a Monetary Phenomenon? Discussion Paper. №2841 CEPR. 2001. June. – Р. 26-29. 1. Graude P., Polan M. Is Inflation Always and Everywhere a Monetary Phenomenon? Discussion Paper. №2841 CEPR. 2001. June. – Р. 26-29. 2. Catao L., Terrones M. Fiscal Deficits and Inflation // Working Paper. 65. IMF. – Р. 63 - 67. 2. Catao L., Terrones M. Fiscal Deficits and Inflation // Working Paper. 65. IMF. – Р. 63 - 67. 3. Hess G., Morris C. The Long-Ruh Costs of Moderate Inflation // Federal Reserve Bank of Kansas City Economic Review. 1996. Second Quarter. – P. 71-88. 4. Пивоварова М.А. Международная научно-практическая конференция: “Инфляция и экономический рост: теория и практика” // Деньги и кредит. – Москва, 2006. - №7. – С. 60. 4. Пивоварова М.А. Международная научно-практическая конференция: “Инфляция и экономический рост: теория и практика” // Деньги и кредит. – Москва, 2006. - №7. – С. 60. 5. Khan M. Senhadji. Thereshild Effects in the Relatioinshop between Inflation and Growth // IMF Staff Papers. 2001. Vol. 48. - P.1-21. 5. Khan M. Senhadji. Thereshild Effects in the Relatioinshop between Inflation and Growth // IMF Staff Papers. 2001. Vol. 48. - P.1-21. 6. Hogan S., Johnson M., Lafleche T. Core Inflation // Bank of Canada Technical Report, 2001. №89. 6. Hogan S., Johnson M., Lafleche T. Core Inflation // Bank of Canada Technical Report, 2001. №89. 7. О. Хмыз. Базовая инфляция и ее измерение (зарубежный опыт) // Экономист. – Москва, 2007.– №7.– С. 65 -77. 7. О. Хмыз. Базовая инфляция и ее измерение (зарубежный опыт) // Экономист. – Москва, 2007.– №7.– С. 65 -77. 8. Kudrin A. Inflyatsiya: rossiyskie i mirovie tendentsii // Voprosi ekonomiki – Moskva, 2007. - №10. – S.19. 8. Kudrin A. Inflyatsiya: rossiyskie i mirovie tendentsii // Voprosi ekonomiki – Moskva, 2007. - №10. – S.19.
https://openalex.org/W2108108277	https://www.scielo.br/j/rpp/a/pv5DBPRkmdq5vj9DYphwPgR/?lang=pt&format=pdf	Portuguese	null	Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica	Revista Paulista de Pediatria	2,009	cc-by	5,885	Instituição: Universidade Federal do Paraná (UFPR), Curitiba, PR, Brasil 1Fisioterapeuta; Doutora em Saúde da Criança e do Adolescente pelo Programa de Pós-graduação do Departamento de Pediatria da UFPR, Curitiba, PR, Brasil 2Médico; Mestre em Saúde da Criança e do Adolescente pelo Programa de Pós-graduação do Departamento de Pediatria da UFPR, Curitiba, PR, Brasil 3Psicóloga; Mestre em Saúde da Criança e do Adolescente pelo Programa de Pós-graduação do Departamento de Pediatria da UFPR, Curitiba, PR, Brasil 4Neuropediatra; Professor do Departamento de Pediatria da UFPR, Curitiba, PR, Brasil RESUMO crepância observada entre os membros superiores e inferiores no lado envolvido (r=0,48) e a discrepância aumenta com a idade (r=0,44). Objetivo: Analisar o crescimento linear, o perímetro ce fálico e as diferenças antropométricas entre o lado envolvido e o não-envolvido de 24 crianças com paralisia cerebral (PC) hemiplégica, comparados à média para a idade. Conclusões: O maior comprometimento no crescimento das crianças com paralisia cerebral estudadas ocorreu nos membros envolvidos pela hemiplegia e, em menor proporção, no perímetro cefálico. Métodos: Estudo transversal com amostragem con secutiva de crianças com PC, classificadas clinicamente como hemiplegia espástica. As medidas antropométricas incluíram: peso, estatura, perímetro cefálico, comprimento total de membro superior, comprimento da mão, largura da palma da mão, comprimento total do membro inferior, comprimento do pé e a circunferência dos membros (braço, coxa e panturrilha). As diferenças antropométricas entre os dimídios foram calculadas em centímetros e como porcen tagem de encurtamento, comparando o lado envolvido com o não-envolvido. Dois referenciais populacionais, tabelas de crescimento e o software ABase®, desenvolvido para sistema PalmOS, foram comparados na classificação das medidas do comprimento da mão e do pé. A análise estatística utilizou o coeficiente de correlação de Spearman para avaliar a asso ciação entre variáveis quantitativas e o teste não-paramétrico de Wilcoxon para comparar as medidas do lado envolvido e não-envolvido. Palavras-chave: antropometria; paralisia cerebral; hemi plegia; crescimento; criança. Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica Growth and anthropometry in hemiplegic cerebral palsy patients Marise Bueno Zonta1, Fábio Agert2, Sandra Regina B. Muzzolon3, Sérgio Antonio Antoniuk4, Neiva Isabel R. Magdalena5, Isac Bruck4, Lú i H l C d S t 6 Marise Bueno Zonta1, Fábio Agert2, Sandra Regina B. Muzzolon3, Sérgio Antonio Antoniuk4, Neiva Isabel R. Magdalena5, Isac Bruck4, Lúcia Helena C. dos Santos6 5Geneticista; Professora do Departamento de Pediatria da UFPR, Curitiba, PR, Brasil 6Pediatra; Neuropediatra; Professora adjunta do Departamento de Pediatria da UFPR, Curitiba, PR, Brasil Artigo Original Artigo Original ABSTRACT Objective: To analyze the linear growth, the head circum ference and the anthropometric differences between involved and non-involved sides of 24 children with hemiplegic cer ebral palsy, comparing them to standard values for age. Methods: This cross-sectional study enrolled 24 consecu tive children with cerebral palsy clinically classified as spastic hemiplegia. The anthropometric measures included: weight, lenght, head circumference, total upper limb length, hand length, palm width, total lower limb length, foot length, and limb circumference of upper-arm, thigh and calf. The anthropometric differences between both sides were cal culated in centimeters and a comparison of the involved and non-involved sides was made. Two different reference values were used to compare the measures of hand and foot length: growth charts and the software ABase® (a PalmOS- based software). The Spearman’s correlation coefficient was Resultados: As médias de peso, estatura e perímetro cefá lico se mostraram dentro dos limites normais para a idade e 21% dos pacientes apresentaram microcefalia. A discrepância entre os dimídios foi evidente em todos os casos, sendo maior na largura e comprimento da mão. Houve correlação da dis Endereço para correspondência: Marise Bueno Zonta Rua Floriano Essenfelder, 81 CEP 80060-270 – Curitiba/PR E-mail: marise.bzonta@terra.com.br Recebido em: 18/12/08 Aprovado em: 10/6/09 Rev Paul Pediatr 2009;27(4):416-23. Marise Bueno Zonta et al Marise Bueno Zonta et al na maturação óssea(12) dos membros envolvidos das crianças com hemiplegia. estimated for the association between quantitative variables and the Wilcoxon non-parametric test was used for age com parisons between involved and noninvolved sides. Devido às relações básicas entre o crescimento de diferen tes partes do corpo, a interferência no crescimento normal pode ser demonstrada pela antropometria, estudo das medi das comparativas do corpo humano(6). Essa técnica é simples, não-invasiva e sem custos adicionais, sendo a ferramenta de escolha para avaliar dismorfismos em crianças, porém pouco utilizada na prática clínica(13). Dentre as possíveis razões para o seu pouco uso, destacam-se: acesso limitado dos profissio nais a valores apropriados de referência, tempo despendido na mensuração e tempo consumido para colocação nas tabelas de crescimento. Atualmente, programas específicos, como o software ABase®, desenvolvidos para sistema PalmOS de computadores de bolso, fornecem dados antropométricos que permitem a comparação rápida das medidas, considerando a idade e o sexo do paciente(13). Results: The mean values of weight, length and head circumference were within the normal range for age and 21% of the children presented microcephaly. Introdução A paralisia cerebral (PC), também denominada encefa lopatia crônica não-progressiva da infância, é consequência de uma lesão no cérebro em fase de maturação estrutural e funcional, no período pré, peri ou pós-natal(1). Crianças com PC em geral crescem menos, apresentando peso e estatura menores que as saudáveis da mesma idade(2). Rotta(3) enfa tiza que estas, além de evoluírem com estatura e peso mais baixos, apresentam também menor resistência às infecções, pontuando a importância do cérebro normal para uma cons tituição física normal. ABSTRACT Discrepancy was noted between both sides in all cases, being the largest discrepancy in hand length and width. There was a positive correlation between the discrepancy observed in superior and inferior affected limbs (r=0.48), and discrepancy increases with age (r=0.44). Conclusion: Growth impairment in children with hemi plegic cerebral palsy was observed on the affected limbs and in smaller proportion in head circumference. Key-words: anthropometry; cerebral palsy; hemiplegia; growth; child. Nesse contexto, propõe-se, neste estudo, uma análise do crescimento linear, do perímetro cefálico e das diferenças antropométricas entre os dimídios de crianças com PC hemiplégica além de se avaliar a relação entre o perímetro cefálico versus função cognitiva e das diferenças antropomé tricas versus função motora. Rev Paul Pediatr 2009;27(4):416-23. Resultados Os valores das aferições de comprimento da mão foram comparados com os dados de Feingold e Bossert(17) e, para o comprimento do pé, com os dados de Blais et al(18) para meninos e meninas, utilizados por Hall et al(15). Essas mesmas medidas foram comparadas com os valores de Freeman por meio do software de distribuição gratuita ABase®(13,19), que oferece valores de referência para 18 medidas antropométricas frequentemente usadas para os dismorfismos, sendo os resul tados expressos em texto e tabelas digitais de crescimento. Neste estudo, o resultado foi expresso na forma de texto. Vinte e quatro crianças com paralisia cerebral tipo hemi plegia espástica participaram deste estudo, sendo 19 (79%) do gênero masculino, com média da idade 49±5 meses. Quanto à lateralidade, 11 (46%) apresentavam o lado direito envolvido. Cinco (21%) foram prematuros e 19 (79%) nas cidos a termo. Três pacientes do gênero masculino tinham estatura abaixo do -2 desvios padrão (-2DP). Desses três, dois apresentavam também déficit ponderal e microcefalia, sendo um deles prematuro, e ambos, ao nascimento, pequenos para a idade gestacional. O status cognitivo foi considerado normal (≥80) em 13 crianças (57%) e havia retardo em dez (43%). A correlação do status cognitivo com o perímetro cefálico não apresentou significância estatística (r=0,04; p=0,85). A avaliação da função motora grossa aferiu um escore médio de 93±12%. As aferições de peso, estatura e perímetro cefálico foram realizadas no período matutino, durante as consultas. Todas as crianças foram medidas com antropômetro de madeira em de cúbito dorsal. As aferições de peso foram realizadas em balança mecânica de marca Fillizola, já que todas as crianças eram nível 1 e 2 do GMFSC, podendo ficar em pé. Os escores Z foram calculados com o software ABase®, que tem como referência a população europeia(19). Todas essas medidas, expressas em percentil ou escore Z, foram consideradas anormais quando diferiam em 2 desvios padrão acima ou abaixo da média ou fora do quinto ou 95º percentis(19). O peso médio aferido foi 15,5±1,5kg e a estatura média, 99,7±4,6cm. O perímetro cefálico médio foi de 48,8±2,3cm e, em 21% das crianças, encontrava-se abaixo de -2DP. Infor mações referentes a esses dados e seus respectivos escores Z e percentis, para cada criança, estão descritas na Tabela 1. Todas as crianças deste estudo apresentaram encurtamento no lado envolvido em pelo menos três das aferições realizadas. Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica intelligence), designado para administração individual e avaliação de diferentes aspectos da inteligência de crianças de 3-7 anos. Contém, em sua composição, 12 subtestes divididos igualmente nas áreas verbal e performance. peso ao nascimento. O estudo teve início após a aprovação do Comitê de Ética em Pesquisa em Seres Humanos do Hos pital de Clínicas da UFPR. Os responsáveis pelas crianças concordaram com sua participação no estudo, assinando um Termo de Consentimento Livre e Esclarecido. Os resultados obtidos no estudo foram expressos por mé dias e desvios padrões, ou por frequências e percentuais. Para avaliar a associação entre variáveis quantitativas, foi estimado o coeficiente de correlação de Spearman. Compararam-se os resultados dos lados (envolvido e não-envolvido) em relação a variáveis quantitativas por meio do teste não-paramétrico de Wilcoxon, considerando-se significante p<0,05. Os dados foram analisados com auxílio do programa computacional Statistica versão 6®. A antropometria foi realizada no período matutino, por um único profissional, de acordo com normas padronizadas(15), aferindo-se o comprimento total do membro superior, o comprimento da mão, a largura da palma da mão, o com primento total do membro inferior, o comprimento do pé e a circunferência dos membros (braço, coxa e panturrilha). A discrepância encontrada entre os dois dimídios foi calculada em centímetros e como porcentagem, pela comparação com o lado não-envolvido, de acordo com fórmula adaptada do estudo de Demir et al(16). Métodos Trata-se de um estudo transversal descritivo. A amostra consecutiva consistiu de crianças com PC do tipo hemiplé gica espástica, em acompanhamento no Ambulatório de Espasticidade em Pediatria (AEP) do Centro de Neurope diatria do Hospital de Clínicas da Universidade Federal do Paraná (UFPR), com faixa etária entre 3 e 5 anos de idade, atendidas no período de junho de 2005 a abril de 2006, com mobilidade independente e comprometimento motor classificado como níveis 1 e 2, de acordo com o Sistema de Classificação da Função Motora Grossa (GMFCS)(14). Nesse ambulatório, que engloba crianças com PC provenientes de diferentes serviços, as avaliações são realizadas por uma equipe multidisciplinar, sendo da responsabilidade da última autora a avaliação neurológica e a classificação funcional. As orientações são realizadas pelos profissionais específicos (fi sioterapeuta, terapeuta ocupacional, psicólogo e enfermeiro) de forma individualizada a cada uma das famílias. Nenhuma das crianças deste estudo apresentou história concomitante de doença genética, metabólica, neurodegenerativa ou qualquer outra alteração que pudesse influenciar o crescimento. Não foram excluídas crianças prematuras ou que tiveram baixo A etiologia do retardo no crescimento de crianças com doenças crônicas como a PC é multifatorial(4), podendo estar relacionada a fatores não-nutricionais resultantes das malfor mações cerebrais ou de lesões responsáveis pela incapacidade da criança(2,5). O momento da agressão que levou à PC(6), o tipo de desordem no movimento(2), a gravidade do envolvi mento da PC, especialmente em relação à autoalimentação e à capacidade de deambulação(7), e o grau de limitação na atividade física(4) têm sido apontados como fatores que in fluenciam as alterações no crescimento. O retardo no crescimento é maior na forma quadriplégica espástica(4,8), mas também é documentado em crianças com diplegia e hemiplegia(2), mesmo na ausência de desnutrição. Em crianças com PC, aproximadamente 35 a 40% são do tipo hemiplégica espástica, com um lado do corpo mais afe tado que o outro(9,10). Estudos prévios apontam alterações no crescimento(10,11) e atraso no desenvolvimento muscular(5,11) e Rev Paul Pediatr 2009;27(4):416-23. 417 Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica Resultados 418 Marise Bueno Zonta et al Tabela 1 – Gênero, dados antropométricos e quociente de inteligência na população do estudo Gênero Peso Estatura Perímetro cefálico QI kg Escore Z cm Escore Z cm Percentila 1 M 18,0 0,95 99 -0,56 51,5 -2DP-Md 45 2 M 16,4 -0,39 102 -0,75 47,5b -3,7 DP 52 3 M 17,5 0,39 104 0,08 50,2 -2DP-Md 81 4 M 17,0 0,07 102 -0,50 49,0 -2DP-Md 80 5* F 16,0 -0,34 103 -0,19 42,0 -3,6DP 56 6 M 15,5 -1,22 102 -1,21 49,5 -2,4DP 74 7 M 14,5 -0,48 100 0,24 49,0 -2DP-Md 77 8* M 16,5 -0,08 102 -0,38 49,0 -2DP-Md 99 9 M 14,8 -1,30 102 -0,75 45,6 b -2DP-Md 67 10 F 17,9 1,32 102 1,03 48,0 -2DP-Md 88 11* F 13,7 -1,76 94 -2,39 46,5 b -4,4DP 101 12* M 16,0 0,45 105 1,60 51,0 -2DP-Md 44 13 M 16,2 0,74 98 0,20 48,4 -2,6DP 126 14 M 15,0 -1,08 100 0,17 47,5 -3,6DP 43 15 M 14,5 -1,03 100 -0,59 49,0 -2DP-Md 98 16 F 13,5 -0,54 96 0,01 49,2 -2DP-Md 61 17 M 15,5 -0,70 88 -3,61 52,8 -2DP-Md 72 18 M 13,6 -2,12 98 -1,54 48,2 -3,0DP 94 19 M 16,2 0,11 104 0,63 53,0 M-+2DPe 76 20 M 13,0 -2,58 91 -3,22 47,7 b -3,7DP 50 21 M 17,7 -0,18 106 -0,45 49,5 -2,4DP 94 22* M 13,0 -2,58 91 -3,22 47,7 b -3,7DP c 23 F 16,0 -0,05 98 -0,70 48,4 -2,9DP 83 24 M 15,0 -1,08 104 -0,17 51,0 -2DP-Md 141 QI: quociente de inteligência; prematuros, 33, 34, 26, 32 e 27 semanas; a: dados obtidos pelo software ABase®; b: dados que correspondem ao -2DP na tabela de crescimento; c: dado não obtido; d: -2DP-M, valor entre o -2 desvio padrão e a média; e: M-+2DP, valor entre a média e o +2 desvio padrão. – Gênero, dados antropométricos e quociente de inteligência na população do estudo QI: quociente de inteligência; prematuros, 33, 34, 26, 32 e 27 semanas; a: dados obtidos pelo software ABase®; b: dados que correspondem ao -2DP na tabela de crescimento; c: dado não obtido; d: -2DP-M, valor entre o -2 desvio padrão e a média; e: M-+2DP, valor entre a média e o +2 desvio padrão. Resultados Todas as médias para as medidas de comprimento, largura e circunferência aferidas foram significativamente menores do lado envolvido, comparadas às mesmas médias do lado não- envolvido (p<0,001). A maior desproporção foi observada no comprimento e na largura da palma da mão (Tabela 2). A análise de correlação mostrou que, quanto maior a diferença no comprimento total do membro superior envolvido, maior a diferença observada no comprimento (r=0,54) e na largura da palma da mão (r=0,52). Quanto maior a diferença na A função motora foi avaliada através da escala medida da função motora grossa (do inglês gross motor function measure, GMFM)(20), desenvolvida e validada para crianças com PC(21), sendo amplamente aceita inclusive no Brasil(22). A escala foi aplicada por um único profissional, habilitado e consiste em 88 itens que avaliam a função relacionada a cinco dimensões de evolução motora: (A) deitar e rolar, (B) sentar, (C) ajoelhar e engatinhar, (D) ficar em pé, (E) andar, correr e pular. O teste WPPSI-R(23) foi utilizado para a avaliação cog nitiva. Esse instrumento clínico é uma revisão completa de 1989 do WPPSI (Wechsler preschool and primary scale of Rev Paul Pediatr 2009;27(4):416-23. Rev Paul Pediatr 2009;27(4):416-23. Resultados Tabela 2 – Relação entre as medidas do lado envolvido e não envolvido Média da diferença entre os lados (cm) Porcentagem de encurtamento do LE (média) Proporção do LE em relação ao LNE (%) Comprimento total do MS 1,22±0,94 2,65 96,66 Comprimento da mão 0,88±0,54 6,10 92,82 Largura da palma da mão 0,36±0,34 5,99 92,50 Comprimento total do MI 0,65±0,65 1,50 97,82 Comprimento do pé 0,73±0,56 4,41 96,06 Circunferência do braço 1,12±0,57 5,75 94,44 Circunferência da coxa 0,88±0,63 2,68 96,87 Circunferência da panturrilha 0,88±0,53 4,25 96,55 MS: membro superior; MI: membro inferior; LE: lado envolvido; LNE: lado não-envolvido. Tabela 2 – Relação entre as medidas do lado envolvido e não envolvido 32% para o comprimento da mão e em 18% para o com primento do pé (Tabelas 3 e 4). Estas divergências mostram que o software ABase® conferiu valores menores, exceto em um caso. Além disso, houve discordância na classificação de normalidade em três pacientes para o comprimento da mão e, em dois, para o comprimento do pé. 32% para o comprimento da mão e em 18% para o com primento do pé (Tabelas 3 e 4). Estas divergências mostram que o software ABase® conferiu valores menores, exceto em um caso. Além disso, houve discordância na classificação de normalidade em três pacientes para o comprimento da mão e, em dois, para o comprimento do pé. largura da palma da mão, maior a do pé (r=0,48). Quanto maior a idade, maior a porcentagem de encurtamento na circunferência do braço, sendo que, quanto maior a idade, maior a discrepância encontrada (r=0,44). largura da palma da mão, maior a do pé (r=0,48). Quanto maior a idade, maior a porcentagem de encurtamento na circunferência do braço, sendo que, quanto maior a idade, maior a discrepância encontrada (r=0,44). Comparando os dados obtidos pela tabela de crescimento e software ABase®, observaram-se resultados divergentes em Rev Paul Pediatr 2009;27(4):416-23. Resultados 419 Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica Tabela 3 – Comparação dos valores do comprimento da mão considerando os dados da tabela de crescimento e software ABase nas 24 crianças Idade Lado não- envolvido (cm) Tabela de crescimentoa (DP) software ABase®b (DP) Lado envolvido (cm) Tabela de crescimentoa (DP) software ABase®b (DP) 1 3a 11m 12,5 75-97 61 11,5 25-50 13* 2 4a 3m 12,0 25-50 48 11,5 3-25 7 3 4a 2m 11,7 25-50 8* 10,5 <3 -3 4 4a 2m 12,5 50-75 50 11,5 3-25 8 5 4a 5m 12,5 50-75 43* 11,0 3-25 6 6 4a 10m 12,5 50-75 31* 12,0 25-50 31 7 3a 9m 10,9 3-25 -2* 10,0 <3 -2 8 4 anos 11,5 25-50 11* 11,0 3-25 11 9 4a 5m 12,2 25-50 43 11,5 3-25 6 10 3a 8m 12,0 50-75 69 10,5 3-25 1* 11 4a 1m 11,0 3-25 9 10,0 <3 -3 12 3a 5m 11,2 25-50 25 11,0 25-50 25 13 3a 3m 12,0 75-97 80 12,0 50-75 80* 14 4a 3m 13,0 75-97 90 11,0 3-25 7 15 3a 11m 11,5 25-50 13* 10,5 3-25 -2* 16 3a 3m 11,0 25-50 29 10,5 3-25 2* 17 4a 3m 11,0 3-25 7 9,3 <3 -4 18 4a 5m 10,5 <3 -3 10,0 <3 -3 19 3a 10m 12,0 50-75 64 10,0 <3 -2 20 4a 5m 10,5 <3 -7 10,2 <3 -7 21 4a 10m 12,8 50-75 31* 12,0 25-50 31 22 4a 6m 11,5 3-25 5 10,8 3-25 -3* 23 4 anos 11,5 25-50 11* 11,0 3-25 11 24 4a 7m 12,5 25-50 45 12,0 25-50 45 DP: desvio padrão; dados discordantes entre os dois referenciais; a: comparação com os dados de Feingold(17); b: dados obtidos pelo software ABase® em forma de texto, baseado nos dados de Freeman et al(19). DP: desvio padrão; dados discordantes entre os dois referenciais; a: comparação com os dados de Feingold(17); b: dados obtidos pelo software ABase® em forma de texto, baseado nos dados de Freeman et al(19). Discussão podem ser aferidos e comparados com valores de referência para a população geral. O crescimento é fundamental no desenvolvimento da criança, sendo um marcador de sua saúde e bem-estar(24). O crescimento anormal entre os dimídios nas crianças com PC de forma hemiplégica é usado como modelo para o estudo da influência dos fatores não-nutricionais no crescimento. O fato de que cada criança é o seu próprio controle permite eliminar fatores como a desnutrição, endocrinológicos, gênero, raça, estatura média dos pais e estádio puberal(5). Embora a antropometria seja a técnica de escolha para avaliar dismorfismos em crianças, na prática ela é pouco uti lizada. O consumo de tempo e a eventual indisponibilidade de tabelas apropriadas para as várias mensurações do corpo poderiam ser responsáveis por este fato. O software ABase®(13) foi idealizado com o intuito de amenizar esse problema. Neste estudo, os resultados obtidos pelos dois métodos, ta belas de crescimento e software ABase®, foram comparados, mostrando algumas diferenças. As tabelas de crescimento oferecem intervalos para classificação das medidas do pa ciente, enquanto o software ABase®, utilizado na forma de texto, fornece um número específico. No presente estudo, em cerca de um quarto dos pacientes houve divergência entre a informação do software ABase® e da curva de crescimento. O fato do software ABase® ter como referência a população europeia(19) pode ser um dos motivos dessas discordâncias. No Uma vez que não existe diferença significativa entre os dimídios na criança normal, já constatado por Demir et al(16), foi utilizado neste estudo o lado não-envolvido para comparação com o envolvido nas crianças com PC hemi plégica, como descrito por Stevensen et al(5) e Van Heest et al(25), que também incluíram ambos os sexos na mesma amostra. As alterações no crescimento linear ou específico dos membros das crianças com PC e o perímetro cefálico Rev Paul Pediatr 2009;27(4):416-23. Discussão 420 Marise Bueno Zonta et al Tabela 4 – Comparação dos valores do comprimento do pé considerando os dados da tabela de crescimento e software ABase nas 24 crianças Idade Lado não- envolvido (cm) Tabela de crescimentoa (DP) software ABase®b (DP) Lado envolvido (cm) Tabela de crescimentoa (DP) software ABase®b (DP) 1 3a 11m 16,5 50-75 50 15,5 25-50 13* 2 4a 3m 17,5 75-97 73* 16,5 25-50 31 3 4a 2m 16,0 25-50 34 16,0 25-50 34 4 4a 2m 16,0 25-50 34 16,0 25-50 34 5 4a 5m 16,5 25-50 26 16,5 25-50 26 6 4a 10m 17,5 50-75 51 16,0 3-25 15 7 3a 9m 16,0 50-75 58 15,0 3-25 19 8 4 anos 16,0 25-50 41 16,0 25-50 41 9 4a 5m 17,7 50-75 67 16,0 25-50 26 10 3a 8m 16,8 50-75 62 15,7 25-50 22* 11 4a 1m 15,8 25-50 7* 14,7 3-25 -2* 12 3a 5m 16,0 50-75 73 15,0 25-50 33 13 3a 3m 16,0 75-97 79 16,0 75-97 79 14 4a 3m 17,0 50-75 73 15,5 3-25 5 15 3a 11m 15,5 3-25 13 14,0 <3 1* 16 3a 3m 15,5 25-50 41 15,0 25-50 41 17 4a 3m 15,0 3-25 5 14,0 <3 -3 18 4a 5m 15,5 3-25 4 15,0 3-25 4 19 3a 10m 16,7 50-75 54 16,3 25-50 54 20 4a 5m 15,5 3-25 4 14,5 <3 -3 21 4a 10m 18,0 75-97 85 17,5 50-75 51 22 4a 6m 15,3 3-25 3 14,4 <3 -3 23 4 anos 17,0 75-97 81 16,5 50-75 41 24 4a 7m 16,5 25-50 21* 16,5 25-50 21* DP: desvio padrão; : dados discordantes entre os dois referenciais; a: comparação com os dados de Blais et al(18); b: dados obtidos pelo software ABase®, baseado nos dados de Freeman et al(19), neste caso em forma de texto. valores do comprimento do pé considerando os dados da tabela de crescimento e software ABase DP: desvio padrão; : dados discordantes entre os dois referenciais; a: comparação com os dados de Blais et al(18); b: dados obtidos pelo software ABase®, baseado nos dados de Freeman et al(19), neste caso em forma de texto. 169 crianças com hemiplegia e observou que as médias do peso e altura também não foram significativamente diferentes do normal. Os dados do presente estudo concordam com a ausência de diferença no crescimento linear dessas crianças em relação à população normal. Rev Paul Pediatr 2009;27(4):416-23. Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica ta amostra, foram 8, 50 e 42%. Observou-se também, na presente amostra, que o encurtamento do braço foi mais marcante do que o da perna. A maior desproporção entre os dimídios na hemiplegia estaria localizada na mão e no pé(10); porém, no presente estudo, a maior desproporção foi observada no comprimento e na largura da palma da mão e, em seguida, na circunferência do braço, concordando com Tizard et al(29). menor comprometimento nos hemiplégicos comparados aos diplégicos e quadriplégicos. O fato de as crianças deste estudo terem apresentado uma função motora próxima do normal indica uma grande probabilidade de virem a ter também uma participação social e indicadores de saúde mais adequados do que as crianças com PC com maiores alterações no cres cimento, como demonstrado por Stevensen et al(24). O perímetro cefálico, considerado uma das medidas mais importantes na infância, reflete o volume intracraniano do cérebro em desenvolvimento. A discrepância em sua propor ção pode indicar processos patológicos(15). Nesse estudo, 21% dos pacientes apresentaram microcefalia. Stewart, citado por Uvebrant(11), encontrou perímetro cefálico reduzido em quase todos os casos de hemiplegia examinados em seu estudo, o que não foi confirmado nesta pesquisa e na de Uvebrant(11), na qual 15% tinham perímetro cefálico menor que -2DP. Recentemente Ibrahim e Hawamdeh(8) observaram uma diminuição significativa do perímetro cefálico nas meninas hemiplégicas. Existe controvérsia sobre a relação entre a discrepância no crescimento do lado envolvido e a idade(5,16,24). Neste estudo, a idade média foi menor e mais homogênea com parada à observada pelos outros pesquisadores que inves tigaram a relação entre a discrepância no crescimento do lado envolvido e a idade(5,16,24); mesmo assim, observou-se que, quanto maior a idade, maior a porcentagem de redução na circunferência do braço envolvido. Isso reforça o termo preferido por Tizard et al(29), ‘undergrowth’, menor cresci mento, pois o processo do encurtamento está relacionado à dificuldade no crescimento, e não a seu retrocesso. Mesmo que tardiamente haja piora pelo desuso, o encurtamento nos membros se dá precocemente durante o crescimento. No desenvolvimento normal, o crescimento de diferentes partes do corpo segue um curso previsível e proporcional(15). A relação observada entre as medidas do membro superior e inferior no lado envolvido sugere existir uma tendência natural para o crescimento proporcional desse dimídio, mesmo que a assimetria em relação ao lado não-envolvido seja evidente. Poucos estudos tentaram associar microcefalia e função cognitiva. Discussão presente estudo, ao verificar a simetria e as proporções de cada indivíduo, as tabelas de crescimento forneceram informações que propiciaram maior facilidade na comparação entre as medidas dos dois dimídios. Por outro lado, dependendo do objetivo da avaliação antropométrica, instrumentos como o software ABase® simplificam a avaliação, considerando-se especialmente o consumo de tempo. O fato dos resultados obtidos por esses dois referenciais não serem intercambiáveis sugere que a escolha do referencial deve ser feita previamente e mantida em todas as avaliações. Talvez um dos fatores que estimule o crescimento linear nos pacientes com hemiplegia seja a possibilidade de um desempenho motor próximo ao normal. O escore médio na GMFM (93%) nessa amostra indicou uma capacidade motora muito próxima da normal, tendo em vista que o esperado, nessa escala, é que aos cinco anos uma criança normal complete 100% dos itens. Estudos têm demonstrado o benefício da atividade física no estímulo ao crescimento e desenvolvimento(27), enquanto outros consideram que a gravidade da alteração no crescimento na PC poderia também estar relacionada à restrição dessa atividade(4). O estudo de Ibrahim e Hawamdeh(8) associou o crescimento mais adequado na PC à melhor função motora e evidenciou Holt(26) analisou o crescimento de 50 crianças com hemi plegia e observou tendência a estarem abaixo do peso e es tatura, comparando-as com a média populacional, enquanto Maekawa et al, citados por Uvebrant(11), não encontraram essa tendência. Na amostra aqui estudada, as médias estavam dentro da normalidade, embora três pacientes apresentassem déficit pôndero-estatural não relacionado a fatores nutricio nais e se encontram em investigação. Uvebrant(11) analisou Rev Paul Pediatr 2009;27(4):416-23. 421 Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica O teste WIPPSI-R(23) aqui utilizado para a ava liação cognitiva é aceito pelo Conselho Federal de Psicologia para essa faixa etária. Esse teste é aplicado há alguns anos no ambulatório de Recém-Nascidos de Risco do Hospital de Clínicas da UFPR, detectando aos 5 anos um quociente de inteligência <80 nas crianças que apresentaram duas ou mais reprovações no Denver II aos 2 anos de idade, com sensibi lidade e especificidade de 70%(28). No presente estudo, não houve correlação entre função cognitiva e perímetro cefálico. Já Uvebrant(11) notou que as crianças com perímetro cefálico <-2DP apresentaram um aumento de 55% na incidência de retardo mental, enquanto aquelas com perímetro >-2DP tiveram um aumento de 21%. A causa da discrepância entre os dimídios na PC hemi plégica não foi totalmente esclarecida. Dentre as hipóteses discutidas, destacam-se: menor fluxo sanguíneo para os membros, lesões no córtex pós-central e consequente desuso, e, provavelmente, a associação desses fatores(11). Stevensen et al(30), investigando os mecanismos do cres cimento anormal na PC, questionaram se intervenções não-nutricionais, como colocação de peso e fisioterapia, poderiam ter impacto positivo no crescimento dessas crianças, o que até o momento não foi esclarecido. Demir et al(16) encontraram diferenças significativas nas medidas de membro superior entre os dimídios de crianças com PC hemiplégica, porém o grau do encurtamento não se relacionou à espasticidade. A influência da fisioterapia não pode ser controlada no presente estudo, visto que esses pacientes realizavam tal tratamento em vários ou tros locais. São necessários novos estudos para verificar a influência de alterações (como o grau de espasticidade, fraqueza muscular e alterações na sensibilidade) no menor Em contraste com a estatura e o peso normais, a assi metria do crescimento entre os dimídios nas crianças com hemiplegia analisadas foi evidente, sendo o lado envolvido menor e mais curto nas medidas de comprimento, largura e circunferência, dados também observados por Stevensen et al(5). Uvebrant(11) observou hipotrofia em 96% dos casos e alteração frequente no crescimento dos membros envolvidos com discrepância média de 15mm no membro superior e 6mm no inferior. No presente estudo, essas médias foram res pectivamente 12,2mm e 6,5mm. Holt(26) observou que 20% das crianças hemiparéticas tinham encurtamento da perna envolvida >2,5%, correspondendo a 20mm; 62% <20mm e 18% nenhum encurtamento. No estudo de Uvebrant(11), esses achados foram 13, 75 e 12% respectivamente e, nes Rev Paul Pediatr 2009;27(4):416-23. Referências bibliográficas 1. World Health Organization, WHO. International classification of function and disability, beta-2 version. Geneva; 1999. 16. Demir SO, Oktay F, Uysal H, Seluk B. Upper extremity shortness in children with hemiplegic cerebral palsy. J Pediatr Orthop 2006;26:764-8. 17. Feingold M, Bossert WH. Normal values for selected physical parameters: a aid to syndrome delineation. Birth Defects Orig Artic Ser 1974;10:1-16. 2. Shapiro BK, Green P, Krick J, Allen D, Capute AJ. Growth of severely impaired children: neurological versus nutritional factors. Dev Med Child Neurol 1986;28:729-33. 18. Blais MM, Green WT, Anderson M. Lengths of the growing foot. J Bone Joint Surg 1956;38:998-1000. 3. Rotta NT. Cerebral palsy, new therapeutic possibilities. J Pediatr (Rio J) 2002;78:S48-54. 19. Freeman JV, Cole TJ, Chinn S, Jones PR, White EM, Preece MA. Cross sectional stature and weight reference curves for the UK, 1990. Arch Dis Child 1995;73:17-24. 4. Stallings VA, Charney EB, Davies JC, Cronk CE. Nutrition-related growth 4. Stallings VA, Charney EB, Davies JC, Cronk CE. Nutrition-related growth failure of children with quadriplegic cerebral palsy. Dev Med Child Neurol 1993;35:126-38. failure of children with quadriplegic cerebral palsy. Dev Med Child Neurol 1993;35:126-38. 20. Russell DJ, Rosenbaum PL, Cadman DT, Gowland C, Hardy S, Jarvis S. The gross motor function measure: a means to evaluate the effects of physical therapy. Dev Med Child Neurol 1989;31:341-52. 5. Stevenson RD, Roberts CD, Vogtle L. The effects of non-nutritional factors on growth in cerebral palsy. Dev Med Child Neurol 1995;37:124-30. 21. Bjornson KF, Graubert CS, Buford VL, McLaughlin J. Validity of the gross motor function measure. Pediatr Phys Ther 1998;10:43-7. 6. Pryor HB, Thelander HE. Growth deviations in handicapped children. An anthopometric study. Clin Pediatr (Phila) 1967;6:501-12. 7. Tobis JS, Saturen P, Larios G, Posniak AO. Study of growth patterns in cerebral palsy. Arch Phys Med Rehabil 1961;42:475-81. 22. Cury VC, Mancini MC, Melo AP, Fonseca ST, Sampaio RF, Tirado MG. The effects of the use of orthoses on the functional mobility of children with cerebral palsy. Rev Bras Fisioter 2006;10:67-74. 8. Ibrahim AI, Hawamdeh ZM. Evaluation of physical growth in cerebral palsied children and its possible relationship with gross motor development. Int J Rehabil Res 2007;30:47-54. 23. Wechsler D. Wechsler preschool and primary scale of intelligence – revised. San Antonio: The Psychological Corporation; 1989. 9. Pellegrino L, Dormans JP. Definitions, etiology and epidemiology of cerebral palsy. In: Dormans JP, Pellegrino L, editors. Crescimento e antropometria em pacientes com paralisia cerebral hemiplégica 422 Marise Bueno Zonta et al Em conclusão, pode-se afirmar que as alterações mais fre quentes encontradas no crescimento de crianças com PC de forma hemiplégica se referem à discrepância entre os dimídios e ao perímetro cefálico, não se observando alteração no cresci mento linear. A discrepância entre os dimídios foi evidente, mais importante na largura e comprimento da mão, e 21% dos pacientes apresentaram microcefalia não-relacionada à função cognitiva. Os referenciais de normalidade utilizados, tabelas de crescimento e software ABase®, não se mostraram intercambiá veis, sendo importante que o pediatra valorize a seleção prévia do instrumento e o mantenha no decorrer das avaliações. crescimento do lado envolvido e sua relação com o uso funcional nas crianças com PC hemiplégica, bem como a investigação de fatores que possam estimular o melhor crescimento do dimídio envolvido e a influência do tra tamento fisioterápico. A inclusão de cinco crianças prematuras pode ser questio nada, porém não existem estudos que façam essa comparação em prematuros, tanto adequados como pequenos para a idade gestacional. Futuros estudos com um maior número de pacientes prematuros serão necessários para comprovar esses achados. Referências bibliográficas Caring for children with cerebral palsy. Baltimore: Paul H Brooks; 1998. p. 3-30. 24. Stevenson RD, Conaway M, Chumlea WC, Rosenbaum P, Fung EB, Henderson RC et al. Growth and health in children with moderate-to-severe cerebral palsy. Pediatrics 2006;118:1010-8. 25. van Heest AE, House J, Putnam M. Sensibility deficiencies in the hands of children with spastic hemiplegia. J Hand Surg (Am) 1993;18:278-81. 10. Swaiman KF, Wu Y. Cerebral palsy. In: Swaiman KF, Ashwal S, Ferriero DM, editors. Pediatric neurology: principles & practice, volume 2. Philadelphia: Mosby Elsevier; 2006. p. 491-504. 26. Holt KS. Growth disturbances. In: Bax M, editor. Hemiplegic cerebral palsy in children and adults. London: Heinemann; 1961. p. 39-53. 11. Uvebrant P. Hemiplegic cerebral palsy. Aetiology and outcome. Acta Paediatr Scand 1988;345:1-100. children and adults. London: Heinemann; 1961. p. 39-53. 27. Alves C, Lima RV. Linear growth and puberty in children and adolescents effects of physical activity and sports. Rev Paul Pediatr 2008;26:383-91. 12. Roberts CD, Vogtle L, Stevenson RD. Effect of hemiplegia on skeletal maturation. J Pediatr 1994;125:824-8. 28. Bruck I, Antoniuk S, Santos LC. Avaliação aos dois anos prevê desenvolvimento neuropsicomotor aos cinco anos? 1º Congresso Brasileiro de Neurologia Infantil; 2003, agosto 15-17; São Paulo, Brasil. p.33. 13. Zankl A. Computer-aided anthropometry in the evaluation of dysmorphic children. Pediatrics 2004;e114:333-6. 29. Tizard JP, Paine RS, Crothers B. Disturbances of sensation in children with hemiplegia. J Am Med Assoc 1954;155:628-32. 14. Palisano R, Rosenbaum P, Walters S, Russell D, Wood E, Galuppi B. Development and reliability of a system to classify gross motor function in children with cerebral palsy. Dev Med Child Neurol 1997;39:214-23. 30. Stevensen RD, Hayes RP, Cater LV, Blackman JA. Clinical correlates of linear growth in children with cerebral palsy. Dev Med Child Neurol 1994;36: 135-42. 15. Hall JG, Froster-Iskenius UG, Allanson JE. A handbook of normal physical measurements. Oxford: Oxford Medical Publications; 1989. 423 Rev Paul Pediatr 2009;27(4):416-23.
https://openalex.org/W2175416579	https://repository.ubn.ru.nl/bitstream/handle/2066/171272/1/171272.pdf	English	null	Prognostic impact of concurrent <i>MYC</i> and <i>BCL6</i> rearrangements and expression in <i>de novo</i> diffuse large B-cell lymphoma	Oncotarget	2,015	cc-by	8,763	Prognostic impact of concurrent MYC and BCL6 rearrangements and expression in de novo diffuse large B-cell lymphoma Prognostic impact of concurrent MYC and BCL6 rearrangements and expression in de novo diffuse large B-cell lymphoma Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/171272 Download date: 2024-10-24 Prognostic impact of concurrent MYC and BCL6 rearrangements and expression in de novo diffuse large B-cell lymphoma Ye, Q.; Xu-Monette, Z.Y.; Tzankov, A.; Deng, L.; Wang, X.; Manyam, G.C.; Visco, C.; Montes- Moreno, S.; Zhang, L.; Dybkaer, K.; Chiu, A.; Orazi, A.; Zu, Y.; Bhagat, G.; Richards, K.L.; Hsi, E.D.; Choi, W.W.; Krieken, J.H. van; Huh, J.; Ponzoni, M.; Ferreri, A.J.; Parsons, B.M.; Moller, M.B.; Piris, M.A.; Winter, J.N.; Medeiros, L.J.; Hu, S.; Young, K.H. 2016, Article / Letter to editor (Oncotarget, 7, 3, (2016), pp. 2401-16) Doi link to publisher: https://doi.org/10.18632/oncotarget.6262 Prognostic impact of concurrent MYC and BCL6 rearrangements and expression in de novo diffuse large B-cell lymphoma Scientific Institute, Milan, Italy 17 Gundersen Lutheran Health System, La Crosse, Wisconsin, USA 18 Odense University Hospital, Odense, Denmark 19 Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA 20 The University of Texas School of Medicine, Graduate School of Biomedical Sciences, Houston, Texas, USA * These authors have contributed equally to this work 20 The University of Texas School of Medicine, Graduate School of Biomedical Sciences, Houston, Texas, USA * e U e s ty o e as Sc oo o ed c e, G aduate Sc oo o o ed ca Sc e ces, ousto * These authors have contributed equally to this work Correspondence to: Ken H. Young, email: khyoung@mdanderson.org Keywords: diffuse large B-cell lymphoma, double-hit, MYC, BCL6, BCL2 Received: August 28, 2015 Accepted: September 09, 2015 Published: November 12, 2015 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Oncotarget, Vol. 7, No. 3 www.impactjournals.com/oncotarget/ Prognostic impact of concurrent MYC and BCL6 rearrangements and expression in de novo diffuse large B-cell lymphoma Qing Ye1,, Zijun Y. Xu-Monette1,, Alexandar Tzankov2,, Lijuan Deng1, Xiaoxiao Wang1, Ganiraju C. Manyam3, Carlo Visco4, Santiago Montes-Moreno5, Li Zhang3, Karen Dybkær6, April Chiu7, Attilio Orazi8, Youli Zu9, Govind Bhagat10, Kristy L. Richards11, Eric D. Hsi12, William W.L. Choi13, J. Han van Krieken14, Jooryung Huh15, Maurilio Ponzoni16, Andrés J.M. Ferreri16, Ben M. Parsons17, Michael B. Møller18, Miguel A. Piris5, Jane N. Winter19, L. Jeffrey Medeiros1 Shimin Hu1 and Ken H. Young1,20 Qing Ye1,, Zijun Y. Xu-Monette1,, Alexandar Tzankov2,, Lijuan Deng1, Xiaoxiao Wang1, Ganiraju C. Manyam3, Carlo Visco4, Santiago Montes-Moreno5, Li Zhang3, Karen Dybkær6, April Chiu7, Attilio Orazi8, Youli Zu9, Govind Bhagat10, Kristy L. Richards11, Eric D. Hsi12, William W.L. Choi13, J. Han van Krieken14, Jooryung Huh15, Maurilio Ponzoni16, Andrés J.M. Ferreri16, Ben M. Parsons17, Michael B. Møller18, Miguel A. Piris5, Jane N. Winter19, L. Jeffrey Medeiros1 Shimin Hu1 and Ken H. Young1,20 1 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Housto 1 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 University Hospital Basel Switzerland 2 University Hospital, Basel, Switzerland 3 Department of Computational Biology and Bioinformatics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 San Bortolo Hospital, Vicenza, Italy 5 H it l U i it i M d V ld ill S t d S i 4 San Bortolo Hospital, Vicenza, Italy 5 Hospital Universitario Marques de Valdecilla, Santander, Spain 6 Aalborg University Hospital, Aalborg, Denmark 7 Memorial Sloan-Kettering Cancer Center, New York, New York, USA 8 Weill Medical College of Cornell University, New York, New York, USA 9 Houston Methodist Hospital, Houston, Texas, USA 0 Columbia University Medical Center and New York Presbyterian Hospital, New York, New York, US 11 University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA 13 University of Hong Kong Li Ka Shing Faculty of Medicine, Hong Kong, China 14 Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 15 Asan Medical Center, Ulsan University College of Medicine, Seoul, Korea 16 San Raffaele H. INTRODUCTION has been developed in recent years referring to DLBCL with coexpression of MYC and BCL2 detected by immunohistochemistry (IHC), regardless of activation mechanisms. MYC/BCL2 DPL is more common than MYC/BCL2 DHL and accounts for 18-44% of DLBCL cases and might result from gene amplification, transcriptional dysregulation or both [11, 22-26]. A series of studies have shown that patients with MYC/ BCL2 DPL have a significantly poorer outcome than patients who express only one or neither protein, with a 5-year progression-free survival (PFS) of 25% following R-CHOP treatment [11, 22]. Interestingly, unlike MYC/ BCL2 DHL which is mainly observed in the GCB subtype, MYC/BCL2 DPL is more common in the activated-B cell- like (ABC) subtype and may largely contribute to inferior survival via NF-κB pathway activation [23]. has been developed in recent years referring to DLBCL with coexpression of MYC and BCL2 detected by immunohistochemistry (IHC), regardless of activation mechanisms. MYC/BCL2 DPL is more common than MYC/BCL2 DHL and accounts for 18-44% of DLBCL cases and might result from gene amplification, transcriptional dysregulation or both [11, 22-26]. A series of studies have shown that patients with MYC/ BCL2 DPL have a significantly poorer outcome than patients who express only one or neither protein, with a 5-year progression-free survival (PFS) of 25% following R-CHOP treatment [11, 22]. Interestingly, unlike MYC/ BCL2 DHL which is mainly observed in the GCB subtype, MYC/BCL2 DPL is more common in the activated-B cell- like (ABC) subtype and may largely contribute to inferior survival via NF-κB pathway activation [23]. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma and has heterogeneous biologic features. Chromosomal rearrangements, the biologic and diagnostic hallmarks of some other types of B-cell lymphoma, also occur in DLBCL. The most common chromosomal rearrangements in DLBCL are those involving chromosomal gene loci 8q24/MYC, 18q21/BCL2, and 3q27/BCL6 [1, 2]. MYC rearrangement, a disease-initiating event in Burkitt lymphoma (BL), can be observed in approximately 10% of de novo DLBCL and correlates with a poorer outcome [3-7]. However, MYC rearrangement alone may not explain the poor prognosis of patients with DLBCL that carry MYC rearrangement plus another chromosomal rearrangement. The designation double-hit lymphoma (DHL) has been used for a B-cell lymphoma carrying a MYC/8q24 rearrangement in combination with a rearrangement involving either BCL2, BCL6, or rarely other known oncogenes [2, 8, 9]. Abstract Double-hit B-cell lymphoma is a common designation for a group of tumors characterized by concurrent translocations of MYC and BCL2, BCL6, or other genes. The prognosis of concurrent MYC and BCL6 translocations is not well known. In this study, we assessed rearrangements and expression of MYC, BCL2 and BCL6 in 898 patients with de novo diffuse large B-cell lymphoma treated with standard chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab). Neither BCL6 translocation alone (more frequent in activated B-cell like www.impactjournals.com/oncotarget Oncotarget 2401 diffuse large B-cell lymphoma) nor in combination with MYC translocation (observed in 2.0% of diffuse large B-cell lymphoma) predicted poorer survival in diffuse large B-cell lymphoma patients. Diffuse large B-cell lymphoma patients with MYC/BCL6 co-expression did have significantly poorer survival, however, MYC/BCL6 co-expression had no effect on prognosis in the absence of MYC/BCL2 co-expression, and had no additive impact in MYC+/ BCL2+ cases. The isolated MYC+/BCL6+/BCL2− subset, more frequent in germinal center B-cell like diffuse large B-cell lymphoma, had significantly better survival compared with the isolated MYC+/BCL2+/BCL6− subset (more frequent in activated B-cell like diffuse large B-cell lymphoma). In summary, diffuse large B-cell lymphoma patients with either MYC/ BCL6 rearrangements or MYC/BCL6 co-expression did not always have poorer prognosis; MYC expression levels should be evaluated simultaneously; and double-hit B-cell lymphoma needs to be refined based on the specific genetic abnormalities present in these tumors. diffuse large B-cell lymphoma) nor in combination with MYC translocation (observed in 2.0% of diffuse large B-cell lymphoma) predicted poorer survival in diffuse large B-cell lymphoma patients. Diffuse large B-cell lymphoma patients with MYC/BCL6 co-expression did have significantly poorer survival, however, MYC/BCL6 co-expression had no effect on prognosis in the absence of MYC/BCL2 co-expression, and had no additive impact in MYC+/ BCL2+ cases. The isolated MYC+/BCL6+/BCL2− subset, more frequent in germinal center B-cell like diffuse large B-cell lymphoma, had significantly better survival compared with the isolated MYC+/BCL2+/BCL6− subset (more frequent in activated B-cell like diffuse large B-cell lymphoma). In summary, diffuse large B-cell lymphoma patients with either MYC/ BCL6 rearrangements or MYC/BCL6 co-expression did not always have poorer prognosis; MYC expression levels should be evaluated simultaneously; and double-hit B-cell lymphoma needs to be refined based on the specific genetic abnormalities present in these tumors. RESULTS observed in two COO subtypes (9 GCB, 5 ABC) without significantly difference in frequency (P = 0.37). Using immunohistochemistry 249 (30.2%), 439 (51.7%), and 555 (62.6%) patients had high levels of MYC (≥70%), BCL2 (≥70%), and BCL6 ( > 50%) expression, respectively. MYC expression was similarly distributed between GCB and ABC subtypes (P = 0.17) whereas BCL2 and BCL6 expression were significantly more common in the ABC and GCB subtypes, respectively (both P < 0.0001), in contrast to the association of their gene translocations with GCB and ABC subtype respectively. MYC+/BCL2+, MYC+/BCL6+, and BCL2+/ BCL6+ coexpression were observed in 146 (17.6%), 178 (21.7%), and 282 (33.4%) cases, respectively. Unlike the predominance of MYC+BCL2+ coexpression in the ABC subtype (P = 0.0079), both MYC+/BCL6+ and BCL2+/ BCL6+ coexpression were equally distributed between the two COO subtypes (P = 0.35 and P = 0.47, respectively). All MYC protein positive patients were further stratified into three subgroups: MYC+/BCL2+/BCL6- (MYC+/BCL2+ coexpression, BCL6-), MYC+/BCL6+/BCL2- (MYC+/ BCL6+ coexpression, BCL2-) and MYC+/BCL2+/BCL6+ coexpression. The MYC+BCL2+BCL6- subgroup was predominantly of ABC subtype (P = 0.0019), whereas the MYC+BCL6+BCL2- subgroup was more commonly of INTRODUCTION According to the concept of DHL used currently, another type of DHL is MYC/BCL6 DHL with concurrent MYC and BCL6 rearrangements [1, 2]. However, there are far less data available for MYC/BCL6 DHL, in part because of its rarity. BCL6 is a transcriptional suppressor required for germinal center formation with numerous transcriptional targets, including the cell cycle regulator CCND2 and MYC, which explains downregulation of MYC in normal germinal center B-cells [27-29]. Studies by others have suggested that BCL6 expression is associated with better survival of DLBCL patients [24, 30]. The frequency and the prognostic impact of concurrent MYC/BCL6 rearrangements and MYC/BCL6 protein coexpression in DLBCL remain unclear. g [ ] By far, the most common and well-studied type of DHL is characterized by concurrent MYC and BCL2 rearrangements (MYC/BCL2 DHL), occurring in about 5% of all cases of DLBCL [10, 11]. As key regulators of cell proliferation and apoptosis, respectively, MYC and BCL2 may act synergistically to drive the pathogenesis of MYC/ BCL2 DHL [12]. Clinically, patients with MYC/BCL2 DHL often exhibit adverse prognostic factors, such as high serum lactate dehydrogenase (LDH) level, advanced stage of disease, extranodal involvement, and a high proliferation index with a median value of 90%. There is a general consensus that MYC/BCL2 DHL represents a treatment-refractory subgroup with a median survival of approximately 8 months [13-20]. Despite the dismal outcome of patients with MYC/BCL2 DHL, almost all of these tumors arise within the germinal center B cell-like (GCB) subtype, a generally favorable prognostic subtype, illustrating a discordance between clinical behavior and cell of origin (COO) subtypes [1, 15, 17, 21]. In this study, we assessed the frequency, clinicopathologic features, and the prognostic impact of concurrent MYC/BCL6 rearrangements or MYC/BCL6 coexpression in a large cohort of de novo DLBCL patients treated with R-CHOP, in comparison to MYC/BCL2 rearrangements and MYC/BCL2 coexpression. The study evaluated the role of each genetic translocation separately and in combinations, providing reliable conclusion and practical recommendations for diagnostic workup and prognostic prediction. As an extension of the concept of MYC/BCL2 DHL, the concept of “double protein lymphoma (DPL)” www.impactjournals.com/oncotarget Oncotarget 2402 MYC, BCL2 and BCL6 gene translocations and protein overexpression, and multivariate Table 1: Frequencies of MYC, BCL2 and BCL6 gene translocations and protein overexpression, and multivariate survival analysis Table 1: Frequencies of MYC, BCL2 and BCL6 gene translocations and protein overexpression, and multivariate survival analysis y Overall frequency and distribution The median age of the study population was 64 years (range, 16-95). The median follow-up time was 58.9 months (range, 1-187 months). Among the 898 cases, 469 (52%) were GCB and 429 (48%) were determined to be GCB and ABC subtype, respectively. The complete response rate to R-CHOP therapy was 75%. As shown in Table 1, rearrangements of MYC, BCL2, and BCL6 were detected in 71 (11.8%) of 600, 94 (13.6%) of 690, and 145 (23.1%) of 628 cases, respectively. MYC and BCL2 rearrangements were detected predominantly in GCB subtype (P = 0.0005 and P < 0.0001, respectively) whereas BCL6 rearrangement was more frequently observed in the ABC subtype (P = 0.0002). MYC/BCL2, MYC/ BCL6, and BCL2/BCL6 concurrent rearrangements were identified in 20 (2.8%), 14 (2%), and 21 (2.9%) patients, respectively. Both MYC/BCL2 and BCL2/BCL6 concurrent rearrangements were observed mostly in the GCB subtype (MYC+/BCL2+: P < 0.0001; BCL2+/BCL6+: P = 0.0045) whereas MYC/BCL6 concurrent rearrangements were www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 2403 GCB subtype (P = 0.011) (Table 1). 67 (P = 0.0002), and the lymphoma was more often of ABC subtype (P = 0.0079). Patients with MYC+/BCL6+ coexpression were associated with advanced disease stage (P = 0.015), extranodal sites (P = 0.011), low complete response rate (P = 0.0023) and high Ki-67 index (P = 0.0017). When MYC+/BCL2+/BCL6- and MYC+/BCL6+/ BCL2- subgroups were isolated from the MYC+/BCL2+ and MYC+/BCL6+ patients respectively, MYC+/BCL2+/ BCL6- tumors were more often of ABC subtype, whereas MYC+/BCL6+/BCL2- tumors were more commonly of GCB subtype (P = 0.0003) (Table 2). Clinicopathologic features of DLBCL with concurrent rearrangement and coexpression The clinicopathologic features of patients in the study cohort with or without concurrent gene rearrangements and protein coexpression are listed in Table 2. DLBCL patients with MYC/BCL2 rearrangements more frequently had large tumors (P = 0.02) and a lower complete response rate (P = 0.0033), and commonly the tumors were of GCB subtype (P < 0.0001). No clinicopathologic features were significantly different between DLBCL patients with concurrent MYC/BCL6 rearrangements versus patients without MYC/BCL6 concurrent rearrangement, although larger tumor size was of borderline significance (P = 0.058). Prognostic impact of concurrent rearrangements of MYC, BCL2, and BCL6 in DLBCL We first assessed the prognostic impact of rearrangements of MYC, BCL2, and BCL6 in DLBCL (Figure 1 and Supplementary Figure S1). Both MYC and BCL2 rearrangements correlated to a poorer survival in the whole cohort (P = 0.003 for MYC, Figure 1A; P = 0.046 for BCL2, Figure 1B) and in the GCB subtype (P < 0.0001 for MYC, Figure 1D; P < 0.0009 for BCL2, Figure 1E) but not in the ABC subtype (Figure 1G-1H). BCL6 gi ( ) Patients with MYC+/BCL2+ coexpression were more often of older age (P = 0.0016) and more often had advanced disease stage (P < 0.0001), extranodal involvement (P = 0.0026), large tumor size (P = 0.03), International Prognostic Index score > 2 (P < 0.0001), low complete response rate (P = 0.0071), and high Ki- Figure 1: Univariate analysis for patients with DLBCL with MYC, BCL2, and BCL6 rearrangemnts in the overall-, GCB, and ABC groups. A.-B., D.-E, G.-H. MYC and BCL2 rearrangements correlated with significantly poorer overall survival in overall and GCB- but not ABC-DLBCL. C., F., I. BCL6 translocation did not correlate with poorer overall survival. Figure 1: Univariate analysis for patients with DLBCL with MYC, BCL2, and BCL6 rearrangemnts in the overall-, GCB, and ABC groups. A.-B., D.-E, G.-H. MYC and BCL2 rearrangements correlated with significantly poorer overall survival in overall and GCB- but not ABC-DLBCL. C., F., I. BCL6 translocation did not correlate with poorer overall survival. www.impactjournals.com/oncotarget Oncotarget 2404 BCL2 translocations had a worse survival than patients with MYC rearrangement alone (overall survival [OS]: P = 0.025; PFS: P = 0.012, Figure 2A-2B). However, no difference was observed in OS and PFS between DLBCL patients with MYC/BCL6 rearrangement versus DLBCL patients with only MYC rearrangement (Figure 2C-2D). BCL2 translocations had a worse survival than patients with MYC rearrangement alone (overall survival [OS]: P = 0.025; PFS: P = 0.012, Figure 2A-2B). However, no difference was observed in OS and PFS between DLBCL patients with MYC/BCL6 rearrangement versus DLBCL patients with only MYC rearrangement (Figure 2C-2D). translocation, by contrast, did not correlate with poorer survival for either the whole cohort (P = 0.33, Figure1C) or in the GCB (P = 0.87, Figure 1F) and ABC (P = 0.32, Figure 1I) subtypes. We then assessed the prognostic impact of concurrent rearrangements in DLBCL. Patients with MYC/ Oncotarget 2405 www.impactjournals.com/oncotarget Figure 2: A.-B. Prognostic impact of concurrent rearrangements of MYC, BCL2, and BCL6 in DLBCL The prognostic significance of MYC rearrangements in DLBCL depends on BCL2 rearrangement. C.-D. BCL6 rearrangement had no additive effect to MYC rearrangements. E.-F. BCL6 translocation had no additive effect to BCL2 rearrangements. G.-H. MYC expression levels appeared to impact the survival of MYC+/BCL6+ rearranged DLBCL with marginal P values probably due to the small case numbers. Figure 2: A.-B. The prognostic significance of MYC rearrangements in DLBCL depends on BCL2 rearrangement. C.-D. BCL6 rearrangement had no additive effect to MYC rearrangements. E.-F. BCL6 translocation had no additive effect to BCL2 rearrangements. G.-H. MYC expression levels appeared to impact the survival of MYC+/BCL6+ rearranged DLBCL with marginal P values probably due to the small case numbers. www.impactjournals.com/oncotarget Oncotarget 2405 Table 2: Clinical characteristics of patients with concurrent MYC, BCL2 or BCL6 translocations and MYC, BCL2 or BCL6 protein co-expression Table 2: Clinical characteristics of patients with concurrent MYC, BCL2 or BCL6 translocations and MYC, BCL2 or BCL6 protein co-expression BCL6 protein co-expression There was also no difference in OS and PFS between patients with DLBCL with BCL2/BCL6 rearrangement versus DLBCL with only BCL2 rearrangement (Figure 2E-2F). Notably the survival of patients with MYC/BCL6 rearrangement appeared to be affected by MYC expression levels (Figure 2G-2H). did not correlate with poorer patient survival, either in the whole group or in COO subtypes (Figure 4C, 4F, and 4I). In contrast, MYC+ or BCL2+ expression in DLBCL correlated with significantly poorer survival for the overall patient cohort (P < 0.0001, Figure 4A-4B) and for patients with GCB (MYC+: P < 0.0001, Figure 4D; BCL2+: P = 0.004, Figure 4E) and ABC subtypes of DLBCL (MYC+: P = 0.032, Figure 4G; BCL2+: P < 0.0001, Figure 4H). The prognostic differences between patients with concurrent rearrangements of MYC/BCL2, MYC/BCL6, BCL2/BCL6 versus the remaining DLBCL patients are shown in Figure 3. Only concurrent MYC/BCL2 rearrangements correlated with significantly poorer survival (Figure 3A-3B). Additional BCL6 translocation (triple-hit, n = 5, 26% of 19 MYC+/BCL2+ cases with BCL6 translocation status available) had no synergistic effect with concurrent MYC/BCL2 rearrangements and, on the contrary, attenuated the adverse impact of concurrent MYC/BCL2 rearrangements (Figure 3G-3H). Prognostic impact of concurrent rearrangements of MYC, BCL2, and BCL6 in DLBCL , g ; , g ) Our results further showed that MYC+/BCL2+ (P < 0.0001, Figure 5A, Supplementary Figure S3A), MYC+/BCL6+ (OS: P = 0.0001, Figure 5C; PFS: P = 0.0002, Supplementary Figure S3C), and BCL2+/ BCL6+ (OS: P = 0.014, Figure 5E; PFS: P = 0.033, Supplementary Figure S3E) coexpression correlated with significantly poorer survival in the overall cohort. The inferior survival of patients MYC+/BCL2+ and MYC+/ BCL6+ DLBCL compared with all other DLBCL patients was significant for both the GCB and ABC subtypes, whereas BCL2+/BCL6+ only correlated with poorer OS for patients with ABC-DLBCL (Supplementary Figure S4). MYC expression showed dependence and synergy only with BCL2 expression (Figure 5B, Supplementary Figure S3B); BCL6 expression had no additive adverse impact in patients with MYC+, BCL2+ or MYC+/BCL2+ DLBCL (Figure 5D, 5F-5G, Supplementary Figure S3D, Prognostic impact of coexpression of MYC, BCL2, and BCL6 in DLBCL The prognostic impact of protein expression of MYC, BCL2, and BCL6 is shown in Figure 4 and Supplementary Figure S2. BCL6 expression in DLBCL www.impactjournals.com/oncotarget Oncotarget 2406 Table 3: GEP signatures of MYC/BCL2 double-hit lymphoma, isolated MYC+BCL2+ (i.e., BCL6- DPL) and MYC+BCL2- BCL6+ (i.e., BCL2-DPL) Table 3: GEP signatures of MYC/BCL2 double-hit lymphoma, isolated MYC+BCL2+ (i.e., BCL6- DPL) and MYC+BCL2- BCL6+ (i.e., BCL2-DPL) S3F-S3G). Patients with MYC+/BCL2+ DLBCL but not MYC+/BCL6+ DLBCL had poorer survival (Figure 5G, Supplementary Figure S3G). The poor prognosis of patients with MYC+/BCL6+ DLBCL was attributable to the poorer survival of MYC+/BCL2+ DLBCL patients. CASP1), transcription factors and genes related to cell adhesion, extracellular matrix, and migration (MYO3B, SLAMF7, SILEC10/12, TPM4, and SRGN). ) Comparison of the GEP of MYC+ BCL6+ versus MYC+BCL2+ DLBCL did not show significant DEGs. We further compared the GEP of isolated MYC+BCL2- BCL6+ DLBCL versus isolated MYC+BCL2+BCL6- DLBCL, which resulted in 36 DEGs at the false discovery rate threshold of 0.05 (Table 3B, Figure 6B). Upregulated genes in MYC+BCL2-BCL6+ compared with MYC+BCL2+BCL6- included BCL6, a proliferation signature (MYBL1, NEK6, BRWD1 [bromodomain and WD repeat domain containing 1], SFRS15, HMGN1, and TMPO), MSH6 involved in DNA repair, DNAJC10 which promotes apoptotic in response to endoplasmic reticulum stress, various signaling genes including PIK3CG (PI3K catalytic subunit gamma), STAP1 (B-cell receptor signaling) SORL1, RFTN1, GNA13, SWAP70, and ANKRD13A, and genes involved in actin cytoskeleton regulation and migration (MARCKSL1, VNN2, OSBPL3, ACTR2 and etc.). Comparably, upregulated genes in MYC+BCL2+BCL6- included signatures of apoptosis (antiapoptotic BCL2, and paradoxically proapoptotic, CASP10), glutamine metabolism (ALDH4A1) , indicating that abnormal dysregulation of apoptotic and proliferation pathways are critical in patients with MYC+BCL6+BCL2−. In contrast, such signaling and molecular defects were not seen in patients with MYC+BCL6−BCL2+. The observations provide molecular basis for the difference of outcome and survival between these two groups of DLBCL patients. We further evaluated the survival of patients with isolated MYC+/BCL2+/BCL6- (i.e., BCL6- DPL) versus isolated MYC+/BCL6+/BCL2- (i.e., BCL2- DPL) DLBCL. Patients with isolated MYC+/BCL6+ DLBCL had favorable survival compared with isolated MYC+/BCL2+ DLBCL patients (OS: P = 0.004, Figure 5H; PFS: P < 0.0001, Supplementary Figure S3H). Gene expression signatures of concurrent MYC, BCL2, and BCL6 rearrangements and isolated MYC+BCL2+ and MYC+BCL6+ coexpression To better understand the molecular mechanisms of the effects on prognosis, we compared the GEP of patients with concurrent MYC/BCL2 or MYC/BCL6 rearrangements with the remaining patients. Only MYC/BCL2 rearranged DLBCL showed a distinctive GEP signature, whereas concurrent MYC/BCL6 rearranged DLBCL did not show DEGs compared with MYC rearranged DLBCL or the remaining DLBCL patients. The GEP signature of concurrent MYC/BCL2 rearranged DLBCL included 24 upregulated genes and 14 downregulated genes at the false discovery rate threshold of 0.01 (Table. 3A, Figure 5A). These genes were involved in signaling (upregulation of BMP3, SWAP70, and CELSR1, and downregulation of PLA2G7 and DOCK10), cell proliferation (upregulated STRBP and MUC4), metabolism (eight upregulated genes), apoptosis (TMEM49, CFLAR, CARD16 and www.impactjournals.com/oncotarget Oncotarget 2407 Discussion know about double-hit B-cell lymphoma is derived from studies of the most common form, MYC/BCL2 DHL. In contrast, very little has been published on DLBCL patients with concurrent MYC/BCL6 rearrangements. The findings in this study for a group of DLBCL patients treated with R-CHOP suggest that patients with concurrent MYC/BCL6 rearrangements do not have a poorer prognosis and that grouping these tumors with other forms of DHL could lead to inappropriate therapy. know about double-hit B-cell lymphoma is derived from studies of the most common form, MYC/BCL2 DHL. In contrast, very little has been published on DLBCL patients with concurrent MYC/BCL6 rearrangements. The findings in this study for a group of DLBCL patients treated with R-CHOP suggest that patients with concurrent MYC/BCL6 rearrangements do not have a poorer prognosis and that grouping these tumors with other forms of DHL could lead to inappropriate therapy. In the literature B-cell lymphomas with concurrent MYC/BCL2 or MYC/BCL6 rearrangements are grouped together as double-hit B-cell lymphomas [1, 2]. Patients with MYC/BCL2 DHL have responded poorly to all traditional chemotherapy regimens and have extremely poor outcomes [1, 10, 11]. However, most of what we l / together as double-hit B-cell lymphomas [1, 2]. Patients with MYC/BCL2 DHL have responded poorly to all traditional chemotherapy regimens and have extremely poor outcomes [1, 10, 11]. However, most of what we in this study for a group of DLBCL patients treated with R-CHOP suggest that patients with concurrent MYC/BCL6 rearrangements do not have a poorer prognosis and that grouping these tumors with other forms of DHL could lead to inappropriate therapy. Figure 3: A.-B. Concurrent MYC/BCL2 rearrangements correlated with significant poorer overall survival. C.-D. Concurrent MYC+/ BCL6+ rearrangements did not correlate with poorer overall survival. E.-F. Concurrent BCL2+/BCL6+ rearrangements did not correlate with poorer overall survival. G.-H. BCL6 attenuated the adverse prognostic impact of MYC+/BCL2+ double-hit lymphoma. Figure 3: A.-B. Concurrent MYC/BCL2 rearrangements correlated with significant poorer overall survival. C.-D. Concurrent MYC+/ BCL6+ rearrangements did not correlate with poorer overall survival. E.-F. Concurrent BCL2+/BCL6+ rearrangements did not correlate with poorer overall survival. G.-H. BCL6 attenuated the adverse prognostic impact of MYC+/BCL2+ double-hit lymphoma. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 2408 In this large cohort of 898 cases of de novo DLBCL, rearrangements of MYC, BCL2, and BCL6 were found in 11.8%, 13.6% and 23.1% of cases, respectively. BCL6 rearrangements were more frequently observed in the ABC subtype whereas MYC and BCL2 rearrangements were more frequently observed in the GCB subtype. Concurrent MYC/BCL6 and MYC/BCL2 rearrangements were observed in 2.0% and 2.8% of DLBCL cases, respectively. MYC/BCL2/BCL6 triple-hit was observed in 38% of MYC/BCL6 and 26% of MYC/BCL2 double- hit cases. The frequencies of MYC, BCL2, and BCL6 rearrangement and concurrent MYC/BCL2 rearrangements are similar to those reported in DLBCL in earlier studies [10, 11, 31-34]. with significantly poorer survival, and was associated with a distinctive GEP signature suggesting increased proliferation, growth and metabolism and decreased apoptosis pathway (our results showed downregulation of both pro- and anti-apoptotic genes). In the MD Anderson Cancer Center experience with 52 DHL patients tested for BCL6, 24 patients with BCL6 gene abnormality (translocation or amplification, n = 15 and n = 9 respectively. Among them, 14 patients had MYC/BCL2/BCL6 triple-hit) showed slightly better survival than other patients with DHL (hazard ratio: 0.59, 95% confidence interval: 0.21-1.69, P = 0.33) [36]. Ueda et al. presented a case report consistent with our results, in which a person having DLBCL with concurrent MYC, BCL2, and BCL6 rearrangements achieved complete remission after chemoradiotherapy for two years [37]. A recent study reported the largest DHL series including 41 cases with BCL6 rearrangement, in which patients’ OS was not significantly affected by whether the DHL was MYC/BCL2 or MYC/BCL6 (P = 0.537). However, 25 (58.5%) of the 41 MYC/BCL6 patients also had BCL2 rearrangement (triple-hit) [38]. In contrast, in another study, MYC/BCL6 (n = 13) showed significantly worse survival than MYC/BCL2 DHL (n = 20) after exclusion of triple-hit lymphoma [39]. These MYC/BCL6 DHL showed a trend toward higher MYC mRNA expression and a distinct gene expression profile compared to MYC/BCL2 DHL. In a smaller study reported by Pillai et al. B-cell In this study, DLBCL patients with concurrent MYC/ BCL6 rearrangements did not have a poorer prognosis. The notion that concurrent MYC/BCL6 rearrangements in DLBCL is not an indication of aggressive lymphoma was further substantiated by the lack of a distinctive GEP signature for MYC/BCL6 rearranged DLBCL although BCL6 was overexpressed in almost all MYC/BCL6 rearranged patients except one case with an IHC of 30%. www.impactjournals.com/oncotarget These phenomena might be explained by a previous finding that up to 43% of BCL6 translocations involved non-IG loci showing complex gene expression patterns [35], limited case numbers, and 36% of MYC/BCL6 cases showing low MYC expression levels (Figure 2G-2H). In contrast, MYC/BCL2 rearranged DLBCL correlated Figure 4: Univariate analysis for DLBCL patients with MYC, BCL2 and BCL6 protein expression in the overall-, GCB, and ABC-DLBCL. A.-B., D.-E., G.-H. MYC and BCL2 protein expression correlated with significantly poorer overall survival in overall, GCB- and ABC-DLBCL. C., F., I. BCL6 overexpression did not correlate with poor survival. Figure 4: Univariate analysis for DLBCL patients with MYC, BCL2 and BCL6 protein expression in the overall-, GCB, and ABC-DLBCL. A.-B., D.-E., G.-H. MYC and BCL2 protein expression correlated with significantly poorer overall survival in overall, GCB- and ABC-DLBCL. C., F., I. BCL6 overexpression did not correlate with poor survival. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 2409 combination with rituximab [40]. In this study, all patients met morphologic and immunophenotypic criteria for DLBCL with a median patient age of 64 years, which is comparable to that of patients with DLBCL without concurrent MYC/BCL6 rearrangements, and all patients were treated with standard R-CHOP therapy. lymphoma with concurrent MYC/BCL6 rearrangements was associated with aggressive clinical course and poor survival [40]. A possible explanation for the discrepancy is patient selection. In the study by Pillai et al, patients with BL, BL-like lymphoma, and primary effusion lymphoma were also included. Moreover, the median age of their patients was 83 years and only one of six patients with adequate information received chemotherapy in Recently, the concept of MYC/BCL2 rearranged DHL has been extended to MYC and BCL2 protein of their patients was 83 years and only one of six patients with adequate information received chemotherapy in Recently, the concept of MYC/BCL2 rearranged DHL has been extended to MYC and BCL2 protein Figure 5: A., C., E. Patients with DLBCL and MYC/BCL2, BCL6/MYC or BCL2/BCL6 co-expression had significantly poorer overall survival in the DBLCL cohort. B. BCL2 overexpression had a synergetic effect with MYC overexpression and the adverse prognostic impact of MYC depended on BCL2 overexpression. D. BCL6 expression had no synergetic effect with MYC expression. F. BCL6 expression appeared to attenuate the adverse prognostic impact of BCL2 overexpression. G. The poorer overall survival of MYC+BCL6+ patients was due to the poor survival of MYC+BCL2+ patients. H. www.impactjournals.com/oncotarget Isolated MYC+BCL6+ versus MYC+BCL2+ double-positive DLBCL had significantly better patient survival. Figure 5: A., C., E. Patients with DLBCL and MYC/BCL2, BCL6/MYC or BCL2/BCL6 co-expression had significantly poorer overall survival in the DBLCL cohort. B. BCL2 overexpression had a synergetic effect with MYC overexpression and the adverse prognostic impact of MYC depended on BCL2 overexpression. D. BCL6 expression had no synergetic effect with MYC expression. F. BCL6 expression appeared to attenuate the adverse prognostic impact of BCL2 overexpression. G. The poorer overall survival of MYC+BCL6+ patients was due to the poor survival of MYC+BCL2+ patients. H. Isolated MYC+BCL6+ versus MYC+BCL2+ double-positive DLBCL had significantly better patient survival. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 2410 [11, 22, 23]. In this study, DLBCL patients with MYC/ BCL6 coexpression showed a significantly poorer survival than DLBCL patients without MYC/BCL6 coexpression. However, this prognostic effect was significant only in coexpression [11, 22, 23]. These studies showed that patients with DLBCL with MYC/BCL2 double-positive (by immunohistochemistry) also have a dismal prognosis, regardless of the status of MYC or BCL2 rearrangement Oncotarge 2411 pactjournals.com/oncotarget e 6: Gene expression signature for MYC/BCL2 double-hit DLBCL (A) and comparison of MYC+BCL2+ BCL6− s MYC+BCL2−BCL6+ translocation in DLBCL (B). Figure 6: Gene expression signature for MYC/BCL2 double-hit DLBCL (A) and comparison of MYC+BCL2+ BCL6− versus MYC+BCL2−BCL6+ translocation in DLBCL (B). Figure 6: Gene expression signature for MYC/BCL2 double-hit DLBCL (A) and comparison of MYC+BCL2+ BCL6− versus MYC+BCL2−BCL6+ translocation in DLBCL (B). Figure 6: Gene expression signature for MYC/BCL2 double-hit DLBCL (A) and comparison of MYC+BCL2+ BCL6− versus MYC+BCL2−BCL6+ translocation in DLBCL (B). www.impactjournals.com/oncotarget Oncotarget 2411 doxorubicin, vincristine and prednisolone (R-CHOP) therapy was collected as part of The International DLBCL Rituximab-CHOP Consortium Program Study [33,46]. All patients were diagnosed according to the World Health Organization (WHO) classification system and received treatment between 1998 and 2010. Cases were excluded if patients had a history of low-grade B-cell lymphoma; human immunodeficiency virus infection; or primary mediastinal, cutaneous B cell lymphoma, or central nervous system DLBCL. This study was approved by the Institutional Review Boards of each participating center, and the comprehensive collaborative study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center. the presence of DLBCL with MYC/BCL2 coexpression. The isolated MYC+BCL2-BCL6+ (from all MYC+BCL6+) subgroup had significantly better patient survival compared with the MYC+BCL2+BCL6- (from all MYC+BCL2+) subgroup. Previous studies also suggested that BCL6 expression is associated with favorable survival in patients with DLBCL [24, 30, 41, 42]. p [ , , , ] There is a biologic basis that may explain the lack of significantly adverse prognostic impact of recurrent MYC/ BCL6 rearrangements and MYC/BCL6 coexpression. BCL6 represses CCND2 and MYC [27-29, 43]; we have recently shown that MYC expression levels significantly impact the prognosis of MYC rearranged DLBCL [44], and our multivariate analysis suggested BCL6 expression correlated with favorable survival (P = 0.048 for OS and P = 0.016 for PFS, Table 1), therefore the adverse prognostic impact of MYC might have been diminished by high BCL6 expression in these cases. www.impactjournals.com/oncotarget In addition to the potential role of BCL6, the GEP signature of MYC+BCL2- BCL6+ DLBCL suggested DNA repair and proapoptosis, in contrast with the upregulation of antiapoptotic BCL2 in MYC+BCL2+BCL6- DLBCL. Interestingly, MYBL1 and LIMD1 were also significantly upregulated in our MYC+BCL2-BCL6+ (associated with GCB) and MYC+BCL2+BCL6- (associated with ABC) DLBCL subgroup respectively, which is in consistent with the correlations between a novel two-gene expression index, “LIMD1-MYBL1 Index”, and GCB/ABC subtypes and clinical outcome [45, 46]. Tissue microarray, immunohistochemistry and fluorescence in situ hybridization Construction of tissue microarrays, IHC staining procedures on tissue microarray sections, and scoring criteria for MYC, BCL2 and BCL6 have been described previously [23, 44, 47]. MYC (clone Y69; Epitomics, Burlingame, CA) and BCL6 (clone LN22; Leica Microsystems, Buffalo Grove, IL) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO, Denmark) expression exhibited a cytoplasmic pattern. Cutoffs for MYC, BCL2 and BCL6 overexpression are determined as ≥70%, ≥70%, and > 50% respectively based on survival analysis as described previously, and cutoffs and positivity rates reported by other study groups.l In summary, DLBCL patients with concurrent MYC/ BCL6 rearrangements are not necessarily associated with an inferior prognosis when treated with R-CHOP therapy, unlike DLBCL patients with concurrent MYC/BCL2 rearrangements, probably due to different pathogenesis and MYC expression levels. In addition, DLBCL patients with MYC/BCL6 coexpression did have an inferior prognosis, but only in the presence of MYC/BCL2 coexpression, and therefore MYC/BCL6 coexpression seems to be of less prognostic importance [46]. These data support the notion that DLBCL with concurrent MYC/ BCL6 rearrangements and DLBCL with MYC/BCL2 DHL are not equivalent prognostically. These results suggest that the concept of double-hit lymphoma needs to be refined. The grouping of cases of DLBCL with concurrent MYC/BCL6 rearrangements with cases of DLBCL with MYC/BCL2 rearrangements may lead to over treatment of MYC/BCL6 rearranged DLBCL patients. Interphase fluorescence in situ hybridization (FISH) analysis was performed using formalin-fixed, paraffin- embedded tissue sections and BCL2 and BCL6 dual-color, break-apart probes (Vysis), IGH/MYC/CEP8 tricolor dual- fusion probes (Vysis) and a locus specific MYC dual-color break-apart probe (Vysis) as described previously [47]. Gene expression profiling and COO classification Gene expression profiling (GEP) was performed on formalin-fixed, paraffin-embedded tissue samples using Affymetrix GeneChip HG-U133 Plus Version 2.0 (Affymetrix, Santa Clara, CA) as described in an earlier study [47]. The CEL files are deposited in the National Center for Biotechnology Information Gene Expression Omnibus repository (GSE#31312).61 The microarray data were quantified and normalized by the frozen robust multiarray analysis (RMA) algorithm [48]. Differential expression gene (DEG) analysis was performed using multiple t-tests [31, 47]. Cell-of-origin classification into either GCB or ABC subtypes was achieved by using either GEP (n = 497) or IHC methods (n = 401) according to the Visco-Young (first selection) and Choi (the second selection) algorithms [49-53]. PATIENTS, MATERIALS AND METHODS PATIENTS, MATERIALS AND METHODS Statistical analysis analysis and interpretation: QY, ZYXM, LD, XW, SH, KHY; Manuscript writing: YQ, ZYXM, LD, XW, LJM, SH, KHY; Final approval of manuscript: All authors. analysis and interpretation: QY, ZYXM, LD, XW, SH, KHY; Manuscript writing: YQ, ZYXM, LD, XW, LJM, SH, KHY; Final approval of manuscript: All authors. Clinical and laboratory features of DLBCL patients at the time of presentation according to different subgroups were compared using the chi-squared test and the Spearman rank correlation test. Overall survival (OS) was calculated from the date of diagnosis to the date of last follow-up or death. Progression-free survival (PFS) was calculated from the date of diagnosis to the time of progression or death. Kaplan-Meier survival curves were used to estimate OS and PFS rates, and the log- rank (Mantel-Cox) test was used to assess differences in survival between groups. Multivariate analysis for survival was performed with IBM statistics SPSS 19 software using the Cox proportional hazards regression model (Chicago, SPSS Inc.). All differences with P≤0.05 were considered to be statistically significant. Editorial note This paper has been accepted based in part on peer- review conducted by another journal and the authors’ response and revisions as well as expedited peer-review in Oncotarget. References 1. Aukema SM, Siebert R, Schuuring E, van Imhoff GW, Kluin-Nelemans HC, Boerma EJ, Kluin PM. Double-hit B-cell lymphomas. Blood. 2011; 117:2319-31. Acknowledgments This study is supported by the National Cancer Institute and National Institutes of Health grants (R01CA138688 and R01CA187415, K.H.Y). QY is the recipient of pathology scholarship award from Jiangsu Province for Oversea Studies (JS-2011-093) and Nanjing Medical Science and Technology Development Foundation (ORX11080).. ZYXM is the recipient of the Harold C. and Mary L. Daily Endowment Fellowships and Shannon Timmins Fellowship for Leukemia Research Award. KHY is supported by The University of Texas MD Anderson Cancer Center Lymphoma Moonshot Program, Institutional Research Grant Award, an MD Anderson Lymphoma Specialized Programs of Research Excellence (SPORE) Research Development Program Award, an MD Anderson Myeloma SPORE Research Development Program Award, MD Anderson Collaborative Research Funds with High-Throughput Molecular Diagnostics, Gilead Pharmaceutical, Adaptive Biotechnology, Seattle Genetics, and Roche Molecular Systems. This work was also partially supported by National Cancer Institute and National Institutes of Health grants (P50CA136411 and P50CA142509), and by the MD Anderson Cancer Center Support Grant CA016672. 2. Sinha P, Hutter G, Kottgen E, Dietel M, Schadendorf D, Lage H. Increased expression of epidermal fatty acid binding protein, cofilin, and 14-3-3-sigma (stratifin) detected by two-dimensional gel electrophoresis, mass spectrometry and microsequencing of drug-resistant human adenocarcinoma of the pancreas. Electrophoresis. 1999; 20:2952-60. 3. Barrans S, Crouch S, Smith A, Turner K, Owen R, Patmore R, Roman E, Jack A. Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab. J Clin Oncol. 2010; 28:3360-5. 4. Savage KJ, Johnson NA, Ben-Neriah S, Connors JM, Sehn LH, Farinha P, Horsman DE, Gascoyne RD. MYC gene rearrangements are associated with a poor prognosis in diffuse large B-cell lymphoma patients treated with R-CHOP chemotherapy. Blood. 2009; 114:3533-7. 5. Klapper W, Stoecklein H, Zeynalova S, Ott G, Kosari F, Rosenwald A, Loeffler M, Trumper L, Pfreundschuh M, Siebert R, German High-Grade Non-Hodgkin’s Lymphoma Study G. Structural aberrations affecting the MYC locus indicate a poor prognosis independent of clinical risk factors in diffuse large B-cell lymphomas treated within randomized trials of the German High-Grade Non-Hodgkin’s Lymphoma Study Group (DSHNHL). Leukemia.2008; 22:2226-9. The authors declare no conflicts of interest. The authors declare no conflicts of interest. Patients A cohort of 898 patients with de novo DLBCL treated with standard rituximab, cyclophosphamide, www.impactjournals.com/oncotarget Oncotarget 2412 Authors' Contributions Conception and design: SH, ZYXM, KHY; Research performance: QY, ZYXM, AT, KHY; Provision of study materials, key reagents and technology: QY, ZYXM, LD, AT, XW, GCM, CV, LZ, SMM, , KD, AC, AO, YZ, GB, KLR, EDH, WWLC, JHK, JH, MP, AJMF, BMP, MBM, MAP, JNW, S.H., LJM, KHY; Collection and assembly of data under approved IRB and MTA: ZYXM, AT, CV, SMM, , KD, AC, AO, YZ, GB, KLR, EDH, WWLC, JHK, JH, MP, AJMF, BMP, MBM, MAP, JNW, KHY; Data 6. Ott G, Rosenwald A, Campo E. Understanding MYC- driven aggressive B-cell lymphomas: pathogenesis and classification. Blood. 2013; 122:3884-91. 7. Valera A, Lopez-Guillermo A, Cardesa-Salzmann T, Climent F, Gonzalez-Barca E, Mercadal S, Espinosa I, Novelli S, Briones J, Mate JL, Salamero O, Sancho JM, Arenillas L, et al. MYC protein expression and genetic alterations have prognostic impact in patients with diffuse large B-cell lymphoma treated with immunochemotherapy. www.impactjournals.com/oncotarget Oncotarget 2413 34:327-40. Haematologica. 2013; 98:1554-62. Haematologica. 2013; 98:1554-62. 20. Tomita N, Tokunaka M, Nakamura N, Takeuchi K, Koike J, Motomura S, Miyamoto K, Kikuchi A, Hyo R, Yakushijin Y, Masaki Y, Fujii S, Hayashi T, et al. Clinicopathological features of lymphoma/leukemia patients carrying both BCL2 and MYC translocations. Haematologica. 2009; 94:935-43. 8. Cheah CY, Oki Y, Westin JR, Turturro F. A clinician’s guide to double-hit lymphomas. Br J Haematol. 2015; 168:784-95. 9. Petrich AM, Nabhan C, Smith SM. MYC-associated and double-hit lymphomas: a review of pathobiology, prognosis, and therapeutic approaches. Cancer. 2014; 120:3884-95. 21. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti GE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000; 403:503-11. 10. Friedberg JW. Double-hit diffuse large B-cell lymphoma. J Clin Oncol. 2012; 30:3439-43. 11. Johnson NA, Slack GW, Savage KJ, Connors JM, Ben- Neriah S, Rogic S, Scott DW, Tan KL, Steidl C, Sehn LH, Chan WC, Iqbal J, Meyer PN, et al. Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. J Clin Oncol. 2012; 30:3452-9. 22. Green TM, Young KH, Visco C, Xu-Monette ZY, Orazi A, Go RS, Nielsen O, Gadeberg OV, Mourits- Andersen T, Frederiksen M, Pedersen LM, Moller MB. Authors' Contributions Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. J Clin Oncol. 2012; 30:3460-7. 12. Patel JH, McMahon SB. BCL2 is a downstream effector of MIZ-1 essential for blocking c-MYC-induced apoptosis. J Biol Chem. 2007; 282:5-13. 13. Johnson NA, Savage KJ, Ludkovski O, Ben-Neriah S, Woods R, Steidl C, Dyer MJ, Siebert R, Kuruvilla J, Klasa R, Connors JM, Gascoyne RD, Horsman DE. Lymphomas with concurrent BCL2 and MYC translocations: the critical factors associated with survival. Blood. 2009; 114:2273-9. 23. Hu S, Xu-Monette ZY, Tzankov A, Green T, Wu L, Balasubramanyam A, Liu WM, Visco C, Li Y, Miranda RN, Montes-Moreno S, Dybkaer K, Chiu A, et al. MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program. Blood. 2013; 121:4021-31; quiz 4250. 14. Kanungo A, Medeiros LJ, Abruzzo LV, Lin P. Lymphoid neoplasms associated with concurrent t(14;18) and 8q24/c- MYC translocation generally have a poor prognosis. Mod Pathol. 2006; 19:25-33. 15. Le Gouill S, Talmant P, Touzeau C, Moreau A, Garand R, Juge-Morineau N, Gaillard F, Gastinne T, Milpied N, Moreau P, Harousseau JL, Avet-Loiseau H. The clinical presentation and prognosis of diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC rearrangement. Haematologica. 2007; 92:1335-42. 24. Horn H, Ziepert M, Becher C, Barth TF, Bernd HW, Feller AC, Klapper W, Hummel M, Stein H, Hansmann ML, Schmelter C, Moller P, Cogliatti S, et al. MYC status in concert with BCL2 and BCL6 expression predicts outcome in diffuse large B-cell lymphoma. Blood. 2013; 121:2253- 63. 16. Li S, Lin P, Fayad LE, Lennon PA, Miranda RN, Yin CC, Lin E, Medeiros LJ. B-cell lymphomas with MYC/8q24 rearrangements and IGH@BCL2/t(14;18)(q32;q21): an aggressive disease with heterogeneous histology, germinal center B-cell immunophenotype and poor outcome. Mod Pathol. 2012; 25:145-56. 25. Valera A, Lopez-Guillermo A, Cardesa-Salzmann T, Climent F, Gonzalez-Barca E, Mercadal S, Espinosa I, Novelli S, Briones J, Mate JL, Salamero O, Sancho JM, Arenillas L, et al. MYC protein expression and genetic alterations have prognostic impact in patients with diffuse large B-cell lymphoma treated with immunochemotherapy. Haematologica. 2013; 98:1554-62. 17. Lin P, Medeiros LJ. High-grade B-cell lymphoma/leukemia associated with t(14;18) and 8q24/MYC rearrangement: a neoplasm of germinal center immunophenotype with poor prognosis. Haematologica. 2007; 92:1297-301. Authors' Contributions 26. Perry AM, Alvarado-Bernal Y, Laurini JA, Smith LM, Slack GW, Tan KL, Sehn LH, Fu K, Aoun P, Greiner TC, Chan WC, Bierman PJ, Bociek RG, et al. MYC and BCL2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with rituximab. Br J Haematol. 2014; 165:382-91. 18. Niitsu N, Okamoto M, Miura I, Hirano M. Clinical features and prognosis of de novo diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC translocations. Leukemia. 2009; 23:777-83. 27. Basso K, Dalla-Favera R. BCL6: master regulator of the germinal center reaction and key oncogene in B cell lymphomagenesis. Adv Immunol. 2010; 105:193-210. 19. Snuderl M, Kolman OK, Chen YB, Hsu JJ, Ackerman AM, Dal Cin P, Ferry JA, Harris NL, Hasserjian RP, Zukerberg LR, Abramson JS, Hochberg EP, Lee H, et al. B-cell lymphomas with concurrent IGH-BCL2 and MYC rearrangements are aggressive neoplasms with clinical and pathologic features distinct from Burkitt lymphoma and diffuse large B-cell lymphoma. Am J Surg Pathol. 2010; 28. Klein U, Tu Y, Stolovitzky GA, Keller JL, Haddad J, Jr., Miljkovic V, Cattoretti G, Califano A, Dalla-Favera R. Transcriptional analysis of the B cell germinal center reaction. Proc Natl Acad Sci U S A. 2003; 100:2639-44. www.impactjournals.com/oncotarget Oncotarget 2414 29. Shaffer AL, Yu X, He Y, Boldrick J, Chan EP, Staudt LM. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity. 2000; 13:199-212. 39. Aukema SM, Kreuz M, Kohler CW, Rosolowski M, Hasenclever D, Hummel M, Kuppers R, Lenze D, Ott G, Pott C, Richter J, Rosenwald A, Szczepanowski M, et al. Biological characterization of adult MYC-translocation- positive mature B-cell lymphomas other than molecular Burkitt lymphoma. Haematologica. 2014; 99:726-35. 30. Iqbal J, Greiner TC, Patel K, Dave BJ, Smith L, Ji J, Wright G, Sanger WG, Pickering DL, Jain S, Horsman DE, Shen Y, Fu K, et al. Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma. Leukemia. 2007; 21:2332-43. 40. Pillai RK, Sathanoori M, Van Oss SB, Swerdlow SH. Double-hit B-cell lymphomas with BCL6 and MYC translocations are aggressive, frequently extranodal lymphomas distinct from BCL2 double-hit B-cell lymphomas. Am J Surg Pathol. 2013; 37:323-32. 31. Choi WW, Weisenburger DD, Greiner TC, Piris MA, Banham AH, Delabie J, Braziel RM, Geng H, Iqbal J, Lenz G, Vose JM, Hans CP, Fu K, et al. Authors' Contributions A new immunostain algorithm classifies diffuse large B-cell lymphoma into molecular subtypes with high accuracy. Clin Cancer Res. 2009; 15:5494-502. 41. Lossos IS, Czerwinski DK, Alizadeh AA, Wechser MA, Tibshirani R, Botstein D, Levy R. Prediction of survival in diffuse large-B-cell lymphoma based on the expression of six genes. N.Engl.J.Med. 2004; 350:1828-1837. 42. Lossos IS, Jones CD, Warnke R, Natkunam Y, Kaizer H, Zehnder JL, Tibshirani R, Levy R. Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma. Blood. 2001; 98:945-51. 32. Tzankov A, Xu-Monette ZY, Gerhard M, Visco C, Dirnhofer S, Gisin N, Dybkaer K, Orazi A, Bhagat G, Richards KL, Hsi ED, Choi WW, van Krieken JH, et al. Rearrangements of MYC gene facilitate risk stratification in diffuse large B-cell lymphoma patients treated with rituximab-CHOP. Mod Pathol. 2014; 27:958-71. 43. Nahar R, Ramezani-Rad P, Mossner M, Duy C, Cerchietti L, Geng H, Dovat S, Jumaa H, Ye BH, Melnick A, Muschen M. Pre-B cell receptor-mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYC. Blood. 2011; 118:4174-8. 33. 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https://openalex.org/W2050139511	https://zenodo.org/records/2493747/files/article.pdf	English	null	The operative treatment of closed fractures of the long bones by metal bands, with a description of a new instrument	British journal of surgery	1,921	public-domain	11,492	INTRODUCTION. one is able to obtain a perfect linear coaptation, and at this moment, and not till then, maintain this union by tightening the band. On the other hand, when using plates and screws, it will be admitted that the slightest error in drilling the holes leads to an imperfect coaptation, and to redrill the holes seriously compromises the solidarity of the fragments. Therefore, in cases of simple oblique fracture we believe that the metal band is superior to the plate. Two points must be noted : from the experience of our cases we believe it is R mistake to employ a single band : two at least must be used, each placed close to the extremity of a frngment (Pig. 240). This is an application of the law of levers ; placed at the centre of the fracture (Fig. 241) the strength is greatly diminished. Nevertheless, one must be careful to leave at least 2 mm. between the band and the extremity of the fragment ; otherwise the latter may disengage itself from the band, as in two of our cases. The coaptation with plate and screw may be perfect ; with bands it always is. 1. The Ease of Application rind the SimpliJcation of Material used.-The introduction of Parham's bands is extremely simple ; the area of application having been chosen, one merely has to pass the hollow curved director described later. The armanicntarium of electrically-driven saws, screw-drivers, drills, etc., finds no place here. But above all, as the reduction takes place little by little under direct visual control. one is able to obtain a perfect linear coaptation, and at this moment, and not till then, maintain this union by tightening the band. On the other hand, when using plates and screws, it will be admitted that the slightest error in drilling the holes leads to an imperfect coaptation, and to redrill the holes seriously compromises the solidarity of the fragments. Therefore, in cases of simple oblique fracture we believe that the metal band is superior to the plate. p q p p Two points must be noted : from the experience of our cases we believe it is R mistake to employ a single band : two at least must be used, each placed close to the extremity of a frngment (Pig. 240). INTRODUCTION. DCRINC recent years much thought has been given to the operative treatment of fractures. At first discussions centred round the mechanical aspect of such treatment, some preferring the osteosynthcqis obtained by extraperiosteal means (Parham-Martin bands, wires, etc.) ; others the union maintained by screws, nails, hooks, etc., penetrating the bone itself. Very soon the problem became complicated by the perfection of sterilized bone-grafts, Albee in America and Nageotte in France leading the way, this work being stimulated by the researches of Leriche and Policard,22 which seemed to discredit fixation by metal plates, whether fixed by screws or bands (as giving rise to the formation of necrosis and superficial sequestra). Later still, operative treatment was shown to be a factor in delaying consolidation, the trauniatism of the intervention itself being the cause. In the present article we present the results of research into the immediate and remote effects obtained by the use of Parham's bands, applied alone in rases of oblique fractures, but associated with plates of metal or bone in transverse fractures. p We desire to express our indebtedness to M. Pierre Duval, Professor at the Faculty of Medicine of Paris, for facilities of research and clinical work, and also for access to thc records of his clinic which he most kindly pnt at our disposition. We first directed our attention to the advantages of, and indications for, the use of Parham's bands, and in this connection especially to the action of metal plates on new bone formation. The general indications for the use of Parham's bands in fractures of the long bones have recently been set forth at length by various authors, and may be generally accepted. The technique has been well described recently by Dipeon,'* especially as regards muscular interposition, methods of reduction, and the protection of nerves in relation to the fracture. We wish especially to claim for Parham's bands several very definite advantages, once the general principles of osteosynthesis by open operation are realized. 1. The Ease of Application rind the SimpliJcation of Material used.-The introduction of Parham's bands is extremely simple ; the area of application having been chosen, one merely has to pass the hollow curved director described later. The armanicntarium of electrically-driven saws, screw-drivers, drills, etc., finds no place here. But above all, as the reduction takes place little by little under direct visual control. THE OPERATIVE TREATMENT OF CLOSED FRACTURES OF TEE LONG BONES BY METAL BANDS, WITH A DESCRIPTION OF A NEW INSTRUMENT. BY E;. GERALD STANLEY AND .JEAN GATELLIER, PARIS. INTRODUCTION. INTRODUCTION. Earhj Passive Movement.-Early passive movement is thc rule in all fracture treatment. Do Parham’s bands give any advantage in this respect ? Martin, of Philadelphia,‘j quotes the case of a patient, operated upon for fracture of the tihia by two Parham’s bands, who became delirious on the night of the opcration, left his bed, and walked : no displacement of the fracture occurred. Now there is no doubt that the mere traumatism of an operation does slightly delay union, especially early callus formation : therefore it must be always kept in mind that the maintenance of apposition is solely secured by whatever mechanical means have been employed, and this for rather longer than in non-operated fractures. Later the con- solidation and union is stronger than in Dactures treated by external splints. Now thc question of mobilization, we believe, turns upon the length of the lever in question and the force that can be exerted on this lever. Thus :- $’I(:. 240. - nniids placed correctly near extremities of frapmerits. a. In fractures of the forearm we allow gentle mobilization at once (second day), the arm in the meantime resting in a plaster ‘gutter splint’. a. In fractures of the forearm we allow gentle mobilization at once (second day), the arm in the meantime resting in a plaster ‘gutter splint’. b. Fractures of the tibiu and the $buZn we mobilizc on the fifteenth day, and allow walking with crutches on the twenty-fifth. b. Fractures of the tibiu and the $buZn we mobilizc on the fifteenth day, and allow walking with crutches on the twenty-fifth. c. Fractures of the humerus must be treated with caution, for the humeral lever supports the weight of the arm and the leverage is powerful. Here mobilization is allowed on the tenth day and active movements on the twentieth, the arm in the mean- time being merely slung and bandaged lightly to the thorax. c. Fractures of the humerus must be treated with caution, for the humeral lever supports the weight of the arm and the leverage is powerful. Here mobilization is allowed on the tenth day and active movements on the twentieth, the arm in the mean- time being merely slung and bandaged lightly to the thorax. Digeonl8 mobilizes these cases as early as the thirtieth day : we cannot agree with him. INTRODUCTION. This is an application of the law of levers ; placed at the centre of the fracture (Fig. 241) the strength is greatly diminished. Nevertheless, one must be careful to leave at least 2 mm. between the band and the extremity of the fragment ; otherwise the latter may disengage itself from the band, as in two of our cases. The coaptation with plate and screw may be perfect ; with bands it always is. THE BRITISH JOURNAL OF SURGERY 260 In comminuted fractures bands are excellent : the band acts as a ferrule, drawing the fragments together, preserving smaller splinters, and forming a veritable ‘conglomeration ’ of bone. Screws and hooks are useless in such cases ; an alternative is the employment of several or branched plates, but these are objectionable by their multiplicity, and often split the smaller fragments, already numerous enough, and thus prejudice their vitality. p g , y g , p j y Lastly, Parham’s bands are equally indicated in transverse fractures, if associate with metal or bone plates. We discuss later the merits of these plates. p g , y g , p j y Lastly, Parham’s bands are equally indicated in transverse fractures, if associated with metal or bone plates. We discuss later the merits of these plates. p 2. Superiority of Parham’s Bands to Other Methods of Circular Ligature.-These bands are flat, and do not cut or damage the periosteum, as is the case where wire is used ; the compression is gradual and controlled at will. Wire certainly damages the periosteurn severely, and we have seen cases where the bone itself has suffered from the force necessary to retain the fragments. Furthermore, in twisting the ends of the wires, one frequently breaks them, or the twisted ends break off flnsh in hammering them snugly to the bone. The twisted end may irritate the tissues and cause a fistula in the absence of sepsis. CrinCo has introduced a ‘safety knot’ : this does not break or twist off, hit p the end may cause irritation. In any case the reduction and its maintenance by wire is more or less guess-work, as com- pared with the easy compression and traction given by Parham’s bands. $’I(:. 240. - nniids placed correctly near extremities of frapmerits. VIO. 211.--Danills placed incorrectly near middle of fracture. 3. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 261 4. What are the Effects of Parham’s Bands on iYew Bone Formation ?-We will now give a brief rCsuniC of the disadvantages of metallic osteosynthesis ; later n e present the results of a study of cases upon which re-operation was necessary for various reasons, and of the study of n series of radiographs taken at various intervals from the time of opcra- tion to several months later. Leriche and PolicardZ1 made recently a careful study of 15 cases of osteosynthesis by means of Lanibotte’s plates; but the fact must be noted that in all these cases the plate was placed beneath the periosteum. According to these authors, microscopic exaniin- ation shows a certain amount of fibrous tissue external to the plate. Around the plate there is sometimes a sheath of new bone, while beneath it the bone immediately subjacent is dry, white. and avascular. If the plate was removed early (20 to 90 days) they found an extremely thin lamelliform sequestrum. Further investigation showed an ischemic necrosis, and, deeper, the bone in the process OP rarefaction. The central callus was slow to appear, and poor at that, and the tissues were impregnated with iron salts. Milletz2 repeated the researches of these investigators and confirmed their findings ; he attributes this superficial necrosis to ischaemia, produced by compression and the destructive action of the body fluids attacking the metal. This writer states further that the plate is rarely covered by new bone. Albecll says that metal plates have no place in osteosynthesis. In the presence of these changes one easily understands that consolidation is delayed if metal plates are used. p Hallopeau,l9 L)ujarier,20 FrCdet et Ronvilloisls vigorously attacked these observa- tions, from the clinical point of view, bringing forward their statistics and results. IIallopeau quotes an interesting case of a double symmetrical fracture in the same patient: one fracture was treated by Parham’s bands, the other by external splints. Clinically the former consolidated very much more rapidly. y y p y CunCo and RollandZ3 examined the action of metal in the tissues, and the tolerance of the latter to the former. They found that the organic iron salts formed had no deleterious action on the tissues, bony or otherwise. With this preliminary, we now proceed to give our own results, as shown by clinical investigation, radiographs, and the microscope. INTRODUCTION. Nlearly all our accidents have occurred in these cases-bending of the callus, delayed union, etc.-and our practice is to keep these cases immobilized for at least fifty days. d. Fractures of the femur require much care and judgement. Digeonl8 mobilizes these cases as early as the thirtieth day : we cannot agree with him. Nlearly all our accidents have occurred in these cases-bending of the callus, delayed union, etc.-and our practice is to keep these cases immobilized for at least fifty days. d. Fractures of the femur require much care and judgement. In order to avoid large plasters we began by using continuous extension-bending and angulation occurred. We then tried the large plaster generally used for tuberculous diseases of the hip-exactly the same results took place while moulding the plaster after operation. We believe that the following method gives the best results : A bivalve plaster-cast is prepared beforehand and applied to the limb, which is maintained in continuous extension. The operation is performed with the thigh resting in the posterior valve; when completed the anterior valve is placed in position and the whole cast held together by bandaging. How should these fractures be immobilized? OPERATIVE TREATMENT OF CLOSED FRACTURES 2 in cases where we have used (1) Parhnrn’s bends alone, or ( 2 ) Parhunt’s brtnds nssociated aith plates. 1. The Employment of Parham’s Bands Alone.-An examination of radiographs taken in series shows, as early as the fifteenth day, irregular shadows, more or less opaque, completely surrounding the operative area and band. These shadows become increasingly distinct, till, by the twenty-fifth day, they extend longitudinally along the bone, the appearance being that of a spindle or tapering sheath. In certain cases-especially in the tibia, clavicle, and bones of the forearm-the shadow remains more localized and does not extend along the bone. In other cases- the humerus, and especially the femur-the callus is excessive ; but if radiographs, taken at the expiration of several months, are examined, one nearly always sees a distinct thinning of this callus, while in many cases it is reduced to a minimum. Further, these radiographs show that the callus surrounds and covers the band, contrary to the assertions of the opponents of metallic plating ; but we would call special attention to the small clear area, well seen in Pig. 262, between the callus and the band. This demonstrates that, in immediate contact with the band, early ossification is delayed ; but radiographs show that later this clear space disappears. On the other hand, I4’rCdet has seen this space persisting at eight months. Radiographs taken in profile and anteroposteriorly after the callus has thinned out and ‘settled down’ reveal at once an irregularity in its development : it is always far better developed and more abundant on the side of the bone opposed to the track of operative approach. Hallopeauls has confirmed this, and believes that the mere exposure of a fracture by operation delays union. In both these cases essentially the same conditions were discovered, a bony mass completely hiding I n two of our cases (femurs) we were obliged to re-operate for pain. THE BRITISH JOURNAL OF SURGERY 262 the bands. After chiselling away this callus, the bands were found completely embedded and firmly fixed. The bands, which were of soft steel, were covered with a black, slightly- adherent layer, but no trace of rust was found, and no sequestrum. The tissue was extremely vascular, and the steady oozing could only be controlled by irrigation with hot saline solution. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 g d. An absence of pain, and perfect toleration of the bands by the tissues. e. Absence of rust or toxic action by organic iron salts. e. Absence of rust or toxic action by organic iron salts. 2. The Employment of Parham’s Bands associated with Bone or Metal Plates in Transverse Fractures.-Parham’s bands find an application in transverse fractures if associated with metal or bone splints. At the beginning we used the metal plates to hand, namely, those of Lane or Lambotte : later, to obviate the bands slipping from the plate, we used Sherman’s plates provided with grooves to receive the bands. Later still, bone-grafts (living and dead) having come to the fore, we used Parham’s bands to fix various forms of bony splints. We wish first to submit certain points which we have found essential in the general technique of osteosynthesis, and then to describe the methods and material which have given us the best results. B 2. The Employment of Parham’s Bands associated with Bone or Metal Plates in Transverse Fractures.-Parham’s bands find an application in transverse fractures if associated with metal or bone splints. At the beginning we used the metal plates to hand, namely, those of Lane or Lambotte : later, to obviate the bands slipping from the plate, we used Sherman’s plates provided with grooves to receive the bands. Later still, bone-grafts (living and dead) having come to the fore, we used Parham’s bands to fix various forms of bony splints. We wish first to submit certain points which we have found essential in the general technique of osteosynthesis, and then to describe the methods and material which have given us the best results. a. METAL PLmEs AND PARMAM’S BANDS.-l$-e have no hesitation in saying that Sherman’s plates are far superior to all others in this combination. Extremely strong, they nevertheless present a very limited area of contact with the bone-a great advantage-at the same time being provided with grooves which render slipping of the showit% pollits (A. B) bands impossible if the technique is correct. Lambotte’s plates have ,,lAte the ba,,d been made with grooves, but being much thinner they necessitate a m liable to snap. larger area of bone contact to give the necessary strength, which is unfavourable to the vitality of the underlying bone. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 The new bone was more developed on the side of the fracture opposed to the wound, and was moderate in amount. , All authors have insisted that callus is more abundant in fractures treated by metallic osteosynthesis than by simple reduction. From this point of view it seems to us that Parham’s bands give better results than other metallic appliances ; but here we must again emphasize the fact that when using plates (Lane’s or Lambotte’s) and other methods of fixation (nails, screws, or hooks) the periosteum is always incised and the metal placed beneath it. We believe that the interference with the periosteum, however carefully carried out, explains this trouble with callus formation. Now Parham’s bands are always placed extraperiosteally. To the objection that the sensitive and vascular periosteum is traumatized and strangled, we reply that it is of little importance. We have only twice seen eases of persistent pain necessitating the removal of the bands, and as for strangling the periosteum it can matter little, for the blood-supply of this membrane is not longi- tudinal and continuous, but of the type of intestinal vascularization, by means of abundant and fine anastomoses. Furthermore, the bands in no way interfere with the periosteum at the line of fracture: it is here intact; while finally, all research, from Macewen to Gallie and Robertson,** shows that the periosteum is a vascularizing membrane primarily. and takes no part in actual bone formation apart from this property, important though it be. p y p g Summing up : our study of cases treated by Parham’s bands alone shows :-- n. A delay in consolidation hardly appreciable even if it exists. p y p g Summing up : our study of cases treated by Parham’s bands alone show n. A delay in consolidation hardly appreciable even if it exists. y y pp b. Complete absence of necrosis at the point of contact of the bands. y y pp b. Complete absence of necrosis at the point of contact of the bands. p p c. A callus formation which is regular, reduced to a minimum, and completely surrounding the bands. c. A callus formation which is regular, reduced to a minimum, and completely surrounding the bands. c. A callus formation which is regular, reduced to a minimum, and completely surrounding the bands. d. An absence of pain, and perfect toleration of the bands by the tissues. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 Can these accidents be explained or remedied ? First, the tissues encircled by the bands are not homogeneous : the resistance offered by the bone and the steel plate is not the same, and there is no doubt that during the few days following operation the encircling force slightly diminishes, whether it be that the plate sinks into the periosteum, or that the band slips more easily on steel than on bone. We have certainly seen cases where the plate has slipped on the band. Another observation is that with mobilization the band may break, and we believe the reason to be as follows. The band after encircling the bone passes ‘‘‘‘(L?~~. on to the plate (Sherman’s), which is quadrmwular. and PI(;. 244. YIG. 24.5. PIG. ?43.-Oblique fracttlre, requiritiq t n o A- YIG. 211;. PI(;. 21i. m;. “48. FIUS. %fi, %i, 24B.-Diacrams illustratiit:: incorrect position of plates a t opposite poles of tlie same axi3 (A B). The axis at rixht all:leS (C D) is ut~protected, imd displacemetit is liable to occur. bauds otilj-. 1:1os. ‘241, ?.iB.-Traner-er;e fracture, reqnir- in- four bands. 0 3 ~~ ~ i n s 0 d 0 i n g bridges a very small triangu- lar space between the former and the latter; it snaps at the edges of the plate (Fig. 242, A, 11). This might be avoided by altering the form of the plate. We observed as an illustra- tion of this the case of a young girl who was moved from hospital to her home because of a scarlet-fever epidemic. During transport both bands broke and the callus bent. Continuous extension produced perfect alinement, and on the seventieth day the plates and bands were removed from a mass of new bone which cont- plrtely surrounded them. p ‘‘‘‘(L?~~. PI(;. 244. YIG. 24.5. PIG. ?43.-Oblique fracttlre, requiritiq t n o bauds otilj-. 1:1os. ‘241, ?.iB.-Traner-er;e fracture, reqnir- in- four bands. ‘‘‘‘(L?~~ PI(;. 244. YIG YIG. 24.5. ‘‘‘‘(L?~~ PI(;. 244. YIG. 24.5. PIG. ?43.-Oblique fracttlre, requiritiq t n o bauds otilj-. 1:1os. ‘241, ?.iB.-Traner-er;e fracture, reqnir- in- four bands. A- YIG. 211;. PI(;. 21i. m;. “48. FIUS. %fi, %i, 24B.-Diacrams illustratiit:: incorrect position of plates a t opposite poles of tlie same axi3 (A B). The axis at rixht all:leS (C D) is ut~protected, imd displacemetit is liable to occur. PI(;. 21i. m;. “48. A- YIG. 211;. YIG. 211;. m;. “48. PI(;. 21i. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 We have already shown the advantages and disadvantages of metal plates, which are still under discussion ; but upon one result of the use of such plates everyone agrees-the volume of the callus formed. Now in the femur, the tibia, and the humerus this excess of callus is of little or no importance, but it is otherwise when the clavicle or bones of the forearm are in B 1’10. 242.-D1agrmi at the edge of the showit% pollits (A. B) ,,lAte the ba,,d m liable to snap. B 1’10. 242.-D1agrmi at the edge of the a. METAL PLmEs AND PARMAM’S BANDS.-l$-e have no hesitation in saying that Sherman’s plates are far superior to all others in this combination. Extremely strong, they nevertheless present a very limited area of contact with the bone-a great advantage-at the same time being provided with grooves which render slipping of the showit% pollits (A. B) bands impossible if the technique is correct. Lambotte’s plates have ,,lAte the ba,,d been made with grooves, but being much thinner they necessitate a m liable to snap. larger area of bone contact to give the necessary strength, which is unfavourable to the vitality of the underlying bone. We have already shown the advantages and disadvantages of metal plates, which are still under discussion ; but upon one result of the use of such plates everyone agrees-the volume of the callus formed. Now in the femur, the tibia, and the humerus this excess of callus is of little or no importance, but it is otherwise when the clavicle or bones of the forearm are in 1’10. 242.-D1agrmi at the edge of the showit% pollits (A. B) ,,lAte the ba,,d m liable to snap. 1’10. 242.-D1agrmi at the edge of the OPERATIVE TREATMENT OF CLOSED FRACTURES 2 263 question. In these bones the exuberant callus is so disadvantageous, and may cause so much trouble, that we believe Parham’s bands with plates to be contra-indicated if the fracture is distinctly transverse, and employ for prefer- ence either Dujarier’s hooks, or drilling and wire. q g , y much trouble, that we believe Parham’s bands with plates to be contra-indicated if the fracture is distinctly transverse, and employ for prefer- ence either Dujarier’s hooks, or drilling and wire. What are the results of bands and plates? We have had several failures, bending of the callus occurring on mobilization. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 FIUS. %fi, %i, 24B.-Diacrams illustratiit:: incorrect position of plates a t opposite poles of tlie same axi3 (A B). The axis at rixht all:leS (C D) is ut~protected imd displacemetit is liable to occur. p D /I I C A --* A. B B FIG. 2-13. lX.. 250. FIGS. 2x1, 25u-Dia:’rarns illu\trat- ~ ~ r ~ ~ ~ ~ ~ , (pAlaF$ky$ w h t w1es to each other. p D /I I C A --* A. B B FIG. 2-13. lX.. 250. There is a D /I great difference in the mechanical distribution of the force which may be applied to oblique or to trar8sverse fractures. of the bone and in the direction of the weight applied, resistance of a plate is to force applied perpendicularly to it, a parallel force tending to cause displacement. We therefore use long plates fixed by four bands: two close to the line of fracture, two towards the extremity of the plate (Figs. 243, 244, 245). In fractures of the femur, I C strain, it is wise to use two plate?. These plates should (Figs. 246, 247, A B), because an axis with feeble resistance (Figs. 246, 247, c D) will be left unguarded. They should, on the contrary, be placed each at the extremity of two axes perpendicular the one to the other (Figs. 349, 250, A,c). b. BONE SPLINTS AND PARHAM’S RANDs.-Stimulated by the remarkable work of Nageotte and Sencert on the revitalization of tissues sterilized in alcohol, a large number In oblique fractures the force is distributed in the length while in transverse fractures the maximum and optimum A --* A. B B where the fixation appliance has to withstand considerable never be placed at the two extremities of the same axis FIG. 2-13. lX.. 250. FIGS. 2x1, 25u-Dia:’rarns illu\trat- ~ ~ r ~ ~ ~ ~ ~ , ’ (pAlaF$ky$ w h t w1es to each other. There is a great difference in the mechanical distribution of the force which may be applied to oblique or to trar8sverse fractures. of the bone and in the direction of the weight applied, resistance of a plate is to force applied perpendicularly to it, a parallel force tending to cause displacement. We therefore use long plates fixed by four bands: two close to the line of fracture, two towards the extremity of the plate (Figs. 243, 244, 245). b. BONE SPLINTS AND PARHAM’S RANDs.-Stimulated by the remarkable work Nageotte and Sencert on the revitalization of tissues sterilized in alcohol, a large numb OPERATIVE TREATMENT OF CLOSED FRACTURES 2 ~ ., resembling that of Hallopeac but bearing lateral ridges which are destined to rest on the edges of the groove cut in the bone (Fig. 253). In the cases of the two latter plates a groove has to be cut to receive them into the bone at the site of fracture by means of an Albee saw. Of Duval's cases, 9 consolidated and were satisfactory; in 2 a fracture of the plate occurred ; and in 1 case severe infec- tion took place ; the wound was re-opened and the platc removed. The latter lay loosely on the bone and was soiled with pus. In each case where the bone plate broke, re-operation was necessary and the plate was removed. In no case was the plate incorporated with the living bone. No vascularization was seen; on the con- trary, the bone appeared rarefied in contact with the plate, which was roughened but not surrounded by callus ; the callus was well developed on the opposite side of the bone. In short, the plate was free and independent, both superficially and deep. The dates after their removal were examined by the kindness of Dr. Rolland, who reports that " histological examination shows that no invasion of osteoblasts from the neighbouring bone has taken place: there is no trace of commencing absorption, nor is the graft vascularized ". Thus the conditions found where bone plates have been used are essentially the same as described by the opponents of metallic osteosynthesis. As to the cases which consolidated well and in which a good result was obtained, we found the same fusiform callus formation as when metal plates were used, especially developed on the side of the bone opposed to the plate, and in about the same period of time. O lt th ith b l t h t b i b i i f h We have had some experience in the use of bone transplants as splints with Parham's bands, and we now present our results and conclusions. By the courtesy of Professor Pierre Duval we are also able to bring forward the results of several of his cases, observed by ourselves. Duval used bone plates sterilized in alcohol in the form of a shuttle, resentbling a Lanibotte plate, provided with grooves for the bands : this plate was always placed upon the periosteum (Pig. 251). OPERATIVE TREATMENT OF CLOSED FRACTURES 2 Rolland, who reports that " histological examination shows that no invasion of osteoblasts from the neighbouring bone has taken place: there is no trace of commencing absorption, nor is the graft vascularized ". Thus the conditions found where bone plates have been used are essentially the same as described by the opponents of metallic osteosynthesis. As to the cases which consolidated well and in which a good result was obtained, we found the same fusiform callus formation as when metal plates were used, especially developed on the side of the bone opposed to the plate, and in about the same period of time. Our results then with bone plates have not been encouraging ; but in view of the FIR 251.- Dural's bone plate FIG. 252.- HallopeiLu's hoiie platc. I I G . 353.- €Ieitz-lioyer's bon plate. ~ . FIR 251.- Dural's bone plate FIG. 252.- HallopeiLu's hoiie platc. I I G . 353.- €Ieitz-lioyer's bone plate. ~ ., Of Duval's cases, 9 consolidated and were satisfactory; in 2 a fracture of the plate occurred ; and in 1 case severe infec- tion took place ; the wound was re-opened and the platc removed. The latter lay loosely on the bone and was soiled with pus. In each case where the bone plate broke, re-operation was necessary and the plate was removed. In no case was the plate incorporated with the living bone. No vascularization was seen; on the con- trary, the bone appeared rarefied in contact with the plate, which was roughened but not surrounded by callus ; the callus was well developed on the opposite side of the bone. In short, the plate was free and independent, both superficially and deep. The dates after their removal were examined by the kindness of Dr. Rolland, who reports that " histological examination shows that no invasion of osteoblasts from the neighbouring bone has taken place: there is no trace of commencing absorption, nor is the graft vascularized ". Thus the conditions found where bone plates have been used are essentially the same as described by the opponents of metallic osteosynthesis. As to the cases which consolidated well and in which a good result was obtained, we found the same fusiform callus formation as when metal plates were used, especially developed on the side of the bone opposed to the plate, and in about the same period of time. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 In fractures of the femur, strain, it is wise to use two plate?. These plates should (Figs. 246, 247, A B), because an axis with feeble resistance (Figs. 246, 247, c D) will be left unguarded. They should, on the contrary, be placed each at the extremity of two axes perpendicular the one to the other (Figs. 349, 250, A,c). b BONES S In oblique fractures the force is distributed in the length while in transverse fractures the maximum and optimum where the fixation appliance has to withstand considerable never be placed at the two extremities of the same axis A. FIG. 2-13. lX.. 250. FIGS. 2x1, 25u-Dia:’rarns illu\trat- ~ ~ r ~ ~ ~ ~ ~ , (pAlaF$ky$ w h t w1es to each other. FIGS. 2x1, 25u-Dia:’rarns illu\trat- ~ ~ r ~ ~ ~ ~ ~ , (pAlaF$ky$ w h t w1es to each other. THE BRITISH JOURNAL OF SURGERY 264 of surgeons attempted the use of sterilized bone transplants in human surgery. Although these bony transplants were totally different from the tissues used by these authors, both in their clinical behaviour and histological character, it was neverthelers hoped they would be vitalized and organized by the invasion of osteoblasts from the adjacent bone- ends. Gallie and Robertson24 have recently shown by careful experiment that boiled bone may be thus invaded, vascularized, and absorbed, while at the same time new bone is formed. bone is formed. We have had some experience in the use of bone transplants as splints with Parham's bands, and we now present our results and conclusions. By the courtesy of Professor Pierre Duval we are also able to bring forward the results of several of his cases, observed by ourselves. Duval used bone plates sterilized in alcohol in the form of a shuttle, resentbling a Lanibotte plate, provided with grooves for the bands : this plate was always placed upon the periosteum (Pig. 251). Hall~peau'~ uses a sterilized beef-bone plate some 10 to 12 cm. long, triangular on section, but with a rounded back, this plate also carrying four grooves for the bands (Fig. 252). Heitz-Royer prefers a sterilized bone plate or splint FIR 251.- Dural's bone plate FIG. 252.- HallopeiLu's hoiie platc. I I G . 353.- €Ieitz-lioyer's bone plate. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 Hall~peau'~ uses a sterilized beef-bone plate some 10 to 12 cm. long, triangular on section, but with a rounded back, this plate also carrying four grooves for the bands (Fig. 252). Heitz-Royer prefers a sterilized bone plate or splint resembling that of Hallopeac but bearing lateral ridges which are destined to rest on the edges of the groove cut in the bone (Fig. 253). In the cases of the two latter plates a groove has to be cut to receive them into the bone at the site of fracture by means of an Albee saw. Pierre Duval we are also able to bring forward the results of several of his cases, observed by ourselves. Duval used bone plates sterilized in alcohol in the form of a shuttle, resentbling a Lanibotte plate, provided with grooves for the bands : this plate was always placed upon the periosteum (Pig. 251). Hall~peau'~ uses a sterilized beef-bone plate some 10 to 12 cm. long, triangular on section, but with a rounded back, this plate also carrying four grooves for the bands (Fig. 252). Heitz-Royer prefers a sterilized bone plate or splint FIR 251.- Dural's bone plate FIG. 252.- HallopeiLu's hoiie platc. I I G . 353.- €Ieitz-lioyer's bone plate. ~ ., resembling that of Hallopeac but bearing lateral ridges which are destined to rest on the edges of the groove cut in the bone (Fig. 253). In the cases of the two latter plates a groove has to be cut to receive them into the bone at the site of fracture by means of an Albee saw. Of Duval's cases, 9 consolidated and were satisfactory; in 2 a fracture of the plate occurred ; and in 1 case severe infec- tion took place ; the wound was re-opened and the platc removed. The latter lay loosely on the bone and was soiled with pus. In each case where the bone plate broke, re-operation was necessary and the plate was removed. In no case was the plate incorporated with the living bone. No vascularization was seen; on the con- trary, the bone appeared rarefied in contact with the plate, which was roughened but not surrounded by callus ; the callus was well developed on the opposite side of the bone. In short, the plate was free and independent, both superficially and deep. The dates after their removal were examined by the kindness of Dr. VOL. IS.-NO. 34. OPERATIVE TREATMENT OF CLOSED FRACTURES 2 FIR 251.- Dural's bone plate FIG. 252.- HallopeiLu's hoiie platc. I I G . 353.- €Ieitz-lioyer's bone plate. ~ ., FIR 251.- Dural's bone plate I I G . 353.- €Ieitz-lioyer's bone plate. ~ ., FIG. 252.- HallopeiLu's hoiie platc. Our results, then, with bone plates have not been encouraging ; but in view of the work of Gallie and Robertson we wish to emphasize that the bone was heterogenous, devoid of periosteum, and dead. In Nageotte's work he insisted on the employment of emhryonic connective tissue, which condition the bone of an ox does not satisfy. l if h f l i i b i d bl i i i f h Lastly, if one hopes for vascularization, absorption, and osteoblastic invasion of the graft, it should undoubtedly be placed beneath the periosteum, while a great advantage of Parham's bands is their extraperiosteal position. To satisfy both these conditions would injure and compress this membrane, seriously compromising the result. OPERATIVE TREATMENT OF CLOSED FRACTURES PERATIVE TREATMENT OF CLOSED FRACTURES 265 We therefore conclude that bone plates give no advantages from the point of view of consolidation, they lack the necessary resistance and solidity, and are apt to break. The operation is much more complicated if one uses bone splints, which need a groove for their reception : they are not incorporated in the callus, and are not converted into living bone. For these reasons it seems to u s that bone plates are not preferable to those of metal, attractive 6 as th.ey may be in theory. I! not incorporated in the callus, and one. For these reasons it seems to erable to those of metal, attractive 6 L. I! Instruments Required for Parham’s Bands.-Those used by Parham and Martin are a tractor, an aneurysm needle to which the band is attached by a ligature, and bands of soft steel. These, with the operative technique, are well described by Digeon.ls They h a v e m a n y disadvantages. Firstly, the method of passinp the aneurysm needle and draw- ing the attached band through after it is clumsy and difficult. Whcre the needle passes, the band may not be able to follow; it becomes caught in the tissucs, loses its direction, and twists. Frequently the ligature between the needle and band breaks. Tanton,17 in face of these difficulties, invented a ‘needle’ provided with a hook for the ‘eye’ of the band ; but, although an improvement, his ‘needle’ does not pass easily. One of us (J. G.) devised a hollow L. I! aneurysm needle (‘rail passe lime’), which seems to us to overcome the drawbacks of previous instruments (Fig. 254). It consists in a hollow curved director, at right anglcs to its handle, and made in two sizes. This director having encircled the bone at the selected point, one introduces the band at the extremity of this instrument, which thus forms a tunnel through which the band slides. The director is withdrawn, leaving the band in accurate position. The latter must now be tightened : here, again. the tractor used by Parham is not all that could be desired, the band frequently twisting and slipping, and necessitating manoeuvres prrjudicial to asepsis. FrCdetI6 suggested a long band with a ‘running knot’ at the centre tightened by traction with pressure forceps, a useless and tiring process. g p P ~ i t t i ~ has invented an excellent instrument, but we prefer that of Gatellier. The threaded stem of the tractor ends in the form of a shell ‘nose-cap’ (Figs. 255, 256, 2579, which exactly fits into the hollow extremity of a cylinder containing this stem. The latter at its extremity is provided with a slit the exact size of the band. The band, passing through this slit, meets an exactly similar slit in the ‘nose-cap’ ; traversing the latter it now meets a cam which lifts the moment the slot of the band engages the former, a spring snapping back the cam into the slot. The band can now be tightened round the bone by a wheel acting on the stem, while the slot is disengaged from the cam by slight pressure on a button. OPERATIVE TREATMENT OF CLOSED FRACTURES Instruments Required for Parham’s Bands.-Those used by Parham and Martin are a tractor, an aneurysm needle to which the band is attached by a ligature, and bands of soft steel. These, with the operative technique, are well described by Digeon.ls They h a v e m a n y disadvantages. Firstly, the method of passinp the aneurysm needle and draw- ing the attached band through after it is clumsy and difficult. Whcre the needle passes, the band may not be able to follow; it becomes caught in the tissucs, loses its direction, and twists. Frequently the ligature between the needle and band breaks. Tanton,17 in face of these difficulties, invented a ‘needle’ provided with a hook for the ‘eye’ of the band ; but, although an improvement, his ‘needle’ does not pass easily. One of us (J. G.) devised a hollow aneurysm needle (‘rail passe lim previous instruments (Fig. 254). to its handle, and made in two selected point, one introduces th forms a tunnel through which t band in accurate position. The used by Parham is not all tha slipping, and necessitating mano band with a ‘running knot’ at a useless and tiring process. (J. G.) devised a hollow aneurysm needle (‘rail passe lime’), which seems to us to overcome the drawbacks of previous instruments (Fig. 254). It consists in a hollow curved director, at right anglcs to its handle, and made in two sizes. This director having encircled the bone at the selected point, one introduces the band at the extremity of this instrument, which thus forms a tunnel through which the band slides. The director is withdrawn, leaving the band in accurate position. The latter must now be tightened : here, again. the tractor used by Parham is not all that could be desired, the band frequently twisting and slipping, and necessitating manoeuvres prrjudicial to asepsis. FrCdetI6 suggested a long band with a ‘running knot’ at the centre tightened by traction with pressure forceps, a useless and tiring process. (J. G.) devised a hollow aneurysm needle (‘rail passe lime’), which seems to us to overcome the drawbacks of previous instruments (Fig. 254). It consists in a hollow curved director, at right anglcs to its handle, and made in two sizes. OPERATIVE TREATMENT OF CLOSED FRACTURES This director having encircled the bone at the selected point, one introduces the band at the extremity of this instrument, which thus forms a tunnel through which the band slides. The director is withdrawn, leaving the band in accurate position. The latter must now be tightened : here, again. the tractor used by Parham is not all that could be desired, the band frequently twisting and slipping, and necessitating manoeuvres prrjudicial to asepsis. FrCdetI6 suggested a long band with a ‘running knot’ at the centre tightened by traction with pressure forceps, a useless and tiring process. P ~ i t t i ~ has invented an excellent instrument, but we prefer that of Gatellier. The threaded stem of the tractor ends in the form of a shell ‘nose-cap’ (Figs. 255, 256, 2579, which exactly fits into the hollow extremity of a cylinder containing this stem. The latter at its extremity is provided with a slit the exact size of the band. The band, passing through this slit, meets an exactly similar slit in the ‘nose-cap’ ; traversing the latter it now meets a cam which lifts the moment the slot of the band engages the former, a spring snapping back the cam into the slot. The band can now be tightened round the bone by a wheel acting on the stem, while the slot is disengaged from the cam by slight pressure on a button. 18 VOL. IS.-NO. 34. THE BRITISH JOURNAL OF SURGERY 266 The apparatus takes to pieces readily for sterilization, and is re-assembled without It has given us every-satisfaction, and is used in the service of Professor Duval. screwing. FIG. 258.--Casc 1. Fracture of tibia and fibula, ei,olit moiitlis after operatioil ; perfect result. a. Rli:.tit votistrictiori a t bald ; callus iiisigiii- ticant. FIG. 259. - Case 2. Supra- mslleolar fracture four days after operatioil. IW. %BO.-Same frncture as in Pig. 259, elevcii months after operatioil ; perfect anatomical result : very littlc callus. a, 81iIit coiistrictioii at band, wliicli is riot surrouiided by callus. FIG. 259. - Case 2. Supra- mslleolar fracture four days after operatioil. IW. %BO.-Same frncture as in Pig. 259, elevcii months after operatioil ; perfect anatomical result : very littlc callus. a, 81iIit coiistrictioii at band, wliicli is riot surrouiided by callus. FIG. 258.--Casc 1. Fracture of tibia and fibula, ei,olit moiitlis after operatioil ; perfect result. a. OPERATIVE TREATMENT OF CLOSED FRACTURES Rli:.tit votistrictiori a t bald ; callus iiisigiii- ticant. OPERATZVE TREATMENT OF CLOSED FRACTURES 267 Operation, Nov. 14, 1920. after failure of non-operative treatment. Radial nerve freed, bone nds trimmed, and I I’arhani’s band placed enclrrling all three fragments. FIG. ?fi?.-Same fr;icture as ill Fig. P O I , seven moiitlrs after operation. a, Jmxe clear space iti contact witti the batid. 1W. 2lil.-Case fi. Fractur of lutmerus two clays after opera tion. FIO. ?63.-Cnse 7. Yracture of femur three montlis after operation ; moderate amouiit of callus on siile oppo5ecl to plate. a, Clear space. b, Callus opposite the plate. FIO. ?63.-Cnse 7. Yracture of femur three montlis after operation ; moderate amouiit of callus on siile oppo5ecl to plate. a, Clear space. b, Callus opposite the plate. FIO. ?63.-Cnse 7. Yracture of femur three montlis after operation ; moderate amouiit of callus on siile oppo5ecl to plate. a, Clear space. b, Callus opposite the plate. 1W. 2lil.-Case fi. Fracture of lutmerus two clays after opera- tion. FIG. ?fi?.-Same fr;icture as ill Fig. P O I , seven moiitlrs after operation. a, Jmxe clear space iti contact witti the batid. Radiogram, Feb. 24, 1921, showed perfect reduction and position. Three months later, voluminous callus formation with dear space round band. Func- tion good. Case ‘i.-(Fig. 263.) D. .J., age 15, seen Sept. 1 , 1920, with fracture middle third of femur. Effusion into knee-joint aspirated several times. Radiogram showed transverse fracture with much dis- placement. Operation Sept. 10. Incision through vastus internus and reduction by Lambotte’s tractor. Two Sherman’s plates and 2 Parham’s bands, with 2 wire loops. Radiogram, Feb. 24, 1921, showed perfect reduction and position. Three months later, voluminous callus formation with dear space round band. Func- tion good. Radiogram, Feb. 24, 1921, showed perfect reduction and position. Three months later, voluminous callus formation with dear space round band. Func- tion good. Case ‘i.-(Fig. 263.) D. .J., age 15, seen Sept. 1 , 1920, with fracture middle third of femur. Effusion into knee-joint aspirated several times. Radiogram showed transverse fracture with much dis- placement. Operation Sept. 10. Incision through vastus internus and reduction by Lambotte’s tractor. Two Sherman’s plates and 2 Parham’s bands, with 2 wire loops. Radiogruw).- Perfect reduc- tion. Four months later, moder- ate callus formation, especially developed on side of bone opposed to plates. at junction of middle and lower t Bands embedded in c Case &-(Figs. 265, 266.) G FIG. ILLUSTRATIVE CASES. Oblique fracture of tibia, junction middle and lower thirds, fracture of fibula. Case ].-(Fig. 258.) H. AI., age 40. First seen July 2, 1920. Operalion, July 5. Discharged Sept. 9. Inter- position of tibialis anticus. Reduction. Two Parham’s bands. Radiogram, July 16, showed perfect position. Anatomical and functional result perfect. Eight months after, Zimar cousolidutiou without appreciable callus. Oblique fracture of tibia, junction middle and lower thirds, fracture of fibula. Case ].-(Fig. 258.) H. AI., age 40. First seen July 2, 1920. Operalion, July 5. Discharged Sept. 9. Inter- position of tibialis anticus. Reduction. Two Parham’s bands. Radiogram, July 16, showed perfect position. Anatomical and functional result perfect. Eight months after, Zimar cousolidutiou without appreciable callus. p First seen Jan. 30, 1920, for supramalleolar fracture with marked displacement of tibia1 fragment. Operation, Feb. 10, 1 Parham’s band produced excellent reduction. Left April 1. Radiogram at I1 months shows perfect anatomical result. Minimum of callus ; functional result excellent. , Case 2.-(Figs. 259, 260.) L. S. Case 3-G. T . Supracondylar fracture of femur involving knee-joint. Considerable back- ward displacement of lower fragment. p g Operalion, Nov. 14, 1920. V-shaped incision, joint washed out, reduction, and 2 bands Radiogram, 4 months later, showed circular callus enclosing bands, and not excessive. Case 4.--RI. M., age 32. Posterior incision, 1 Parham’s band, May 31, 1920. Radiogram, Dec. 4 : Excellent reduction. Moderate amount of fusiform callus surrounding band. Fracture lower third humerus. Spiral fracture of tibia and fibula in lower third, with moderate displacement. Operalion, Oct. 6, 1920. Reduction and 2 Parham’s bands. Rodiogram three months later, showed good position ; modcrate fusiform callus formation ; bands surrounded with small clear space at point of contact with callus. Case 5.-G. H., age 60. Case 6.-(Figs. 261, 262.) P. C. Admitted Oct. 30, 1920, fracture of middle third of humerus, and of both bones of both forearms. Extreme disptacement and comminuted fracture (3 pieces). developed on side of bone opposed to plates. at junction of middle and lower third. Bands embedded in callus, with small clear space around. Case &-(Figs. 265, 266.) G. S. Seen July 7, 1920. Transverse fracture of tibia and fibula OPERATZVE TREATMENT OF CLOSED FRACTURES 267 26 I.--Fracture of clavicle after operation ; perfect resilt. Case ‘i.-(Fig. 263.) D. .J., age 15, seen Sept. 1 , 1920, with fracture middle third of femur. Effusion into knee-joint aspirated several times. Radiogram showed transverse fracture with much dis- placement. Operation Sept. 10. Incision through vastus internus and reduction by Lambotte’s tractor. Two Sherman’s plates and 2 Parham’s bands, with 2 wire loops. Radiogruw).- Perfect reduc- tion. Four months later, moder- ate callus formation, especially FIG. 26 I.--Fracture of clavicle after operation ; perfect resilt. developed on side of bone opposed to plates. at junction of middle and lower third. Bands embedded in callus, with small clear space around. Case &-(Figs. 265, 266.) G. S. Seen July 7, 1920. Transverse fracture of tibia and fibula FIG. 26 I.--Fracture of clavicle after operation ; perfect resilt. THE BRITISH JOURNAL OF SURGERY Opemtion.-July 9. Reduction. Stcrilized bone-plate a Radio~ram.--.July 11. Excellent rcc‘uction and position. and a half months later some pain. Opemtion.-July 9. Reduction. Stcrilized bone-plate a Radio~ram.--.July 11. Excellent rcc‘uction and position. and a half months later some pain. Opemtion. July 9. Reduction. Stcrilized bone plate Radio~ram.--.July 11. Excellent rcc‘uction and position. and a half months later some pain. Nov. 2. Bone-plate broken, callus formation on side of bone opposed to bone plate, band hidden by callus, and usual clear spare round band. FIG. ?(i(i.-Sanie fracture as in Fig. 265, four motitlis after opern- tioii ; callus dereloped 01: ride opposit,e phte : clear spare vrrll alionti. a, Fracture of boiie plate. b, Clear since. PIG. 205.-Casc 8. Fracture of tibia aiid fibula forty d;iys after olmation ; bone plate broketi, cdlus Iniuimum. Re-operation, Nov. 3. The two pieces of bone plate lay free, red appearance, and not invaded by new bone. X o callus round plate, but entirely on other side of the hone. where it was excessive. Bands were rovered by rallus, bnt were rasily cut and withdrawn. Small clear space around band. V. A. Admitted to hospital Sept. 11, 1920, with a traiz.werse fracture of upper third of thigh, marked overlapping and displacement. Put on con- tinuous extension. Case 9.-( Figs. 268, 269.) V. A. Admitted to hospital Sept. 11, 1920, with a traiz.werse fracture of upper third of thigh, marked overlapping and displacement. Put on con- tinuous extension. Case 9.-( Figs. 268, 269.) Not satisfied with reduction. Operation, Ort. 4, 1920. OPERATZVE TREATMENT OF CLOSED FRACTURES 267 Bone-ends trimmed, 2 Sherman’s plates and 4 Parham’s bands placed. Radiogram Reduction and position satisfartory. Ward was closed on account of a serious srarlet-fever epidemir. Patient returned Dec. 7 with bow- ing of the thigh. Not satisfied with reduction. Operation, Ort. 4, 1920. Bone-ends trimmed, 2 Sherman’s plates and 4 Parham’s bands placed. Radiogram Reduction and position satisfartory. Ward was closed on account of a serious srarlet-fever epidemir. Patient returned Dec. 7 with bow- ing of the thigh. t,,,ird FIR. of 267.-Casc femur, 10. This Fracture i,lul;trates of lower bad technique, the bone plate havitig brokeu owing to its beiug too short. PIG. 205.-Casc 8. Fracture of tibia aiid fibula forty d;iys after olmation ; bone plate broketi, cdlus Iniuimum. FIG. ?(i(i.-Sanie fracture as in Fig. 265, four motitlis after opern- tioii ; callus dereloped 01: ride opposit,e phte : clear spare vrrll alionti. a, Fracture of boiie plate. b, Clear since. Rudiogrorn-The bone is bent ; the bands are intact, but one Sherman’s plate has slipped from beneath a band allowing this bending. Der. 11, continuous extension applied. Jan. 20, re-operation. Bands and plates rcmoved. Latter not covered by rallus, former well embedded. April 21, Radiogram. Sound consolidation ; callus moderate in amount ; result fairly good. Case 10.-(Fig. 267.) L. L., age 23. Fracture lower third of thigh. Upper fragment displaced externally, and lower markedly backwards. Case 10.-(Fig. 267.) L. L., age 23. Fracture lower third of thigh. Upper fragment displaced externally, and lower markedly backwards. Bone extremities trimmed and regularized. Bone plate fixed to external surface of femur by 4 Parham’s bands. Plaster. Operation, Sept. 20, 1920. Radiogram next day. To note: Technique was bad. (1) Bone-plate much too short. On the twenty-fifth day the plaster was removed for mas- sage, the patient made a sudden muscular effort with the leg, and the bone bent. Very good position and reduction. (2) Bands too near ‘centre of plate. Radiogram next day. To note: Technique was bad. (1) Bone-plate much too short. Very good position and reduction. (2) Bands too near ‘centre of plate. On the twenty-fifth day the plaster was removed for mas- sage, the patient made a sudden muscular effort with the leg, and the bone bent. p t,,,ird FIR. of 267.-Casc femur, 10. This Fracture i,lul;trates of lower bad technique, the bone plate havitig brokeu owing to its beiug too short. Fio. Sil.--ltiotber example of bad tecliiiique, shoainn callus de- veloped oti the siGde of the hoiie opposite the plate. Here again four harids slioiild have heen wed, and Slierrriaii’s plate iirat.ead of Lam- botte’a. 2, Clear space. h, Callus opposite the plate. OPERATZVE TREATMENT OF CLOSED FRACTURES 267 Radiogram.-The bone-plate has broken owing to faulty position of bands and powerful leverage overcoming weak resistance of too short a plate. technique, the bone plate havitig broke owing to its beiug too short. Continuous extension for thirty days produced good result. Radiogram.-The bone-plate has broken owing to faulty position of bands and powerful leverage overcoming weak resistance of too short a plate. Continuous extension for thirty Radiogram.-The bone-plate has broken owing to faulty position of bands and powerful leverage overcoming weak resistance of too short a plate. technique, the bone plate havitig brok owing to its beiug too short. Continuous extension for thirty days produced good result. OPERATIVE TREATMENT OF CLOSED FRACTURES OPERATIVE TREATMENT OF CLOSED FRACTURES 2 FIG. 270.- Case 11. Transverse fracture of loner third of femur slioaiiig anot,tier iiistarire of faulty technique. 13ratiori is bad owir!? to the emllloymelit of two hand only iiistesd of four. Pliernmn‘s plate, too. is hettrr tliaii Lain- Botte’r. n, 11, Clear spnces. FIG. 268.- Case 9. Traiis- Terse fracture of upper third of fernur in wliicli a Bliermaii‘s plate lias escaped from a hand, allon itig iiiflerioti of the femitr. 1%:. 26!).-Saiiie case iis iii Fig. 2 G S . 1%:. 26!).-Saiiie case iis iii Fig. 2 G S . FIG. 270.- Case 11. Transverse fracture of loner third of femur, slioaiiig anot,tier iiistarire of faulty technique. 13ratiori is bad owir!? to the emllloymelit of two hands only iiistesd of four. Pliernmn‘s plate, too. is hettrr tliaii Lain- Botte’r. n, 11, Clear spnces. FIG. 270.- Case 11. Transverse fracture of loner third of femur, slioaiiig anot,tier iiistarire of faulty technique. 13ratiori is bad owir!? to the emllloymelit of two hands only iiistesd of four. Pliernmn‘s plate, too. is hettrr tliaii Lain- Botte’r. n, 11, Clear spnces. 9. Traiis- per third of rmaii‘s plate nd, allon itig r. otber example of shoainn callus de- e siGde of the hoiie ate. Here again four have heen wed, and te iirat.ead of Lam- ear space. h, Callus ate. of e- e ur d m- us FIG. 268.- Case 9. Traiis- Terse fracture of upper third of fernur in wliicli a Bliermaii‘s plate lias escaped from a hand, allon itig iiiflerioti of the femitr. Fio. Sil.--ltiotber example of bad tecliiiique, shoainn callus de- veloped oti the siGde of the hoiie opposite the plate. CONCLUSIONS. 1. The simplicity and the ease of application of the Parham-Martin bands establishes their superiority for closed fractures to all other means of operative splinting. Their application is carried out with the minimum of operative manipulation, and perfect apposition is ensured and maintained. They are better than wirc for encircling the bone. 1. The simplicity and the ease of application of the Parham-Martin bands establishes their superiority for closed fractures to all other means of operative splinting. Their application is carried out with the minimum of operative manipulation, and perfect apposition is ensured and maintained. They are better than wirc for encircling the bone. 2. We have been able to observe the remote results of fractures thus treated in cases where we have been obliged to re-operate, and in a series of radiographs. They show that the objections made to metallic osteosynthesis, which are very real, cannot be applied to the use of Parham’s bands. The consolidation is certainly not delayed, there is no necrosis at the point of contact of the band, and it is surrounded by callus (a small clear space may remain). Furthermore, any organic iron salts that may be formed have no toxic effect on the tissues ; the callus is not excessive in quantity, and is frequently reduced to a minimum. Lastly, the bands very rarely give trouble from their presence. and may with confidence be left buried. y 3. Transverse fractures require to be treated with metal or bone plates (or splints) encircled by the bands. Our results with bone plates have been very disappointing, though we admit Nageotte’s principles in theory, and believe the work of Gallie and Robertson to be most valuable, though needing clinical confirmation in its application to recent fractures. 3. Transverse fractures require to be treated with metal or bone plates (or splints) encircled by the bands. Our results with bone plates have been very disappointing, though we admit Nageotte’s principles in theory, and believe the work of Gallie and Robertson to be most valuable, though needing clinical confirmation in its application to recent fractures. We are satisfied with our results with Sherman’s metal plates and bands, but our failures corrected our technique. Certain principles in technique must be adhered to if perfect results are to be secured. 4. SYNOPSIS OF THE ELEVEN CASES. (’uses 1 and 2.-Perfect anatomical result. Callus hardly appreciable, and linear union. Cnses 3, 4, and 5.-Very satisfactory anatomical result. Callus not exuberant, circular Bands embedded in callus, and a small clear area is visible around them. and fusiform. Case 6.-Moderate callus surrounding band ; well marked clear space. Case 7.-Two Sherman’s plates and four Parham’s bands-certain amount of callus Bands surrounded with clear space. on opposite side to plates. Cases 8 and 10.-Bone plates and Parham’s bands. Bone plate broken and callus Bands badly placed, and bone plate too short to developed on opposite side to plate. resist. Case 9.-Sherman’s plates and Parham’s bands. Bands loosened. Plate disengaged and slipped. Case 9.-Sherman’s plates and Parham’s bands. Bands loosened. Plate disengaged and slipped. Case 9.-Sherman’s plates and Parham’s bands. Bands loosened. Plate disengage and slipped. Case 11.-Lambotte’s plate and tw-o Parham’s bands. Failure of resistance because Faulty fixation by two bands only one plate was used (resistance in one axis only). when four should have been used. Case 11.-Lambotte’s plate and tw-o Parham’s bands. Failure of resistance because Faulty fixation by two bands only one plate was used (resistance in one axis only). when four should have been used. Case 11.-Lambotte’s plate and tw-o Parham’s bands. Failure of resistance because Faulty fixation by two bands only one plate was used (resistance in one axis only). when four should have been used. OPERATZVE TREATMENT OF CLOSED FRACTURES 267 Here again four harids slioiild have heen wed, and Slierrriaii’s plate iirat.ead of Lam- botte’a. 2, Clear space. h, Callus opposite the plate. applied; and resulted in consolidation in a good position by Ju FIG. 268.- Case 9. Traiis- Terse fracture of upper third of fernur in wliicli a Bliermaii‘s plate lias escaped from a hand, allon itig iiiflerioti of the femitr. FIG. 268.- Case 9. Traiis- Terse fracture of upper third of fernur in wliicli a Bliermaii‘s plate lias escaped from a hand, allon itig iiiflerioti of the femitr. Case 11.-(Fig. 2’10.) D. F., age 52, seen in consultation April 4, 1920, with transverse fracture of the femur in lower third, with much overlapping of fragments. Operratiou, April 10. External incision ; the bone extremities were trimmed and 1 Lambotte’s plate was fixed by 2 Parham’s bands. Case 11.-(Fig. 2’10.) D. F., age 52, seen in consultation April 4, 1920, with transverse fracture of the femur in lower third, with much overlapping of fragments. Fio. Sil.--ltiotber example of bad tecliiiique, shoainn callus de- veloped oti the siGde of the hoiie opposite the plate. Here again four harids slioiild have heen wed, and Slierrriaii’s plate iirat.ead of Lam- botte’a. 2, Clear space. h, Callus opposite the plate. pp g g Operratiou, April 10. External incision ; the bone extremities were trimmed and 1 Lambotte’s plate was fixed by 2 Parham’s bands. Radiogrmn.-Redriction and position good. April 22, slight bending noted. g Observe also the bad terh- nique. Two fragments are dis- placed, the plate only offering resistance in a single axis : two plates should have been em- ployed. Also four bands should have been used. Continuous extension was applied; and. resulted in consolidation in a good position by July 1. THE BRITISH JOURNAL OF SURGERY 270 ’JONAS, A. F., ‘‘ Indications for, and Results of, the use of Metal Bone Plates and Bone Grafts, and Conservative Methods in t.he Treatment of Fractures ”, Nebraska Mrd. Jour., Norfolk, 1918, iii, 329. * F R ‘‘ O h M d M h d h T f F ” C d d A CONCLUSIONS. The improved instrument devised by Gatellier which we have described is a great impovenlent on all others, and is as near mechanical perfection as possible. * Ex’Gr.AND, F. R., ‘‘ On the Modern Methods of the Treat.ment of Fractures ”, Canad. Med. Assoe. Jozcr., Toronto, 1917, vii, 1072. MARTIN, Fracture Clinic, Philadelphia, 1917, 72. p , ’ O ASA F ‘‘ di i f d l LUND, F. B., “ The Parham and Martin Band in Oblique Fract.ures ”, Trans. Amer. Sury. z4ssoe., MARTINF Cli i Phil d l hi 1917 72 , Philadelphia, 1916, xsxiv, 422. BIBLIOGRAPHY. PARHAM, F. W., ‘‘ A New Device for the Treatment of Fractures,” h’ew Orleans Med. and Swq. * PARHAMd MARTIN ‘D i f h T f F ” M d S L i 1916 i 75 Jour., 1913-14, xxi, 461, 4G6. * PARHAM and MARTIN, .‘ Device for the Treatment, of Fmctures,” Mod. Hosp., St. Louis, 1916, vi, 75. PUTTI, ’‘ Tin Novo Metodo di Ostrosintesi ”, Clin. chir. di Milano, 1014, ssii, June 30, 1021. , , , , , , LUND, F. H., ‘‘ The Parham and Martin Band in Oblique Fracture ”, Surg. G?jnecol. and Obst, 1916, LUND F B Th P h d M i B d i Obli F ” T A S xxiii, 545. LUND, F. B., “ The Parham and Martin Band in Oblique Fract.ures ”, Trans. Amer. Sury. z4ssoe., MARTINF Cli i Phil d l hi 1917 72 Philadelphia, 1916, xsxiv, 422. OPERATIVE TREATMENT OF CLOSED FRACTURES 27 DODGE, R., ‘‘ Advantages and Disadvant,a:es of the use of nfctal and Bone Plates for Fractures ” Winconsin Med Jour Milwaukee 1918 xvii 10 g Winconsin Med. ,Jour., Milwaukee, 1918, xvii, 10. DUVAL, PIERRE, Pi6cas d‘Os traiteis par 1‘Akool pour les Interventions osReuaes. ’‘ ” , , , , , , , p p l1 ATSEE, ’‘ The Fundamental Principles involved in the use of Bone-grafts in Surgery ”, -4nn. of Surg. 1913, cxlix, 313. BARTLZTT and HEWITT, “ Experimental and Clinical Works to Determine the Value of Lane’s Bon , , Platinq,” Joiw. AVMT. Med. Assoc., 1912, ix, 51. Is BAILLEUL, “ Quinze Cas d’OstBosynt,h&se mi.tallique par Fil de Bronze, Plaque de Lambotte, et Cerclap DE BOUCHER‘‘ O t th h i iti d l F t P j til d G q, J , , , de Parham ’ I , Reunion -%fkd~CO-ChirUrgiCal de la 20me Rdgion, 1917, Nov. 10. ‘4 DE BOUCHER, ‘‘ Osteosynthche primitive precoce dans les Fractures par Projectil COTTONF d DUFF J ‘‘ Wi B di f F t ith th D i ti f N de Parham I , Reunion %fkd CO ChirUrgiCal de la 20me Rdgion, 1917, Nov. 10. principalement par la M6thode de Parham ”, ThLe de Pa&, 1918. ‘4 DE BOUCHER, ‘‘ Osteosynthche primitive precoce dans les Fractures par Projectiles de Guerre, ‘‘ ’* f g g , , principalement par la M6thode de Parham ”, ThLe de Pa&, 1918. l 5 COTTON, F. and DUFF, J., ‘‘ Wire Banding for Fractures, with the Description of a New Instrument ’, ‘‘ ” p p p , , Surg. Gynecol. and Obst., 1917, xxv, 557. 16 FR~DET, ‘‘ Sur le Traitement des Fractures de Jamhe par OstBosynthAse ”, Bull. Soc. de Park, 1918 ‘‘ g y , , , Nov. 13, 1703. I 7 TANTON, ‘‘ P n nouveau Procede d’OstAosprth8se par Cerclaee ”, Mid. de Paris, 191R, 317. ’‘ ” , I 7 TANTON, ‘‘ P n nouveau Procede d’OstAosprth8se par Cerclaee ”, Mid. de Paris, DIGEON’‘ T i d F d 0 l l MB h d d P h p p , DIGEON, ’‘ Traitement des Fractures des 0 s longs par la MBthode de Parham et Martin ”, Thha de ” PrCriQ, 1920. l9 HALLOPEAU, “ Des Lames m6talliques dans le Traitement des Fractures ”, Bnll. SOC. de Ghk. de Puris Q, 1920, :Tune 13. ROLLAND. Contribution k la Pratiqu : de la Chirurgie osseuso , ThLe de PUT , 19 GALLIE and ROBERTSOX, ‘‘ The Repair of Bone ”, Brit. Jowr. S U T ~ . , 1919, vii, 211. OPERATIVE TREATMENT OF CLOSED FRACTURES 27 ” 20 DUJARIER, ‘‘ , Des Lames m6talliques dans le Traiteinent des Fractureq ”, Zbid., .June 29, 990. 21 LERICHE and POLICARD, ‘‘ Recherches biologiques s u p I’Ost&osynth&e et la Plaque de Lambotte ” Ibid,, Julv 2, 1145. q q 2 MILLET, “ Co&ibut.ions histo.pat.hologiqnes Q !’itude den Protheses osseuses ”, Thk.se de Lyon, 1919. ROLLAND C ib i k l P i ’ d l Chi i ” ThL d PUT^ 1920 ,, , *2 MILLET, “ Co&ibut.ions histo.pat.hologiqnes Q !’itude den Protheses osseuses ”, Thk.se de Ly p 3 ROLLAND. “ Contribution k la Pratiqu’: de la Chirurgie osseuso ”, ThLe de PUT^, 1920. ” ,, , q g , , 24 GALLIE and ROBERTSOX, ‘‘ The Repair of Bone ”, Brit. Jowr. S U T ~ . , 1919, vii, 211.
W2964253343.txt	https://www.mdpi.com/2410-3896/3/4/40/pdf?version=1553256598	en		Seeking to Develop Global SYK-Ness	Condensed matter	2,018	cc-by	5,264	Article Seeking to Develop Global SYK-Ness Dmitri V. Khveshchenko Department of Physics and Astronomy, University of North Carolina, Chapel Hill, NC 27599, USA; khvesh@physics.unc.edu, Tel.: +1-919-962-7213 Received: 4 October 2018; Accepted: 13 November 2018; Published: 15 November 2018 Abstract: Inspired by the recent interest in the Sachdev–Ye–Kitaev (SYK) model, we study a class of multi-flavored one- and two-band fermion systems with no bare dispersion. In contrast to the previous work on the SYK model that would routinely assume spatial locality, thus unequivocally arriving at the so-called ‘locally-critical’ scenario, we seek to attain a spatially-dispersing ‘globally-SYK’ behavior. To that end, a variety of the Lorentz-(non)invariant space-and/or-time dependent algebraically decaying interaction functions is considered and some of the thermodynamic and transport properties of such systems are discussed. Keywords: strongly correlated fermions; SYK model; holography 1. Introduction The recent rise of the asymptotically solvable 0 + 1-dimensional SYK model [1–4] possessing a genuine (albeit, still debated over its fine details) holographic dual has also rekindled the long-standing interest in analytically solvable examples of non-Fermi liquid (NFL) behavior. To that end, References [5–23] have extensively explored the idea of combining multiple quantum dot-like copies of the SYK model and/or hybridizing them with some itinerant fermions. However, all those works operated under the common assumption of a spatially local nature of the SYK propagator which limitation resulted in a number of the NFL scenarios exhibiting ‘(ultra-)local’ criticality characterized by the lack of any spatial dispersion. In view of its formal affinity to the well established dynamical mean-field theory and some resemblance to the observed properties of such families of strongly correlated compounds as the cuprates, pnictides, and heavy fermion materials the (ultra-)local regime was claimed to be physically relevant and possibly providing a new evidence in support of the intriguing, yet still largely speculative, ‘bottom-up’ holographic phenomenology [24–26]. However, it would seem that a firm justification of the general holographic approach as a viable phenomenological scheme should require one to venture off the beaten path by attempting to extend it to the multitude of more general, including spatially dispersing, NFLs. A host of such states were generated in the previous holographic studies involving the so-called Lifshitz and hyperscaling-violating background geometries and numerous opportunistic proposals for utilizing them in the studies of materials were put forward (although the seemingly endless flurry of those look-alike exercises in classical Einstein–Maxwell-dilaton relativity has been finally withering out, as of lately). In that regard, a further investigation into the various generalizations of the SYK model and its non-random cousins [27–30] could pave the way for gaining insight into such largely uncharted territory as the various ‘super-strongly coupled’ systems with a nearly—or even completely—flat dispersion. Their dynamics is then governed solely by the (in general) space- and/or time-dependent q-fermion couplings which can, among other things, endow the bare flat-band fermions with a non-trivial dispersion e( p). Condens. Matter 2018, 3, 40; doi:10.3390/condmat3040040 www.mdpi.com/journal/condensedmatter Condens. Matter 2018, 3, 40 2 of 9 The pertinent interaction functions—-which play the role of disorder correlations in the (generalized) SYK models [2–23]—could then be distinguished on the basis of such important properties as their short- vs long-ranged nature, combined vs. independent dependence on space-time separation, etc. Depending on such important details, the systems in question might demonstrate an entire variety of novel (non)critical regimes. As the SYK-related examples show, the key requirement that enables asymptotically exact solutions of such systems—as well as their non-random counterparts [27–30]—is a large number N of the different species (see, however, Equation (6) below for a further clarification). 2. Results 2.1. Model In what follows, we consider the d + 1-dimensional non-random action S= Z L N iq/2 ∑ ∑ χiα † ∂τ χiα − N q−1 τ i α Z L N ∑ ∑ τk i α ,β m n l α ...α β ...β q 1 Fi1 1...iq ,jq1 ...j q [q/2] ∏ k =1 χiαk k † [q/2] q ∏ l =[q/2]+1 χiαl l ∏ m =1 β † χ jmm q ∏ n=[q/2]+1 β χ jnn , (1) where the Greek indexes stand for the N-valued colors, while the Latin ones run over the L sites of a d-dimensional cubic lattice. In addition, for the sake of simplicity and in order to focus on the most interesting regime, we choose the chemical potential of the complex fermions χ to be µ = 0, thus enforcing particle-hole symmetry. To introduce spatial (and/or additional temporal) dispersion into the problem, while keeping it tractable, we consider a broad family of interaction functions α1 ...αq β 1 ...β q (τ ) 1 ...iq j1 ,...jq Fi q = Fxij (τ ) ∏ δαa β a δia i δja j , (2) a which depend algebraically on the separation in space and/or time between the common locations of simultaneously (dis)appearing q-fermion complexes, which is Fx (τ ) = F . \|τ \|2α \|x\|2β (3) Since in a lattice model the distance xij takes discrete values, Equation (3) needs to be carefully defined at its shortest values. In the generalization of the original SYK model proposed in Reference [31], action (1) results from taking the Gaussian average over the (completely antisymmetric under the simultaneous permutations α a ↔ αb and i a ↔ ib ) random amplitudes that entangle the groups of q fermions. By choosing Fxij (τ ) ∼ δij , one obtains L decoupled copies of the original N-colored SYK model, while by allowing for non-zero nearest-neighbor terms Fxij (τ ) ∼ δi,j+e one can describe the various SYK lattice (chain) models of References [5–23]. In contrast, an instantaneous and/or contact interaction corresponds to choosing α = 1/2 and/or β = d/2, which power count is similar to that of a δ-function in the time- and/or space-domain. It should be pointed out, though, that a uniform and spacetime-independent correlation function Fxij (τ ) = const (α = β = 0) does not reproduce the original (single-site) SYK model of the total of NL fermions where the entangling correlations would be equally strong among any 2q orbitals chosen arbitrarily from any of the L sites and N colors alike. Notably, in the limit of β → 0, the theory (1) becomes symmetric under the permutations among the L sites, so the very notion of a spatial distance, alongside an underlying lattice structure, becomes ill-defined. Nevertheless, as long as the correlation amplitude (3) remains distance-dependent, the fermion correlations acquire an emergent non-trivial dispersion, as demonstrated below. Condens. Matter 2018, 3, 40 3 of 9 2.2. Schwinger–Dyson Equations and Their Solutions Analogously with the previous analyses of the SYK-type models [2–31], the partition function of the theory (1) can be written as the path integral over a pair of bosonic fields G and Σ whose expectation values yield the fermion propagator and self-energy, respectively: Z= Z DG (τ, x) DΣ(τ, x)Det(δ(x)∂τ + Σ(τ, x)), exp( N 2 Z 1 ( Fx (τ ) G q (τ, x) − G (τ, x)Σ(τ, x))), (4) τ,x q where the determinant results from integrating out the fermions and, with a focus on the IR regime, the discrete sum over the lattice sites can be replaced with the integral over the spatial coordinate x. The saddle points in theory (4) corresponds to the various solutions of the Schwinger–Dyson (SD) equation Z ∂τ1 G (τ12 , x12 ) + τ3 ,x3 Σ(τ13 , x13 ) G (τ32 , x32 ) = δ(x12 )δ(τ12 ), (5) where the self-energy is given, to leading order in 1/N, by the sum of the so-called ‘watermelon’ diagrams while ignoring any vertex corrections Σ(τ, x) = Fx (τ ) G q−1 (τ, x). (6) It must be noted, though, that a solid justification of the approximation behind Equation (6) may require some adjustments to the action (1), thus effectively making it conform to one of the non-random ‘(non)colored tensor’ models of References [27–30]. In practice, it can be achieved by decorating each of the L sites of the underlying lattice with a properly designed unit cell composed of N sites which are occupied by the q fermions at split locations (see Reference [23] for an explicit example of such construction). However, this technical complication appears to have no bearing on the robust algebraically decaying amplitudes that we are going to study. The Fourier transform of Equations (5) and (6) then reads q −1 Z G −1 (e, p) = ie + ∏ i =1 ωi ,ki G (ωi , ki ) Fp+∑i ki (e + ∑ ωi ). (7) i A relative importance of the interaction-induced self-energy can be ascertained by the standard arguments. Under a scaling of the temporal, ω → sω, and spatial, k → s1/z k, dimensions, where z is the dynamical critical exponent, one finds that the self-energy dominates over the kinetic term or, at least, remains marginally relevant in both the UV and IR regimes, provided that the condition d(q − 2) + 2β + 2α − 2 ≤ 0 z (8) is met [31]. We leave a systematic investigation into all the viable solutions of Equation (7) to future work. Such solutions should ultimately be selected on the basis of their (minimal) energies—for a reliable evaluation of which a proper ansatz first needs to be chosen. Such choice is likely to depend on the details of the action (1) and, therefore, may not be universally applicable. As was first argued in the case of the random SYK-type models of Reference [31], the customary ultra-local solution G (τ, x) = 0 for x 6= 0 (hence, G (e, p) = G (e)) would generally be favored by the Hartree-type terms in the overall fermion energy, whereas the Fock-type ones tend to support non-local ones. Moreover, while being finite when evaluated on the ultra-local solution in the case of short-ranged couplings, the Hartree terms develop IR divergences, once the fermion interactions become sufficiently long-ranged. For instance, the lattice sum ∑x Fx (τ ) appearing in the Hartree terms with Fx (τ ) given by Equation (3) diverges for all β ≤ d/2 (in contrast, a spurious UV divergence for β > d/2 is absent as Condens. Matter 2018, 3, 40 4 of 9 long as the separately defined amplitude F0 (τ ) remains finite). This observation alone suggests that, at least for β ≤ d/2, the ultra-local solution becomes unstable, as compared to a non-local one. Therefore, in the vicinity of an emergent fermion dispersion (‘on-shell’), we seek the solution of Equation (7) in the general form A(p) G (e, p) = , (9) (ie − Bpz )η for which ansatz includes all the important ingredients: effective dispersion relation characterized by the critical exponent z and prefactor B, anomalous exponent η controlling the ‘on-shell’ singularity, and the ‘wave-function renormalization’ A(p). Away from the ‘on-shell’ regime (be it at an extended Fermi surface or near an isolated nodal Dirac point), the ansatz (9) is no longer applicable, so its use can only be justified if the integrals in Equation (7) are dominated by the ’on-shell’ contributions—which indeed appear to be the case. Analyzing Equation (7) in the ’on-shell’ regime \|e − Bpz \| ω, Bpz and equating the powers of e and p, alongside dimensionfull prefactors, on both sides, one finds the anomalous dimension η η = 1− 1 − 2α , q (10) together with the dynamical critical exponent z= d(q − 2) + 2β 2 − 2α as well as the residue (11) (2α−1) A(p) ∝ ( Fpd(q−2)+2β ) q(2−2α) (12) 1 and the coefficient B ∼ F 2−2α in the emergent fermion dispersion. A non-vanishing value of the latter implies that the propagator G (e, p) acquires a non-trivial momentum dependence, thus signaling that its real-space Fourier transform is no longer ultra-local. Alternatively, this can be viewed as a spontaneous breaking of the local Z2 symmetry χiα → −χiα of the action given by Equations (1) and (2) which, if preserved, would have prohibited any spatial non-locality, thus enforcing the condition of ultra-locality, < χi† χ j >= 0 for i 6= j. When contemplating the general possibility of such symmetry breaking, it is worth noting that it is, in fact, specific to the two-point interaction function given by Equation (2), whereas for a generic α1 ...αq β 1 ...β q 1 ...iq j1 ,...jq Fi Equation (1) would not possess this symmetry in the first place. As regards the appicability of the above solution, the criterion (8) appears to be satisied as equality, thus signifying a marginally relevant nature of the 2q-fermion interaction given by Equations (2) and (3). Among other things, this makes it difficult to find a numerical prefactor in Equation (12), as both, the self-energy and bare kinetic, terms in Equation (7) turn out to be important. In what follows, we restrict our analysis to the parameter values 0 ≤ α ≤ 1/2 for which η < 1 and the propagator (9) readily conforms to the anticipated ’no quasiparticle’ regime. Moreover, the above restriction also guarantees that the physical condition z > 0 will be fulfilled for any d ≥ 0 and q > 1 as long as β ≥ 0. In particular, for q = 2 and in the limit α = β → 0, the theory (1) is equivalent to the disorder-averaged action for a non-interacting single-particle state of a fixed energy. Then, Equation (7) becomes merely algebraic and features an exact momentum-independent solution G (e, p) = 2 ie + p (ie)2 − F , (13) which, upon being expanded in the on-shell regime (\|ie − F1/2 p0 \| \|ie\|), manifests the exponents η = 1/2 and z = 0, as well as the coefficients A ∼ F −1/4 p0 and B ∼ F1/2 , in full agreement with Condens. Matter 2018, 3, 40 5 of 9 Equations (10)–(12). Notably, though, in the special case of q = 1, action (1) becomes Gaussian and the exact fermion propagator can be immediately obtained as the inverse of the quadratic kernel G (e, p) = (ie)1−2α . (ie)2−2α + Fp2β−d (14) This expression exhibits the dynamical index z = (2β − d)/(2 − 2α) which is again consistent with (11), although, under the previously imposed restriction 0 ≤ α ≤ 1/2, the condition z > 0 now requires a convergence of the spatial Fourier transform of Equation (3), i.e., β ≥ d/2. Thermodynamics of the system described by Equation (1) can be studied with the use of the Luttinger–Ward functional which yields the free energy F = N Z τ,x 1 (ln G −1 + GΣ − FG q ) ∼ T 1+d/z . q (15) For d = 0 and/or z = ∞, it manifests such a salient feature of the SYK physics as finite zero-temperature ∂F ∼ T d/z , whereas for d > 0 and z < ∞ the result complies with the 3rd law entropy, S( T ) = − ∂T of thermodynamics. Transport coefficients such as longitudinal optical conductivity can also be evaluated in the ‘no vertex correction’ approximation (cf. References [32–34]) σ(ω ) = z2 dω Z e,p e(e + ω ) G (e + ω, p) G (e, p) ∼ ω 2−2η +(d−2)/z . p2 (16) For α = β = 0 and z → ∞, the exponent in Equation (16) becomes 4/q which contains the extra factor of ω 2 as compared to the result of Reference [34] where the fermions were endowed (despite their purportedly localized nature) with a bare dispersion characterized by a finite velocity v F . However, Equation (16) does pass one important check: for d = 2, T = 0, and in the presence of well-defined quasiparticles (η = 1) the conductivity σ (ω ) becomes a dimensionless constant as in this case there are no relevant energy scale that could be combined with the frequency into one dimensionless ratio. In turn, the vanishing in the D.C. limit (‘soft gap’) behavior of Equation (16) for d ≥ 2 and η < 1 would be consistent with the highly correlated nature of the system in question. Another potentially interesting class of models is represented by the manifestly Lorentz-invariant interaction functions F Fx (τ ) = 2 . (17) \| τ − x2 \| γ In this case, the fermion propagator inherits the same symmetry through Equation (7), thus forcing the dynamical exponent z = 1 upon the solution G (e, p) = ((ie)2 A , − p2 )η/2 (18) which is manifestly spatially non-local. This time around, equating the powers of e and p on both sides of the Equation (10) yields d+1−γ η = d+1−2 , (19) q while the strong-coupling regime can now be attained under the condition d(q − 2) + 2γ − 2 ≤ 0, (20) which turns out to be rather restrictive. In particular, a contact instantaneous coupling of the Thirring or Gross–Neveu variety which scales similarly to the power-law with γ = (d + 1)/2 limits the applicability of Equation (19) to d = 1, q = 2 where it appears to be, at most, marginal Condens. Matter 2018, 3, 40 6 of 9 (cf. References [35–37]). Nonetheless, for q = 2 and γ ≤ 1, the ’no quasiparticle’ condition can hold for all d. In addition, for the maximally (space-time) long-ranged 3-particle couplings (q = 3) with γ = 0, the upper critical dimension that fulfills the criterion (20) can be as high as d = 2. 2.3. Hybrid Models In addition of interest are the two-band models where the SYK fermions (χ) are coupled to some ‘itinerant’ (ψ) ones, the latter possessing a dispersion ξ k ≈ v F (k − k F ) S= Z L N ∑ ∑[δij χiα † ∂τ χαj + ψiα † (δij ∂τ − ξ ij )ψαj ] τ i,j α − − iq/2 N ( q −1) i p/2 N ( p −1) Z N L Z ∑ ∑ τk i α ,β m n l L N ∑ ∑ τk i α ,β m n l α1 ...αq β 1 ...β q 1 ...iq ,j1 ...jq Fi [q/2] k =1 [ p/2] α ...α p β 1 ...β p 1 1 ...j p ∏ Hi 1...i p ,j k =1 ∏ l m =1 [ p/2] p α † ∏ α χi l l =[ p/2]+1 l ∏ m =1 q β † ∏ χ jmm α χi l l =[q/2]+1 χi k k [q/2] q α † ∏ χikk β n=[q/2]+1 χ jnn (21) p β † ψjmm ∏ ∏ n=[ p/2]+1 β ψjnn , where the functions F and H describe, respectively, a q-particle self-interaction of the SYK fermions (chi) and a p-particle coupling between the SYK and itinerant (ψ) ones. The coupled SD equations now read q −1 Σχ (τ, x) = Fx (τ ) Gχ p/2−1 (τ, x) + Hx (τ ) Gχ and p/2−1 Σψ (τ, x) = Hx (τ ) Gψ p/2 (τ, x) Gψ (τ, x) p/2 (τ, x) Gχ (τ, x). (22) (23) In what follows, for simplicity, we choose the function Hx (τ ) to be given by the same Equation (3), albeit with an independent prefactor. For p = 2, the corresponding self-energies generated by the H-coupling can be evaluated as H Σχ,ψ (ω, k) = Z Gψ,χ (ω + e, p + k) Hp (e). (24) e,p A self-consistent solution of the coupled Equation (24) shows that the contribution ΣχH ∼ ω η towards the total self-energy of the χ-fermions is of the same functional form (9) as that of (ΣχF ) due to the self-interaction between them. Concomitantly, the spectrum of the ψ-fermions gets strongly affected by their coupling to the χ-ones ΣψH (e) ∼ e2α−η +2β/z . (25) In the case of Fk (ω ), Hk (ω ) ∝ δ(ω )const(k) (α = 0, β = d/2) and by putting z = ∞ as in the SYK model, one can readily reproduce the results of Reference [32]: ΣχH ∼ ω 1−2/q and ΣψH ∼ ω 2/q−1 which manifest a markedly incoherent behavior of the ψ-fermions. In the other practically important case of p = 4, one obtains H Σχ,ψ (ω, k) = Z Ω,e,q,p Gχ,ψ (Ω + e + ω, p + k + q)Πψ,χ (Ω, q) Hp (e), (26) where the polarization operators Πψ,χ (ω, k) = Z Gψ,χ (e + ω, p + k) Gψ,χ (e, p) e,p have to be evaluated self-consistently with the use of the exact propagators. (27) Condens. Matter 2018, 3, 40 7 of 9 Performing this procedure under the assumption that the correction (24) does not alter the functional form of the overall self-energy ΣχH + ΣχF which is still given by Equation (9), one obtains ΣψH (e) ∼ e1−η +α+(d+ β)/z . (28) Conversely, with the use of (28), one can confirm that the dynamics of the χ-fermions is governed by both their self-interaction and coupling to the ψ-ones, thus justifying the above assumption. In the case of q = 4, Fk (ω ), Hk (ω ) ∝ δ(ω )const(k) (α = 0, β = d/2) and z = ∞, the above predictions can be contrasted against the results of Reference [29]: Σχ ∼ ω and Σψ ∼ ω ln ω. In turn, the results of Reference [34], Σχ ∼ ω 1+2/q and Σψ ∼ ω 4/q ln ω, pertain to the case of q ≥ 4, Fk (ω ), Hk (ω ) ∝ δ(ω )δ(k) (α = β = 0), and z = ∞. It should be noted, though, that the√above estimates originate from using the free-fermion polarization operator Πψ (ω, k) ∝ 1 + ω/ k2 + ω 2 instead of the proper one at all (rather than only small) momenta in Equation (26). Such approximation is prone to lacking self-consistency which would be pertinent to the asymptotic strong-coupling regime. In fact, had one chosen to use the bare polarization function Πψ in (24), while maintaining z = ∞, it would have resulted in the expressions ΣχH ∼ ω 1−η and ΣψH ∼ ω 2−2η which get augmented by the factors of ln ω if the corresponding integrals are logarithmic, just as in the aforementioned cases. Thermodynamics of the two-band model is described by the Luttinger–Ward functional that includes Equation (15) written in terms of Gχ , alongside the additional term ∆F = N Z ω,k (ln Gψ−1 + Gψ Σψ − 1 p/2 p/2 HGψ Gχ ). p (29) Computing (29) with the use of the exact propagators that utilize Equations (9) and (28), one arrives at the same behavior (15), which, once again, signifies the lack of a competing energy scale in the IR strong-coupling regime T F1/2 . As to the conductivity of the ψ-fermions, one now obtains σψ (ω ) = 1 ωd Z e,p v2p Gψ (e + ω, p) Gψ (e, p) ∼ ω 2η −2−2α−(d+2β)/z , (30) which diverges at ω → 0 for η < 1, consistent with the general expectation of a system with a non-flat dispersion but no source of momentum relaxation. In addition, should the customary substitution ω → T prove to be justifiable (which might indeed be true in the ‘no momentum drag’ regime), the resistivity would then show a rising (‘metallic’) behavior with increasing temperature. In particular, the above results imply that, for α = β = 0 and z = ∞, the exponent in Equation (30) equals −4/q, thus suggesting an ‘accidentally linear’ temperature dependence of resistivity for q = 4 (cf. References [34]). However, the general possibility of a host of other values of this exponent should be viewed as a caution against invoking the generalized SYK models to explain the ostensibly ‘universal’ linear resistivity observed in a variety of strongly correlated systems [32,33]. Once a (non-universal) mechanism of momentum relaxation is specified and added to the Hamiltonian, then frequency- and temperature-dependent conductivity as well as other transport coefficients can be evaluated and tested for a compliance (or a lack thereof) with such hallmarks of the Fermi liquid regime as the Wiedemann–Franz, Mott, and other standard relations. Such a systematic analysis would help one to ascertain a potential viability of the ‘globally SYK’ models for describing the phenomenology of certain strongly correlated materials. We leave these tasks to future work. In the two-band models, the potentially competing long-ranged q- and p- body interactions might drive various transitions from the parent—diffusive and highly chaotic (‘incoherent metallic’)—state to either a (‘heavy’) Fermi liquid, a (many-body) localized insulator, or some ordered states, all with a varying temperature and/or rates of decay and strengths of the couplings. Therefore, it would be interesting to carry out a systematic analyses of the potential instabilities of action (21). Condens. Matter 2018, 3, 40 8 of 9 In addition, as far as the current global quest into holography is concerned, it would be an interesting challenge to come up with a plausible conjecture for a holographic dual of such models (see Reference [38] for a preliminary attempt in that direction). For one thing, the viable background geometry is expected to be rid of the ubiquitous near-extremal black hole with its universal AdS2 × Rd metric as in its presence the boundary state would likely remain maximally chaotic. Indeed, the multi-dimensional and long-ranged generalization of the random SYK model studied in Reference [31] was found to be less chaotic than the original one and also sub-diffusive, thus diminishing the chances of its having an AdS2 (or, for that matter, any) type of a holographic dual. 3. Conclusions To summarize, in the present communication, an exploratory attempt was made to investigate the properties of ‘super-strongly coupled’ fermion systems with no bare dispersion and long-ranged interactions. Certain Lorentz (non-)invariant solutions of the corresponding SD equations were proposed and some basic properties of such systems discussed, including the continuously varying exponents in the power-law behavior of spectral function, entropy, and optical conductivity. This study continues the investigation of Reference [31] into the question of whether or not the ’holography-friendly’ properties of the original SYK model can survive such an important generalization as a non-trivial time/space dependence. If that were the case, one could argue that the SYK model describes generic properties of a whole universality class of systems, thereby providing much needed support for a possible applicability of the tantalizing ’bottom-up’ holographic approach to a broad variety of quantum systems. Instead, the pristine SYK behavior appears to be fragile and hardly extendable beyond the original SYK model and, possibly, its minimal (irrelevant-operator-driven) modifications, thus leaving the status of the overreaching holographic conjecture as undetermined as it was before the rise of the SYK model. Funding: This research received no external funding. Conflicts of Interest: The author declares no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Sachdev, S.; Ye, J. Gapless spin-fluid ground state in a random quantum Heisenberg magnet. Phys. Rev. Lett. 1993, 70, 3339. [CrossRef] [PubMed] Sachdev, S. Holographic metals and the fractionalized Fermi liquid. Phys. Rev. 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https://openalex.org/W3200742618	https://www.frontiersin.org/articles/10.3389/fneur.2021.728111/pdf	English	null	Dual-Antiplatelet Therapy May Not Be Associated With an Increased Risk of In-hospital Bleeding in Patients With Moderate or Severe Ischemic Stroke	Frontiers in neurology	2,021	cc-by	5,789	Dual-Antiplatelet Therapy May Not Be Associated With an Increased Risk of In-hospital Bleeding in Patients With Moderate or Severe Ischemic Stroke Ossama Khazaal 1, Aaron Rothstein 1, Muhammad R. Husain 2, Matthew Broderick 1, Daniel Cristancho 1, Sahily Reyes-Esteves 1, Farhan Khan 1, Christopher G. Favilla 1, Steven R. Messé 1 and Michael T. Mullen 1 1 Department of Neurology, University of Pennsylvania, Philadelphia, PA, United States, 2 Department of Neurology, Camden Clark Medical Center/WVU Medicine, Parkersburg, WV, United States Keywords: dual antiplatelet therapy, moderate stroke, severe stroke, secondary prevention, bleeding risk, bleeding rate, hemorrhagic transformation ORIGINAL RESEARCH published: 20 September 2021 doi: 10.3389/fneur.2021.728111 Background and Purpose: Dual antiplatelet therapy (DAPT), compared to single antiplatelet therapy (SAPT), lowers the risk of stroke or death early after TIA and minor ischemic stroke. Prior trials excluded moderate to severe strokes, due to a potential increased risk of bleeding. We aimed to compare in-hospital bleeding rates in SAPT and DAPT patients with moderate or severe stroke (deﬁned by NIHSS ≥4). Edited by: Peter Sporns, University Hospital of Basel, Switzerland Edited by: Peter Sporns, University Hospital of Basel, Switzerland Reviewed by: Anita Ante Arsovska, Saints Cyril and Methodius University of Skopje, North Macedonia Anna Christina Alegiani, University Medical Center Hamburg-Eppendorf, Germany Methods: We performed a retrospective cohort study of ischemic stroke over a 2-year period with admission NIHSS ≥4. The primary outcome was symptomatic intracranial hemorrhage (ICH) with any change in NIHSS. Secondary outcomes included systemic bleeding and major bleeding, a composite of serious systemic bleeding and symptomatic ICH. We performed analyses stratiﬁed by stroke severity (NIHSS 4–7 vs. 8+) and by preceding use of tPA and/or thrombectomy. Univariate followed by multivariate logistic regression evaluated whether DAPT was independently associated with bleeding. Correspondence: Ossama Khazaal Ossama.khazaal@ pennmedicine.upenn.edu Specialty section: This article was submitted to Stroke, a section of the journal Frontiers in Neurology Received: 20 June 2021 Accepted: 20 August 2021 Published: 20 September 2021 Specialty section: This article was submitted to Stroke, a section of the journal Frontiers in Neurology Results: Of 377 patients who met our inclusion criteria, 148 received DAPT (39%). Symptomatic ICH was less common with DAPT compared to SAPT (0.7 vs. 6.4%, p < 0.01), as was the composite of major bleeding (2.1 vs. 7.6%, p = 0.03). Symptomatic ICH was numerically less frequent in the DAPT group, but not statistically signiﬁcant, when stratiﬁed by stroke severity (NIHSS 4–7: 0 vs. 5.9%, p = 0.06; NIHSS 8+: 1.5 vs. 6.6%, p = 0.18) and by treatment with tPA and/or thrombectomy (Yes: 2.6 vs. 9.1%, p = 0.30; No: 0 vs. 2.9%, p = 0.25). DAPT was not associated with major bleeding in either the univariate or the multivariate regression. Received: 20 June 2021 Accepted: 20 August 2021 Published: 20 September 2021 Citation: Khazaal O, Rothstein A, Husain MR, Broderick M, Cristancho D, Reyes-Esteves S, Khan F, Favilla CG, Messé SR and Mullen MT (2021) Dual-Antiplatelet Therapy May Not Be Associated With an Increased Risk of In-hospital Bleeding in Patients With Moderate or Severe Ischemic Stroke. Front. Neurol. 12:728111. doi: 10.3389/fneur.2021.728111 Conclusions: In this single center cohort, symptomatic ICH and the composite of serious systemic bleeding and symptomatic ICH was rare in patients on DAPT. Relative to single antiplatelet therapy DAPT was not associated with an increased risk of in-hospital bleeding in patients with moderate and severe ischemic stroke. September 2021 \| Volume 12 \| Article 728111 Frontiers in Neurology \| www.frontiersin.org Khazaal et al. DAPT in Moderate/Severe Stroke INTRODUCTION SAPT (n = 229) DAPT (n = 148) P-value Age, mean 66.4 67.3 0.58 Sex, % female 53.3 44.6 0.10 Medical history (%) Hypertension 73.4 86.5 <0.01 Hyperlipidemia 50.7 62.8 0.02 Diabetes mellitus 32.8 43.9 0.03 Coronary artery disease 15.7 27.0 <0.01 Active smoking 25.8 28.4 0.58 Chronic kidney disease 13.1 17.6 0.23 Congestive heart failure 10.5 12.2 0.61 Atrial ﬁbrillation 11.8 3.4 <0.01 Antiplatelet prior to admission (%) None 61.6 38.5 <0.01 SAPT 35.8 39.9 0.43 DAPT 2.6 21.6 <0.01 Admission NIHSS, median (IQR) 9 (6–17) 7 (5–11) <0.01 Acute stroke treatments (%) tPA 38.9 15.5 <0.01 Thrombectomy 29.3 19.6 0.04 Stroke mechanism (%) Large artery atherosclerosis 11.8 46.0 <0.01 Small vessel disease 9.2 8.8 0.90 Cardioembolic 22.7 6.1 <0.01 Cryptogenic 39.3 17.6 <0.01 >1 mechanism 7.0 10.1 0.28 Other 10.0 11.5 0.66 Length of stay, median (IQR) 5 (3–10) 5 (3–9.5) 0.63 Disposition (%) Home 28.8 23.0 0.21 Acute rehab 56.8 71.0 <0.01 Skilled nursing facility 9.2 3.4 0.03 SAPT, single antiplatelet therapy; DAPT, dual antiplatelet therapy; tPA, tissue plasminogen activator. Abbreviations: DAPT, Dual antiplatelet therapy; SAPT, Single antiplatelet therapy; sICH, Symptomatic intracranial hemorrhage. p g METHODS This was a single center retrospective institutional review board approval, we s stroke registry to identify all patients adm 1st 2017 and November 1st 2019 with were age >18 years, and who had an TABLE 2 \| Bleeding rates by antiplatelet therapy at th Not DAPT (N = 236) Any systemic bleeding 3.8% Gastrointestinal 1.3% Genitourinary 0.8% Other 1.7% Serious systemic bleeding 1.3% Major bleeding 7.6% Asymptomatic ICH 11.0% Symptomatic ICH (NIHSS any change) 6.4% Symptomatic ICH (NIHSS change >/=4) 1.3% ICH classiﬁcation HI1 5.1% HI2 3.4% PH1 2.1% PH2 4.2% Other (Any SAH, IVH, SDH) 8.9% DAPT, dual antiplatelet therapy; ICH, intracranial hemorr of Health stroke scale; HI1, hemorrhagic transformat petechiae without mass effect); HI2, hemorrhagic tran more conﬂuent petechiae without mass effect); PH1, 1 (deﬁned as hematoma <30% of infarct bed with s parenchymal hematoma type 2 (deﬁned as hematoma >3 mass effect); SAH, subarachnoid hemorrhage; IVH, intr subdural hemorrhage. TABLE 1 \| Baseline characteristics. INTRODUCTION SAPT (n = 229) DAPT (n = 148) P-value Age, mean 66.4 67.3 0.58 Sex, % female 53.3 44.6 0.10 Medical history (%) Hypertension 73.4 86.5 <0.01 Hyperlipidemia 50.7 62.8 0.02 Diabetes mellitus 32.8 43.9 0.03 Coronary artery disease 15.7 27.0 <0.01 Active smoking 25.8 28.4 0.58 Chronic kidney disease 13.1 17.6 0.23 Congestive heart failure 10.5 12.2 0.61 Atrial ﬁbrillation 11.8 3.4 <0.01 Antiplatelet prior to admission (%) None 61.6 38.5 <0.01 SAPT 35.8 39.9 0.43 DAPT 2.6 21.6 <0.01 Admission NIHSS, median (IQR) 9 (6–17) 7 (5–11) <0.01 Acute stroke treatments (%) tPA 38.9 15.5 <0.01 Thrombectomy 29.3 19.6 0.04 Stroke mechanism (%) Large artery atherosclerosis 11.8 46.0 <0.01 Small vessel disease 9.2 8.8 0.90 Cardioembolic 22.7 6.1 <0.01 Cryptogenic 39.3 17.6 <0.01 >1 mechanism 7.0 10.1 0.28 Other 10.0 11.5 0.66 Length of stay, median (IQR) 5 (3–10) 5 (3–9.5) 0.63 Disposition (%) Home 28.8 23.0 0.21 Acute rehab 56.8 71.0 <0.01 Skilled nursing facility 9.2 3.4 0.03 SAPT, single antiplatelet therapy; DAPT, dual antiplatelet therapy; tPA, tissue plasminogen activator. Abbreviations: DAPT, Dual antiplatelet therapy; SAPT, Single antiplatelet therapy; sICH, Symptomatic intracranial hemorrhage. INTRODUCTION Not DAPT (N = 236) DAPT (N = 141) P-value Any systemic bleeding 3.8% 5.0% 0.59 Gastrointestinal 1.3% 0.7% 1.00 Genitourinary 0.8% 2.1% 0.37 Other 1.7% 2.1% 0.72 Serious systemic bleeding 1.3% 1.4% 1.00 Major bleeding 7.6% 2.1% 0.03 Asymptomatic ICH 11.0% 1.4% <0.01 Symptomatic ICH (NIHSS any change) 6.4% 0.7% <0.01 Symptomatic ICH (NIHSS change >/=4) 1.3% 0.7% 1.00 ICH classiﬁcation HI1 5.1% 0.7% 0.04 HI2 3.4% 0% 0.03 PH1 2.1% 0% 0.16 PH2 4.2% 0.7% 0.06 Other (Any SAH, IVH, SDH) 8.9% 1.4% <0.01 DAPT, dual antiplatelet therapy; ICH, intracranial hemorrhage; NIHSS, National Institute of Health stroke scale; HI1, hemorrhagic transformation type 1 (deﬁned as small petechiae without mass effect); HI2, hemorrhagic transformation type 2 (deﬁned as more conﬂuent petechiae without mass effect); PH1, parenchymal hematoma type 1 (deﬁned as hematoma <30% of infarct bed with some mild mass effect); PH2, parenchymal hematoma type 2 (deﬁned as hematoma >30% of infarct bed with signiﬁcant mass effect); SAH, subarachnoid hemorrhage; IVH, intraventricular hemorrhage; SDH: subdural hemorrhage. Patients with moderate or severe ischemic stroke were excluded from these trials because of a perceived increased risk of hemorrhagic complications. Nonetheless, these patients are at high short-term risk of recurrent ischemic stroke, death, and other major vascular events (5). With this background, we completed a single center retrospective cohort comparing the safety of single and dual antiplatelet therapy in moderate and severe ischemic stroke, deﬁned by a National Institute of Health Stroke Scale (NIHSS) of 4 or greater. We hypothesized that bleeding rates in patients on DAPT would be similar to patients on single antiplatelet therapy. TABLE 1 \| Baseline characteristics. INTRODUCTION Recent studies evaluating the safety of DAPT in moderate to severe ischemic stroke have had limitations. Two studies using a nationwide registry in Korea compared patients with non-minor strokes who were treated with DAPT to those on Aspirin alone. Recent studies evaluating the safety of DAPT in moderate to severe ischemic stroke have had limitations. Two studies using a nationwide registry in Korea compared patients with non-minor strokes who were treated with DAPT to those on Aspirin alone. They found that hemorrhagic stroke rates did not signiﬁcantly diﬀer between groups, and that recurrent vascular events were lower in the DAPT group (6, 7). These studies did not report details about the type of hemorrhagic transformation, did not report systemic bleeding, and were conducted in a homogenous patient population which might limit generalizability. Three randomized controlled trials found that early dual antiplatelet therapy (DAPT), compared to single antiplatelet therapy, lowers the risk of stroke or death after TIA or minor ischemic stroke with only a small absolute increase in the risk of major bleeding (1–3). As a result, guidelines recommend a short course of treatment with DAPT for patients after high risk TIA and minor non-cardioembolic ischemic stroke (4). They found that hemorrhagic stroke rates did not signiﬁcantly diﬀer between groups, and that recurrent vascular events were lower in the DAPT group (6, 7). These studies did not report details about the type of hemorrhagic transformation, did not report systemic bleeding, and were conducted in a homogenous patient population which might limit generalizability. course of treatment with DAPT for patients after high risk TIA and minor non-cardioembolic ischemic stroke (4). Patients with moderate or severe ischemic stroke were excluded from these trials because of a perceived increased risk of hemorrhagic complications. Nonetheless, these patients are at high short-term risk of recurrent ischemic stroke, death, and other major vascular events (5). TABLE 1 \| Baseline characteristics. INTRODUCTION SAPT (n = 229) DAPT (n = 148) P-value Age, mean 66.4 67.3 0.58 Sex, % female 53.3 44.6 0.10 Medical history (%) Hypertension 73.4 86.5 <0.01 Hyperlipidemia 50.7 62.8 0.02 Diabetes mellitus 32.8 43.9 0.03 Coronary artery disease 15.7 27.0 <0.01 Active smoking 25.8 28.4 0.58 Chronic kidney disease 13.1 17.6 0.23 Congestive heart failure 10.5 12.2 0.61 Atrial ﬁbrillation 11.8 3.4 <0.01 Antiplatelet prior to admission (%) None 61.6 38.5 <0.01 SAPT 35.8 39.9 0.43 DAPT 2.6 21.6 <0.01 Admission NIHSS, median (IQR) 9 (6–17) 7 (5–11) <0.01 Acute stroke treatments (%) tPA 38.9 15.5 <0.01 Thrombectomy 29.3 19.6 0.04 Stroke mechanism (%) Large artery atherosclerosis 11.8 46.0 <0.01 Small vessel disease 9.2 8.8 0.90 Cardioembolic 22.7 6.1 <0.01 Cryptogenic 39.3 17.6 <0.01 >1 mechanism 7.0 10.1 0.28 Other 10.0 11.5 0.66 Length of stay, median (IQR) 5 (3–10) 5 (3–9.5) 0.63 Disposition (%) Home 28.8 23.0 0.21 Acute rehab 56.8 71.0 <0.01 Skilled nursing facility 9.2 3.4 0.03 SAPT, single antiplatelet therapy; DAPT, dual antiplatelet therapy; tPA, tissue plasminogen activator. Abbreviations: DAPT, Dual antiplatelet therapy; SAPT, Single antiplatelet therapy; sICH, Symptomatic intracranial hemorrhage. details about the type of hemorrhagic transformation, did not report systemic bleeding, and were conducted in a homogenous patient population which might limit generalizability. With this background, we completed a single center retrospective cohort comparing the safety of single and dual antiplatelet therapy in moderate and severe ischemic stroke, deﬁned by a National Institute of Health Stroke Scale (NIHSS) of 4 or greater. We hypothesized that bleeding rates in patients on DAPT would be similar to patients on single antiplatelet therapy. METHODS This was a single center retrospective cohort study. After institutional review board approval, we searched our hospital’s stroke registry to identify all patients admitted between January 1st 2017 and November 1st 2019 with ischemic stroke who were age >18 years, and who had an admission NIHSS ≥4. TABLE 2 \| Bleeding rates by antiplatelet therapy at the time of event. METHODS NIHSS 4–7 (n = 160) ≥8 (n = 217) Not DAPT (n = 85) DAPT (n = 75) P-value Not DAPT (n = 151) DAPT (n = 66) P-value Major bleeding 5.9% 0% 0.06 8.6% 4.6% 0.40 Asymptomatic ICH 5.9% 2.7% 0.45 13.9% 0% <0.01 Symptomatic ICH (NIHSS any change) 5.9% 0% 0.06 6.6% 1.5% 0.18 Symptomatic ICH (NIHSS change >/=4) 1.2% 0% 1.00 1.3% 1.5% 1.00 ICH classiﬁcation HI1 3.5% 1.3% 0.62 6.0% 0% 0.06 HI2 3.5% 0% 0.25 3.3% 0% 0.33 PH1 0% 0% 1.00 3.3% 0% 0.33 PH2 2.4% 0% 0.50 5.3% 1.5% 0.28 Other (Any SAH, IVH or SDH) 4.7% 1.3% 0.37 11.3% 1.5% 0.02 DAPT, dual antiplatelet therapy; ICH, intracranial hemorrhage; NIHSS, National Institute of Health stroke scale; HI1, hemorrhagic transformation type 1 (deﬁned as small petechiae without mass effect); HI2, hemorrhagic transformation type 2 (deﬁned as more conﬂuent petechiae without mass effect); PH1, parenchymal hematoma type 1 (deﬁned as hematoma <30% of infarct bed with some mild mass effect); PH2, parenchymal hematoma type 2 (deﬁned as hematoma >30% of infarct bed with signiﬁcant mass effect); SAH, subarachnoid hemorrhage; IVH, intraventricular hemorrhage; SDH, subdural hemorrhage. DAPT, dual antiplatelet therapy; ICH, intracranial hemorrhage; NIHSS, National Institute of Health stroke scale; HI1, hemorrhagic transformation type 1 (deﬁned as small petechiae without mass effect); HI2, hemorrhagic transformation type 2 (deﬁned as more conﬂuent petechiae without mass effect); PH1, parenchymal hematoma type 1 (deﬁned as hematoma <30% of infarct bed with some mild mass effect); PH2, parenchymal hematoma type 2 (deﬁned as hematoma >30% of infarct bed with signiﬁcant mass effect); SAH, subarachnoid hemorrhage; IVH, intraventricular hemorrhage; SDH, subdural hemorrhage. TABLE 4 \| Bleeding rates among those who received tPA or thrombectomy and those who didn’t subcategorized by antiplatelet therapy groups. METHODS This was a single center retrospective cohort study. After institutional review board approval, we searched our hospital’s stroke registry to identify all patients admitted between January 1st 2017 and November 1st 2019 with ischemic stroke who were age >18 years, and who had an admission NIHSS ≥4. September 2021 \| Volume 12 \| Article 728111 Frontiers in Neurology \| www.frontiersin.org 2 DAPT in Moderate/Severe Stroke Khazaal et al. Patients who received at least one dose of any form of therapeutic anticoagulation during their hospitalization were excluded. Patients who received at least one dose of any form of therapeutic anticoagulation during their hospitalization were excluded. Variables were summarized by means and standard deviations or medians and interquartile range for continuous variables and frequencies/proportions for categorical variables. Diﬀerences between groups were evaluated using chi-squared tests for categorical variables and parametric (t-test) or non-parametric tests (Kruskal Wallis, Wilcoxon rank sum) as appropriate. We evaluated for diﬀerences in bleeding outcomes among patients on DAPT and those not on DAPT. We also performed secondary analysis stratiﬁed by NIHSS (4–7 vs. 8+) and based on whether patients received tPA and/or thrombectomy. Finally, multivariate logistic regression was used to determine if DAPT was independently associated with bleeding. Age, NIHSS, and variables signiﬁcant in univariate analysis with p < 0.10 were included. Analysis was performed using Stata 15.0 (StataCorp LP. College Station, TX, 2017). g g p We collected demographic characteristics, vascular risk factors, NIHSS, stroke mechanism, antiplatelet treatments throughout admission, and bleeding outcomes. Given the retrospective nature of this analysis, there was no pre-speciﬁed criteria for the use of DAPT. The primary outcome was symptomatic intracranial hemorrhage (sICH) with any change in NIHSS. Secondary outcomes included asymptomatic ICH, sICH with a change in NIHSS of 4 or more points, ICH classiﬁed according to the Heidelberg criteria (8), any systemic bleeding, and major bleeding (sICH or systemic bleeding that required transfusion, pressors, escalation of care, or causing death) (9). For each bleeding event it was determined if the bleeding occurred before or after initiation of DAPT. Only bleeding that occurred after initiation of DAPT was attributed to DAPT. TABLE 3 \| Bleeding rates among lower vs. higher NIHSS groups subcategorized by antiplatelet therapy groups. tPA, tissue plasminogen activator; DAPT, dual antiplatelet therapy; ICH, intracranial hemorrhage; NIHSS, National Institute of Health stroke scale; HI1, hemorrhagic transformation type 1 (deﬁned as small petechiae without mass effect); HI2, hemorrhagic transformation type 2 (deﬁned as more conﬂuent petechiae without mass effect); PH1, parenchymal hematoma type 1 (deﬁned as hematoma <30% of infarct bed with some mild mass effect); PH2, parenchymal hematoma type 2 (deﬁned as hematoma >30% of infarct bed with signiﬁcant mass effect); SAH, subarachnoid hemorrhage; IVH, intraventricular hemorrhage; SDH, subdural hemorrhage. Frontiers in Neurology \| www.frontiersin.org METHODS Variable OR 95% CI P-value Age <60 Ref 60–79 1.70 0.53–5.48 0.37 80+ 2.30 0.63–8.45 0.21 Male gender 0.59 0.24–1.47 0.26 Stroke etiology Large vessel disease Ref Small vessel disease No bleeding events Cardioembolic 2.72 0.09 Other 0.46 0.85–8.73 0.49 More than 1 mechanism 1.24 0.05–4.08 0.80 Cryptogenic 0.81 0.23–2.89 0.745 Cardioembolic etiology 3.52 1.39–8.90 <0.01 NIHSS 4–7 Ref 8+ 2.47 0.88–6.88 0.08 Past medical history Hypertension 1.68 0.48–5.86 0.413 Hyperlipidemia 1.65 0.65–4.19 0.29 Diabetes mellitus 1.04 0.42–2.59 0.93 Coronary artery disease 1.25 0.44–3.54 0.67 Chronic kidney disease 1.87 0.66–5.32 0.24 Congestive heart failure 1.35 0.38–4.81 0.64 Atrial ﬁbrillation 6.62 2.45–17.89 <0.01 End stage liver disease 7.39 1.35–40.59 0.02 Prior GI Bleeding 1.75 0.49–6.26 0.39 Prior GU Bleeding 1.57 0.19–12.76 0.67 Prior hemorrhagic transformation 9.09 3.09–26.74 <0.01 Current smoking 1.39 0.55–3.56 0.49 tPA or thrombectomy 3.21 1.22–8.45 0.02 Ref, Reference; tPA, tissue plasminogen activator; GI, gastrointestinal; GU, genitourinary. TABLE 6 \| Multivariate analysis for variables associated with Major Bleeding. Variable OR 95% CI P-value Age <60 Ref 60–79 1.42 0.41–4.89 0.58 80+ 1.44 0.31–6.71 0.64 Male gender 0.73 0.26–2.04 0.55 Cardioembolic etiology 1.36 0.36–5.09 0.65 NIHSS 4–7 Ref 8+ 1.84 0.59–5.76 0.30 Past medical history Atrial ﬁbrillation 1.99 0.41–9.62 0.39 End stage liver disease 6.73 0.63–71.78 0.12 Prior hemorrhagic transformation 2.95 0.76–11.50 0.12 tPA or thrombectomy 1.63 0.56–4.72 0.37 DAPT at time of bleed 0.34 0.08–1.50 0.15 Ref, Reference; tPA, tissue plasminogen activator; DAPT, dual antiplatelet therapy. DAPT group were loaded with clopidogrel (either 300 mg or 600 mg). Bleeding outcomes are shown in Table 2. Of the 148 patients that received DAPT during their hospitalization, 7 had ICH before DAPT was initiated. For this analysis these 7 subjects are included in the “Not DAPT” group, as their bleeding event occurred while on single or no antiplatelet therapy. Asymptomatic, sICH with any change in NIHSS, and sICH with NIHSS change ≥4 were all less common in the DAPT group. Only 1 of 141 subjects in the DAPT group had a sICH with NIHSS change ≥4. This patient had a PH2 hemorrhagic transformation which occurred on the same day that the patient was loaded with 600 mg of clopidogrel. There were 15 subjects in the Not DAPT group with symptomatic ICH, of which 12 (80%) occurred within 1 day of hospital admission. RESULTS In the stratiﬁed analysis (Tables 3, 4), sICH was numerically less common in the DAPT group, but the diﬀerences were not signiﬁcant (NIHSS 4–7: 0 vs. 5.9%, p = 0.06; NIHSS 8+: 1.5 vs. 6.6%, p = 0.18). Similarly, when stratiﬁed by treatment with tPA and/or thrombectomy, sICH was less frequent in the DAPT group but the diﬀerences were not signiﬁcant (Yes: 2.6 vs. 9.1%, p = 0.30; No: 0 vs. 2.9%, p = 0.25). Of the 13 sICH that occurred in patients treated with tPA or thrombectomy 10 (77%) occurred within 1 day of admission. A total of 377 patients met our inclusion criteria. Patients were divided into two groups based on the antiplatelet treatment they received throughout their admission. There were 148 patients who were treated with DAPT at any time during the hospitalization and 229 who were not (single antiplatelet therapy). All the DAPT subjects were treated with the combination of aspirin and clopidogrel. Of these 148 patients, 28.4% were loaded with 600 mg of clopidogrel and then maintained on 75 mg daily, 20.3% were loaded with 300 mg and then maintained on 75 mg daily, and 50.7% were started on 75 mg of clopidogrel daily without receiving a loading dose. Baseline demographics are summarized in Table 1. Patients in the DAPT group were more likely to have traditional vascular risk factors and large artery atherosclerosis as the mechanism of stroke. The median admission duration was 5 days and the median time from admission to start of DAPT was 1 day (IQR 0–2). About half (49%) of the patients in the DAPT was not associated with major bleeding in either the univariate (Table 5) or the multivariate regression analysis (Table 6). METHODS The composite outcome of major bleeding was seen in 2.1% of subjects on DAPT and 7.6% of patients not on DAPT (p = 0.03). METHODS Variable OR 95% CI P-value Age <60 Ref 60–79 1.70 0.53–5.48 0.37 80+ 2.30 0.63–8.45 0.21 Male gender 0.59 0.24–1.47 0.26 Stroke etiology Large vessel disease Ref Small vessel disease No bleeding events Cardioembolic 2.72 0.09 Other 0.46 0.85–8.73 0.49 More than 1 mechanism 1.24 0.05–4.08 0.80 Cryptogenic 0.81 0.23–2.89 0.745 Cardioembolic etiology 3.52 1.39–8.90 <0.01 NIHSS 4–7 Ref 8+ 2.47 0.88–6.88 0.08 Past medical history Hypertension 1.68 0.48–5.86 0.413 Hyperlipidemia 1.65 0.65–4.19 0.29 Diabetes mellitus 1.04 0.42–2.59 0.93 Coronary artery disease 1.25 0.44–3.54 0.67 Chronic kidney disease 1.87 0.66–5.32 0.24 Congestive heart failure 1.35 0.38–4.81 0.64 Atrial ﬁbrillation 6.62 2.45–17.89 <0.01 End stage liver disease 7.39 1.35–40.59 0.02 Prior GI Bleeding 1.75 0.49–6.26 0.39 Prior GU Bleeding 1.57 0.19–12.76 0.67 Prior hemorrhagic transformation 9.09 3.09–26.74 <0.01 Current smoking 1.39 0.55–3.56 0.49 tPA or thrombectomy 3.21 1.22–8.45 0.02 Ref, Reference; tPA, tissue plasminogen activator; GI, gastrointestinal; GU, genitourinary. TABLE 6 \| Multivariate analysis for variables associated with Major Bleeding. Variable OR 95% CI P-value Age <60 Ref 60–79 1.42 0.41–4.89 0.58 80+ 1.44 0.31–6.71 0.64 Male gender 0.73 0.26–2.04 0.55 Cardioembolic etiology 1.36 0.36–5.09 0.65 NIHSS 4–7 Ref 8+ 1.84 0.59–5.76 0.30 Past medical history Atrial ﬁbrillation 1.99 0.41–9.62 0.39 End stage liver disease 6.73 0.63–71.78 0.12 Prior hemorrhagic transformation 2.95 0.76–11.50 0.12 tPA or thrombectomy 1.63 0.56–4.72 0.37 DAPT at time of bleed 0.34 0.08–1.50 0.15 Ref, Reference; tPA, tissue plasminogen activator; DAPT, dual antiplatelet therapy. DAPT group were loaded with clopidogrel (either 300 mg or 600 mg). Bleeding outcomes are shown in Table 2. Of the 148 patients that received DAPT during their hospitalization, 7 had ICH before DAPT was initiated. For this analysis these 7 subjects are included in the “Not DAPT” group, as their bleeding event occurred while on single or no antiplatelet therapy. Asymptomatic, sICH with any change in NIHSS, and sICH with NIHSS change ≥4 were all less common in the DAPT group. Only 1 of 141 subjects in the DAPT group had a sICH with NIHSS change ≥4. This patient had a PH2 hemorrhagic transformation which occurred on the same day that the patient was loaded with 600 mg of clopidogrel. There were 15 subjects in the Not DAPT group with symptomatic ICH, of which 12 (80%) occurred within 1 day of hospital admission. The composite outcome of major TABLE 5 \| Univariate analysis. METHODS Received tPA or Thrombectomy Yes (n = 171) No (n = 206) Not DAPT (n = 132) DAPT (n = 39) P-value Not DAPT (n = 104) DAPT (n = 102) P-value Major bleeding 10.6% 2.6% 0.20 3.9% 2% 0.68 Asymptomatic ICH 15.2% 2.6% 0.05 5.8% 1.0% 0.12 Symptomatic ICH (NIHSS any change) 9.1% 2.6% 0.30 2.9% 0% 0.25 Symptomatic ICH (NIHSS change >/=4) 2.3% 2.6% 1.00 0% 0% 1.00 ICH classiﬁcation HI1 3.8% 2.6% 1.00 6.7% 0% 0.01 HI2 5.3% 0% 0.35 1.0% 0% 1.00 PH1 3.8% 0% 0.59 0% 0% 1.00 PH2 6.8% 2.6% 0.46 1.0% 0% 1.00 Other (Any SAH, IVH or SDH) 13.6% 2.6% 0.08 2.9% 1.0% 0.62 tPA, tissue plasminogen activator; DAPT, dual antiplatelet therapy; ICH, intracranial hemorrhage; NIHSS, National Institute of Health stroke scale; HI1, hemorrhagic transformation type 1 (deﬁned as small petechiae without mass effect); HI2, hemorrhagic transformation type 2 (deﬁned as more conﬂuent petechiae without mass effect); PH1, parenchymal hematoma type 1 (deﬁned as hematoma <30% of infarct bed with some mild mass effect); PH2, parenchymal hematoma type 2 (deﬁned as hematoma >30% of infarct bed with signiﬁcant mass effect); SAH, subarachnoid hemorrhage; IVH, intraventricular hemorrhage; SDH, subdural hemorrhage. ng rates among those who received tPA or thrombectomy and those who didn’t subcategorized by antiplatelet therapy groups. tPA, tissue plasminogen activator; DAPT, dual antiplatelet therapy; ICH, intracranial hemorrhage; NIHSS, National Institute of Health stroke scale; HI1, hemorrhagic transformation type 1 (deﬁned as small petechiae without mass effect); HI2, hemorrhagic transformation type 2 (deﬁned as more conﬂuent petechiae without mass effect); PH1, parenchymal hematoma type 1 (deﬁned as hematoma <30% of infarct bed with some mild mass effect); PH2, parenchymal hematoma type 2 (deﬁned as hematoma >30% of infarct bed with signiﬁcant mass effect); SAH, subarachnoid hemorrhage; IVH, intraventricular hemorrhage; SDH, subdural hemorrhage. September 2021 \| Volume 12 \| Article 728111 Frontiers in Neurology \| www.frontiersin.org 3 DAPT in Moderate/Severe Stroke Khazaal et al. TABLE 5 \| Univariate analysis. Frontiers in Neurology \| www.frontiersin.org DISCUSSION Early treatment with DAPT has been shown to be beneﬁcial in high-risk TIA and minor ischemic stroke, although the beneﬁt September 2021 \| Volume 12 \| Article 728111 Frontiers in Neurology \| www.frontiersin.org 4 DAPT in Moderate/Severe Stroke Khazaal et al. DAPT. Additionally, only about half of the patients in this study were loaded with clopidogrel, which may also have contributed to lower bleeding rates in the DAPT group. We deﬁned non-minor stroke using NIHSS, rather than infarct volume. This deﬁnition is consistent with published randomized trials which used NIHSS to deﬁne minor stroke. NIHSS is correlated with infarct volume (11) but nonetheless, direct volume measurements would have a stronger correlation with bleeding risk (12). Outcomes were deﬁned as bleeding during hospitalization. Median time from admission to start of DAPT was 1 day, with an average length of stay of 5 days. Hemorrhagic transformation risk is likely highest early after ischemic stroke, but it is possible that there were bleeding events after discharge that were missed. Finally, the relatively small number of outcome events limited the statistical power. in moderate to severe stroke is uncertain. In this single center retrospective study, we found that patients with non-minor stroke deﬁned by NIHSS ≥4 who were treated with DAPT did not have higher rates of major bleeding during their hospitalization than those who were not treated with DAPT. Both asymptomatic and sICH were less common in the DAPT group than the group not on DAPT. We observed relatively high rates of ICH in the single antiplatelet therapy group. This outcome was biologically unexpected and we believe it reﬂects, in part, careful screening for asymptomatic ICH at our institution. It is probable that clinicians were less likely to use dual antiplatelet therapy in patients with asymptomatic ICH and this selection bias may have contributed to the observed diﬀerence in asymptomatic ICH between groups. Importantly, Table 4 shows that 77% of asymptomatic ICH and 80% of sICH in the single antiplatelet group occurred in patients treated with tPA and/or thrombectomy. These hemorrhages likely reﬂect complications of those treatments and further biased the results toward a higher overall ICH rate in the single antiplatelet therapy group. While this bias impacts the comparison between groups in this study, it is still reassuring that ICH in the DAPT group was very low. ETHICS STATEMENT Major bleeding was seen in 2.1% of patients treated with DAPT. This is numerically higher than the major bleeding rates reported in the CHANCE (0.2%), POINT (0.9%), and THALES (0.5%) trials (1–3), but similar to the major bleeding rate reported in the medical arm of the SAMMPRIS Trial (2.2%) (10). It may be that the overall higher rates of bleeding in our study reﬂect a higher risk in this population, compared to a TIA and minor stroke population, rather than an eﬀect speciﬁc to DAPT. The studies involving human participants were reviewed and approved by Penn IRB. The Ethics Committee waived the requirement of written informed consent for participation. CONCLUSION In this single center retrospective study, we found that DAPT was associated with a very low rate of sICH in patients with moderate and severe ischemic stroke deﬁned by NIHSS ≥4. A randomized trial is needed to conﬁrm this ﬁnding and to determine if DAPT is eﬀective at preventing recurrent stroke in this population. AUTHOR CONTRIBUTIONS OK contributed to the writing of the paper. AR, MH, MB, DC, SR-E, FK, CF, SM, and MM contributed to data collection and review of written portion. MM contributed to the statistical analysis and writing of the paper as well as being the senior author. All authors contributed to the article and approved the submitted version. As a retrospective cohort, this study has limitations. The most important is the potential for selection bias. As discussed above, clinicians likely selected patients for DAPT who they felt were at a lower, or at least acceptable, risk of ICH, biasing the results toward a higher overall hemorrhage rate in the single antiplatelet group. We did not record the rationale for using DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors upon request. DISCUSSION Only 1 of the subjects had sICH after initiation of DAPT, and there was with no signal of increased ICH risk in the DAPT group in the stratiﬁed analyses. A prospective, randomized trial is needed to determine if DAPT is eﬀective in moderate and severe stroke. Such a trial would likely exclude patients treated with tPA and/or thrombectomy, particularly if those treatments were complicated by ICH, and our data suggests that in such a population treatment with DAPT is associated with very low rates of sICH. REFERENCES stroke: 2019 update to the 2018 guidelines for the early management of acute ischemic stroke: A guideline for healthcare professionals from the american heart association/american stroke association. Stroke 1970. (2019) 50:e344–418. doi: 10.1161/STR.0000000000000211 1. Wang Y, Wang Y, Zhao X, Liu L, Wang D, Wang C, et al. Clopidogrel with aspirin in acute minor stroke or transient ischemic attack. N Engl J Med. (2013) 369:11–9. doi: 10.1056/NEJMoa1215340 1. Wang Y, Wang Y, Zhao X, Liu L, Wang D, Wang C, et al. Clopidogrel with aspirin in acute minor stroke or transient ischemic attack. N Engl J Med. 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REFERENCES Comparative eﬀectiveness of dual antiplatelet therapy with aspirin and clopidogrel versus aspirin monotherapy in mild-to-moderate acute ischemic stroke according to the risk of recurrent stroke: an analysis of 15,000 patients from a nationwide, multicenter registry. Circ Cardiov Quality Outcomes. (2020) 13:853–63. doi: 10.1161/CIRCOUTCOMES.119.006474 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 8. Von Kummer R, Broderick JP, Campbell BCV, Demchuk A, Goyal M, Hill MD, et al. The heidelberg bleeding classiﬁcation: classiﬁcation of bleeding events after ischemic stroke and reperfusion therapy. Stroke 1970. (2015) 46:2981–6. doi: 10.1161/STROKEAHA.115.010049 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 9. Schulman S, Kearon C on behalf of the subcommittee on control of anticoagulation of the Scientiﬁc and Standardization committee of the International Society on Thrombosis and Haemostasis. Deﬁnition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients. J Thromb Haemo. (2005) 3:692– 4. doi: 10.1111/j.1538-7836.2005.01204.x 9. Schulman S, Kearon C on behalf of the subcommittee on control of anticoagulation of the Scientiﬁc and Standardization committee of the International Society on Thrombosis and Haemostasis. Deﬁnition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients. J Thromb Haemo. (2005) 3:692– 4. doi: 10.1111/j.1538-7836.2005.01204.x Copyright © 2021 Khazaal, Rothstein, Husain, Broderick, Cristancho, Reyes-Esteves, Khan, Favilla, Messé and Mullen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 10. Chimowitz MI, Lynn MJ, Derdeyn CP, Turan TN, Fiorella D, Lane BF, et al. Stenting versus aggressive medical therapy for intracranial arterial stenosis. N Engl J Med. (2011) 365:993–1003. doi: 10.1056/NEJMoa1105335 11. Behymer T, Vagal A, Sucharew H, Yeluru V, Minhas A, Hazenﬁeld JM, et al. Frontiers in Neurology \| www.frontiersin.org REFERENCES NIHSS and variation of infarct volume by hemisphere. International Stroke Conference Poster Abstracts Session Title: Acute neuroimaging Posters II. Houston, TX (2017). September 2021 \| Volume 12 \| Article 728111 Frontiers in Neurology \| www.frontiersin.org 6
https://openalex.org/W1986955643	https://zenodo.org/records/1818080/files/article.pdf	English	null	WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOGICAL EFFECTS OF ALCOHOL?	School science and mathematics	1,909	public-domain	3,232	EFFECTS OF ALCOHOL EFFECTS OF ALCOHOL 455 WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? BY-HARRY CLIFFORD DOANE. EDITORIAL NOTE: This article will serve to remind us that a few ye^ars ago there was an abundance of evidence relative to the physiological effects of alcohol. But few people cared to consider it dispassionately then, owing to the obviously untrust- worthy claims made by the chief exploiters of the evidence. Now, however, we may return with equanimity to the consid- eration of experimental data from all sources and ultimately hope to come at the whole truth. The teaching of physiology and hygiene is far from being in a satisfactory condition in our schools. The agitation so vig- orously prosecuted’ by some against the various state laws re- quiring the teaching of the effects of alcohol and narcotics and the criticisms of the accuracy of the statements on this subject found in our school physiologies seem to be in a measure re- sponsible for a serious neglect of physiology teaching in many schools. There appears to have grown up a widespread feeling that physiology is an unfit subject for grade schools, and even for high schools unless it can be pursued in a thoroughly scien- tific manner by laboratory methods. I do not wish to be understood as minimizing in any degree the value or need of making scientific subjects the means of teaching scientific methods of thought, nor would I decry the place of the laboratory in such instruction; but physiology oc- cupies a peculiar position among the sciences as a subject of school study. It is essential to the physical welfare of the indi- vidual and of -.the community that the pupils of our schools ac- quire a knowledge of the more important principles of hygiene. These principles of hygiene are dependent for their truth on the facts of physiology, and cannot be successfully taught with- out some reference to ’physiology as a basis. The value of a knowledge of physiology and hygiene for their own sake makes it important that they be taught definitely and systematically in all schools, even though laboratory methods cannot be used, beginning as low in the grades as possible. Many facts of physiology and hygiene can be successfully taught in the first and second grades. SCHOOL SGIENGE AND MATHEMATICS 456 The claim that place should be given in our teaching of physiology and hygiene to the effects of alcohol and narcotics needs no defense. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? In the year ending June 30, 1907, the people of the United States consumed 2,019,690,911 gallons of distilled spirits, wines and malt liquors, according to the "Statistical Abstract of the United States," published by the government. A careful estimate gives the retail cost of this liquor as $2,275,- '70,857. The per capita consumption was as follows: Distilled liquors ....................... 1.63 gallons Wines ............................... 0.67 " Malt liquors .........................21.23 £t Total per capita ....................23.53 " This gives a per capita cost of $26.50. This gives a per capita cost of $26.50. The people can be brought to get rid of this enormous, worse than useless, expense only by educating them as to the true facts in the case. We who are not specialists in this subject must accept and teach the conclusions of those who have scientifically and ex- perimentally investigated the effects of alcohol upon the human body and its processes. In the following paragraphs I have col- lected some of the testimony of such investigation. Effect of Alcohol on Muscular Effort.Professor Destree of Belgium measured the weight he could lift by flexing the middle finger. In six tests he could lift nearly seventeen pounds. After fatigue came on he could lift only nine pounds in the same number of tests. When he had taken some wine and rested thirty minutes, he could raise but three and one half pounds. During a period of ten years Professor Kraepelin performed .some 3,000 experiments on the effects of alcohol. He found by means of a dynamometer that the pull that could be given with the hand was weakened by taking small quantities of alcohol. Mountain climbers usually recommend that no alcoholic drinks be used just before or during the climb. Professor A. Durig reports the results of experiments performed in 1906 to deter- mine the effect of alcohol on mountain climbing. Careful ob- servation of the subject of the experiment’ in climbs when "2 to 2.5 fluid ounces of alcohol, equivalent to about a pint of wine, were taken before the ascent" and also when no alcohol was taken, showed that "alcohol diminished the amount of work performed per minute by about one-sixth." Effect of Alcohol on Muscular Effort.Professor Destree of Belgium measured the weight he could lift by flexing the middle finger. In six tests he could lift nearly seventeen pounds. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? After fatigue came on he could lift only nine pounds in the same number of tests. When he had taken some wine and rested thirty minutes, he could raise but three and one half pounds. During a period of ten years Professor Kraepelin performed .some 3,000 experiments on the effects of alcohol. He found by means of a dynamometer that the pull that could be given with the hand was weakened by taking small quantities of alcohol. y g q Mountain climbers usually recommend that no alcoholic drinks be used just before or during the climb. Professor A. Durig reports the results of experiments performed in 1906 to deter- mine the effect of alcohol on mountain climbing. Careful ob- servation of the subject of the experiment’ in climbs when "2 to 2.5 fluid ounces of alcohol, equivalent to about a pint of wine, were taken before the ascent" and also when no alcohol was taken, showed that "alcohol diminished the amount of work performed per minute by about one-sixth." 457 EFFECTS OF ALGOHOL In "Physiological Aspects of the Liquor Problem/’ Professor Hodge of dark University describes many of his own experi- ments showing the effect of alcohol on animals. He trained four selected puppies to recover a ball thrown across a gym- nasium. To two of the dogs he gave food mixed with dietetic doses of alcohol while the others were fed normally. The ball was thrown 100 feet as rapidly as recovered. This was re- peated 100 times each day for fourteen successive days. Out of the 1,400 times- the dogs to which alcohol had been given brought back the ball only 478 times, while the others secured it 922 times. Dr. Parkes experimented with two gangs of men, selected to be as nearly similar as possible, in mowing. He found that with one gang abstaining from alcoholic drinks and the other not, the abstaining gang could accomplish more. On transpos- ing the gangs the same results were repeatedly obtained. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? Sim- ilar results were obtained by Professor Aschaffenburg of Heidel- burg University, who found experimentally that men "were able to do 15 per cent less work after taking alcohol." Pro- fessor Abel of Johns Hopkins University says: "Both science and the experience of life have exploded the pernicious theory that alcohol gives any persistent increase of muscular power.51 Alcohol and the Circulation.As a result of experiments per- formed in 1869, Zimmerberg declares: "In the light of these ex- periments one is not only justified in denying to alcohol any stimulating power whatever for the heart, but, on the contrary, in declaring that it lowers the working capacity of that organ." Dr. J. H. Kellogg, head of the Battle Creek Sanitarium, says: "The full bounding pulse usually produced by the administra- tion of an ounce or two of brandy properly diluted, gives the impression of an increased vigor of heart action; but it is only necessary to determine the blood pressure by means of a Riva- Rocci instrument, or Gaertner^s tonometer, to discover that the blood pressure is lowered instead’of raised. This-lowering may amount to twenty or thirty millimeters, or even more. * * * It can readily be seen, then, that the bounding pulse is not the result of increased heart vigor, but indicates rather a weakened state of the heart, combined with a dilated condition of the small vessels." In an address before the Liverpool Medical Association,- Dr. James Barr, president of the association, discussing the effects SCHOOL SCIENCE AND MATHEMATICS 458 of medicinal doses of alcohol upon the circulation remarked: "It causes dilatation of the arterioles and of all arteries well suppled with muscular fibers, owing to its paretic effect upon the vasomotor nervous system, and its direct action as a proto- plasmic poison on the muscular fiber. It has a similar, though less marked, action on the cardiac muscle. From these causes the systolic blood pressure is lowered, the systolic output from the heart is diminished, and the cardiac energy is wasted in pumping blood into relaxed vessels; the large bounding pulse with comparatively short systolic period, which gives a decep- tive appearance of vigor and force in the circulation, is due to the large wave in the dilated vessels." Effects of Alcohol on the Nervous System.In an article, in the Journal of Inebriety, Dr. J. W. Grosvener of Buffalo says: "Alcohol is a paralyzer. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? The truth of this proposition has been demonstrated experimentally scores of times by world-famed physiologists. Says Forel: ’Through all parts of nervous ac- tivity from the innervation of the muscles and the simplest sen- sation of the highest activity of the soul the paralyzing effect of alcohol can be demonstrated." Several experimenters of un- doubted ability have noted the paralyzing effect of alcohol even in small doses. By the use of delicate instruments of precision, Ridge tested the effect of alcohol on the senses of smell, vision, and muscular sense of weight. He found that two drams of absolute alcohol produced a positive decrease in the sensitiveness of the nerves of feeling, that so small a quantity as one half dram of absolute alcohol diminished the power of vision and the muscular sense of weight. Kraepelin and Kurz by experi- ment determined that the acuteness of the special senses of sight, ’hearing, touch, taste, and smell was diminished by an ounce of alcohol, the power of vision being lost "to one third of its extent’and a similar effect being ^produced on the other spe- cial senses. Other investigators, as Crothers, Madden, Kellogg, Frey, von Bunge have reached like conclusions." Dr. Kellogg, previously quoted, in an article giving conclu- sions from experiments by himself and others says-: "Kleefeld has shown that alcohol, when taken into the blood, produces .almost immediately a marked change in the minute structures of the brain. The dendrons and contact globules are shriveled, thus’ breaking contact and thus interrupting the normal nerve circuits. This fact explains, to a large degree, the mental and EFFECTS OF ALCOHOL 459 moral effects of alcohol, especially its effect in destroying in- hibition. When often repeated, this toxic effect gives rise to degenerative changes which are seen in their full development in general paresis. The flushing of the face seen after the ad- ministration of alcohol, and the exhilaration felt by a person in ordinary health, are due, not to a stimulating effect, but rather to a paralyzing or sedative effect. * * * A person who is fatigued, after taking alcohol feels relieved, not because he is rested or because his ’muscles have been reinforced, but because the nerves of fatigue are paralyzed so that he no longer appreciates the fact that he is fatigued. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? A person who is cold, after taking a dram, no longer suffers from cold or chilliness, and imagines that the alcohol has warmed him. This is not the case, however. The alcohol has only lessened the sensibility of his thermic nerves, so that he is less sensitive to cold, while at the same time increasing the flow of blood to the skin by par- alyzing the vasomotor centers." With regard to the supposed quickening of the mental proc- esses Horsley and Sturge, in their recent book, "Alcohol and the Human Body," say: "Kraepelin found that the simple reac- tion period, by which is meant the time occupied in making a mere response to a signal, as, for instance, to the sudden ap- pearance of a flag, was, after the ingestion of a small quantity of alcohol (J4 to y-2. ounce), slightly accelerated; that there was; in fact, a slight shortening of the time, as though the brain were enabled to operate more quickly than before. But he found that after a few minutes,. in most cases, a slowing of mental action began, becoming more and more marked, and enduring as long as the alcohol was in active operation in the body, i. e., four to five hours. * * * Kraepelin found that it was only more or less automatic work, such as reading aloud, which was quickened by alcohol, though even this was rendered less trust- worthy and accurate." Again: "Kraepelin had always shared the popular belief that a small quantity of alcohol (one to two tcaspoonfuls) had an accelerating effect on the activity of his mind, enabling him to perform test operations, as the adding and- subtracting and learning of figures more quickly. But when he came to measure with his instruments the exact period and time occupied, he found, to his astonishment, that he had accomplished these mental operations not more, but less, quickly than before. * * * Numerous further experiments were SCHOOL SCIENCE AND MATHEMATICS 460 carried out in order to test this matter, and these proved that alcohol lengthens the time taken to perform complex mental processes, while by a singular illusion the person experimented upon imagines that his psychical actions are rendered more rapid." p Relation of Digestion and Nutrition to Alcohol.As a result of his own experiments Prof. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? * * * p Alcohol behaves in this regard just as does opium or any other drug. It has no resemblance to a food. "3. When alcohol is withdrawn from a person who has been accustomed to its daily use, most distressing effects are ex- perienced. * * * Who ever saw a man’s hand trembling or his nervous system unstrung because he could not get a potato or a piece of cornbread’ for breakfast? In this respect, also, alcohol behaves like opium, cocaine, or any other enslaving drug. "3. When alcohol is withdrawn from a person who has been accustomed to its daily use, most distressing effects are ex- perienced. * * * Who ever saw a man’s hand trembling or h ld "3. When alcohol is withdrawn from a person who has been accustomed to its daily use, most distressing effects are ex- perienced. * * * Who ever saw a man’s hand trembling or his nervous system unstrung because he could not get a potato or a piece of cornbread’ for breakfast? In this respect, also, alcohol behaves like opium, cocaine, or any other enslaving drug. "4. Alcohol lessens the appreciation and the value of brain and nerve activity, while food reinforces nervous and mental energy. "5. Alcohol as a protoplasmic poison lessens muscular power, whereas food increases energy and endurance. "6. Alcohol lessens the power to endure cold. This is true to such a marked degree that its use by persons accompanying Arctic expeditions is absolutely prohibited. Food, on the other hand, increases ability to endure cold. The temperature after taking food is raised. After taking alcohol the temperature, as shown by the thermometer, is lowered. "6. Alcohol lessens the power to endure cold. This is true to such a marked degree that its use by persons accompanying Arctic expeditions is absolutely prohibited. Food, on the other hand, increases ability to endure cold. The temperature after taking food is raised. After taking alcohol the temperature, as shown by the thermometer, is lowered. "7. ^Alcohol cannot be stored in the body for future use, whereas all food substances can be so stored. "8. Food burns slowly in the body, as it is required to sat- isfy the body’s needs. Alcohol is readily oxidized and elim- inated, the same as any other oxidizable drug." The superintendent of the Northern Illinois Hospital for the Insane, Dr. Vaclav H. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? Chittenden of Yale University remarks: "We believe that the results obtained justify the con- clusion that gastric digestion as a whole is not materially modi- fied by the introduction of alcoholic fluids with the food. In other words, the unquestionable acceleration of gastric secretion which follows the ingestion of alcoholic beverages is^ as a rule, counterbalanced by the inhibitory effect of the alcoholic fluids upon the chemical process of gastric digestion, with perhaps at times a tendency towards preponderance of inhibitory action." Kellogg, Sir William Roberts and others have come to the same or stronger conclusions as to the undesirable action of alcohol on digestion, as a result of their own experiments. Horsley and Sturge, in their book previously mentioned, say: "Hundreds of men and women who haunt the out-patient de- partments of hospitals suffer from chronic atony and slight dilatation of the stomach, which arise in part from the ba.dly cooked food they eat, but chiefly owe their origin to the debili- tating effect of alcohol upon the muscular walls of this organ and the fermentation of its retained contents." The experiments of Professor Atwater demonstrated that a certain amount of alcohol is oxidized in the body. As the ox- idation produces energy he held that alcohol was a food. To call alcohol a food is to distort the meaning of food as ordi- narily used and few of the physiologists who can properly be considered authorities agree with Atwater in calling alcohol a food instead of a poison. Kellogg points out that strychnine, quinine and many other drugs are oxidized in the body, but surely cannot be called foods. The following reasons for not considering alcohol a food are taken from Kellogg: "i. A habitual user of alcohol has an intense craving for his accustomed dram. Without it he is entirely unfitted for business. One never experiences such an insane craving for bread, potatoes, or any other particular article of food. * * * "3. By continuous use the body acquires a tolerance for al- EFFECTS OF ALCOHOL 461 cohol. That is, the amount which may be imbibed and the amount required to produce the characteristic effects first ex- perienced gradually increase until very great quantities are sometimes required to satisfy the craving which its habitual use often produces*. This is never the case with true foods. WHAT SHALL WE TEACH CONCERNING THE PHYSIOLOG- ICAL EFFECTS OF ALCOHOL? Podstata, says: "Alcohol is unquestion- ably a direct and powerful poison to the nervous structure. It not only temporarily impairs and prevents the activity of nerv- ous tissue/but in a measure, though at times very minute and almost imperceptible, every dose of it permanently disables the functional activity of those organs." (To be continued.)
https://openalex.org/W3147357222	https://hal.inrae.fr/hal-03212645/file/fpls-03-2021.pdf	English	null	Trait Diversity of Pulse Species Predicts Agroecosystem Properties Trade-Offs	Frontiers in plant science	2,021	cc-by	9,672	To cite this version: Julie Guiguitant, Denis Vile, Hélène Marrou. Trait Diversity of Pulse Species Predicts Agroecosystem Properties Trade-Offs. Frontiers in Plant Science, 2021, 12, pp.636915. 10.3389/fpls.2021.636915. hal-03212645 Distributed under a Creative Commons Attribution 4.0 International License Edited by: Eric Von Wettberg, University of Vermont, United States Edited by: Eric Von Wettberg, University of Vermont, United States Reviewed by: Edward Marques, University of Vermont, United States Agnieszka Klimek-Kopyra, University of Agriculture in Krakow, Poland Anoob Prakash, University of Vermont, United States Correspondence: Denis Vile denis.vile@inrae.fr Reviewed by: Edward Marques, University of Vermont, United States Agnieszka Klimek-Kopyra, University of Agriculture in Krakow, Poland Anoob Prakash, University of Vermont, United States Correspondence: Denis Vile denis.vile@inrae.fr Correspondence: Denis Vile denis.vile@inrae.fr Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Received: 02 December 2020 Accepted: 08 March 2021 Published: 31 March 2021 Citation: Guiguitant J, Vile D and Marrou H (2021) Trait Diversity of Pulse Species Predicts Agroecosystem Properties Trade-Offs. Front. Plant Sci. 12:636915. doi: 10.3389/fpls.2021.636915 Trait Diversity of Pulse Species Predicts Agroecosystem Properties Trade-Offs Julie Guiguitant1,2, Denis Vile2 and Hélène Marrou3 1 SYSTEM, Montpellier SupAgro, INRAE, CIRAD, Institut Agronomique Méditerranéen de Montpellier, CIHEAM, University of Montpellier, Montpellier, France, 2 LEPSE, Univ Montpellier, INRAE, Institut Agro, Montpellier, France, 3 African Integrated Plant and Soil Research Group (AiPlaS), AgroBioSciences, Mohammed VI Polytechnic University, Ben Guerir, Morocco Julie Guiguitant1,2, Denis Vile2* and Hélène Marrou3 1 SYSTEM, Montpellier SupAgro, INRAE, CIRAD, Institut Agronomique Méditerranéen de Montpellier, CIHEAM, University of Montpellier, Montpellier, France, 2 LEPSE, Univ Montpellier, INRAE, Institut Agro, Montpellier, France, 3 African Integrated Plant and Soil Research Group (AiPlaS), AgroBioSciences, Mohammed VI Polytechnic University, Ben Guerir, Morocco 1 SYSTEM, Montpellier SupAgro, INRAE, CIRAD, Institut Agronomique Méditerranéen de Montpellier, CIHEAM, University of Montpellier, Montpellier, France, 2 LEPSE, Univ Montpellier, INRAE, Institut Agro, Montpellier, France, 3 African Integrated Plant and Soil Research Group (AiPlaS), AgroBioSciences, Mohammed VI Polytechnic University, Ben Guerir, Morocco Crop diversity management in agriculture is a fundamental principle of agroecology and a powerful way to promote resilient and sustainable production systems. Pulses are especially relevant for diversiﬁcation issues. Yet, the speciﬁc diversity of legumes is poorly represented in most cropping systems. We used the trait-based approach to quantify the functional diversity of 30 pulses varieties, belonging to 10 species, grown under common ﬁeld conditions. Our aim was to test relationships between traits, yield, and supporting agroecosystem properties. Our experimental results highlighted trade-offs between agroecosystem properties supported by different combinations of traits. Also, results demonstrated the relevance of leaf nitrogen content (LNC), leaf area ratio (LAR), and reproductive phenology to predict most of the trade-offs observed between agroecosystem properties. A comparison with a previous analysis based on literature data collected in diverse agronomic situations suggested that some traits are more plastic than others and therefore contribute differently to frame legumes diversity depending on the conditions of observation. Present results suggested that the implementation of such trait-based approach would rapidly beneﬁt the selection of species/varieties for speciﬁc targeted agroecosystem services provisioning under speciﬁc (environmental or management) conditions. Keywords: agroecosystem services, functional diversity, leaf nitrogen content, legumes, pulse crops, trait-based agroecology, traits–services trade-offs HAL Id: hal-03212645 https://hal.inrae.fr/hal-03212645v1 Submitted on 29 Apr 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ORIGINAL RESEARCH published: 31 March 2021 doi: 10.3389/fpls.2021.636915 INTRODUCTION Received: 02 December 2020 Accepted: 08 March 2021 Published: 31 March 2021 Crop diversity management in agriculture is a fundamental principle of agroecology and a powerful way to promote resilient and sustainable production systems (Gaba et al., 2015). Agroecosystem management oﬀers multiple alternatives to monoculture, such as diversiﬁcation with pulses, through intercropping or rotation. Their well-known ability to ﬁx atmospheric nitrogen reduces fossil energy consumption and makes them particularly suitable for low-input systems. They are also particularly important in human nutrition as a source of proteins to complement cereal-based diets (Singh et al., 2003; Tharanathan and Mahadevamma, 2003). Yet, the speciﬁc diversity of Citation: Front. Plant Sci. 12:636915. doi: 10.3389/fpls.2021.636915 March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 1 Guiguitant et al. Functional Diversity of Pulses Plant Material and Growing Conditions Plant Material and Growing Conditions A ﬁeld experiment was conducted at UCOSEM’s experimental farm in Lectoure (lat./long 43.911158/0.666182) on a deep clay- limestone soil. Three varieties for each one of the 10 pulses species (Table 1) were planted in two sequential batches/sub- experiments on contiguous ﬁelds of approximately 1 ha each. According to their temperature requirements: Cool season legumes were sown after the last negative temperatures, on March 14, 2018, and warm season legumes were sown on June 7, 2018, when daily temperature was over 15◦C. Each batch/sub- experiment was laid out in split plot design with four replicates, varieties being subplots and species the main plot. Plots were 6 m long and counted four rows. Preceding crop was sunﬂower, and ﬁelds were fertilized with 0/22/8 NPK (350 kg ha−1) fertilizer before sowing. Each species was manually inoculated before sowing with an adequate strain of rhizobia (provider: UMR LSTM, Montpellier, France). Sowing density was chosen according to agronomical recommendations for each species and ranged from 10 to 150 plants m−2. Manual weeding and drip irrigation were performed on a regular basis from sowing. Soil moisture was recorded weekly in each block and for each species at three depths (0–20, 20–40, and 40–60 cm) using TDR probes installed vertically at sowing (mini TRASETM system with buriable probes, Soilmoisture Equipment Corp., CA) and conﬁrmed that the plants did not experiment any water deﬁcit in none of the two sub-experiments. To increase diversity within cropping systems, choosing appropriate species is an important prerequisite (Brooker et al., 2015). The trait-based approach is well suited for designing diversiﬁed cropping systems capable of providing speciﬁc sets of services. In ecology, the approach has been increasingly employed to link biodiversity to ecosystem functioning and services (Lavorel and Garnier, 2002; Violle et al., 2007; Garnier and Navas, 2012); a methodology that could be applied to cropping systems (Litrico and Violle, 2015; Martin and Isaac, 2015; Wood et al., 2015; Damour et al., 2018). However, trait-based approach requires extensive data collection to develop trait databases for crop species or varieties and document relationships between traits and services (Barot et al., 2017). As a neglected group of species, pulses are particularly diﬃcult to document (Barot et al., 2017), for implementing a trait-based approach. In a previous study, Guiguitant et al. (2020) set up a database suitable for functional trait approach on pulses. Frontiers in Plant Science \| www.frontiersin.org Plant Material and Growing Conditions The database gathered disparate data from multiple published and/or public sources and contained data on plant functional traits for 43 pulse species, and agroecosystem properties recorded at canopy level for a subset of these species. The analysis of the functional trait space highlighted three ecological strategies among cultivated pulses that were similar to those already described in spontaneous species in the leaf-seed-height (LSH) scheme (Westoby, 1998). However, reduced data on agroecosystem properties and data disparity did not allow to identify clear link between traits and agroecosystem properties. In an applied perspective of supporting the integration of pulses in cropping systems, a consolidated analysis of trait–properties relationships is still needed to identify which traits would make legume species more productive, competitive, or adapted to low nitrogen or water conditions and favor production services. Trade-oﬀs between plant traits and how they determine the diﬀerent strategies also require more attention. MATERIALS AND METHODS legumes is poorly represented in most cropping systems. While more than 80 pulses species are known to contribute to human diet, the FAO database includes only 11 of them (Tiwari et al., 2011). This lack of attention creates a risk of continued erosion of knowledge and genetic resources that could further hamper the integration of these crops into more diversiﬁed cropping systems (Dansi et al., 2012). legumes is poorly represented in most cropping systems. While more than 80 pulses species are known to contribute to human diet, the FAO database includes only 11 of them (Tiwari et al., 2011). This lack of attention creates a risk of continued erosion of knowledge and genetic resources that could further hamper the integration of these crops into more diversiﬁed cropping systems (Dansi et al., 2012). Measurements of Individual Plant Traits Measurements of Individual Plant Traits Occurrence of phenological stages (date of 50% ﬂowering, date of physiological maturity, i.e., when more than 50% of the pods were yellow-brown) was recorded in calendar days and then converted into thermal units using a base temperature of 0◦C for Cicer arietinum and Vicia faba, 2◦C for Lens culinaris, 3.5◦C for Lathyrus sativus, 4.1◦C for Trigonella foenum-graecum (Covell et al., 1986; Lamichhane et al., 2020), 4.7◦C for Lotus tetragonolobus (Moot et al., 2000), 6◦C for Phaseolus vulgaris (López et al., 2003), and 8.5◦C for Vigna sp. (Craufurd et al., 1996). This allowed to compute degree days from sowing to ﬂowering (FLO) and degree days from sowing to maturity (MAT). The aim of the present study was to identify pulses traits and/or trait combinations that can be used as predictors of yield and supporting agroecosystem services, further accounting for the relationships between traits and between agroecosystem properties. Original data were collected on 30 pulse varieties from 10 diﬀerent species monitored in similar environmental conditions. Agroecosystem properties were measured at the canopy level, whereas functional traits, i.e., morphological, physiological, or phenological traits supposed to have direct or indirect eﬀects on agroecosystem properties, were measured at the individual or organ level. We combined these original data collected within uniform environmental conditions with literature data collected in diverse agronomic situations (see Guiguitant et al., 2020) to assess the eﬀect of trait measurement conditions on trait values and whether it could distort the relationships between traits and agroecosystem properties. Biomass was harvested at ﬂowering stage and maturity. At each sampling date, plants were collected on 0.5 m2 quadrats, in the two central rows to avoid border eﬀect. Plant number was recorded, and then leaves, stems, ﬂowers, and pods were detached and then dried separately at 60◦C until constant weight. Leaf and stem dry mass ratio over total aboveground plant mass (LMR and SMR, respectively) was calculated at ﬂowering, and harvest index (HI) was calculated at maturity. Leaf area ratio (LAR; calculated as total plant leaf area (LA) over total aboveground mass), total LA (mm2 plant−1), speciﬁc LA (SLA, cm2 g−1), and leaﬂet length (LL, mm) as well as leaﬂet width (LW, mm) were measured at ﬂowering (mean of the measurement of every leaf of a plant sub-sample), whereas thousand seed weight (TSW, g) was determined at harvest. Measurements of Individual Plant Traits March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 2 Functional Diversity of Pulses Guiguitant et al. TABLE 1 \| List of the 10 species included in the experiment with a description of the three varieties associated to each species, the abbreviation (Abb.) used in the study, sources of the seeds, and the ß value used for %Ndfa computation. TABLE 1 \| List of the 10 species included in the experiment with a description of the three varieties associated to each species, the abbreviation (Abb.) used in the study, sources of the seeds, and the ß value used for %Ndfa computation. Species Varieties Abb. Source β (h) Vigna mungo ISRA 58-77 (V1), ISRA 66-41 (V2), ISRA 67-30 (V3) Vmu ISRA, Sénégal −1,75 Vigna unguiculata YACINE (V1), SAM (V2), IT98K-1092-1-1 (V3) Vun ISRA, Sénégal −1,61 Vigna aconitifolia SH_VM (V1), SH_VA (V2), Tvu 45 35 (V2) Vac ISRA, Sénégal −0,91 Lotus tetragonolobus Landrace Lte Palmbeach medicinal herb, rareexoticseed, hobbyseed −0,12 Phaseolus vulgaris Red kidney (V1), zorro (V2), michelet (V3) Pvu Epi de Gascogne, France −2,16 Cicer arietinum Elmo (V1), billy bean (V2), orion (V3) Car Epi de Gascogne, France; USDA Pullman, WS −1,75 Lens culinaris Belezana (V1), Richela (V2), Anicia (V3) Lcu Epi de Gascogne, USDA Pullman, WS −0,56 Trigonnella foenum-graecum FENUSOL, landrace Tfo Epi de Gascogne, France; ICARDA, Morroco −0,42 Lathyrus sativus Wassie (V1), Almenaza (V2), Chicharo (V3) Lsa Epi de Gascogne, France −0,38 Vicia faba Aguadulce (V1), alﬁa (V2), lobaba (V3) Vfa ICARDA, Morroco −0,5 TABLE 2 \| List of agroecosystem properties and traits measured in the experiment with their respective abbreviation and unit. TABLE 2 \| List of agroecosystem properties and traits measured in the experiment with their respective abbreviation and unit. Total nitrogen content was determined (Vario PyroCube auto- analyzer and Precision mass spectrometer) on leaves, stems, and grain samples collected at maturity and at ﬂowering. Total plant nitrogen content was computed both at ﬂowering and maturity. Leaf nitrogen content (LNC, mg g−1), and seed nitrogen content (SNC, mg g−1) were determined at ﬂowering and at maturity, respectively. TABLE 2 \| List of agroecosystem properties and traits measured in the experiment with their respective abbreviation and unit. Measurements of Individual Plant Traits Variable Abbreviation Unit Agroecosystem property Grain yield GY t ha−1 Biomass yield BY t ha−1 Leaf area index LAI % Nitrogen derived from atmosphere at maturity Ndfa_MAT % % Nitrogen derived from atmosphere at ﬂowering Ndfa_FLO % Water use efﬁciency WUE kg ha−1 mm−1 Biomass at ﬂowering B_FLO t ha−1 Maximal soil cover Maximum soil cover % Soil cover duration Soil cover duration Thermal unit Traits Leaf area LA mm2 Speciﬁc leaf area SLA cm2 g−1 Leaﬂet length LL mm Leaﬂet width LW mm Leaf nitrogen content LNC mg g−1 Plant N content at ﬂowering Nplant_FLO g plant−1 Plant N content at maturity Nplant_MAT g plant−1 Phyllochron Phyllochron Thermal unit Soil cover rate Cover rate Degree days to maturity MAT Thermal unit Degree days to ﬂowering FLO Thermal unit Thousand seed weighed TSW G Leaf area ratio LAR – Leaf mass ratio LMR – Stem mass ratio SMR – Harvest index HI – Seed nitrogen content SNC mg g−1 Phyllochron was calculated from observations of three tagged plants on each plot for which emerged leaves were counted weekly (cool season legumes) or twice a week (warm season legumes). The rate of soil coverage was estimated by measuring soil cover weekly, using nadiral photos taken at the same location within each plot, with a large angle camera at chest height, and then analyzed with ImageJ software (Schneider et al., 2012). Soil cover was modeled as a logistic function of thermal time. The maximal rate of soil coverage was deﬁned as the slope at the inﬂexion point of the logistic curve. Consistency of Pulses Functional Trait Space A PPCA was performed on the 43-species-database from Guiguitant et al. (2020) using only traits that were also measured in the present ﬁeld experiment. The ﬁrst three PC axes of this PPCA explained 70% of total variance observed in the literature (Figure 1). PC1 (34%) was associated with morphological traits, especially leaf traits (LL, LW, LA, and to a lesser extend TSW). PC2 (24%) was positively associated with late ﬂowering (DF) and maturity (DM) and to a lesser extend HI. PC3 (12%) was strongly associated to SLA and LNC. Experimental data variability on PC axes was in the same range as the literature variability. Experimental variance was close to 100 and 60% of literature variance on PC1 and PC2, respectively. On the contrary, the variance of supplementary individuals on the third axis (driven by SLA and LNC) was almost three times higher than the variance of active data from the literature (Appendix 1). For all other traits, ﬁeld measurements range was always narrower than the one observed in Guiguitant et al. (2020), for a larger set of species (Appendix 1). y Second, a principal component analysis (PCA) was performed on experimental data including all traits measured. Agroecosystem properties were added as supplementary variables, in order to investigate how they ﬁt into the phenotypic space deﬁned by the traits. The appropriate number of PCs was determined with Kaiser’ criterion which selects components that correspond to eigenvalues larger than 1. A between and within-class correspondence analysis was carried out to evaluate the percentage of variance captured by the intra and inter-speciﬁc diversity (Doledec, 1987). p Distance between the barycenters of each species as tested in the present experiment and from the literature database was short in the ﬁrst plane of the PPCA (Figure 1). Species ranking in literature versus present data remained conserved on PC1 (ρ = 0.85, p = 0.003) and to a lesser extent on PC2 (ρ = 0.53, p = 0.10). Species rankings were rather inconsistent on PC3 (ρ = 0.24, p = 0.12). Cool season species had barycenters closely positioned along the three dimensions of the PPCA (Euclidian distance lower than 2; Table 3). Among them, C. arietinum and T. foenum-graecum had the highest Euclidian distance mostly because of the divergence with literature on the third axis, while V. faba diverged mostly on the second axis. RESULTS Data were ﬁrst analyzed through the prism of the framework presented in Guiguitant et al. (2020) in order to compare the variability observed with the global functional diversity of pulses species. Data of the present ﬁeld measurements were projected as supplementary individuals in a probabilistic principal component analysis (PPCA) (Tipping and Bishop, 1999) computed with nine common functional traits (DM, DF, HI, LL, LW, LA, SLA, LNC, and TSW) values taken from Guiguitant et al. (2020) database. We used Euclidian distance to quantify the distance between the scores of barycenters of each species as calculated from ﬁeld data and previously published in Guiguitant et al. (2020). The consistency of species ranking in trait values across literature and experimental data was tested with Spearman’s rank correlation analysis. Consistency of Pulses Functional Trait Space For warm season species, distances between barycenters from the literature and from the present experiment were larger, on PC3 but also on PC2 for Vigna sp. Finally, trade-oﬀs among agroecosystem properties were analyzed using a hierarchical clustering analysis (HCA). A classiﬁcation and regression tree (CART) analysis was done on raw data (n = 120) to identify how functional traits could allow to predict the clusters of varieties obtained from the HCA. Regression trees are prediction models obtained through machine-learning algorithms that recursively partition the data space in order to ﬁt the simplest prediction model within each partition. The resulting partitioning can be represented graphically as a decision tree. The random forest aggregation method, which consists in adding a random component to the choice of variables in the model, was also used. This method does not allow to generate a visual decision tree, but it provides indices proportional to the importance of each variable in the aggregated model and thus its participation to the discrimination between groups of individuals (here, varieties). Mean decrease in Gini impurity (MDG) can be deﬁned as the total decrease in node impurity (weighted by the proportion of samples reaching a given node) averaged across all of the trees that make up the forest. The most important variables to the model will have the largest MDG values; conversely, the least important variable will have the smallest MDG values. Measurement of Agroecosystem Properties p Agroecosystem properties were measured on the same experimental plots (Table 2). Grain yield (GY, t ha−1), biomass yield (BY, t ha−1), and biomass measured at ﬂowering (t ha−1) were determined from plant sampling on 0.5 m2 quadrats. Water use eﬃciency (WUE) was computed as grain mass produced per mm of water input (rainfall and irrigation). Maximum soil cover (%), LA index (LAI), and the duration (in thermal units) of the phase when soil coverage is above 80% of the maximum (soil cover duration) were recorded. Finally, the capacity to ﬁx nitrogen from the atmosphere was estimated through the percent of nitrogen derived from biological ﬁxation at ﬂowering and at maturity, measured with the natural isotopic dilution method, using barley and maize grown in the experiment outer borders as control plants for cool season and warm season legumes, respectively. From the 15N abundance (d15N) of the samples, the proportion of plant N derived from atmosphere (%Ndfa) was calculated from Eq. 1 (Shearer and Kohl, 1986): Where β is the 15N natural abundance of the N derived from biological nitrogen ﬁxation (BNF) in the aerial tissue of the legume, δ15Nref is the 15N abundance in the control plant (maize or barley) tissues, and δ15Nlegume is the 15N abundance in legume tissues. The β values used are detailed in Table 1 (Unkovich et al., 2008). %Ndfa = 100 δ15Nref −δ15Nlegume δ15N−β ref (1) March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 3 Guiguitant et al. Functional Diversity of Pulses Frontiers in Plant Science \| www.frontiersin.org Characterization of Functional Trait Diversity The ﬁrst four axes of the PCA performed on 30 individuals and 16 traits from the present experiment (Table 2) captured 80% of the total variance (Figure 2). Interspecies variance explained 81% of the total variance, while intra-species explained 19%. Leaf attributes were strongly associated with PC1 (40%). On the ﬁrst principal component (PC1), LA, LL, and plant N content at ﬂowering were negatively correlated with SLA, LNC, SNC, and LAR. This axis discriminated cool season species which were characterized by small leaﬂets and small LA with high leaf and seed nitrogen content and a large LAR, from warm season species characterized by large leaﬂet, high LA, and high plant nitrogen content. PC2 (20%) was positively correlated to LMR and negatively correlated to SMR and cover rate. The cover rate was signiﬁcantly negatively correlated to MAT (ρ = –0.72, p < 0.001). SMR was also signiﬁcantly negatively correlated to the phyllochron (ρ = –0.60, p < 0.001). L. tetragonolobus and All statistical analyses were performed in the computing environment R 3.5.2 (R Core Team, 2018) using pcaMethods package (Stacklies et al., 2007), rpart package (Therneau et al., 2015), ade4 package (Dray and Dufour, 2007), and randomForest package (Breiman, 2001). March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 4 Functional Diversity of Pulses Guiguitant et al. FIGURE 1 \| Principal component analysis (PCA) performed on nine functional traits collected on 43 pulses species. (A,C) Individual species projection on the ﬁrst three axes of the PCA; light gray points represent the 43 species from literature (Guiguitant et al., 2020) and colored points are from the current experimental study projected as supplementary individuals. Transparent points are the data of the four plots for the three varieties of the 10 species, the larger opaque points represent the barycenter of each species, and the horizontal and vertical bars represent the standard deviation along axes. Visualization of the variables and correlation circles on (B) PC1–PC2 and (D) PC2–PC3 planes. See Table 2 for abbreviations of traits. FIGURE 1 \| Principal component analysis (PCA) performed on nine functional traits collected on 43 pulses species. (A,C) Individual species projection on the ﬁrst three axes of the PCA; light gray points represent the 43 species from literature (Guiguitant et al., 2020) and colored points are from the current experimental study projected as supplementary individuals. Characterization of Functional Trait Diversity Transparent points are the data of the four plots for the three varieties of the 10 species, the larger opaque points represent the barycenter of each species, and the horizontal and vertical bars represent the standard deviation along axes. Visualization of the variables and correlation circles on (B) PC1–PC2 and (D) PC2–PC3 planes. See Table 2 for abbreviations of traits. positively correlated to plant N content at ﬂowering (ρ = 0.41, p = 0.02). Ndfa at maturity was not correlated to Ndfa at ﬂowering (ρ = 0.04, p = 0.83) but was signiﬁcantly positively correlated to LNC (r = 0.49, p = 0.005) and SLA (ρ = 0.57, p = 0.001) as well as with SMR (ρ = 0.43, p = 0.02). The soil cover duration was inversely correlated to LAI (ρ = –0.21, p = 0.0428) and biomass at ﬂowering (ρ = –0.22, p = 0.033) and signiﬁcantly positively correlated with FLO (ρ = 0.47, p = 0.009) and to a lesser extent with MAT (ρ = 0.33, p = 0.08). Maximum soil cover was not correlated to soil cover duration (ρ = –0.03, p = 0.86) and was slightly associated with SMR (ρ = 0.35, p = 0.05). Finally, GY and WUE were positively correlated to PC3 which was mostly determined by FLO, whereas BY, and to a lesser extent GY and WUE, were positively associated to PC4 alongside TSW. P. vulgaris were apart from all other species on PC2, and both expressed a high LMR at ﬂowering. PC3 (11%) and PC4 (9%) were associated to FLO and TSW, respectively. PC3 separated V. faba from L. culinaris and L. tetragonolobus, whereas PC4 discriminated C. arietinum and P. vulgaris. All agroecosystem properties, except LAI, biomass at ﬂowering and Ndfa at maturity were not well represented in the factor planes formed by the fourth PCs constructed with traits (Figure 2). LAI and biomass at ﬂowering were relatively well represented in the ﬁrst plane and were signiﬁcantly positively correlated to LL (ρ = 0.46, p < 0.001; ρ = 0.63, p = 0.01, respectively) and LA (ρ = 0.67, p < 0.001; ρ = 0.83, p < 0.001, respectively) as well as cover rate (ρ = 0.59, p < 0.001; ρ = 0.60, p < 0.001, respectively). Trade-Offs Between Agroecosystem Properties The classiﬁcation analysis revealed seven clusters of species and varieties based on their agroecosystem properties (Figure 3). All varieties of a species generally grouped into the same cluster. V. aconitifolia varieties were all in cluster 3 together with one variety of Vigna unguiculata. This cluster is apart from a group of close clusters composed essentially by cool season species; L. sativus varieties were grouped with L. tetragonolobus (cluster 4), L. culinaris was alone in cluster 5, cluster 6 was dominantly occupied by T. foenum-graecum, and cluster 7 grouped both V. faba and Vigna mungo. These groups were distant from cluster 1 essentially represented by C. arietinum and cluster 2 that contained varieties of P. vulgaris, and of V. unguiculata. Relying on MDG, plant N content at maturity was the second most important trait to discriminate the clusters. Phyllochron, SNC, SLA, and LL were not selected in the tree and had very low mean decrease in Gini value. LAR was not used in the trees, but had a higher mean decrease in Gini value than LA and LMR. Yet, LA and LMR captured more variability in the PCA than LAR. Finally, cover rate did not appear on the tree despite its high mean decrease in Gini value, probably due to its negative correlation with DDR. Each cluster was characterized by a combination of values of agroecosystem properties. Cluster 1 grouped species with high production (GY and BY) as well as a high maximum soil cover and soil cover duration, but a low Ndfa and a low biomass and LAI at ﬂowering. Cluster 2 was characterized by a low biomass at ﬂowering as well as a low LAI at ﬂowering and maximum soil cover but a high Ndfa at ﬂowering and a long soil cover duration. Cluster 3 varieties expressed the lowest yield as well as the lowest WUE but high Ndfa at maturity, a high LAI at ﬂowering, and a long cover duration. Cluster 4 varieties expressed the highest WUE concomitantly with a high GY and a low Ndfa at both stages. Varieties of cluster 5 expressed high Ndfa both at ﬂowering and maturity and high maximum soil cover. Yet, they showed a low maximum soil cover and soil cover duration. Traits Related to Resource Acquisition Are More Plastic Across Growing Conditions and Varieties Than Architectural Traits Through functional traits, researchers usually seek to make generalizable predictions across organizational and spatial scales (Adler et al., 2013) and therefore usually use data collected in diverse environment and management contexts. On the contrary, ﬁeld measurements sharing similar environment as in the present study deliver a local picture of functional diversity which variations can be solely attributed to genotypic determinants. Several studies have demonstrated that despite the signiﬁcant plasticity of most plant traits across environments, the classiﬁcation of species for a given trait remains fairly consistent in space and time (Garnier et al., 2001; Kazakou et al., 2014; Tribouillois et al., 2015). Yet, other studies have reported a signiﬁcant eﬀect of plasticity on species ranking across environments, particularly with respect to traits associated with light acquisition and nitrogen nutrition (Lipowsky et al., 2015; Siebenkäs et al., 2015; Keenan and Niinemets, 2017). These inconsistencies may impair the use of simple aggregation functions (means, medians, etc.) for generalized trait-based approaches at the species level. Trade-Offs Between Agroecosystem Properties Cluster 6 was characterized by a low BY and GY, low Ndfa both at ﬂowering and maturity, and poor soil covering capacity, yet LAI and soil cover at ﬂowering were high. Finally, cluster 7 was composed of varieties producing the highest biomass at ﬂowering, along with the highest maximum soil cover, LAI, and Ndfa at ﬂowering. Characterization of Functional Trait Diversity Although Ndfa at ﬂowering was poorly represented on the ﬁrst PC, this property was signiﬁcantly March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 5 Functional Diversity of Pulses Guiguitant et al. TABLE 3 \| Euclidian distance between literature and current study coordinates of the barycenter of each species on the ﬁrsts three PCA axes. TABLE 3 \| Euclidian distance between literature and current study coordinates of the barycenter of each species on the ﬁrsts three PCA axes. allowed to gain purity in the process of classiﬁcation for most of the nodes in the CART it was associated with (Figure 4B). LNC was assigned to the ﬁrst division node and allowed to sort cluster 1 varieties apart from the remaining ones. Cluster 1 varieties have an LNC between 2.7 and 3 mg N g−1. Varieties of cluster 4 were separated. Half of them fell on the same side of the tree as cluster 1 but exhibited lower LNC value (below 2.7 mg N g−1), while the other half had an average LNC above 3 mg N g−1. Varieties in this second cluster 4 half also had an LMR above 43% concomitantly with low plant N content at ﬂowering and at maturity and a longer cycle. LMR, DDR, and plant N content at maturity had strong MDG unlike the plant N content at ﬂowering. Varieties with an LMR under 0.43 and having high MAT were assigned either to cluster 3 (characterized by a high plant N content but only at ﬂowering), or cluster 2 or 5 (both characterized by high plant N content at ﬂowering and maturity). Varieties in cluster 2 ﬂowered later than those in cluster 5. Short cycle varieties were dominantly associated to cluster 6 but were also found in cluster 5 if they also expressed a high LA. Finally, cluster 7 was characterized by an LMR under 43%. the barycenter of each species on the ﬁrsts three PCA axes. Species PC1 PC2 PC3 Three PCs Cicer arietinum 1.1 0.39 1.25 1.68 Lathyrus sativus 1.0 0.18 0.11 1.03 Lens culinaris 0.4 0.25 1.29 1.39 Lotus tetragonolobus 0.9 1.41 1.74 2.40 Phaseolus vulgaris 0.4 0.92 3.74 3.87 Trigonella foenum-graecum 0.8 0.44 1.73 1.97 Vicia faba 1.1 1.20 0.22 1.64 Vigna aconitifolia 1.0 2.61 2.96 4.08 Vigna mungo 0.6 1.21 1.76 2.23 Vigna unguiculata 0.7 1.23 2.39 2.77 Relevant Traits for Prediction of Agroecosystem Properties and Structuration of Functional Diversity Classiﬁcation and regression tree analysis showed very diverse trait combinations to predict the cluster aﬃliation (Figure 4A). LNC was identiﬁed as the most important trait to discriminate the clusters as it had the highest MDG value, which means that it March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 6 Functional Diversity of Pulses Guiguitant et al. FIGURE 2 \| Principal component analysis (PCA) performed on traits measured on three varieties of 10 species. Visualization of the variables and correlation circles on (A,C) PC1–PC2 and (B,D) PC3–PC4 planes. Gray scale represents the contribution of each variable to axes construction. Orange variables are agroecosystem properties, used as supplementary variables. See Table 2 for abbreviations of traits and properties. Soil cover stands for maximal soil cover. FIGURE 2 \| Principal component analysis (PCA) performed on traits measured on three varieties of 10 species. Visualization of the variables and correlation circles on (A,C) PC1–PC2 and (B,D) PC3–PC4 planes. Gray scale represents the contribution of each variable to axes construction. Orange variables are agroecosystem properties, used as supplementary variables. See Table 2 for abbreviations of traits and properties. Soil cover stands for maximal soil cover. FIGURE 2 \| Principal component analysis (PCA) performed on traits measured on three varieties of 10 species. Visualization of the variables and correlation circles on (A,C) PC1–PC2 and (B,D) PC3–PC4 planes. Gray scale represents the contribution of each variable to axes construction. Orange variables are agroecosystem properties, used as supplementary variables. See Table 2 for abbreviations of traits and properties. Soil cover stands for maximal soil cover. In the present study, we compared common ﬁeld measurements to the meta-analysis by Guiguitant et al. (2020) to analyze how environmental and management conditions could alter trait values and distort their relationships. When using the experimental dataset, where all data were acquired in a common environment, species rankings on the PC axis summarizing the variation of architectural traits were fairly consistent with literature ranking. However, rankings on the axis summarizing SLA and LNC variation diverged between the two datasets. This contrast mainly concerned warm-season species, particularly Vigna sp. for which divergence between the two datasets was also noted in terms of phenology. Number of days required to reach maturity was generally lower in literature than in experimental data. Relevant Traits for Prediction of Agroecosystem Properties and Structuration of Functional Diversity This comparison between local measurements and literature averages suggests that some traits are more plastic than others and therefore contribute diﬀerently to frame legumes diversity depending on the conditions of observation. Frontiers in Plant Science \| www.frontiersin.org Intraspeciﬁc Variation Contributed Only Marginally to Global Variability Results from the PCA showed that the total trait variance was mainly explained by the variance between species. Variation in intraspeciﬁc traits is generally considered of marginal importance compared to diﬀerences in interspeciﬁc traits (McGill et al., 2006; Hulshof and Swenson, 2010). However, several recent studies have shown that intraspeciﬁc variation in traits accounts for a substantial proportion of overall plant community variability (Jung et al., 2010; Albert et al., 2011; Roscher et al., 2015; March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 7 Guiguitant et al. Functional Diversity of Pulses FIGURE 3 \| Hierarchical clustering analysis (HCA) of the 30 pulse varieties based on mean value of agroecosystem properties (n = 3). The names of varieties belonging to the same species are written in the same color. Warm and cool season species are depicted with yellow and green bars, respectively. Radars represent the mean (standardized to maximum) value of agroecosystem properties of each cluster. Colors in radar plots correspond to branch color in the HCA. FIGURE 3 \| Hierarchical clustering analysis (HCA) of the 30 pulse varieties based on mean value of agroecosystem properties (n = 3). The names of varieties belonging to the same species are written in the same color. Warm and cool season species are depicted with yellow and green bars, respectively. Radars represent the mean (standardized to maximum) value of agroecosystem properties of each cluster. Colors in radar plots correspond to branch color in the HCA. Siefert et al., 2015). The small number of varieties considered in this study may have not been able to highlight such diversity. Siefert et al., 2015). The small number of varieties considered in this study may have not been able to highlight such diversity. The experimental data conﬁrmed that LL is an important determinant of legume diversity. This trait was strongly related to LNC and to a lesser extent SLA. This ﬁnding can be related to the well know leaf economic spectrum (LES; Wright et al., 2004). The LES reﬂects a trade-oﬀbetween a leaf’s lifespan and its maximum net photosynthetic rate. Yet, relations between LNC and photosynthesis in grain legumes have been questioned (Adams et al., 2016). Thus, the fact that this axis reﬂects a gradient in leaf photosynthetic capacity is uncertain. Intraspeciﬁc Variation Contributed Only Marginally to Global Variability However, a high LNC could have other biological implications such as remobilization to the reproductive organs (Davies et al., 2000; Schiltz et al., 2005). Indeed, SNC was correlated with LNC in our experiment. Small leaﬂet associated with a high LNC could support the growth of N-rich legume seeds through remobilization. CONCLUSION Integrating pulses in resilient and sustainable production systems requires a better understanding of their functional diversity and how this functional diversity scales up to provide speciﬁc sets of agroecosystem services. Previous studies demonstrated the usefulness of trait value aggregation at the species-level to implement trait-based approaches for a better knowledge of the plant-agroecosystem functioning relationships and increasing our predictive capacity (Lavorel and Garnier, 2002; Violle et al., 2007; Garnier and Navas, 2012; Kazakou et al., 2014; Martin and Isaac, 2015; Wood et al., 2015; Guiguitant et al., 2020). Our experimental results highlighted trade-oﬀs and synergies between agroecosystem properties supported by diﬀerent pulses species. We were able to demonstrate the relevance of LNC, LAR, and phenology to predict most of the trade-oﬀs observed for agroecosystem properties in those conditions. Integrating pulses in resilient and sustainable production systems requires a better understanding of their functional diversity and how this functional diversity scales up to provide speciﬁc sets of agroecosystem services. Previous studies demonstrated the usefulness of trait value aggregation at the species-level to implement trait-based approaches for a better knowledge of the plant-agroecosystem functioning relationships and increasing our predictive capacity (Lavorel and Garnier, 2002; Violle et al., 2007; Garnier and Navas, 2012; Kazakou et al., 2014; Martin and Isaac, 2015; Wood et al., 2015; Guiguitant et al., 2020). Our experimental results highlighted trade-oﬀs and synergies between agroecosystem properties supported by diﬀerent pulses species. We were able to demonstrate the relevance of LNC, LAR, and phenology to predict most of the trade-oﬀs observed for agroecosystem properties in those conditions. The multi-faceted beneﬁts of cover crops are well known (Blanco-Canqui et al., 2015), but land cover is often not considered for cash crops and the interaction between cover capacity and yield is rarely discussed. Maximum cover and cover duration appeared to be related to biomass at ﬂowering and LAI as well as to yield properties. Cluster 7 expressed an important cover at ﬂowering as a result of an important biomass and a large LA. However, cluster 1 and 3 had a low biomass at ﬂowering and a relatively high maximum cover that lasts long, probably because of a delayed senescence. Except for cluster 1, most of those species had low yield. Delayed senescence, by prolonging photosynthesis, is usually expected to contribute to increasing the yield of high- carbon crops such as cereals (Gregersen et al., 2013). Leaf Morphological Traits Conﬁrmed as Variability Drivers Among Pulses Guiguitant et al. (2020) found that seed and leaf traits exhibited the greatest variability among 43 pulse species. In the present study, leaf traits (LA and LL) remained an important variability driver that was correlated to the ﬁrst PCA axis, whereas seed size, associated to the ﬁrst axis in the literature analysis, poorly contributed to the total variance across varieties (associated only with the fourth PCA axis) and was decoupled from leaf size, which is consistent with LHS scheme (Westoby, 1998). March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 8 Guiguitant et al. Functional Diversity of Pulses FIGURE 4 \| (A) Classiﬁcation and regression tree (CART) regression for prediction of membership to one of the seven clusters based a hierarchical clustering analysis of the 30 pulse varieties based on agroecosystem properties (see Figure 3). Root nodes represent single input variables (traits) and related split point used to make the prediction; the best discriminant is chosen at each step of the classiﬁcation procedure. Leaf nodes contain mean, number, and percentage of observations of the predicted variable (cluster membership). (B) Gini decrease value for each trait obtained through a random forest procedure. A high Gini decrease value means the variable considered allowed to gain purity in the process of classiﬁcation for most of the nodes in the CART it was associated with during the random forest procedure. FIGURE 4 \| (A) Classiﬁcation and regression tree (CART) regression for prediction of membership to one of the seven clusters based a hierarchical clustering analysis of the 30 pulse varieties based on agroecosystem properties (see Figure 3). Root nodes represent single input variables (traits) and related split point used to make the prediction; the best discriminant is chosen at each step of the classiﬁcation procedure. Leaf nodes contain mean, number, and percentage of observations of the predicted variable (cluster membership). (B) Gini decrease value for each trait obtained through a random forest procedure. A high Gini decrease value means the variable considered allowed to gain purity in the process of classiﬁcation for most of the nodes in the CART it was associated with during the random forest procedure. March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 9 Functional Diversity of Pulses Guiguitant et al. Trait-Based Predictions of Agroecosystem Properties Understanding the links between functional traits and agroecosystem functioning is fundamental for optimal management of agroecosystem services (Zhang et al., 2007; Litrico and Violle, 2015; Wood et al., 2015). As noted earlier, leaf characteristics and especially LNC were crucial in describing pulse diversity. Other traits such as LAR, total nitrogen, seed size, and cycle length are also important, which is consistent with the study of Guiguitant et al. (2020). The decision tree did not highlight all these traits but discriminated speciﬁc functional characteristics for each cluster. Clusters 1 and 4 (high yielding species) were characterized by low LNC values which is consistent with their low N ﬁxation. Cluster 7 (high biomass at ﬂowering) was characterized by a large LA but a low biomass investment in leaves. Clusters 6 and 5 (medium maximum soil cover and low value for every other properties) were characterized by a short cycle, with cluster 5 (high nitrogen DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author/s. Yield Is Inversely Related With Ndfa and Soil Cover ﬁxation) having a higher LA. Clusters 2 (medium value for every properties) and 3 (high N ﬁxation at maturity and long cover duration) were characterized by a long cycle and high nitrogen content at maturity or ﬂowering, respectively. Nitrogen ﬁxation by legumes is generally expected to contribute to improving soil fertility through the mineralization of crop residues such as senescent leaves or stems, or roots. In the current study, Ndfa at ﬂowering and Ndfa at harvest in grain tissue varied almost independently. Clustering reveled three types of “high ﬁxing species”: cluster 7, cluster 3, and cluster 5. On the other hand, cluster 1 or 6 expressed a low N ﬁxation capacity. “High yielding” species were clustered in cluster 1 and, to a lesser extent, cluster 4 both concerned by low ﬁxation. Cluster 5 ﬁxed a lot of N during ﬂowering and produced a decent yield. However, yield in cluster 5 was lower than that of clusters 1 or 4. Conversely, the very low yielding species (cluster 3) expressed a signiﬁcant increase in relative Ndfa between ﬂowering and maturity. Other multiple species comparisons conﬁrmed the negative relation between BNF and seed yield (Liu et al., 2019). Some studies reported BNF over a certain level results in a reduction of yield (Mesﬁn et al., 2020). To conclude on the highlighted linkages, a high LNC value does not translate into higher yields but is generally associated with a high N ﬁxation. Investment in LA is important for N2 ﬁxation at ﬂowering but is associated with rapid senescence and low yield. Biomass investment in leaves and a short cycle coincide with low values for almost every property except maximum soil cover. Finally, long cycle and high N content at maturity are related to delayed senescence and maintenance of N2 ﬁxation along pod ﬁlling. Frontiers in Plant Science \| www.frontiersin.org CONCLUSION However, studies suggest that the stay-green trait may have limited value for high N species (Ismail et al., 2000; Kumudini, 2002) as its value lies in an improvement of remobilization capacity. This corroborates the absence of relation we found between yield and duration of ground cover. However, the present study advocates further for a systematic characterization of the functional diversity (and its plasticity), including intra-speciﬁc variability. We argue that the trait-based approach may be easier to implement in crop species where varieties origin and genetics are often better described and accessible, compared with the huge genetic variation encountered in populations of spontaneous species. Application of such trait-based approach would rapidly beneﬁt the selection of species/varieties for speciﬁc targeted agroecosystem services provisioning under speciﬁc (environmental or management) conditions. 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FUNDING This work was funded by the European Programme ARIMNet/ERA-NET, FP7-KBBE, session 2017–2020, SEMIARID, grant agreement no. 618127. ACKNOWLEDGMENTS of the manuscript. DV and HM contributed substantially to the manuscript through extensive revision. HM designed the experiment. DV participated to data analyses. The authors thank Laurent Palau and UCOSEM team in Lectoure for having the ﬁeld experiment on their farm and ensuring proper crop husbandry, and for providing genetic resources. The authors are also grateful to the LSTM lab (B. Brunel) and the USDA ARS winter legume breeding program at Pullman (WS) (R. McGuee) for providing, respectively, rhizobial inoculum as well as plant genetic resources. The authors also thank ISRA Senegal for sharing Vigna accessions. Programme 2017–2020, REFERENCES Trends Plant Sci. 20, 604–613. doi: 10.1016/j.tplants.2015.07.007 Doledec, S. (1987). Rythmes Saisonniers et Composantes Stationnelles en Milieu Aquatique. 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(2020). Mineral fertilizer demand for optimum biological nitrogen ﬁxation and yield potentials of legumes in Northern Ethiopia. Sustainability 12:6449. doi: 10.3390/ su12166449 Tiwari, B. K., Gowen, A., and McKenna, B. M. (2011). Pulse Foods: Processing, Quality and Nutraceutical Applications. Amsterdam: Academic Press. Tribouillois, H., Fort, F., Cruz, P., Charles, R., Flores, O., Garnier, E., et al. (2015). A functional characterisation of a wide range of cover crop species: growth and nitrogen acquisition rates, leaf traits and ecological strategies. PLoS One 10:e0122156. doi: 10.1371/journal.pone.012 2156 Moot, D. J., Scott, W. R., Roy, A. M., and Nicholls, A. C. (2000). Base temperature and thermal time requirements for germination and emergence of temperate pasture species. N. Z. REFERENCES J. Agric. Res. 43, 15–25. doi: 10.1080/00288233.2000. 9513404 R Core Team (2018). R: The R Project for Statistical Computing. 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Phenotypic plasticity to light and nutrient availability alters functional trait ranking across eight perennial grassland species. AoB Plants 7:lv029. doi: 10.1093/aobpla/plv029 Zhang, W., Ricketts, T. H., Kremen, C., Carney, K., and Swinton, S. M. (2007). Ecosystem services and dis-services to agriculture. Ecol. Econ. 64, 253–260. doi: 10.1016/j.ecolecon.2007.02.024 Siefert, A., Violle, C., Chalmandrier, L., Albert, C. H., Taudiere, A., Fajardo, A., et al. (2015). A global meta-analysis of the relative extent of intraspeciﬁc trait variation in plant communities. Ecol. Lett. 18, 1406–1419. doi: 10.1111/ele. Frontiers in Plant Science \| www.frontiersin.org March 2021 \| Volume 12 \| Article 636915 REFERENCES 12508 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Singh, B. B., Ajeigbe, H. A., Tarawali, S. A., Fernandez-Rivera, S., and Abubakar, M. (2003). Improving the production and utilization of cowpea as food and fodder. Field Crops Res. 84, 169–177. doi: 10.1016/S0378-4290(03)00148-5 Copyright © 2021 Guiguitant, Vile and Marrou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Stacklies, W., Redestig, H., Scholz, M., Walther, D., and Selbig, J. (2007). pcaMethods a bioconductor package providing PCA methods for incomplete data. Bioinformatics 23, 1164–1167. doi: 10.1093/bioinformatics/btm069 Tharanathan, R. N., and Mahadevamma, S. (2003). Grain legumes – a boon to human nutrition. Trends Food Sci. Technol. 14, 507–518. doi: 10.1016/j.tifs. 2003.07.002 March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 12 Functional Diversity of Pulses Guiguitant et al. Frontiers in Plant Science \| www.frontiersin.org March 2021 \| Volume 12 \| Article 636915 APPENDIX Appendix 1 \| Comparison of traits range for data measured in the present ﬁeld experiment over 30 varieties issued from 10 species of pulses (violet) and species-level data collected in literature in Guiguitant et al. (2020) for 43 species of pulses (yellow). Appendix 1 \| Comparison of traits range for data measured in the present ﬁeld experiment over 30 varieties issued from 10 species of pulses (violet) and species-level data collected in literature in Guiguitant et al. (2020) for 43 species of pulses (yellow). March 2021 \| Volume 12 \| Article 636915 Frontiers in Plant Science \| www.frontiersin.org 13
https://openalex.org/W2999307335	https://sciforum.net/paper/download/6497/manuscript	Faroese	null	Theoretical study on cation–π interaction in graphene fragments	null	2,019	cc-by	7,425	Theoretical study of cation–π interaction in graphene fragments Jorge A. Carrazana‐García1, Enrique M. Cabaleiro‐Lago1 and Jesús Rodríguez‐Otero 1 Departamento de Química Física, Facultade de Ciencias, Universidade de Santiago de Compostela. Campus de Lugo. Avenida Alfonso X El Sabio s/n, Lugo 27002, Spain. 2 Departamento de Química Física, Facultade de Química, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain. 1 Departamento de Química Física, Facultade de Ciencias, Universidade de Santiago de Compostela. Campus de Lugo. Avenida Alfonso X El Sabio s/n, Lugo 27002, Spain. 2 Departamento de Química Física, Facultade de Química, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain. Correspondence: jorge.carrazana@usc.com * Correspondence: jorge.carrazana@usc.com Keywords: cation‐π interaction, non‐bonding interaction, graphene 1 Departamento de Química Física, Facultade de Ciencias, Universidade de Santiago de Compostela. Campus de Lugo. Avenida Alfonso X El Sabio s/n, Lugo 27002, Spain. 2 Departamento de Química Física, Facultade de Química, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain. * Correspondence jo ge ca a a a@usc co 1. Introduction The cation‐π interaction is a very important non‐covalent interaction because of its presence in several fields of chemistry, biology and industry. Many inclusion complexes and other supra‐ molecular structures are formed via cation‐π contacts.1,2 The participation of cation‐π interactions in catalysis includes the stabilization of reactive intermediates and transition states. 3 , 4 This interaction plays a central role both in the function and in the structure5,6 of proteins and other macromolecules 7 , 8 and determines the action of enzymes, 9 biological receptors 10 and ionic channels.11 The understanding that adhesion of mussel proteins to underwater surfaces occurs via cation–π interactions in aqueous media12 provided new insights for the development of biomimetic underwater adhesives. Recently it has been reported that the presence of alkali metal cations in carbonaceous adsorbents intensifies the adsorption of hydrogen.13 As well, the replacement of metal electrodes by lithium inserted in carbon‐based anodes allowed the production of safer Li‐metal ion rechargeable batteries.14 These findings stimulate the interest of the rechargeable batteries and fuel cells industries in cation‐π interaction. From the theoretical point of view, understanding the nature of a cation‐π interaction is essential for its introduction into reliable and accurate classical force fields used in a wide sort of areas ranging from biomolecule simulations15 and drug design investigations16 to the modeling of metallic‐doped carbon cathodes17 and graphene‐based membranes.18 Since the discovering of this interaction many theoretical approaches explaining its physical origin have been devised.19 The interaction of the cation with the large, permanent quadrupole moment of aromatic rings was initially established as the driving force of the cation‐π interaction.20,21,22 Other investigations revealed that repulsion and induction contributions are important too.23,24 Finally, the recognition that also dispersion plays a very important role in the stability of cation‐π complexes of practical interest25 serves as a guide for their study, indicating that a proper description of dispersive effects is required in their theoretical investigation. In this study, the interaction of some metallic and organic cations with planar conjugated molecules that model graphene fragments of increasing size is theoretically investigated using DFT level of theory with an empirical description of dispersion. For gaining understanding in the nature of the cation‐π interaction a rigorous method of energy analysis is also applied, separating the interaction energy in physically meaningful contributions. The results obtained with organic fragments with an increasing number of conjugated rings can be extrapolated to extended π‐systems as graphene. Abstract: The interaction between cations and delocalized electronic clouds (the cation‐π interaction) occupies a very important place within non‐binding interactions. Its presence has long been recognized as fundamental for both, the structure and function, of proteins and other important biological molecules. Rechargeable batteries and fuel cells industries are also interested in cation‐π interaction and the use of graphene and similar carbon allotropes are investigated as promising alternatives in their technological applications. Reliable and practically applicable theoretical models of cation‐π interaction are needed for guiding these researches. In this work, the interaction of cations (Li+, Na+, K+, ammonium and guanidinium) with graphene fragments (from benzene to circumcoronene) is modeled using DFT level of theory. Linear scans (TPSS+D3/Def2TZVPP) that follow trajectories perpendicular to the central ring of the graphene fragments allow the location of the distance at which the strongest interaction takes place. Using the geometry of the minima, the interaction energy is decomposed in physically meaningful contributions using a SAPT(DFT) method. It is observed that benzene complexes systematically deviate from the trend followed by complexes with larger fragments, so this system does not constitute a good model for the study of cation‐π interaction in graphenes or other large conjugated molecules. While induction is the main contribution in complexes with Li+ and Na+, the stability of most of the complexes investigated depends on a balanced combination of the three contributions: electrostatic, induction and dispersion. Following the tendencies observed with organic fragments with an increasing number of conjugated rings, the results can be extrapolated to extended π‐systems as graphene. Keywords: cation‐π interaction, non‐bonding interaction, graphene Theoretical study of cation–π interaction in graphene fragments 2 of 11 2. Methods The structure of the polyatomic cations (Fig.1) and the graphene fragments (Fig. 2) were optimized at the TPSS+D3/Def2TZVPP level of theory using the Gaussian 09 suite of programs,26 confirming that the stationary points found in the potential energy surface correspond to minima (frequency calculations were avoided in the case of B19 = circumcoronene due to resource limitations). The same level of calculation and program were used in the computation of the Molecular Electrostatic Potential (Figs. 2 and 3). The cation‐π interaction was first studied by rigid scans performed by moving the cation along a path that passes through the molecular center and that it is perpendicular to the plane of the graphene fragment. The orientations of the polyatomic cations during these scans are shown in Fig. 1. BSSE‐free interaction energies were computed with the ORCA (ver 3.0.3) electronic structure package27 using the supermolecule approach also at the J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero ECSOC23 – 2019 Theoretical study of cation–π interaction inn graphene fragm gments 3 of 11 TPSS+D3/D the positio separation the interac intramono MOLPRO PBE0/cc‐pV terms, cor considered a term rep calculation with ΔEHF contributio set of tripl Therefore, suggested values are the dispers Def2TZVPP ons of the m n correspond ction energ omer correla (ver. 2015 VTZ levels rresponding d that Rep = presenting n employing F being the ons to the in le‐zeta qua dispersion by Helgake a combinat sion contrib P level of ca minima are ding to the gy were ob ation effect 5.1). 32 This of calculati g to repuls = E1 exch; Elec contributio g Hartree−F e counterpo nteraction e ality, disper values are er employin tion of the c bution extra alculation. O obtained b minima fou btained usi ts described s energy a ion. The con sion, electro c = E1 pol; Ind ons higher Fock wavefu oise‐ correc energy alrea rsion contri extrapolate ng cc‐pVDZ cc‐pVTZ ele apolated to b Once the po by interpola und in the s ing the sym d at the DF analysis w ntributions ostatic, ind d = E2 ind + E than secon unctions: δH cted interac ady reach v ibution con ed to the ba Z and cc‐pV ectrostatic, basis limit.3 otential cur ation. 2. Methods Using scans, phys mmetry‐ad FT level SA as perform obtained fr duction, and E2 exch‐ind + δH nd order a HF = ΔEHF − ction energ values close nverges very sis set limit VTZ basis se repulsion, a 35,36,37 rves for the g the cation sically signi apted pertu APT(DFT)28, med at the rom SAPT d dispersio HF; Disp = E nd it is ob (E1 exch + E1 gy at the H to the basis y slowly w t assuming a ets.33,34 Thus and inducti e dimers are n – graphen ificant contr turbation th ,29,30,31 imple e PBE0/cc‐p are groupe on contribu E2 disp + E2 exc btained fro 1 pol + E2 ind + HF level. W s set limit w with the bas a cubic dep s, the final S ion contribu e available, ne fragment ributions to heory with emented in pVDZ and ed into four utions. It is h‐disp. δHF is om a SAPT + E2 exch‐ind), While most with a basis sis set size. pendence as SAPT(DFT) utions, plus , t o h n d r s s T , t s . s ) s Figure 1. Or graphen rganic cations ne fragments s s: NH4 + = amm studied. monium and GGua+ = guaniddinium, showing their orien ntations respeect to the Figure 1. Or graphen rganic cations ne fragments s s: NH4 + = amm studied. monium and GGua+ = guaniddinium, showing their orienntations respeect to the J. A. Carrazanna‐García; E. M. Cabaleiro‐Laggo; J. Rodríguezz‐Otero 3. Results and Discussion The st fragments (MEP) dist this interac the electro can be qua tructures of of increasin tribution aro ction is the p static poten antified by t f the conjug ng size are p ound each m primary cau ntial of the π the number gated molec presented in molecule. C use for de fo π‐cloud is in of benzene cules emplo n Fig. 2 alon Cations are e ormation of nfluenced b e rings in ea oyed in this ng with the electrostatic f cation‐π co by the size o ach molecul study for r e Molecular cally attract omplexes. T of the molec e. representin Electrostat ted by the π The (negativ cule that in g graphene tic Potential π‐cloud and ve) value of this family e l d f y MEP v are represe the coordin of Fig. 3, t depth of th not good m the minimu values calcu ented in Fig nates of the there is an he MEP pro models for e um MEP va ulated follo g. 3 as a func e minimum important i ofiles, indic extended gr alue in the t wing the lin ction of the are determ influence o cating that s raphene frag trajectory st ne normal distance to mined by int of the size o small molec gments. For tudied appr to the centr o the plane o erpolation. of the π‐sys cules (anthr r molecules roaches slow ral ring of t of the molec As it can be stems on th racene and, s with four o wly to aroun the molecul cule. In each e observed he position , mainly, be or more ben nd ‐8 … ‐9 k les of Fig. 2 h trajectory in the inset and on the enzene) are nzene rings kcal/mol. 2 y t e e s ECCSOC23 – 2019 J. A. Carrazanna‐García; E. M. Cabaleiro‐Laggo; J. Rodríguezz‐Otero Theoretical study of cation–π interaction inn graphene fragm gments 4 of 11 Figure 2. St identify the Mol shown. tructure of the y the molecule lecular Electro e molecules u e reflects the n ostatic Potenti used in this st umber of benz ial (MEP) map tudy as mode zene rings in i apped on the e ls of graphen its structure. F lectronic dens e fragments. 3. Results and Discussion or each molec sity isosurface The acronym cule the distrib e of 0.0004 a.u m used to bution of u. is also Figure 2. St identify the Mol shown. tructure of the y the molecule lecular Electro e molecules u e reflects the n ostatic Potenti used in this st umber of benz ial (MEP) map tudy as mode zene rings in i apped on the e ls of graphen its structure. F lectronic dens e fragments. or each molec sity isosurface The acronym cule the distrib e of 0.0004 a.u m used to bution of u. is also Figure 3. Mo a functi in the sc rings in olecular electr ion of r, the di cans shown in n the graphene MEP /a.u. rostatic potent istance to the p n Fig. 4. The po e fragments ar 1 1.2 1. 0.025  0.02  0.015  0.01  0.005  tial (MEP) alo plane of the m osition and the e presented in .4 1.6 1.8 2 2. ong the line no molecule. This e energy of the n the inset. 2 2.4 2.6 2.8 3 r /Å B01 B03 B04 B05 B07 B B B B m ormal to the ce line is also th e MEP minim 3 3.2 3.4 3.6 3. Å B09 B11 B13 B19 min 0 2 4 17  15  13  11  9  7  MEP min (kcal/mol) entral ring of g he trajectory fo a ordered by th 8 4 4.2 4.4 4.6 4 6 8 10 12 14 1 MEP min (l r min (right) number of rings graphene fragm ollowed by the the number of 6 4.8 5 B01 B03 B04 B05 B07 B09 B11 B13 B19 min 6 18 20 1.8 2 2.2 2.4 2.6 2.8 left) ) r min (Å) ments as e cations benzene Figure 3. Mo a functi in the sc rings in olecular electr ion of r, the di cans shown in n the graphene rostatic potent istance to the p n Fig. 4. The po e fragments ar tial (MEP) alo plane of the m osition and the e presented in ong the line no molecule. This e energy of the n the inset. J. A. Carrazanna‐García; E. M. Cabaleiro‐Laggo; J. Rodríguezz‐Otero 3. Results and Discussion ormal to the ce line is also th e MEP minim entral ring of g he trajectory fo a ordered by th graphene fragm ollowed by the the number of ments as e cations benzene J. A. Carrazanna‐García; E. M. Cabaleiro‐Laggo; J. Rodríguezz‐Otero ECCSOC23 – 2019 Theoretical study of cation–π interaction in graphene fragments 5 of 11 The energy of the interaction between the molecules of Fig. 2 and the cations studied are shown in the left‐side plots of both columns of Fig. 4. The interaction energy and the separation between the cation and the π‐system corresponding to the minima of these representations are listed in Table I and represented in the right‐side plots of both columns of Fig. 4. In all the cases very stable cation‐π complexes are formed and, for a particular cation, the stability of the complex increases with the size of the conjugate molecule. The separation between the cation and the conjugated molecule in the minima of the scans decreases slightly with the extent of the π‐system, corresponding to the formation of increasingly stable complexes as the graphene fragment grows. As expected from the MEP study, complexes with benzene are clearly apart from the complexes formed by the bigger conjugated molecules. For graphene fragments with four or more benzene rings the interaction energies of the complexes are grouped within a window of 3 to 5 kcal/mol, and for B13 and B19 the Eint value can be considered stabilized. The same is observed in the cation – π‐system separation in the minima of Eint. Thanks to its strong polarizing power, the more stable complexes are formed by Li+. As the cation size increases the contribution of induction is lower and for bigger cations Eint is progressively less negative. This tendency is kept in the complexes with organic cations but when comparing the same cation, guanidinium, in two different orientations it is observed that the complexes in the stacked configuration are more stable than the corresponding complexes with the configuration “in T”. This reveals the importance of another contribution which plays a minor role in the cases previously analyzed: dispersion. Table I. Coordinates of the minima found in the TPSS-D3/Def2TZVPP scans. These values are represented in the right-side plots of both columns in Fig. 4 as function of the size of the π-system. 3. Results and Discussion Eint / kcalꞏmol-1 r (catꞏꞏꞏπ) / Å B01 B03 B04 B05 B07 B09 B11 B13 B19 Li+ -38.98 1.84 -43.57 1.80 -43.92 1.80 -44.01 1.80 -45.41 1.80 -47.70 1.79 -48.91 1.78 -51.20 1.77 -50.55 1.77 Na+ -24.81 2.45 -29.02 2.37 -30.54 2.34 -30.96 2.33 -31.53 2.33 -33.02 2.32 -34.18 2.31 -35.68 2.31 -35.44 2.31 K+ -16.94 2.89 -21.15 2.79 -22.88 2.76 -23.22 2.76 -23.81 2.77 -25.08 2.75 -26.02 2.74 -27.33 2.74 -27.14 2.74 NH4 + -18.92 2.98 -23.14 2.91 -24.66 2.89 -24.97 2.89 -25.76 2.89 -27.22 2.88 -28.23 2.87 -29.80 2.86 -29.50 2.87 Gua+ in T -10.74 4.37 -14.07 4.27 -15.86 4.23 -16.46 4.22 -16.75 4.23 -17.71 4.22 -18.57 4.21 -19.64 4.20 -19.64 4.20 Gua+ stacked -7.47 3.57 -13.61 3.29 -16.95 3.23 -17.74 3.20 -19.21 3.19 -20.37 3.18 -21.41 3.18 -22.54 3.18 22.90 3.17 Coordinates of the minima found in the TPSS-D3/Def2TZVPP scans. These values are represented in the t-side plots of both columns in Fig. 4 as function of the size of the π-system. To quantify more precisely the importance of the different contributions to Eint and gaining understanding about the nature of the cation‐π in these complexes, the SAPT(DFT) energy analysis procedure is applied. With this method and using the cation – π‐system separation corresponding to the minima found in the scans of Fig. 4, the interaction energy is separated in its repulsion, electrostatic, induction and dispersion contributions. The results of the energy analysis are presented in Table II and in Fig. 5. J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero ECSOC23 – 2019 Theoretical study of cation–π interaction in graphene fragments 6 of 11 Figure 4. For both columns: Left = interaction energy as function of the distance between the cations and the π-system. Only the points around the minimum of each dependency are shown. In all the cases the trajectory explored is perpendicular to the center of the graphene fragment. Right = position and interaction energy corresponding to the minima found in the scans, ordered by the number of benzene rings in the graphene fragment. 3. Results and Discussion 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 37  36  35  34  33  32  31  30  29  28  27  26  25  24  23  22  1 3 5 7 9 11 13 15 17 19 37  36  35  34  33  32  31  30  29  28  27  26  25  24  23  22  2.3 2.31 2.32 2.33 2.34 2.35 2.36 2.37 2.38 2.39 2.4 2.41 2.42 2.43 2.44 2.45 number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 52  51  50  49  48  47  46  45  44  43  42  41  40  39  38  37  1 3 5 7 9 11 13 15 17 19 52  51  50  49  48  47  46  45  44  43  42  41  40  39  38  37  1.74 1.75 1.76 1.77 1.78 1.79 1.8 1.81 1.82 1.83 1.84 1.85 1.86 1.87 1.88 1.89 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 29  28  27  26  25  24  23  22  21  20  19  18  17  16  15  14  1 3 5 7 9 11 13 15 17 19 29  28  27  26  25  24  23  22  21  20  19  18  17  16  15  14  2.74 2.75 2.76 2.77 2.78 2.79 2.8 2.81 2.82 2.83 2.84 2.85 2.86 2.87 2.88 2.89 number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å Eint min r min B01 B03 B04 B05 B07 B09 B11 B13 B19 min Li+ Na+ K+ number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 20  19  18  17  16  15  14  13  12  11  10  9  8  7  6  5  1 3 5 7 9 11 13 15 17 19 23  22  21  20  19  18  17  16  15  14  13  12  11  10  9  8  4.2 4.21 4.22 4.23 4.24 4.25 4.26 4.27 4.28 4.29 4.3 4.31 4.32 4.33 4.34 4.35 number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å 1 3 5 7 9 11 13 15 17 19 31  30  29  28  27  26  25  24  23  22  21  20  19  18  17  16  2.84 2.85 2.86 2.87 2.88 2.89 2.9 2.91 2.92 2.93 2.94 2.95 2.96 2.97 2.98 2.99 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 31  30  29  28  27  26  25  24  23  22  21  20  19  18  17  16  2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 23  22  21  20  19  18  17  16  15  14  13  12  11  10  9  8  1 3 5 7 9 11 13 15 17 19 23  22  21  20  19  18  17  16  15  14  13  12  11  10  9  8  3.16 3.17 3.18 3.19 3.2 3.21 3.22 3.23 3.24 3.25 3.26 3.27 3.28 3.29 3.3 3.31 B01 B03 B04 B05 B07 B09 B11 B13 B19 min number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å Eint min r min NH4 + Gua+ in T Gua+ stacked 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 37  36  35  34  33  32  31  30  29  28  27  26  25  24  23  22  1 3 5 7 9 11 13 15 17 19 37  36  35  34  33  32  31  30  29  28  27  26  25  24  23  22  2.3 2.31 2.32 2.33 2.34 2.35 2.36 2.37 2.38 2.39 2.4 2.41 2.42 2.43 2.44 2.45 number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 52  51  50  49  48  47  46  45  44  43  42  41  40  39  38  37  1 3 5 7 9 11 13 15 17 19 52  51  50  49  48  47  46  45  44  43  42  41  40  39  38  37  1.74 1.75 1.76 1.77 1.78 1.79 1.8 1.81 1.82 1.83 1.84 1.85 1.86 1.87 1.88 1.89 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 29  28  27  26  25  24  23  22  21  20  19  18  17  16  15  14  1 3 5 7 9 11 13 15 17 19 29  28  27  26  25  24  23  22  21  20  19  18  17  16  15  14  2.74 2.75 2.76 2.77 2.78 2.79 2.8 2.81 2.82 2.83 2.84 2.85 2.86 2.87 2.88 2.89 number of rings Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint min (kcal/mol) r min (Catꞏꞏꞏπ) /Å Eint min r min B01 B03 B04 B05 B07 B09 B11 B13 B19 min Li+ Na+ K+ 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 37  36  35  34  33  32  31  30  29  28  27  26  25  24  23  22  Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 52  51  50  49  48  47  46  45  44  43  42  41  40  39  38  37  Li+ Na+ Eint (kcal/mol) 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 20  19  18  17  16  15  14  13  12  11  10  9  8  7  6  5  Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 31  30  29  28  27  26  25  24  23  22  21  20  19  18  17  16  NH4 + Gua+ in T Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 29  28  27  26  25  24  23  22  21  20  19  18  17  16  15  14  Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å B01 B03 B04 B05 B07 B09 B11 B13 B19 min K+ r (Catꞏꞏꞏπ) /Å 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 23  22  21  20  19  18  17  16  15  14  13  12  11  10  9  8  B01 B03 B04 B05 B07 B09 B11 B13 B19 min Eint (kcal/mol) r (Catꞏꞏꞏπ) /Å Gua+ stacked             Eint min (kcal/mol) r (Catꞏꞏꞏπ) /Å r (Catꞏꞏꞏπ) /Å Figure 4. J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero 3. Results and Discussion For both columns: Left = interaction energy as function of the distance between the cations and the π-system. Only the points around the minimum of each dependency are shown. In all the cases the trajectory explored is perpendicular to the center of the graphene fragment. Right = position and interaction energy corresponding to the minima found in the scans, ordered by the number of benzene rings in the graphene fragment. The only destabilizing contribution in all of these cases is repulsion, which values (not considering the complexes with benzene) are grouped in the 8 to 14 kcal/mol range. The trends observed by Eint are determined mainly by the balance between the stabilizing contributions. As in most of cation‐π complexes, the electrostatic contribution is important in all of these systems, but induction is the leading contribution to the stability in the complexes formed by Li+, Na+, K+ and NH4+. Complexes formed by the guanidinium cation perpendicular to the conjugated molecule (configuration “in T”) owe their stability to a combination of electrostatic, induction and dispersion that contribute in almost the same proportion. Dispersion, that has a minor effect in the complexes formed by Li+ and Na+, is an important contribution in the complexes with K+ and bigger cations, especially in the complexes formed by the guanidinium cation in the stacked configuration. The influence of the π‐system size is observed mostly in induction: as the conjugated molecule grows, its polarizability increases and the value of the induction contribution becomes more negative. This is reflected in the interaction energy values but when the conjugated molecule has 13 – 19 benzene rings the values of Eint and its contributions can be considered essentially stabilized. J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero ECSOC23 – 2019 Theoretical study of cation–π interaction in graphene fragments 7 of 11 Table II. Interaction energy analysis performed with the SAPT(DFT) method using the geometries of the minima found in the scans presented in Fig. 4. J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero ECSOC23 – 2019 3. Results and Discussion B01 B03 B04 B05 B07 B09 B11 B13 B19 Li+ r 1.84 1.82 1.82 1.82 1.82 1.81 1.78 1.79 1.77 Eint -38.35 -42.38 -42.11 -42.11 -43.04 -45.80 -46.62 -49.15 -47.70 Rep 12.74 12.57 11.79 11.99 12.35 12.83 13.95 13.64 14.37 Elec -17.83 -13.34 -10.62 -10.28 -10.21 -10.49 -9.59 -10.78 -7.67 Ind -32.17 -40.46 -42.16 -42.69 -43.99 -46.90 -49.67 -50.71 -53.04 Disp -1.10 -1.16 -1.13 -1.13 -1.19 -1.24 -1.31 -1.30 -1.36 Na+ r 2.42 2.38 2.36 2.36 2.37 2.36 2.36 2.35 2.31 Eint -23.13 -27.08 -28.32 -28.78 -28.92 -30.49 -31.73 -32.68 -32.17 Rep 7.90 8.14 7.97 8.00 8.02 8.41 8.36 8.78 9.81 Elec -15.30 -13.83 -12.86 -12.71 -12.41 -12.58 -12.31 -12.66 -11.00 Ind -14.85 -20.38 -22.38 -23.00 -23.44 -25.19 -26.65 -27.63 -29.72 Disp -0.89 -1.02 -1.06 -1.07 -1.09 -1.13 -1.13 -1.17 -1.27 K+ r 2.84 2.77 2.74 2.74 2.75 2.74 2.73 2.72 2.74 Eint -17.60 -21.79 -23.34 -23.70 -23.97 -25.49 -26.55 -27.55 -27.14 Rep 9.16 10.27 10.43 10.45 10.39 10.88 11.16 11.71 10.86 Elec -13.34 -13.22 -13.08 -13.03 -12.71 -12.88 -12.86 -13.20 -11.37 Ind -10.10 -14.77 -16.37 -16.75 -17.20 -18.88 -20.15 -21.22 -21.89 Disp -3.31 -4.07 -4.33 -4.37 -4.45 -4.61 -4.71 -4.85 -4.74 NH4 + r 2.97 2.91 2.90 2.90 2.90 2.88 2.88 2.87 2.87 Eint -17.89 -21.62 -22.74 -22.97 -23.62 -25.29 -26.21 -27.42 -27.37 Rep 11.42 12.61 12.25 12.31 12.60 13.47 13.43 13.95 13.85 Elec -12.20 -12.00 -11.70 -11.58 -11.63 -11.95 -11.86 -12.24 -11.12 Ind -11.52 -15.63 -16.54 -16.91 -17.57 -19.45 -20.39 -21.54 -22.46 Disp -5.58 -6.60 -6.75 -6.80 -7.02 -7.36 -7.39 -7.59 -7.64 Gua+ in T r 4.37 4.29 4.25 4.24 4.26 4.24 4.23 4.24 4.20 Eint -10.22 -13.3 -14.92 -15.5 -15.69 -16.76 -17.4 -18.52 -18.52 Rep 8.09 9.47 9.98 10.23 9.81 10.57 10.85 10.70 11.94 Elec -8.09 -8.80 -9.49 -9.81 -9.49 -9.91 -9.92 -10.51 -10.12 Ind -5.01 -7.27 -8.03 -8.32 -8.37 -9.38 -10.09 -10.47 -11.49 Disp -5.21 -6.71 -7.38 -7.60 -7.63 -8.05 -8.25 -8.25 -8.86 Gua+ stacked r 3.49 3.27 3.21 3.19 3.16 3.16 3.16 3.15 3.17 Eint -6.99 -13.22 -16.35 -17.17 -18.60 -19.82 -20.69 -21.73 -20.95 Rep 5.15 9.96 12.03 12.57 13.48 13.58 13.65 14.20 13.22 Elec -4.96 -9.60 -11.97 -12.52 -13.24 -13.34 -13.46 -14.07 -13.01 Ind -1.93 -4.27 -5.34 -5.67 -6.25 -7.22 -7.87 -8.55 -9.20 Disp -5.25 -9.31 -11.08 -11.55 -12.59 -12.85 -13.01 -13.31 -11.95 Table II. J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero 3. Results and Discussion Interaction energy analysis performed with the SAPT(DFT) method using the geometries of the minima found in the scans presented in Fig. 4. Table II. Interaction energy analysis performed with the SAPT(DFT) method using the geometries of the minima found in the scans presented in Fig. 4. J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero ECSOC23 – 2019 Theoretical study of cation–π interaction in graphene fragments 8 of 11 Figure 5. Analysis of the interaction energy of the complexes formed between the graphene fragments and the cations studied. The interaction energy and its repulsion, electrostatic, induction and dispersion contributions are plotted in function of the number of benzene rings in the graphene fragments. number of rings Energy (kcal/mol) 55  45  35  25  15  5  5 15 0 55  45  35  25  15  5  5 15 0 55  45  35  25  15  5  5 15 0 1 3 5 7 9 11 13 15 17 19 55  45  35  25  15  5  5 15 0 1 3 5 7 9 11 13 15 17 19 55  45  35  25  15  5  5 15 0 55  45  35  25  15  5  5 15 0 Eint Rep Elec Ind Disp NH4 + Gua+ in T Gua+ stacked Li+ Na+ K+ Figure 5. Analysis of the interaction energy of the complexes formed between the graphene fragments and the cations studied. 3. Results and Discussion Rodríguez‐Otero Theoretical study of cation–π interaction in graphene fragments 9 of 11 benzene rings. The effect of the size of the π‐system in the stability of these complexes consists in the progressive interchange between the role of electrostatic (more important in the smaller π‐systems) and induction (more important in the extended π‐systems). For the complexes with guanidinium in the stacked configuration the dispersion contribution is even more important than for the complexes analyzed previously and the effect of the size of the π‐system is also more complicated but systematic. benzene rings. The effect of the size of the π‐system in the stability of these complexes consists in the progressive interchange between the role of electrostatic (more important in the smaller π‐systems) and induction (more important in the extended π‐systems). For the complexes with guanidinium in the stacked configuration the dispersion contribution is even more important than for the complexes analyzed previously and the effect of the size of the π‐system is also more complicated but systematic. Among the 54 complexes studied, 25 systems are stable mostly because the induction contribution (%Ind > 50) and the other 29 are stable thanks to a balanced combination of electrostatic, induction and dispersion contributions. The complexes of B01 with all of the cations analyzed are located apart from the other points of the corresponding cluster, confirming that benzene is not a good choice for modeling the cation‐π interaction in graphene fragments. Figure 6. Contributions to the stability of the Electrostatic, Induction and Dispersion, calculated from the interaction energy analysis. Li+ Na+ K+ NH4+ Gua+ in T Gua+ stacked Disp Ind Elec B01 B19 10% 20% 30% 40% 60% 70% 80% 90% 50% 10% 20% 30% 40% 60% 70% 80% 90% 50% 10% 20% 30% 40% 60% 70% 80% 90% 50% Figure 6. Contributions to the stability of the Electrostatic, Induction and Dispersion, calculated from the interaction energy analysis Figure 6. Contributions to the stability of the Electrostatic, Induction and Dispersion, calculated from the interaction energy analysis. 3. Results and Discussion The interaction energy and its repulsion, electrostatic, induction and dispersion contributions l tt d i f ti f th b f b i i th h f t number of rings Energy (kcal/mol) 55  45  35  25  15  5  5 15 0 55  45  35  25  15  5  5 15 0 55  45  35  25  15  5  5 15 0 1 3 5 7 9 11 13 15 17 19 55  45  35  25  15  5  5 15 0 1 3 5 7 9 11 13 15 17 19 55  45  35  25  15  5  5 15 0 55  45  35  25  15  5  5 15 0 Eint Rep Elec Ind Disp NH4 + Gua+ in T Gua+ stacked Li+ Na+ K+ Figure 5. Analysis of the interaction energy of the complexes formed between the graphene fragments and the cations studied. The interaction energy and its repulsion, electrostatic, induction and dispersion contributions are plotted in function of the number of benzene rings in the graphene fragments. The relative importance of induction, electrostatic and dispersion to the stability of the complexes studied is emphasized in the triangular plot of Fig. 6. For each system, the electrostatic contribution is transformed in the %Elec coordinate calculated as follow: Elecꞏ100% %Elec Elec Ind Disp    This coordinate transformation is applied similarly to induction and dispersion and the points are represented in the triangular plot. In Fig. 6 it can be observed that the complexes of the different cations are clearly grouped in clusters. Dispersion has a minor effect in the stability of Li+ and Na+ complexes, but confers more than 10% of the stability to the K+ complexes, about the 20% to the NH4+ complexes and 30% to the guanidinium complexes with the configuration “in T”. The clusters of points corresponding to all of these cations are located almost parallel to the Ind – Elec side of the plot, indicating that the proportional effect of dispersion in the stability is essentially the same in the complexes of Li+, Na+, K+ and NH4+ cations with conjugated molecules of different number of ECSOC23 – 2019 J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. 4. Conclusions Cation‐π interaction is theoretically studied in complexes formed between Li+, Na+, K+, NH4+ and guanidinium cations and planar molecules with 1 to 19 fused benzene rings used as models of graphene fragments. Rigid scans performed at the TPSS+D3/ Def2TZVPP level of calculation show that all of the complexes studied are very stable and its interaction energy depends significantly on both, the size of the π‐system and the nature of the cation. The energy analysis applied using the SAPT(DFT) method reveals that induction is the leading contribution to the stability of the complexes formed by Li+ and Na+ cations, even when electrostatic is also important in their complexes with small conjugated molecules. The contribution of dispersion is important in the complexes formed by K+ and larger cations, especially in those formed by guanidinium in the stacked conformation. More than half of the complexes studied owe its stability to a balanced combination of electrostatic, induction and dispersion. Benzene (mainly) and anthracene are not good models for graphene fragments but following the tendencies observed with the molecules with four or more of benzene rings the results obtained can be extrapolated to extended π systems as graphene. J. A. Carrazana‐García; E. M. Cabaleiro‐Lago; J. Rodríguez‐Otero ECSOC23 – 2019 10 of 11 Theoretical study of cation–π interaction in graphene fragments 5. 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https://openalex.org/W2512599872	https://interactive-plus.ru/e-articles/221/Action221-81157.pdf	Russian	null	Postmodernity situation and modern education	Pedagogičeskij opyt: teoriâ, metodika, praktika	2,016	cc-by	1,470	Center for Scientific Cooperation "Interactive plus" Егорова Юлия Рабисовна канд. филос. наук, доцент ФГБОУ ВПО «Уфимский государственный авиационный технический университет» г. Уфа, Республика Башкортостан СИТУАЦИЯ ПОСТМОДЕРНА И СОВРЕМЕННОЕ ОБРАЗОВАНИЕ Аннотация: в статье определяется специфика современного общества постмодерна в его влиянии на образование. Критический и аналитический под- ход к проблеме образования в современных условиях позволяет сопоставить цели образования с основными тенденциями социальной реальности, противо- речащими им, что само по себе приводит к конструированию модели образова- ния, нацеленной на воспитание целостной личности в условиях демократиче- ского общества. Ключевые слова: постмодерн, современное общество, образование, просве- щение, капиталистическое общество, кризис идеологий, рефлексивность, раци- ональность, мудрость. Образование испокон веков являлось ценным завоеванием человеческой ци- вилизации, с которым связывались все упования и надежды на преобразование общества, на его переход из варварского состояния к состоянию культурному, собственно человеческому. Конфуций, Сократ, Платон, Ф. Аквинский, фило- софы эпохи Просвещения, отечественная философия так много уделяли внима- ния в своих трудах теме просвещенного человека. Почему сегодня образование не нацелено на воспитание просвещенного человека? Что происходит с образо- ванием сегодня? Любят ли учиться дети? Учат ли школы и университеты мыс- лить? Попытаемся ответить на эти вопросы. Специфика современного образования связана с основными характеристи- ками современного общества. Первой характеристикой современного россий- ского общества является то, что это капиталистическое общество, ориентирован- 1 Центр научного сотрудничества «Интерактив плюс» ное на либерально-демократические ценности. Вторая характеристика современ- ного общества это техногенность, информационность общества. И третья – со- временное общество есть общество постмодерна, или, как считают некоторые исследователи, незавершенного модерна (Э. Гидденс, Ю. Хабермас, А. Турен и др.). Понятно, что все три характеристики связаны между собой диалектиче- ской связью, и порождают друг относительно друга не только детерминирован- ные отношения и следствия, но и явления совершенно оппозиционные. Эти оп- позиции порождают неизбежную зависимость одной стороны от другой. Так увя- зываются в современности такие понятия, как «цивилизованность» и «варвар- ство», общество «безопасности, рутинных технологий» и «общество неопреде- ленности и риска», «светскость» и «религиозность», «глобализм» и «традицио- нализм», «общество благоденствия», «качества жизни» и «общество кризиса». Постмодернисты выступили с критикой следующих сторон общества мо- дерна. Во-первых, они решили деконструировать универсалистские концепции общества, которые, по их мнению, приводили к диктату тоталитарного разума, который сращивался с социальной системой, и являлся в конечном итоге сред- ством подавления индивида. Отчуждение разума -тема знакомая в нашей отече- ственной философии (М. Мамардашвилли, Э. Ильенков). Последствием развен- чания такого рода идеологий явилась ситуация неопределенности или кризиса идеологий. Сегодня мы видим, что несмотря на состояние «заката идеологий» или их выбор, общность идей может воспроизводиться в соответствии с локаль- ными пространственно-временными, а также исторически сложившимися усло- виями. Так, Э. 2 www.interactive-plus.ru 4 www.interactive-plus.ru СИТУАЦИЯ ПОСТМОДЕРНА И СОВРЕМЕННОЕ ОБРАЗОВАНИЕ Гидденс, например, настаивал, на неактуальности в наше время идеи дифференцирования общества, настаивая на идее пространственно-времен- ных характеристик социальных практик [1, с. 5]. Мифологическое мышление в наше время подвергается либо деконструкции, либо трансгрессии, либо продол- жает существовать в рамках возвращения субъекта в лоно традиционалистских культур. 2 www.interactive-plus.ru 2 www.interactive-plus.ru Center for Scientific Cooperation "Interactive plus" Другой чертой современного общества является его рефлексивность. Здесь мы можем констатировать то, что рефлексия осуществляется не всегда на выс- шем уровне. Рефлексируют все, как угодно и когда угодно, и на совершенно раз- личных ценностных основаниях. Постоянная рефлексия порождает динамизм общественных состояний. Динамизм порождает неподконтрольность социаль- ных практик. Вместе с тем, неподконтрольность не всегда является чем-то отри- цательным. В обществе постмодерна существует также идея конца истории, то есть конца прогрессивной модели истории, которая неизбежно представлялась до этого как телеологическая модель истории, где в итоге нас ожидает светлое бу- дущее или грядущее царство разума. Сегодня будущее предстоит нам как откры- тое, неизвестное и неопределенное. Четвертая идея постмодерна – идея смерти субъекта. Идея смерти субъекта является ничем иным, как признанием того, что субъект является во многом, результатом структуры языка и культуры. Мы ви- дим здесь идею того, что, с одной стороны, человек формируется культурой, с другой – может сформировать любую идентичность. Означает ли это все, что мы должны забыть о том, что в процессе образова- ния мы стремимся сделать человека целостной личностью? Не является ли низ- кий уровень рефлексии следствием того, что просвещение сегодня начинает быть лишней задачей? Социальный строй сегодня с его общественной диффе- ренциацией оказывается в эпицентре силовых полей, и нельзя не заметить его влияния, например, на сферу образования. Рациональность как направленность современной системы образования, ориентировано на «постав» рабочей силы специалистов узкого профиля. Опти- мизация образования приводит к редукции знания, которое лишается своей уни- версальной силы. По Г.В. Гегелю разум отличается от рассудка тем, что содер- жит в себе две составляющие. Разум соединяет в себе способность мыслить о внешнем мире со способностью мыслить и познавать себя. В современном рос- сийском образовании забыли о второй способности. Забвение второй способно- сти приводит к исключению разума из образования. 3 3 Центр научного сотрудничества «Интерактив плюс» Центр научного сотрудничества «Интерактив плюс» Время кризиса идеологий приводит, с одной стороны к отсутствию веры в высшие ценности и принципы, с другой – происходит трансгрессия этих прин- ципов и ценностей, выхолащивание многих понятий, и наделение их смыслами, служащими вполне определенным интересам, связанным с культом потребле- ния. Прогресс, успех, стиль жизни, досуг, свобода, энергия, знание, счастье и т. д. приобретают сегодня вполне прозаически очерченные формы, становятся частичными, частными, ограниченными обществом потребления. На этом фоне образование становится неизбежно чем-то вторичным, превращается из цели в средство. Это первая опасность, которая нуждается в противостоянии, постоян- ном разоблачении симулякров, восполнении и рефлексии. Идея либерализма сопровождалась верой в то, что человеческая личность получит должную оценку в глазах общества, возможность развиваться, творить, строить цивилизованный мир, то есть мир, в котором ценные и важные завоева- ния культуры, такие, как гуманизм, право, нормы, уважение к другому, совесть, достоинство человека, его свобода не являются вторичными. Казалось бы, вот здесь то и вспомнят о человеке, о воспитании творческой личности. Ведь, если вспомнить основные упования философов-западников, то именно в этой соци- альной форме мы можем воплотить этот проект. Вместе с тем, капиталистическая идеология вполне способна сегодня похо- ронить общество просвещения, и, если мы не хотим погрузиться в бессознатель- ное варварство, то мы должны заботиться о постоянной гуманизации, возвраще- нии человека от вещи к человеку, от бессознательного к смыслу, от материи к духу, к устроению социального капитализма. Идеология капитализма обуславливает общество с преобладанием чув- ственной культуры, той культуры, где за тебя все решили, где дали тебе рецепт счастья, и ты должен быть рад, и забыть о Другом. Человек экономический, пред- сказуемый потребитель под контролем, нацелен на то, чтобы устроиться на рынке труда, получить своё место, и дорожить им, как премудрый пескарь. Свято место пусто не бывает. Отсутствие идеологии обернулось созданием симулякров, ориентацией на культивирование чувственного, бессознательного, Center for Scientific Cooperation "Interactive plus" на соблазн и строго очерченные экономическими условиями пути и проекты че- ловека, на создание «человека-функции». Подобно тому, как тестирование тре- бует от нас ответа, заранее ограниченного рамками теста. Оно не учит порождать конструкции. Не учит оно и творчеству. Знание сегодня перестает занимать ме- сто света. Человек уже не стоит в просвете бытия, он не собирает себя в целост- ную личность. Ребенок теперь ищет, как увернуться от знания, так как оно пере- росло в количество, в ничем не ограниченные масштабы, в отсутствие контроля за их соизмеримостью с человеком, его возможностями. Можно ли найти вдохновляющие смыслы в идеологии капитализма? Ко- нечно. Центр научного сотрудничества «Интерактив плюс» Ведь, если бы там не было каких-то идеалов, общественный строй бы по- терпел поражение в истории. Несомненно, что это свобода индивида, идея пре- образования мира в цивилизацию, и культуру, где целью является человек, идеи честного, преобразовательного труда, рациональности, веры в победу добра и разума. Образ позитивного капиталиста воплощен в романе Д. Дефо «Робинзон Крузо». Вместе с тем, есть оборотная сторона цивилизации, её оппозиция – вар- варство, дикость. И эта пропасть станет нам ближе, если мы не изменим нашего отношения к знанию. Система образования призвана создавать «образ» человека. Чтобы учиться, нужно активно созидать духовное, а для этого нужен план свехреального, сверхчувственного мира, мира подлинности, где истина, добро и красота совпадают в единстве, вдохновляют и озаряют человека на самосозида- ние. «Мудрость, пишет Г.В. Гегель, не что иное, как просвещение, рассуждение. Но мудрость – это не наука. Мудрость есть возвышение души, благодаря опыту, соединенному с размышлением мудрость поднялась над зависимостью от мне- ний и впечатлений чувственности, …она приобретает своё убеждение не на обычном рынке, где знание дается каждому, кто заплатил причитающееся, она не могла расплачиваться блестящей монетой, имеющей хождение валютой, а го- ворит из полноты сердца» [2, с. 62]. В связи с этим перед нами сегодня стоит очень сложная задача – задача возвращения и воспитания мудрого человека, и 5 5 Центр научного сотрудничества «Интерактив плюс» это возможно, если мы точно осознаем, каким угрозам подвергается сегодня со- временное общество, а вместе с ним, и образование. это возможно, если мы точно осознаем, каким угрозам подвергается сегодня со- временное общество, а вместе с ним, и образование. 6 www.interactive-plus.ru Список литературы Список литературы 1. Фурс В.Н. «Критическая теория позднего модерна» Энтони Гидденса [Электронный ресурс]. – Режим доступа: //www.i-ru/biblio/ archive/furs_critical_pheory 2. Гегель Г.В. Народная религия и христианство. Работы разных лет: В 2-х томах. Т. 1. – М.: Мысль, 1970. 6 www.interactive-plus.ru 6 www.interactive-plus.ru
https://openalex.org/W2809231499	https://www.preprints.org/manuscript/201805.0426/v1/download	English	null	Hyaluronic Acid in the Third Millennium	Polymers	2,018	cc-by	19,868	Review Hyaluronic acid in the 3rd millennium Arianna Fallacara 1, Erika Baldini 1, Stefano Manfredini 1,* and Silvia Vertuani 1 Arianna Fallacara 1, Erika Baldini 1, Stefano Manfredini 1,* and Silvia Vertuani 1 1 Department of Life Sciences and Biotechnology, Master Course in Cosmetic Science and Technology (COSMAST), University of Ferrara, Via L. Borsari 46, 44121 Ferrara, Italy; arianna.fallacara@unife.it (A.F.); erika.baldini@student.unife.it (E.B.); smanfred@unife.it (S.M.); vrs@unife.it (S.V.). * Correspondence: smanfred@unife.it (S.M.); Tel.: +39-0532-455294; Fax: +39-0532-455378 Received: date; Accepted: date; Published: date Received: * Correspondence: smanfred@unife.it (S.M.); Tel.: +39-0532-455294; Fax: +39-0532-455378 date; Accepted: date; Published: date Abstract: Since its first isolation in 1934, hyaluronic acid (HA) has been studied across a variety of research areas. This unbranched glycosaminoglycan consisting of repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid is almost ubiquitary in humans and in other vertebrates. HA is involved in many key processes -including cell signaling, wound reparation, tissue regeneration, morphogenesis, matrix organization and pathobiology-, and has unique physico-chemical properties -such as biocompatibility, biodegradability, mucoadhesivity, hygroscopicity and viscoelasticity. For these reasons, exogenous HA has been investigated as drug delivery system and treatment in cancer, ophthalmology, arthrology, pneumology, rhinology, urology, aesthetic medicine and cosmetic. To improve and customize its properties and applications, HA can be subjected to chemical modifications: conjugation and crosslinking. The present review gives an overview regarding HA, describing its history, physico-chemical, structural and hydrodynamic properties, and biology –occurrence, biosynthesis (by hyaluronan synthases), degradation (by hyaluronidases and oxidative stress), roles, mechanisms of action and receptors. Furthermore, both conventional and recently emerging methods developed for the industrial production of HA and its chemical derivatization are presented. Finally, the medical, pharmaceutical and cosmetic applications of HA and its derivatives are reviewed, reporting examples of HA-based products which currently are on the market or are undergoing further investigations. Keywords: biological activity; crosslinking; drug delivery; cosmetic; food-supplement; functionalization; hyaluronan applications; hyaluronan derivatives; hyaluronan synthases; hyaluronic acid; hyaluronidases; physico-chemical properties. doi:10.20944/preprints201805.0426.v1 doi:10.20944/preprints201805.0426.v1 1. Introduction and historical background of HA Research on hyaluronic acid (HA) expands over more than one century. Indeed, the first study that can be referred to HA dates from 1880: the French scientist Portes observed that mucin from vitreous body was different from other mucoids in cornea and cartilage, and called it “hyalomucine” [1]. Nevertheless, only in 1934, Meyer and Palmer isolated from bovine vitreous humor a new polysaccharide containing an aminosugar and an uronic acid, and named it HA - from “hyaloid” (vitreous) and “uronic acid” [2]. During the 1930s and 1950s, HA was isolated also from human umbilical cord, rooster comb, and Streptococci [3, 4]. The physico-chemical properties of HA were widely studied from the 1940s [5-9], and its chemical structure was solved in 1954 by Meyer and Weissmann [10]. During the second half of the twentieth century, the progressive understanding of HA biological roles [11-13] determined an increasing interest in its production and development as medical product for a number of clinical applications. Hence, the extraction processes from animal tissues were progressively optimized, but still carrying several problems of purification from unwanted contaminants (i.e. microorganisms, proteins). The first studies on HA doi:10.20944/preprints201805.0426.v1 production through bacterial fermentation and chemical synthesis were carried out before the 1970s [1]. The first pharmaceutical grade HA was produced in 1979 by Balazs, who developed an efficient method to extract and purify the polymer from rooster combs and human umbilical cords [14]. Balazs procedure set the basis for the industrial production of HA [14]. Since the early 1980s, HA was widely investigated as raw material to develop intraocular lenses for implantation, becoming a major product in ophthalmology for its safety and protective effect to corneal endothelium [15-22]. Additionally, HA was found to be beneficial also for the treatment of joint [23- 27] and skin diseases [28, 29], for wound healing [30-33] and for soft tissue augmentation [34, 35]. Since the late 1980s, HA has also been used to formulate drug delivery systems [36-41], efforts still today to develop HA-based vehicles to improve therapeutic efficacy [42-45]. During the 1990s and 2000s, particular attention was paid to identify and characterize the enzymes involved in HA metabolism, and to develop bacterial fermentation techniques to produce HA with controlled size and polydispersity [1]. Nowadays, HA represents a key molecule in a variety of medical, pharmaceutical, nutritional and cosmetic applications. 1. Introduction and historical background of HA For this reason, HA is still widely studied to elucidate its biosynthetic pathways and molecular biology, optimize its biotechnological production, synthetize derivatives with improved properties, optimize and implement its therapeutic and aesthetic uses [1, 42-44, 46-58]. Because of the great interest from different fields of this biopolymer, the fast growing number of studies and our interest in this topic we decide to provide a comprehensive overview regarding HA and its potentialities, giving a concise update on the latest progresses. As an example a search on the most common public data bases with the key “hyaluron*”: Pubmed, Scopus, Isi Web of Science, ScienceDirect, Google Scholar, ResearchGate and Patent Data Base Questel; gave a feed back of a total of 161863 hits of which 142575 papers and 19288 patents. This huge amount of data is in continuous growth, thus in the aims to give a clearer picture of where researches and applications in the field are going, the present work starts with the update on HA physico-chemical, structural and hydrodynamic properties, and proceeds with the discussion of HA biology –occurrence, biosynthesis (by hyaluronan synthases), degradation (by hyaluronidases and oxidative stress), roles, mechanisms of action and receptors. Furthermore, both conventional and recently emerging methods developed for the industrial production of HA and its chemical derivatization are described. Finally, the medical, pharmaceutical, cosmetic and dietary applications of HA and its derivatives are reviewed, reporting examples of HA-based products which currently are on the market or are undergoing further investigations. Literature search: we searched the Cochrane Controlled Trials Register (Central), Medline, EMBase, and Cinahl from inception to November 2006 using truncated variations of preparation names including brand names combined with truncated variations of terms related to osteoarthritis all as text word. No methodologic filter for controlled clinical trials was applied (the exact search strategy is available from the authors). We entered relevant articles into Science Citation Index to retrieve reports that have cited these articles, manually searched conference proceedings and textbooks, screened reference lists of all obtained articles, and checked the proceedings of the US Food and Drug Administration advisory panel related to relevant approval applications. Finally, we asked authors and content experts for relevant references, and contacted manufacturers known to have conducted trials on viscosupplementation. 2. Physico-chemical, structural and hydrodynamic properties of HA HA is a natural and unbranched polymer belonging to a group of heteropolysaccharides named glycosaminoglycans (GAGs), which are diffused in the epithelial, connective and nervous tissues of vertebrates [46, 59, 60]. All the GAGs -HA, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin sulfate and heparin- are characterized by the same basic structure consisting of disaccharide units of an amino sugar (N-acetyl-galactosamine or N-acetyl-glucosamine) and an uronic sugar (glucuronic acid, iduronic acid or galactose). However, HA differs as it is not sulfated and it is not synthetized by Golgi enzymes in association with proteins [46, 59, 60]. Indeed, HA is produced at the inner face of the plasma membrane without any covalent bond to a protein core. Additionally, HA can reach very high molecular weight (HMW, 108 Da), while the other GAGs are relatively smaller in size (< 5 x 104 Da, usually 1.5-2 x 104 Da) [46, 59, 60]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 May 2018 doi:10.20944/preprints201805.0426 Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 doi:10.20944/preprints201805.0426.v1 Additionally, HA can reach very high molecular weight (HMW, 108 Da), while the other GAGs are relatively smaller in size (< 5 x 104 Da, usually 1.5-2 x 104 Da) [46, 59, 60]. Additionally, HA can reach very high molecular weight (HMW, 108 Da), while the other GAGs are relatively smaller in size (< 5 x 104 Da, usually 1.5-2 x 104 Da) [46, 59, 60]. The primary structure of HA is a linear chain containing repeating disaccharide units linked by ß- 1,4-glycosidic bonds. Each disaccharide consists of N-acetyl-D-glucosamine and D-glucuronic acid connected by ß 1,3-glycosidic bonds (Figure 1) [10, 61]. When both the monosaccharides are in the ß configuration, a very energetically stable structure is formed, as each bulky functional group (hydroxyl, carboxyl, acetamido, anomeric carbon) is in the sterically favourable equatorial position, while each small hydrogen atom occupies the less energetically favourable axial position [62]. Thus, the free rotation around the glycosidic bonds of HA backbone is limited, resulting into a rigid conformation where hydrophobic patches -CH groups- are alternated to polar groups [63, 64], which are linked by intra- and inter-molecular hydrogen bonds (H-bonds) (Figure 1) [65]. At physiological pH, each carboxyl group has an anionic charge, which can be balanced with a mobile cation such as Na+, K+, Ca2+ and Mg2+. 2. Physico-chemical, structural and hydrodynamic properties of HA Hence, in aqueous solution, HA is negatively charged and forms salts generally referred to as hyaluronan or hyaluronate (HA) [66, 67]- which are highly hydrophilic and, consequently, surrounded by water molecules. More precisely, water molecules link HA carboxyl and acetamido groups with H-bonds that stabilize the secondary structure of the biopolymer, described as a single-strand left-handed helix with two disaccharide residues per turn (2-fold helix) [68]. In aqueous solution, HA 2-fold helices form duplexes –i.e. a ß-sheet tertiary structure- due to hydrophobic interactions and inter-molecular H-bonds, which enable the aggregation of polymeric chains with the formation of an extended meshwork [64, 65]. The establishment of this network depends on HA molecular weight (MW) and concentration; for example, HMW native HA (> 106 Da) forms an extended network even at a very low concentration of 1 µg/mL [64, 69]. With increasing MW and concentration, HA networks are strengthened, and, consequently, HA solutions display progressively increased viscosity and viscoelasticity [70]. Since hyaluronan is a polyelectrolyte [71], its rheological properties in aqueous solutions are influenced also by ionic strength, pH and temperature [46, 70, 72]: as these factors increase, HA viscosity declines markedly, suggesting a weakening of the interactions among the polymer chains [73]. In particular, HA is highly sensitive to pH alterations: in acidic and alkaline environments, a critical balance between repulsive and attractive forces occurs [74], and when the pH is lower than 4 or higher than 11, HA is degraded by hydrolysis [75]. In alkaline condition this effect is more pronounced, due to the disruption of H bonds which take part in the structural organization of HA chains [74,76, 77]. Therefore, both the structural properties and the polyelectrolyte character of HA determine its rheological profile [65, 73, 78, 79]. HA solutions are characterized by a non- Newtonian, shear-thinning and viscoelastic behaviour. The shear-thinning (or pseudoplastic) profile of HA is due to the breakdown of the inter-molecular hydrogen bonds and hydrophobic interactions under increasing shear rates: HA chains deform and align in the streamlines of flow, and this results in a viscosity decrease [74, 78]. Additionally, HA solutions are non-thixotropic: as the shear rate decreases and ends, they recover their original structure and viscosity proceeding through the same intermediate states of the breakdown process [73]. Hence, the destructuration of the polymeric network is transient and reversible. Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 Figure 1. Chemical structures of HA disaccharide unit (A) and HA tetrasaccharide unit where the hydrophilic functional groups and the hydrophobic moieties are respectively evidenced in blue and yellow, while the hydrogen bonds are represented by green dashed lines (B). 3 Biology of HA Figure 1. Chemical structures of HA disaccharide unit (A) and HA tetrasaccharide unit where the hydrophilic functional groups and the hydrophobic moieties are respectively evidenced in blue and yellow, while the hydrogen bonds are represented by green dashed lines (B). 3. Biology of HA 3.1. HA occurrence in living organism and diffusion in human body 2. Physico-chemical, structural and hydrodynamic properties of HA This unique rheological behaviour is peculiar and extremely important as it determines many physiologic roles and pharmaceutical, medical, food and cosmetic applications of hyaluronan. doi:10.20944/preprints201805.0426.v1 3.1. HA occurrence in living organism and diffusion in human body 3.1. HA occurrence in living organism and diffusion in human body Hyaluronan is widely diffused in nature: it is present in humans, animals -such as, rabbits, bovines, roosters-, bacteria -such as Streptococcus equi, Streptococcus zooepidermicus, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Pasteurella multocida- [49, 80-82], algae –such as the green algae Chlorella sp. infected by the Chlorovirus [49, 83]-, yeasts such as Cryptococcus neoformans- [49] and molluscs [84]. However, it is not found in fungi, plants and insects [85]. In human body, the total content of HA is about 15 g -for a 70 kg adult [86]. HA is prevalently distributed around cells, where it forms a pericellular coating, and in the extracellular matrix (ECM) of connective tissues [61, 82]. Approximately the 50% of the total HA resides in the skin, both in the dermis and the epidermis [82]. Synovial joint fluid and eye vitreous body, being mainly composed of ECM, contain important amount of hyaluronan: 3-4 mg/mL and 0.1 mg/mL (wet weight), respectively [61, 82]. Moreover, HA is also abundant in the umbilical cord (4 mg/mL), where it represents the major component of Wharton’s jelly together with chondroitin sulfate [87, 88]. The turnover of HA is fast (5 g/die), and is finely regulated through enzymatic synthesis and degradation [86]. 3.2. HA synthesis in human body doi:10.20944/preprints201805.0426.v1 In human body, HA is synthetized as free linear polymer by three transmembrane glycosyltransferase isoenzymes named hyaluronan synthases -HAS: HAS1, HAS2 and HAS3 , whose catalytic sites are located on the inner face of the plasma membrane. HA growing chains are extruded onto the cell surface or into the ECM through the plasma membrane and HAS protein complexes [89, 90]. The three HAS isoforms share the 50-71% of their amino acid sequences (55% HAS1/HAS2, 57% HAS1/HAS3, 71% HAS2/HAS3) and, indeed, they are all characterized by seven membrane-spanning regions and a central cytoplasmic domain [50, 86, 89]. However, HAS gene sequences are located on different chromosomes (hCh19-HAS1, hCh8-HAS2, and hCh16-HAS3) [91, 92], and the expression and the activity of HAS isoforms are controlled by growth factors, cytokines and other proteins such as kinases in different fashions which appear cell and tissue specific [50, 90, 93, 94]. Hence, the three HAS genes may respond differently to transcriptional signals: for example, in human fibroblasts like synoviocytes, transforming growth factor ß up- regulates HAS1 expression, but down-regulates HAS3 expression [95]. 3.1. HA occurrence in living organism and diffusion in human body Moreover, HAS biochemical and synthetic properties are different: HAS1 is the least active isoenzyme and produces HMW hyaluronan (from 2 x 105 to 2 x 106 Da). HAS2 is more active, and synthetizes HA chains greater than 2 x 106 Da. It represents the main hyaluronan synthetic enzyme in normal adult cells, and its activity is finely regulated [96]. HAS2 also regulates developmental and reparation processes of tissue growth, and it may be involved in inflammation, cancer, pulmonary fibrosis and keloid scarring [55, 86, 97-99]. HAS3 is the most active isoenzyme, and produces HA molecules with MW lower than 3 x 105 Da [60]. Dysregulation and misregulation of HAS genes expression results in abnormal production of HA and, therefore, in increased risk of pathological events, altered cell responses to injury, and aberrant biological processes such as malignant transformation and metastasis [47, 48, 50, 100]. Even if the exact regulation mechanisms and functions of each HAS isoenzyme have not been fully elucidated yet [96], all the before mentioned studies suggest that HAS are critical mediators of physiological and pathological processes, as they are involved in development, injury and disease. 3.3. HA degradation in human body 3.3. HA degradation in human body HA degradation in human body is accomplished by two different mechanisms: one is specific, mediated by enzymes (hyaluronidases –HYAL), while the other is nonspecific, determined by oxidative damage due to reactive oxygen species (ROS). Together, HYAL and ROS locally degrade roughly the 30% of the 15 g HA present in human body. The remaining 70% is catabolized systemically: hyaluronan is mostly transported by the lymph to the lymph nodes, where it is internalized and catabolized by the endothelial cells of the lymphatic vessels. Additionally, a small part of HA is carried to the bloodstream and degraded by liver endothelial cells [50]. HYAL have a pivotal regulatory function in the metabolism of hyaluronan. These enzymes predominantly degrade HA, even if they are able to catabolize also chondroitin sulfate and chondroitin [101]. Randomly cleaving the β-N-acetyl-D-glucosaminidic linkages (β-1,4 glycosidic bonds) of HA chains, HYAL are classified as endoglycosidases. In human genome, six HYAL gene sequences have been identified in two linked triplets: HYAL 1, HYAL 2, HYAL 3 genes, clustered on chromosome 3p21.3; HYAL-4 and PH20/SPAM1 genes, similarly located on chromosome 7p31.3, together with HYAL-P1 pseudogene [102]. HYAL have a consistent amino acid sequence in common: in particular, HYAL 1, HYAL 2, HYAL 3, HYAL 4 and PH20/SPAM1 share about the 40% of their identity [101]. The expression of HYAL appears tissue specific. Nowadays, still much is unknown about HYAL activity, functions, and posttranslational processing. HYAL-1, HYAL 2 and PH20/SPAM1 are the most characterized human HYAL. Both HYAL-1 and HYAL 2 have an optimal activity at acidic pH (≤ 4) [103, 104] and are highly expressed in human somatic tissues [102]. HYAL 1 was the first human HYAL to be isolated: it was purified from serum (60 ng/mL) [105] and, successively, from urine [106]. HYAL 1 was found to regulate cell cycle progression and doi:10.20944/preprints201805.0426.v1 apoptosis: it is the main HYAL expressed in cancers, and therefore it may regulate tumor growth and angiogenesis [107]. HYAL 1 works together with HYAL 2 to degrade HA, possibly according to the following mechanism, which is still object of study. HYAL 2 is anchored on the external side of the cell surface: here it cleaves into oligosaccharides (oHA; approximately 25 disaccharide units, 2 x 104 Da) the extracellular HMW¬ HA (≥ 106 Da), which is linked to its receptor cluster of differentiation-44 (CD44). 3.3. HA degradation in human body These intermediate fragments are internalized, transported first to endosomes and then to lysosomes, where they are degraded into tetrasaccharide units (800 Da) by HYAL 1 [51]. Differently from HYAL-1 and HYAL 2, PH20/SPAM1 shows not only endoglycosidase activity both at acidic and neutral pH, but also a role in fertilization [108]. Hence, PH20/SPAM1 is unique among HYAL, as it behaves as multifunctional enzyme. HMW hyaluronan can also be naturally degraded in the organism by ROS, including superoxide, hydrogen peroxide, nitric oxide, peroxynitrite and hypohalous acids, which are massively produced during inflammatory responses, tissue injury and tumorigenesis [60, 109]. The depolymerization of HA occurs through mechanisms of reaction which are dependent on the ROS species, but always involve the scission of the glycosidic linkages [86, 110]. Studies have shown that oxidation related inflammatory processes, determining HA fragmentation, can increase the risk of injury in the airways and determine loss of viscosity in synovial fluid, with consequent cartilage degeneration, joint stiffness and pain [111-113]. ROS-induced degradation of HA might suggest why its antioxidant activity is one of possible role in reducing inflammation; however, so far, this biological function of HA has only been hypothesized, as it is not sufficiently supported by experimental data. Due to these degradation mechanisms which continuously occur in vivo, it has been estimated that the half-life of HA in the skin is about 24 h, in the eye 24-36 h, in the cartilage 1-3 weeks and in the vitreous humor 70 days [82]. 3.4. Biological roles of HA in relation to its MW The equilibrium between HA synthesis and degradation plays a pivotal regulatory function in human body, as it determines not only the amount, but also the MW of hyaluronan. Molecular mass and circumstances of synthesis/degradation are the key factors defining HA biological actions [50, 51, 100]. Indeed, high molecular weight (HMW) and low molecular weight (LMW) hyaluronan can even display opposite effects [51, 60], and when they are simultaneously present in a specific tissue, they can exert actions different from the simple sum of those of their separate size-related effects [51]. Extracellular HMW HA (≥ 106 Da) is anti-angiogenic, as it is able to inhibit endothelial cell growth [51, 60, 114]. Additionally, due its viscoelasticity, it acts as lubricating agent in the synovial joint fluid, thus protecting the articular cartilage [115]. HMW HA has also important and beneficial roles in inflammation, tissue injury and repair, wound healing and immunosuppression: it binds fibrinogen and controls the recruitment of inflammatory cells, the levels of inflammatory cytokines and the migration of stem cells [60, 93, 114]. During some environmental and pathological conditions –such as asthma, pulmonary fibrosis and hypertension, chronic obstructive pulmonary disease and rheumatoid arthritis-, HMW HA is cleaved into LMW HA (2 x 104 - 106 Da), which has been shown to possess pro-inflammatory and pro-angiogenic activities [51, 100]. Indeed, LMW hyaluronan is able to stimulate the production of proinflammatory cytokines, chemokines and growth factors [51], and to promote ECM remodeling [50]. Moreover, LMW HA can also induce tumor progression, exerting its influence on cell [51, 116] and provoking ECM remodeling. doi:10.20944/preprints201805.0426.v1 Both anti- and pro-inflammatory properties have been displayed by oHA and HA fragments (≤ 2 x 104 Da), depending on cell type and disease. Certain studies have shown that oHA are able to reduce Toll-like receptors (TLRs) mediated inflammation [117], inhibit HA-CD44 activation of kinases [118], and retard the growth of tumors [119]. However, oHA have been also found to promote inflammation in synovial fibroblasts [120], stimulate cell adhesion [121] and enhance angiogenesis during wound healing [53]. Therefore, HA is clearly a key molecule involved in a number of physiological and pathological processes. 3.5.1. HA cell surface receptors 3.4. Biological roles of HA in relation to its MW However, despite the intensive studies carried out so far, still little is known about HA biological roles, the factors determining HA accumulation in transformed connective tissues and the consequent cancer progression, and much less is known about their dependence on hyaluronan molecular size and localization (intra- or extra-cellular). Further researches focusing on HA molecular biology and mechanisms of action are necessary to clarify all these aspects and may facilitate the development of novel HA-based therapies. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 May 2018 doi:10.20944/preprints201805.0426.v1 Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 doi:10.20944/preprints201805.0426.v1 HA interactions with its cell surface receptors mediate three biological processes: signal transduction, formation of pericellular coats and receptor-mediated internalization [60]. The present section describes HA cell surface receptors and the biological actions that they control when linked by HA. The principal receptor for HA is CD44, which is a multifunctional transmembrane glycoprotein. It is expressed in many isoforms diffused in almost all human cell types. CD44 can interact not only with HA, but also with different growth factors, cytokines and extracellular matrix proteins as fibronectin [96]. CD44 intracellular domain interacts with cytoskeleton; hence, when its extracellular domain binds ECM hyaluronan, a link between the cytoskeletal structures and the biopolymer is created [46]. HA-CD44 interaction is involved in a variety of intracellular signaling pathways which control cell biological processes: receptor-mediated hyaluronan internalization/degradation, angiogenesis, cell migration, proliferation, aggregation and adhesion to ECM components [46, 51, 60, 100, 122]. Hence, CD44 plays a critical role in inflammation and wound healing [46, 96]. However, abnormal activation of HA-CD44 signaling cascades as well as over-expression and up-regulation of CD44 –due to pro-inflammatory cytokines such as interleukin-1, and growth factors such as epidermal growth factors- can result into development of pathological lesions and malignant transformation [60, 100]. Indeed, CD44 is overexpressed in many solid tumors, such as pancreatic, breast and lung cancer [54]. The receptor for HA-mediated cell motility (RHAMM) is also known as CD168, and it was the first isolated cellular hyaloadherins. It exists in several isoforms which can be present not only in the cell membrane, but also in the cytoplasm and in the nucleus [96]. When liked by HA, cell surface RHAMM mediates and promotes cell migration, while intracellular RHAMM modulates the cell cycle, the formation and the integrity of mitotic spindle [46, 60]. Interactions of HA with RHAMM play important roles in inflammation and tissue repair, by triggering a variety of signaling pathways and thus controlling cells such as macrophages and fibroblasts [96]. Hyaluronan receptor for endocytosis (HARE) was initially isolated from endothelial cells in the liver, lymph nodes and spleen, and successively found also in endothelial cells of eye, brain, kidney and heart [96]. It is able to bind not only HA, but also other GAGs, with the exception of keratin sulfate, heparin sulfate and heparin. It is involved in the clearance of GAGs from circulation [96]. 3.5. Mechanisms of action of HA 3.5. Mechanisms of action of HA HA performs its biological actions (section 3.4.) according with two basic mechanisms: it can act as a passive structural molecule, and as a signaling molecule. Both these mechanisms of action have shown to be size-dependent [51,86]. The passive mechanism is related to the physico-chemical properties of HMW HA. Due to its macromolecular size, marked hygroscopicity and viscoelasticity, HA is able to modulate tissue hydration, osmotic balance and the physical properties of ECM, structuring a hydrated and stable extracellular space where cells, collagen, elastin fibers and other ECM components are firmly maintained [59, 86, 88]. HA also acts as signaling molecule by interacting with its binding proteins. Depending on HA MW, location and on cell-specific factors (receptor expression, signaling pathways and cell cycle), the binding between HA and its proteins determines opposite actions: pro- and anti-inflammatory activities, promotion and inhibition of cell migration, activation and blockage of cell division and differentiation. All the factors which determine HA activities as signaling molecule could be related: MW may influence HA uptake by cells, and may affect receptor affinity. Additionally, receptor complexes may cluster differently depending on HA MW [51]. HA binding proteins can be distinguished into HA-binding proteoglycans (extracellular or matrix hyaloadherins) and HA cell surface receptors (cellular hyaloadherins) [51]. HA has shown two different molecular mechanisms of interaction with its hyaloadherins. First, HA can interact in an autocrine fashion with its receptors on the same cell [60]. Second, it can behave as a paracrine substance which binds its receptors on neighboring cells and thus activates different intracellular signal cascades. If HA has a HMW, a single chain can interact simultaneously with several cell surface receptors and can bind multiple proteoglycans. These structures, in turn, can aggregate with additional ECM proteins to form complexes which can be linked to the cell surface through HA receptors [60, 100]. Hence, HA acts as a scaffold which stabilizes ECM structure not only through its passive structural action, but also through its active interaction with several extracellular hyaloadherins -such as aggrecan (prominent in the cartilage), neurocan and brevican (prominent in the central nervous system) and versican (present in different soft tissues) [60]. For these reasons, pericellular HA is involved in the preservation of the structure and functionality of connective tissues, and in their protection from environmental factors [88]. 3.5.1. HA cell surface receptors 4. Industrial production of HA These plethora of activities in a food contained molecule, has raised important interest for public health: the global market of HA was of USD 7.2 billion in 2016, and it is expected to reach a value of USD 15.4 billion by 2025 [123]. Indeed, hyaluronan is gaining an exponentially growing interest for many pharmaceutical, medical, food and cosmetic applications, due to its important activities - anti-inflammatory, wound healing, and immunosuppressive- and its numerous and incomparable biological and physico-chemical properties biocompatibility, biodegradability, non- immunogenicity, mucoadhesivity, hygroscopicity, viscoelasticity and lubricity. Hence, there is a strong interest in optimizing HA production processes to obtain products that fulfill high quality standards and are characterized by great yield and accessible costs. Both the source and the purification process concur to determine the characteristics of the produced HA in terms of purity, MW, yield and cost [124, 125]. Therefore, producing high quality HA with high yield and less costly methods represents one of the biggest challenges in the field of hyaluronan applied research. The first production process applied at an industrial scale consisted in HA extraction from animal sources, such as bovine vitreous and rooster combs [46, 49, 124]. Despite the extraction protocols were improved over the years, this methodology was always hampered by several technical limitations which led to the production of highly polydispersed HA (MW ≥ 106 Da) with low yield [1, 46]. This was due to the polymer intrinsic polydispersity, its low concentration in the tissues, and its uncontrolled degradation caused by the endogenous HYAL and the harsh isolation conditions [46, 49]. Additional disadvantages of animal-derived HA were represented by the risk of biological contamination –presence of proteins, nucleic acids and viruses- and by the high purification costs [46, 49, 124]. Therefore, alternative methodologies for the industrial production of HA have been developed. Currently, commercial hyaluronan is principally produced by biotechnology (microbial fermentation). Microorganisms-derived HA is biocompatible with human body because HA structure is highly conserved among the different species [1, 49]. Streptococci strains A and C have been the first bacteria used for HA production, and, nowadays, many commercial products are derived from Streptococcus equi (such as Restylane® by Q-med AB and Juvederm® by Allergan). Optimum bacterial culture conditions to obtain HMW HA (3.5-3.9 x 106 Da) have been determined at 37°C, pH 7, in presence of lactose or sucrose [125, 126]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 May 2018 doi:10.20944/preprints201805.0426 Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 May 2018 doi:10.20944/preprints201805.0 Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 doi:10.20944/preprints201805.0426.v1 increased accessibility of the cell receptors to their ligands, in the initiation of the innate immune response and in the enhancement of the inflammatory reaction [52]. For this reason, HA can also be involved in the pathogenesis of diseases sustained by immunological processes [46]. increased accessibility of the cell receptors to their ligands, in the initiation of the innate immune response and in the enhancement of the inflammatory reaction [52]. For this reason, HA can also be involved in the pathogenesis of diseases sustained by immunological processes [46]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 May 2018 doi:10.20944/preprints201805.0426.v1 Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 Also lymphatic vessel endothelial hyaluronan receptor 1 -LYVE1, a HA-binding protein expressed in lymph vascular endothelium and macrophages-, controls HA turnover by mediating its adsorption from tissues to the lymph [60, 96]. In this way, LYVE1 is involved in the regulation of tissue hydration and their biomechanical properties [46, 96]. Additionally, LYVE1 forms complexes with growth factors, prostaglandins and other tissue mediators, which are implicated in the regulation of lymphangiogenesis and intercellular adhesion [46, 96]. Finally, HA is involved in the regulation of the activity of TLRs which, recognizing bacterial lipopolysaccharides and lipopeptides, are able to initiate the innate immune response [96]. Two possible mechanisms have been proposed to explain how HA can influence TLRs. According to the first theory, LMW hyaluronan act as agonist for TLR2 and TLR4, thus provoking an inflammatory reaction [51, 114]. On the contrary, according to the second theory, hyaluronan does not bind to TLRs, but it is able to regulate TLRs interactions with their ligands through the pericellular jelly barrier that it forms [52]. Indeed, in physiological conditions, HMW HA creates a dense and viscous protective coat around the cells, thus covering surface receptors such as TLRs and limiting their interactions with ligands. During inflammation, an unbalance between HA synthesis and degradation occurs, and this alters the thickness and the viscosity of HA pericellular barrier [52]. More precisely, HA is rapidly degraded due to pH reduction, ROS increase and possible presence of pathogens producing HYAL [46, 109]. Hence, HA MW decreases, reducing the polymer water binding ability and the thickness and the viscosity of its pericellular shield [52]. This results in an 4. Industrial production of HA Hyaluronan yield has been optimized up to 6-7 g/L, which is the upper technical limit of the process due to mass transfer limitation caused by the high viscosity of the fermentation broth [1]. As Streptococci genus includes several human pathogens, an accurate and expensive purification of the produced HA is necessary [46, 49]. Hence, other microorganisms have been and are currently investigated to synthetize HA. An ideal microorganism for HA biosynthesis should be generally regarded as safe (GRAS), not secrete any toxins and be able to produce at least 106 Da HA, as the polymer quality and market value increase with its purity and MW –which affect rheological and biological properties, and define suitable applications [49, 127]. Since the natural hyaluronan-producing organisms are mostly pathogenic, metabolic engineering is currently representing an interesting opportunity to obtain HA from non- pathogenic, GRAS microorganism. Endotoxin-free HA has already been synthetized by recombinant hosts including Lactococcus lactis [128], Bacillus subtilis [129], Escherichia coli [130] and Corynebacterium glutamicum [131]. However, up to now, there has been no heterologous bacterial host producing as much HA as the natural ones. Hence, there is an increasing effort to find an ideal bioreactor for HA production: in addition to bacteria, also eukaryotic organisms such as yeasts –like Saccharomyces cerevisiae [132] and Pichia pastoris [133]- and plant cell cultures –like transformed tobacco-cultured cells [134]- have been explored since the last years. doi:10.20944/preprints201805.0426.v1 Finally, to obtain HA of defined MW and narrow polydispersity, other approaches have been used. For example, to produce monodisperse oHA, chemoenzymatic synthesis has been performed [135]. This technique has successfully led to a product commercialized under the name Select HA™ (Hyalose LLC), characterized by a low polydispersity index value. Moreover, other studies have shown the possibility to prepare HA monodisperse fragments by controlling the degradation of HMW hyaluronan using different methods, including acidic, alkaline, ultrasonic and thermal degradation [110]. 5. Synthetic modifications of HA HA has several interesting medical, pharmaceutical, food and cosmetic uses in its natural occurring linear form. However, chemical modifications of HA structure represent a strategy to extend the polymer possible applications, obtaining more performing products which can satisfy specific demands and can be characterized by longer half-life. During the design of novel synthetic derivatives, particular attention is paid to avoid the loss of native HA properties such as biocompatibility, biodegradability and mucoadhesivity [46]. 5.1. General introduction on the chemical approaches to modify HA HA chemical modifications mainly involve two functional sites of the biopolymer: the hydroxyl (probably the primary alcoholic function of the N¬ acetyl D glucosamine) and the carboxyl groups. Furthermore, synthetic modifications can be performed after the deacetylation of HA N-acetyl groups, strategy which allows to recover amino functionalities [136]. All these functional groups of HA can be modified through two techniques which are based on the same chemical reactions, but lead to different products: conjugation and crosslinking (Figure 2). Conjugation consists in grafting a monofunctional molecule onto one HA chain by a single covalent bond, while crosslinking employs polyfunctional compounds to link together different chains of native or conjugated HA by two or more covalent bonds [136]. Crosslinked hyaluronan can be prepared from native HA (direct crosslinking) [56, 58, 137] or from its conjugates by synthesis (crosslinking of functionalized HA) [146]. Conjugation and crosslinking are generally performed for different purposes. Conjugation permit next crosslinking with a variety of molecules; to obtain carrier systems with improved drug delivery properties with respect to native HA; to develop pro-drugs by covalently linking active molecules to HA [136]. On the other hand, crosslinking is normally intended to improve the mechanical, rheological and swelling properties of HA, and reduce its degradation rate, in order to develop derivatives with a longer residence time in the site of application and greater release properties [58, 138, 139]. A recent trend is to conjugate and crosslink HA chains using bioactive molecules in order to develop derivatives with improved and customized activities [58] for a variety of applications in medicine, aesthetic and bioengineering –including cell and molecule delivery, tissue engineering, and development of scaffolds [46, 56, 58, 140-144]. doi:10.20944/preprints201805.0426.v1 Figure 2. Chemical modifications of HA: conjugation and crosslinking (A). HA forms used for pharmaceutical, medical, food and cosmetic applications: native, conjugated and crosslinked (B). Figure 2. Chemical modifications of HA: conjugation and crosslinking (A). 5.2. Modification of HA hydroxyl groups By modifying HA’s hydroxyl groups, the carboxyl groups remain unchanged, thus preserving HA’s natural recognition by its degradative enzymes [136]. Over the years, different derivatives of HA -ethers, hemiacetals, esters and carbamates- have been produced through reactions that occur between the polymeric hydroxyl groups and mono- or bi-functional agents. Epoxides and bisepoxides like butanediol-diglycidyl ether (BDDE) [137], ethylene glycol-diglycidyl ether, polyglycerol polyglycidyl ether [156], epichlorohydrin and 1,2,7,8 diepoxyoctane [157] have been widely used to synthetize ether derivatives of hyaluronan in alkaline aqueous solution. Currently, HA-BDDE ether represents one of the most marketed HA derivative: it can be obtained through simple synthetic procedures in aqueous ambient, and it is degraded into non-cytotoxic fragments [136]. Other efficient methods to form ether derivatives of HA involve the use of divinyl sulfone (DVS) [158] or ethylene sulphide [159] in basic water. Many studies showed that hemiacetal bonds can be formed between the hydroxyl groups of HA and glutaraldehyde in an acetone-water medium. Since glutaraldehyde is toxic, a particular handling is required during the reaction and the purification of the final product [160, 161]. The hydroxyl groups of HA can be also esterified by reacting with octenyl succinic anhydride [162] or methacrylic anhydride [163] under alkaline conditions. Alternatively, HA can be converted into a DMSO-soluble salt which can undergo esterification with activated compounds such as acyl- chloride carboxylates [164]. Finally, the activation of HA hydroxyl groups to cyanate esters, and the subsequent reaction in basic water with amines, allows to synthetize carbamate derivatives with high degrees of substitution, in a reaction time of only 1 hour [165]. 5.3. Modification of HA carboxyl groups Strategies for the derivatization of HA also involve esterification and amidation, which can be performed after the activation of the polymeric carboxyl groups using different reagents. By modifying HA’s carboxyl groups, derivatives more stable to HYAL degradation can be synthetized: hence, if a drug is conjugated on the carboxyl groups of HA, a slow drug release may occur [136]. Esterification can be performed by alkylation of HA carboxyl groups using alkyl halides [166] or tosilate activation [167]. Moreover, HA esters can be synthetized using diazomethane as activator of the carboxyl groups [168]. All these reactions proceed in DMSO from the TBA salt of HA. Alternatively, HA can undergo esterification also in water using epoxides such as glycidyl methacrylate and excess trimethylamine as a catalyst [169]. 5. Synthetic modifications of HA HA forms used for pharmaceutical, medical, food and cosmetic applications: native, conjugated and crosslinked (B). Figure 2. Chemical modifications of HA: conjugation and crosslinking (A). HA forms used for pharmaceutical, medical, food and cosmetic applications: native, conjugated and crosslinked (B). A number of synthetic approaches have been developed to produce conjugated or crosslinked hyaluronan [136]. Generally, HA is chemically modified in liquid phase. Since it is hydrophilic, several reactions are performed in aqueous media [145-147]: however, they are pH-dependent and, therefore, require acidic or alkaline conditions, which, if too strong, can determine HA degradation [75, 145]. Other synthetic methods, involving the use of reagents sensitive to hydrolysis, are performed in anhydrous organic solvents such as dimethylsulfoxide (DMSO) [146] or dimethylformamide (DMF) [148]. These approaches necessarily introduce a preparation step to convert native HA into tetrabutylammonium (TBA) salt, soluble in organic ambient [148]: this increases the reaction time and cost, and the chances of HA chain fragmentation due to physico-chemical treatments. Additionally, when HA modifications take place in organic solvents, longer final purification processes are necessary [136]. The basic and classic chemistry which underlies the possible modifications of HA functional groups in liquid phase is overviewed in the following subsections (5.2., 5.3., 5.4.). Since HA derivatives of high quality and purity are necessary to develop injectable products, implantable scaffolds, drug delivery systems, and 3D hydrogel matrices encapsulating living cells, techniques for efficient, low-cost and safe modification of HA are continuously being explored [46, 147, 149]. Hence, in the last years, several efforts have been done to introduce one-pot reactions which preferably proceed in aqueous ambient, under mild and, possibly, environmental-friendly conditions, and without the use of toxic catalysts and reagents [147, 149]. Additionally, alternative approaches to efficiently modify HA have been introduced: solvent-free methods –i.e. reactions in doi:10.20944/preprints201805.0426.v1 solid phase [57]-; “click chemistry” syntheses, which are simple and chemoselective, proceed with fast kinetics in aqueous ambient, under mild conditions, leading to quantitative yields, without appreciable amounts of side products –i.e. the thiol-ene reaction [150], the Dies-Alder cycloaddition [151] and the azide-alkyne cycloaddition [152]; in situ crosslinking of functionalized HA through air oxidation [153]; photocrosslinking of functionalized HA in presence of photosensitizers [154, 155]. 5.2. Modification of HA hydroxyl groups The conversion of HA carboxyl groups into less hydrophilic esters represents a strategy to decrease the water solubility of HA, with the aim to reduce its susceptibility to HYAL degradation and enhance its in situ permanence time [46]. A well-known biopolymer synthetized to this end is HA benzylester (HYAFF 11), whose properties are finely regulated by its degree of functionalization [36, 170]. Amidation represents a further approach to modify HA: over the years, several synthetic procedures have been developed. However, some of these present important drawbacks: for example, Ugi condensation –useful to crosslink HA chains through diamide linkages- requires a doi:10.20944/preprints201805.0426.v1 strongly acidic pH (3), the use formaldehyde, which is carcinogenic, and cyclohexyl isocyanide, which determines a pending undesired cyclohexyl group in the final product [145, 160]. HA amidation with 1,1’-carbonyldiimidazole [171] or 2-chloro-1-methylpyridinium iodide [148] as activating agents are performed in DMSO and DMF respectively: hence, HA conversion into TBA salt and longer purification steps are needed. Contrarily, other methods are based on reaction conditions that meet the modification requirements for HA. Particularly efficient is the activation of HA carboxyl groups by carbodimide (i.e. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride -EDC) and co-activators (i.e. N-hydroxysuccinimide -NHS- or 1- hydroxybenzotriazole) in water: proceeding under mild conditions, this reaction does not lead to HA chains cleavage, and it is suitable also for the derivatization with biopolymers easily susceptible to denaturation, such as protein or peptides [146, 172, 173]. Another promising method to synthetize HA derivatives with high grafting yields, in mild conditions, is based on triazine- activated amidation, typically performed with 2-chloro-dimethoxy-1,3,5-triazine [174] or (4-(4,6- dimethoxy-1,3,5-triazin-2- yl)-4-methylmorpholinium (DMTMM) [175]. A recent study made a systematic comparison of EDC/NHS and DMTMM activation chemistry for modifying HA via amide formation in water [175]. Results showed that DMTMM is more efficient than EDC/NHS for ligation of amines to HA, and does not require accurate pH control during the reaction to be effective [175]. Using these mild conditions of amidation, it is possible to synthetize highly hydrophilic and biocompatible derivatives, such as urea-crosslinked HA, which has already shown interesting applications in ophthalmic and aesthetic field [56, 58]. 5.4. Modification of HA N-acetyl groups 5.4. Modification of HA N-acetyl groups The deacetylation of the N-acetyl groups of HA recovers amino functionalities which can then react with activated acids using the same amidation methods described here above. 5.2. Modification of HA hydroxyl groups However, this approach is not frequently used to synthetize HA derivatives for two main reasons: first of all, even the mildest deacetylation techniques have been shown to induce chain fragmentation [146, 171, 176]. Moreover, the deacetylation is a strong structural modification which could importantly change the unique biological properties typical of native HA: indeed, it has been recently found that it reduces the interactions with the receptor CD44 [177]. 6. Applications of HA and its derivatives Due to their unique biological and physico-chemical properties, and to their safety profile, native HA and many of its derivatives represent interesting biomaterials for a variety of medical, pharmaceutical, food and cosmetic applications (Figure 3). Some HA-based products are already on the market and/or have already a consolidated clinical practice, while other are currently undergoing further investigations to confirm their effectiveness. Since the literature concerning HA derivatives and their applications is very extensive, only some examples are reported hereafter. doi:10.20944/preprints201805.0426.v1 Figure 3. Medical, pharmaceutical, cosmetic and dietary applications of HA and its derivatives. Figure 3. Medical, pharmaceutical, cosmetic and dietary applications of HA and its derivatives. 6.1. Drug delivery systems HA and its derivatives of synthesis represent useful and emerging tools to improve drug delivery. They have been used alone or in combination with other substances to develop pro-drugs, surface- modified liposomes, nanoparticles, microparticles, hydrogel, and other drug carriers. All these delivery systems are undergoing a continuous optimization as they are object of intensive researches. However, the industrialization and extensive clinical application of HA and its derivatives as drug carriers are still a long way to go, as many scientific studies are only at an in vitro experimental stage. The conjugation of active ingredients to HA is intended to develop pro-drugs with improved physico-chemical properties, stability and therapeutic efficacy compared to free drugs. Considering that hyaluronan plays several biological functions, HA-drug conjugates can exert their activities as such. Alternatively, their therapeutic actions are accomplished when the drugs are released, i.e. when the covalent bonds, which link drugs and HA, are broken down in the organism, ideally at the specific target sites. A variety of active ingredients can be conjugated to HA for topical or systemic uses. For example, HA can be conjugated with antidiabetic peptides such as exedin 4: this derivative shows prolonged half-life, protracted hypoglycemic effect and improved insulinotropic activity compared to the free exedin 4 in type 2 diabetic mice [178]. However, since HA has a short half-life in the blood, the majority of HA-drug conjugates have been developed for local –i.e. intraarticular, intratumoral, subcutaneous, intravesical, intraperitoneal- rather than systemic administration [179]. For instance, a variety of antinflammatory drugs, including hydrocortisone, prednisone, prednisolone and dexamethasone, have been conjugated to HA and investigated for intra-articular therapy of arthritis [166]. Furthermore, a recent study has displayed the potential of an emulsion containing a novel HA-P40 conjugate to treat a mouse model of dermatitis induced by oxazolone [180]. P40 is a particulate fragment isolated from Corynebacterium granulosum (actually doi:10.20944/preprints201805.0426.v1 known as Propionibacterium acnes) which has immunomodulatory, antibacterial, antiviral and antitumor properties. The conjugation of P40 to HA successfully prevents its systemic absorption and, therefore, improves its topical therapeutic effect [180]. Other researches have shown that conjugation of curcumin with HA represents a strategy to enhance water solubility and stability of curcumin [181]. Moreover, HA-curcumin conjugate displays improved healing properties compared to free curcumin and free HA, both in vitro -wound model of human keratinocytes- and in vivo -wound model of diabetic mouse. 6.1. Drug delivery systems Hence, HA conjugated curcumin may be useful to treat diabetic wounds [182]. Finally, the most promising and thoroughly studied conjugations of active ingredients to HA involve antitumoral agents, which can be strategically carried to malignant cells by hyaluronan, as explained in the subsection 6.2 [179, 183]. HA can be also conjugated to phospholipids in order to develop surface-modified liposomes: the chemical modification can be performed prior to liposome formulation [184] or after, on the outside shell [185, 186]. Moreover, HA can be also non-covalently linked on liposome surface: indeed, liposomes can be covered by HA through ionic interaction mechanism [187, 188] or simple lipid film hydration technique [189]. HA-modified liposomes appear promising carriers, as they have been shown to enhance the stability of drugs in the bloodstream, prolong their half-life time, reduce their systemic toxicity [186], enhance their tissue permeability, sustain their prolonged release [189] and ameliorate their therapeutic effects through synergistic actions [190]. HA-coated liposomes could improve the safety and the efficacy of antitumoral therapies: they appear proficient in mediating site-specific delivery of siRNA [191] and anticancer drugs such as doxorubicin [185], gemcitabine [186], imatinib mesylate [188] and docetaxel [184], via CD44 cell receptors. Additionally, HA-surface-modified liposomes have been investigated as delivery systems also in ophthalmology [189], pneumology [190] and topical treatment of wounds and burns [40]. Another promising type of drug delivery system, which can be formulated with HA and its derivatives of synthesis, is represented by nanoparticles. Hyaluronan can be a constituent element of nanoparticles [192, 193], but can also be used to cover nanoparticles, in order to improve the targeting efficiency and the therapeutic action of the encapsulated drugs [194]. HA-nanoparticles are being investigated for a number of administration routes and customized applications: for example, HA-Flt1 peptide conjugate nanoparticles might represent a next-generation pulmonary delivery carrier for dexamethasone in the management of asthma [193], while chitosan nanoparticles coated with HA and containing betamethasone valerate have displayed a great potential for the topical treatment of atopic dermatitis [194]. Hyaluronan nanoparticles included in polymeric films have shown potential as innovative therapeutic system for the prolonged release of vitamin E for the management of skin wounds [195]. Moreover, HA-poly(N-isopropylacrylamide) conjugate appear a promising candidate to treat osteoarthritis: once injected subcutaneously or intra-articularly, it spontaneously forms biocompatible nanoparticles able to control inflammation with a long-lasting action [192]. Finally, other HA surface-modified nanoparticles have been investigated for cancer targeted therapies [196-198]. 6.2. Cancer therapy It has been shown that the receptor CD44 is overexpressed in a variety of tumor cells which, consequently, display an increase of HA binding and internalization [46, 54, 208]. Hence, the receptor CD44 has been identified as potential target in cancer therapy, and hyaluronan, its primary ligand, has been recognized as powerful tool to develop targeted therapies [54, 198]. Many researches have shown that HA can act as drug carrier and targeting agent at the same time, under the form of polymer-antitumoral conjugates, or delivery systems encapsulating anticancer drugs [179]. Additionally, hyaluronan can be employed to design surface-modified nanoparticles [196- 198] or liposomes [184-188]. After CD44 receptor-mediated cell internalization, all these HA derivatives are hydrolyzed by intracellular enzymes and, therefore, drugs are released inside the cancer target cells [179]. This should improve the pharmacokinetic profile and the delivery process of many anticancer drugs, overcoming the limitations that reduce their clinic potential, such as low solubility, short in vivo half-life, lack of discrimination between healthy and malignant tissues, consequent off-target accumulation and side effects [54, 179]. Up to now, there are no HA- antitumoral conjugates and anticancer loaded carriers of HA on the market; however, the promising results of many researches and clinical trials outline their potential and encourage further studies [54, 179]. For example, it has been shown that HA-modified polycaprolactone nanoparticles encapsulating naringenin enhance drug uptake by cancer cells in vitro, and inhibit tumor growth in rat with urethane-induced lung cancer [198]. Additionally, HA-coated chitosan nanoparticles have been found to promote 5-fluorouracil delivery into tumor cells that overexpress CD44 receptor [196]. Further studies have displayed that a novel unsaturated derivative of HA [209] and different types of HA-paclitaxel conjugates [179, 183] have a great potential as anticancer therapies. 6.1. Drug delivery systems Additionally, over the years, HA microspheres and microparticles have been explored as formulations to improve the biomucoadhesive property and the drug release profile, and to ameliorate the texturing feeling in the case of dermal formulations. For example, it has been shown that spray-dried HA microspheres allow favourable ofloxacin delivery to the lung via inhalation, determining a superior pharmacological effect compared to free ofloxacin and to other routes of ofloxacin administration [199]. Similarly, inhaled HA microparticles have displayed prolonged pulmonary retention of salbutamol sulphate and reduced systemic exposure and side effects in a rat model [200]. HA microspheres have been evaluated also as possible materials for bone supplementation: indeed, they could be introduced in mineral bone cements to extend the release of active compounds [201]. A recent work has showed the potential of caffeine-loaded HA microparticles dispersed in a lecithin organogel as dermal formulation for the long-term treatment of cellulite: this drug delivery system is not only effective in repairing cellulite tissue damage, but doi:10.20944/preprints201805.0426.v1 has also an intrinsic moisturizing action [202]. Besides the microparticles prepared from native HA, scientific literature also describes microspheres formulated from HA derivatives of synthesis such as hyaluronan benzyl esters [203] and DVS-crosslinked hyaluronan [138] as topical drug delivery systems. has also an intrinsic moisturizing action [202]. Besides the microparticles prepared from native HA, scientific literature also describes microspheres formulated from HA derivatives of synthesis such as hyaluronan benzyl esters [203] and DVS-crosslinked hyaluronan [138] as topical drug delivery systems. Finally, hydrogels prepared from linear HA and its chemical derivatives are 3D polymeric networks which can be well-suited systems for topical delivery of cells [204] and many active ingredients, such as anti-inflammatories [44], anti-bacterials [205], antibodies and proteins in general [42]. To implement the mechanical and release properties, HA hydrogels can incorporate thermoresponsive polymers [42] or other drug carriers such as liposomes [43, 44]. Up to now, HA hydrogels have shown a great potential for intraocular [42, 204], intratympanic [44], intraarticular [206] and dermal delivery [207]. A topical 2.5% HA hydrogel containing 3% diclofenac has been commercialized for the treatment of actinic keratosis under the tradename of Solaraze® (Pharmaderm) in Europe, USA and Canada [207]. 6.3. Wound treatment As previously explained (section 3.4.), endogenous HA sustains wound healing and re- epithelialization processes thanks to several actions including the promotion of fibroblast proliferation, migration, and adhesion to the wound site, and the stimulation of collagen production [210]. For this reason, HA is used in topical formulations (such as Connettivina® by Fidia) to treat skin irritations and wounds such as abrasions, post-surgical incisions, metabolic and vascular ulcers, and burns [82, 210-213]. Currently, HA derivatives [173, 182, 214, 215] and HA- based wound dressings, films or hydrogels enriched with other therapeutic agents [216, 217] are being evaluated in order to understand if the cicatrisation process could be further enhanced. The wound healing properties of HA and its derivatives are being explored not only in dermatology, but also in other medical fields such as ophthalmology [56, 218], otolaryngology [142], rhinology [219] and odontology [220]. doi:10.20944/preprints201805.0426.v1 6.4. Ophthalmologic surgery and ophthalmology HA is a natural component of the human eye: it has been found in vitreous body, lacrimal gland, corneal epithelium and conjunctiva and tear fluid [56]. Therefore, ophthalmic products based on HA are fully biocompatible and do not trig foreign body reactions [136]. HA is a natural component of the human eye: it has been found in vitreous body, lacrimal gland, corneal epithelium and conjunctiva and tear fluid [56]. Therefore, ophthalmic products based on HA are fully biocompatible and do not trig foreign body reactions [136]. HA solutions are the most used viscosurgical devices to protect and lubricate the delicate eye tissues, replace lost vitreous fluid and provide space for manipulation during ophthalmic interventions [221]. Indeed, the viscosity of HA permits to maintain the tissues in place, reducing the risk of displacement that can potentially compromise both the surgery and the reparation process [46]. The first ophthalmic viscosurgical device containing HA was approved by the FDA in 1980 and is still marketed under the trademark Healon® (Abbott). Moreover, HA is the active ingredient of many eye drops –such as DropStar® by Bracco and Lubristil® by Eyelab- which, hydrating the ocular surface, and improving the quality of vision, are the mainstay to treat diseases such as dry eye syndrome and are useful in increasing the comfortability of contact lenses [56, 222, 223]. Many studies have proved the safety and the efficacy of native HA solutions as artificial tears [222, 224-226]. 6.3. Wound treatment More recently, also novel derivatives of hyaluronan with improved mechanical and biological properties are being investigated to formulate eye drops with enhanced ocular residence times. For example, promising preliminary results have been obtained with solutions of HA-cysteine ethyl ester [227] and urea-crosslinked HA (HA-CL) [56]. 6.5. Arthrology HA is one of the major lubricating agents of the ECM of synovial joint fluid: due to its viscoelasticity, it absorbs mechanical impacts and avoids frictions between the bone-ends [61, 82, 115]. When the synovial fluid is reduced or inflamed, and HA level decreases, disorders such as rheumatoid arthritis and osteoarthritis occur. Viscosupplementation represents an approach to treat and slow down the progression of these conditions: intraarticular injections of HMW HA - such as Supartz FX® by Bioventus and Hyalgan® by Fidia- allow to maximize the topical effect and the reduction of pain, and to minimize systemic adverse effects [140]. Locally injected HA has been shown to provide long-term clinical benefits, suggesting that it acts with more than one mechanism [140, 228], as the restoration of synovial fluid viscoelasticity is only temporary because HA is degraded within 24 hours [229]. Hence, the therapeutic effect of HA intra-articular injection appears prevalently due to biological activities: induction of the synthesis of new HA in synovial cells, stimulation of chondrocyte proliferation and resulting reduction of cartilage degradation [140, 228, 230]. In order to increase the half-life after injection and, consequently, the therapeutic efficacy, crosslinked HA derivatives have been investigated and introduced on the market (such as Synvisc® by Genzyme) as viscosupplementation agents [140, 231]. 6.6. Rhinology and pneumology Endogenous HMW HA plays a pivotal role in the homeostasis of the upper and the lower airways: it is an important component of the normal airway secretions, exerts anti-inflammatory and anti- angiogenic actions, promotes cell survival and mucociliary clearance, organizes extracellular matrix, stabilizes connective tissues, sustains healing processes and regulates tissues hydration [144, 232-234]. Hence, exogenous HMW HA represents a promising therapeutic agent for the treatment of nasal and lung diseases that involve inflammation, oxidative stress and epithelial remodeling, such as allergic and non-allergic rhinitis, asthma, chronic obstructive pulmonary disease and cystic fibrosis [143, 233, 235-238]. Examples of marketed formulations containing HA to treat respiratory diseases are Ialoclean® (Farma-Derma) a nasal spray to treat nasal dryness and rhinitis, and to promote nasal wound healing-, Hyaneb® (Chiesi Farmaceutici) - a hypertonic saline doi:10.20944/preprints201805.0426.v1 solution containing HA to hydrate and reduce mucus viscosity in cystic fibrosis patients [239]- and Yabro® (Ibsa Farmaceutici) -a high viscosity nebuliser solution of HA to treat bronchial hyper- reactivity [143]. solution containing HA to hydrate and reduce mucus viscosity in cystic fibrosis patients [239]- and Yabro® (Ibsa Farmaceutici) -a high viscosity nebuliser solution of HA to treat bronchial hyper- reactivity [143]. 6.8. Soft tissue regeneration HA skin content decreases with aging and the most visible effects are the loss of facial skin hydration, elasticity and volume, which are responsible of wrinkles [88]. Over the last years, HA has been widely used as biomaterial to develop dermal fillers (DFs), which are class III medical device that, injected into or under the skin, restore lost volumes and correct facial imperfections such as wrinkles or scars [58]. Being characterized by most of the properties that an ideal DF should have -biocompatibility, biodegradability, viscoelasticity, safety, versatility-, HA DFs have become the most popular agents for viscoaugmentation, i.e. for soft tissue contouring and volumizing [58]. Indeed, according to data from the American Society of Plastic Surgeons (ASPS), in 2017, out of a total of 2,691,265 treatments with soft tissue fillers, 2,091,476 were performed with HA DFs [242]. One of the reasons of this success resides in the reversibility of HA DFs effect: they correct wrinkles in a reversible manner, as a hypothetical medical error or complication can be remedied through the injection of HYAL (Vitrase®, ISTA Pharmaceuticals; Hylenex®, Halozyme Therapeutics) [58]. The duration of the corrective effect of HA DFs varies between 3 and 24 months, depending prevalently on HA concentration, crosslinking (degree and type), the treated area and the individual [58, 243]. For example, Hylaform® (Genzyme Biosurgery) contains 4.5-6 mg/mL HA crosslinked with DVS (20% degree), and its effect lasts 3 to 4 months, while the Juvederm® DFs family (Allergan) contains 18 to 30 mg/mL HA crosslinked with 9-11% BDDE, and it effect lasts 6 to 24 months [58]. Finally, DFs that combine BTX-A and HA have been developed to ensure an optimal correction even in patients with extremely deep wrinkles [58]. 6.7. Urology Recently, the possible therapeutic uses of HA in urology are being explored. Preliminary evidences have shown that intravesical HA, administered alone or in combination with chondroitin sulfate or alpha blockers, could be able to reduce the recurrence of urinary tract infections such as bacterial cystitis, to alleviate the symptoms of these diseases, and to protect the mucosa of urinary bladder [240, 241]. However, further clinical studies are necessary to confirm the effectiveness of HA treatment in urology. 6.9. Cosmetics In another study, the absorption, distribution and excretion of HMW (1 MDa) labeled HA were evaluated after oral administration in rats and dogs: for the first time, it was showed that dietary HMW HA can be distributed to connective tissues [251]. In particular, these reproducible results suggested that orally administered HMW HA may reach joints, bones and skin, even if in small amounts [251], thus highlighting that a rational may exist in the use of HA-based food supplements designed for joint and skin health. In these regards, several studies have reported on the safety of HA as food supplement, confirming its possible use as food ingredient itself. HA is marketed as food supplement in the USA, Canada, Europe and Asia (particularly in Korea and Japan) with some difference in the suggested use: to treat joint pain in USA and in Europe; to treat wrinkle and to moisturize in Japan, even if the involvement of this polymer in the skin moisture retention effect needs to be further elucidated in the future [252]. In the present review, we focused our attention prevalently on studies that exclude complex mixtures, in order to evidence just HA effects; however, several researches describe the use of HA as food ingredient in enriched extracts or mixtures with collagen and other supplements. For example, in a preliminary double-blind, controlled, randomized, parallel trial over 12 weeks, rooster comb extract was added to low fat yoghurt which was given to mild knee pain patients (n: 40) resulting in significative improvement in muscle strength in men [253]. In another recent clinical study, an oral HA preparation diluted in a cascade fermented organic whole food concentrate supplemented with biotin, vitamin C, copper and zinc (Regulatpro Hyaluron) led to a significant cutaneous antiaging effect in twenty female subjects after 40 days of daily consumption [254]. As written above from mixtures is difficult to elucidate HA specific contribution. Regarding supplementation with HA alone, some studies have demonstrated a direct correlation between ingestion and body effects. In a recent review, Kawada et al. underlined a number of studies in support of the contribution of ingested HA to hydrate skin, thus improving the quality of life for people who suffer from skin dryness induced by UV, smoking and pollutants, responsible of HA cutaneous reduction [255]. 6.9. Cosmetics HA represents a moisturizing active ingredient widely used in cosmetic formulations (gels, emulsions or serums) to restore the physiological microenvironment typical of youthful skin. HA- based cosmetics such as Fillerina® (Labo Cosprophar Suisse) claims to restore skin hydration and elasticity: this is reported to exert an antiwrinkle effect, although no rigorous scientific proof is able to fully substantiate this claim [82, 141, 244]. It has to be considered that HA hydrating effect largely depends on its MW, and its longevity depends on HA stability to hyaluronidases. Indeed, HMW HA mainly works as a film forming polymer: it reduces water evaporation, with an occlusive-like action. On the other hand, medium MW and LMW-HA mainly work by binding moisture from the environment, do to their high hygroscopicity [141, 244]. In some cases this capacity may reverse HA expected hydrating activity as at high concentration HA may even extract humidity from the skyn. Furthermore, also sunscreens containing hyaluronan may contribute to maintain a youthful skin, protecting it against the harmful effects of ultraviolet irradiation, due to the possible free radical scavenging properties of HA [245, 246]. The same capability has been demonstrated by dietary intake of HA (see below). 6.10. Dietary doi:10.20944/preprints201805.0426.v1 HA can also represent an interesting ingredient in enriched food and food supplements: it has gained the unofficial definition of nutri-cosmetic because of its capability to improve skin youth appearance [247]. For long time, the fact that HA can cross in its “intact” form the intestinal barrier has been debated; recently, few studies have appeared in literature to give some highligt to this question. Kimura et al. have evaluated the degradation and absorption of HA (300 KDa and 2 KDa) after oral ingestion in rats, demonstrating intestinal degradation to oligosaccharides, which are subsequently absorbed in the large intestine, translocated into the blood and distributed in the skin [248]. Orally ingested LMW HA has shown opposite effects: some studies have reported inflammatory properties with activation of the immune response [249], while other researches have highlighted the efficacy to reduce knee joint pain without inflammation [250]. Nowadays, the mechanisms at the base of these different actions of LMW ingested HA remain still unclear. 6.9. Cosmetics Indeed, the review of Kawada and coworkers reported that, in different randomized, double-blind, placebo-controlled trials, amounts of HA ranging from 37.52- 240 mg per day, ingested in a period comprised between 4-6 weeks, significantly improved cutaneous moistness [255]. These results, as always it happens with biopolymer as food supplements, are difficult to correlate with quantitative effects, as polymer sources and MW always vary. However, qualitatively, the correlation between HA consumption and decrease of skin dryness is evident. The authors suggest that partially digested HA, regardless of its MW, is adsorbed in the gut, while intact HA is absorbed by the lymphatic system; both are distributed to the skin, where they can work as inducers of fibroblasts proliferation and endogenous HA synthesis [255]. In a more recent study, the same authors have investigated, in a double blind, placebo controlled, randomized study on 61 subjects with dry skin, the effects of HA (120 mg/day) of two different MW (800 KDa and 300 KDa) over six weeks [256]. Both the HA were effective, but the 300 KDa group showed the best improvements in skin dryness and moisture content [256]. Again Kawada et al., in an experiment on hairless mice, demonstrated that the oral administration of 200 mg/kg body weight per day of two different MW HA (300 KDa and less than 10 KDa) for 6 doi:10.20944/preprints201805.0426.v1 weeks reduced epidermal thickness and improved skin hydration upon UV irradiation [257]. The effect was stronger for the less than 10 KDa HA [257]. The authors also demonstrated that the less than 10 KDa orally administered HA also stimulated HAS2 expression, thus highlighting an overall role of LMW HA in the prevention of skin photoageing with different mechanisms [257]. Thus, combining HA oral treatment with HA topical and injective administration could be very succesfull in the control of skin ageing. A further study suggesting that orally administered HA can migrate into the skin of rats, thus possibly reducing skin dryness, was conducted by Oe et al., who demonstrated that about the 90% of the ingested HA was absorbed from the digestive tract and was used as an energy source or a structural component [252]. Dietary HA can be beneficial not only for skin, but also for joints, as evidenced by a number of randomized, double-blinded, placebo-controlled studies relative to the treatment of knee pain, relief of synovial effusion or inflammation, and improvement of muscular knee strength [258]. 7. Conclusions, future trends and perspectives The present review underlines the interest of academic and industrial researches in HA: a comprehensive overview of this polymer is provided through the description of its structural, physico-chemical and hydrodynamic properties, occurrence, metabolism, biological roles, mechanisms of action, methods of production and derivatization, pharmaceutical, biomedical, food supplement and cosmetic applications. During the last decades, HA has gained a great success due to its numerous and unique properties -such as biodegradability, biocompatibility, mucoadhesivity, hygroscopicity and viscoelasticity- and to the broad spectrum of chemical modifications which it can undergo, allowing the development of derivatives with specific targeting and long-lasting drug delivery. Several in vitro and in vivo studies have been showing the beneficial actions of HA treatment, with anti- inflammatory, wound healing, chondroprotective, antiangiogenic, anti-ageing and immunosuppressive effects, among the others [51, 56, 60, 115]. This supported the development of a great number of HA-based commercial products, which started with native HA for ophthalmic and arthritic therapy to food supplement, esthetic and cosmetic application. More recently, also some chemical derivatives of HA have received FDA approval and have been successfully introduced on the market, especially as DFs. As a proof of the great potential of this molecule in terms of real benefits for health, we also conducted a search on a patent data base (Questel, Paris, France) which gave bach (search conducted on 20th April 2018) the following table that nicely illustrate the interest toward this class of biopolimers. Total 13684 Alive 8749 Dead 4935 1st application year After 2015 2717 2011-2015 4568 2006-2010 2647 2001-2005 1694 Before 2001 2058 ints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 29 May 2018 doi:10.20944/preprints201805.042 Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 doi:10.20944/preprints201805.0426.v1 In our opinion, although hyaluronan displays a great number of potential applications, further investigations and technological improvements are required, as there are still some questions to be answered and some issues to be addressed. First of all, many aspects of HA metabolism, receptor clustering and affinity still need to be explored to fully understand the different biological actions that hyaluronan plays through changes in MW. Additional insight needs to be gained in understanding whether there is a relation between HA size and localization, and how concomitant use of different HA sizes may modulate signaling. The comprehension of all this mechanisms could provide opportunities to extend and improve hyaluronan pharmaceutical, biomedical and salutistic (cosmetics and food supplements) applications, obtaining more targeted effects. 7. Conclusions, future trends and perspectives Isolation of hyaluronic acid from the cock's comb. J. Biol. Chem. 1949, 181, 573-575. 5. Kaye, M. A.; Stacey, M. Observations on the chemistry of hyaluronic acid. Biochem. J. 1950, 2, 13. A.; Stacey, M. Observations on the chemistry of hyalu 6. Meyer, K. Highly viscous sodium hyaluronate. J. Biol. Chem. 1948, 176, 993. y g y y 7. Varga, L. Studies on hyaluronic acid prepared from the vitreous body. J. Biol. Chem. 1955, 217, 651 8. Fletcher, E.; Jacobs. J.H.; Markham, R.L. Viscosity studies on hyaluronic acid of synovial fluid rheumatoid arthritis and osteoarthritis. Clin. Sci. 1955, 14, 653-660. 9. Blumberg, B. S.; Ogston, A.G.; Lowther, D.A.; Rogers, H.J. Physicochemical properties of hyaluronic acid formed by Streptococcus haemolyticus. Biochem. J. 1958, 70, 1-4. 10. Weissmann, B.; Meyer, K. The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord. J. Am. Chem. Soc. 1954, 76, 1753-1757. 11. Meyer, K. The biological significance of hyaluronic acid and hyaluronidase. Physiol Rev. 1947, 27, 335-359. 12. Ogston, A. G.; Stanier, J.E. The physiological function of hyaluronic acid in synovial fluid; viscous, ela and lubricant properties. J. Physiol. 1953, 119, 244-252. 13. Pinkus, H.; Perry, E.T. The influence of hyaluronic acid and other substances on tensile strength of healing wounds. J. Invest. Dermatol. 1953, 21, 365-374. g 14. Balazs, E. A. Ultrapure hyaluronic acid and the use thereof. U.S. Patent, US4141973 1979. 15. Miller, D.; Stegmann, R. Use of Na-hyaluronate in anterior segment eye surgery. J. Am. Intraocul. Implant. Soc. 1980, 6, 13-15. 16. Binkhorst, C. D. Advantages and disadvantages of intracamerular Na-hyaluronate (Healon intraocular lens surgery. Doc. Ophthalmol. 1981, 50, 233-235. 17. Binkhorst, C. D. Inflammation and intraocular pressure after the use of Healon in intraocular surgery. J. Am. Intraocul. Implant. Soc. 1980, 6, 340-341. 18. Percival, P. Results of a clinical trial of sodium hyaluronate in lens implantation surgery. J. Am Intraocul. Implant Soc. 1985, 11, 257-259. 19. Graue, E. L.; Polack, F.M.; Balazs, E.A. The protective effect of Na-hyaluronate to corneal endothelium. Exp. Eye. Res. 1980, 31, 119-127. 20. Percival, P. Protective role of Healon during lens implantation. Trans. Ophthalmol. Soc. U.K. 1981, 101, 77- 78. 21. Regnault, F.; Bregeat, P. Treatment of severe cases of retinal detachment with highly viscous hyaluronic acid. Mod. Probl. Ophthalmol. 1974, 12, 378-383. 22. Kanski, J. J. Intravitreal hyaluronic acid injection. A long-term clinical evaluation. Br. J. Ophthalmol. 1975, 59, 255-256. 23. 7. Conclusions, future trends and perspectives Towards this aim, the key mechanisms which control MW during HA biotechnological synthesis should be clarified to develop methods to produce more uniform-size defined HA. Additionally, progresses in metabolic engineering are necessary to improve HA yield and find biosynthetic strategies with good sustainability and acceptable production cost. Also the preparation of hyaluronan chemical derivatives need to be optimized, using strategies such as one-pot reactions, chemo-selective synthesis, solvent-free methods and “click chemistry” approaches. Furthermore, the reproducibility of HA-derivatives during scale-up, their pharmacokinetic and pharmacodynamic properties must be improved to allow their successful commercialization. Finally, all the HA-based next generation products –such as innovative crosslinked derivatives, polymer-drug conjugates and delivery systems- should be developed enabling high biocompatibility, prolonged half-life and improved in situ permanence: hence, in vivo and clinical studies are required to fully characterize their safety and efficacy. Acknowledgments: This work was supported by a PhD grant (to Arianna Fallacara) from IRAlab Srl (Usmate- Velate, Monza-Brianza, Italy) and by Ambrosialab s.r.l. (Ferrara, Italy, Grant 2017 to Stefano Manfredini and Silvia Vertuani). Conflicts of Interest: The authors declare no conflict of interest. Abbreviations: BDDE: butanediol-diglycidyl ether;CD44: cluster of differentiation-44;CDMT: 2-chloro- dimethoxy-1,3,5-triazine;DFs: dermal fillers;DMF: dimethylformamide;DVS:divinyl sulfone; DMTMM: 4-(4,6- dimethoxy-1,3,5-triazin-2- yl)-4-methylmorpholinium; DMSO: dimethylsulfoxide;DVS: divinyl sulfone;ECM: extracellular matrix;EDC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; HA: hyaluronic acid; hyaluronate; hyaluronan;HA-CL: urea-crosslinked hyaluronic acid;HARE: hyaluronan receptor for endocytosis;HAS: hyaluronan synthases;H-bonds: hydrogen bonds;HYAL: hyaluronidases;HMW: high molecular weight;GAGs: glycosaminoglycans;GRAS: generally regarded as safe;LYVE1: lymphatic vessel endothelial hyaluronan receptor 1;LMW: low molecular weight;MW: molecular weight;NHS: N- hydroxysuccinimide; oHA: oligosaccharides of hyaluronic acid;RHAMM: receptor for HA-mediated cell motility;ROS:reactive oxygen species; TBA: tetrabutylammonium; TLRs: Toll-like receptors. doi:10.20944/preprints201805.0426.v1 Peer-reviewed version available at Polymers 2018, 10, 701; doi:10.3390/polym10070701 References 1. 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https://openalex.org/W4253386706	https://edoc.unibas.ch/76887/1/20200604205707_5ed9440392c7d.pdf	English	null	Poor mental health of livestock farmers in Africa: a mixed methods case study from Ghana	Research Square (Research Square)	2,019	cc-by	10,751	Nuvey et al. BMC Public Health (2020) 20:825 https://doi.org/10.1186/s12889-020-08949-2 Nuvey et al. BMC Public Health (2020) 20:825 https://doi.org/10.1186/s12889-020-08949-2 Open Access Poor mental health of livestock farmers in Africa: a mixed methods case study from Ghana Francis Sena Nuvey1,2* , Katharina Kreppel3, Priscilla Awo Nortey1, Adolphina Addo-Lartey1, Bismark Sarfo1, Gilbert Fokou4, Donne Kofi Ameme1,2, Ernest Kenu1,2, Samuel Sackey1, Kennedy Kwasi Addo5, Edwin Afari1, Dixon Chibanda6 and Bassirou Bonfoh4,7,8 © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Agriculture represents the mainstay of African economies and livestock products are essential to the human population’s nutritional needs. However, in many developing countries, including Ghana, livestock production fails to meet demand due to population growth and negative effects of climate change. One of the challenges to production is livestock loss affecting farmers. However, despite stressful events experienced, livestock farmers’ mental health is poorly documented. This study aims to identify the root causes of livestock losses and their influence on pastoralists’ mental health. Methods: We conducted a mixed methods study in two districts in the Northern and Southern Belts of Ghana. Using the Depression Anxiety and Stress Scale–21 and guided interviews, we collected quantitative and qualitative data from 287 livestock farmers and 24 key-informants respectively. Mental health scores were categorized using standard guidelines. We evaluated the factors that explained variations in mental wellbeing using general linear models (α = 0.05). Results: About 85% (240/287) of the livestock farmers lost cattle within 1 year. Of these, 91% lost cattle to animal diseases, 50% to theft and 27% to pasture shortages. Qualitative findings reveal that due to poor access to veterinary services, farmers treat livestock diseases themselves with drugs from unregulated sources and often sell diseased cows for meat to recover losses. Findings showed that 60% of livestock farmers had poor mental health. Of those, 72% were depressed, 66% anxious and 59% stressed. Mental wellbeing was negatively associated with the number of adverse events experienced, proportion of livestock lost to most of the major loss factors, emotional attachment to livestock and self-reported physical illnesses in farmers, but positively associated with increasing herd size [F (8,278) = 14.18, p < 0.001, R2 = 0.29]. * Correspondence: fsnuvey@gmail.com 1University of Ghana, Accra, Ghana 2Ghana Health Service, Ministry of Health, Accra, Ghana Full list of author information is available at the end of the article * Correspondence: fsnuvey@gmail.com 1University of Ghana, Accra, Ghana 2Ghana Health Service, Ministry of Health, Accra, Ghana Full list of author information is available at the end of the article * Correspondence: fsnuvey@gmail.com 1University of Ghana, Accra, Ghana 2Ghana Health Service, Ministry of Health, Accra, Ghana Full list of author information is available at the end of the article Background [10]. A nationally representative survey conducted in Ghana between 2009 and 2010 also found that about 20% of the general population have psychological distress [11]. The gov- ernment of Ghana passed the Mental Health Act 2012 (Act 846) in the same year, with the goal of improving the mental healthcare delivery in the country [12]. Agricultural activities represent the main source of liveli- hood for a vast majority of the poor in developing coun- tries, where it is mainly operated on a subsistence basis and contributes to the local and global economy through trade. Livestock production is a big part of this and animal products serve as a key protein source in the human diet [1]. In spite of agriculture’s contribution to the economies of developing countries, it is linked to different forms of adverse events [2] which are a source for psychosocial problems to farmers. Mental problems arise when people are confronted by adverse events that are left unaddressed, affecting their normal day-to-day functioning. Against this background, the United Nations’ Sustain- able Development Goals (SDGs) One (1) through Three (3) were formulated, to improve health outcomes, includ- ing mental health, food security, and to reduce poverty globally. In spite of the strategies implemented to attain the set indicators of these goals, the United Nations Department of Economic and Social Affairs reports slow progress of some of the indicators in Africa and proposed a proper execution of agricultural practices as the key so- lution to self-sustainability on the continent [13]. Livestock farmers in Africa face multiple adverse events, all of which have increased in incidence recently [1], including disease outbreaks, drought, and conflict. The increased incidence of adverse event has been at- tributed to climate change [3]. Due to this, farmers often lose livestock and are left in a traumatic situation [4]. According to the International Labour Organization (ILO), occupational pressures in any workplace repre- sent the leading source of psychological problems in adults, with high stress levels at work negatively affecting productivity [5]. The mental health of individuals is therefore a key predictor of their productivity. In a farm- ing context, the loss of livestock, thus, affect the produc- tion levels of livestock farmers in the midst of increasing demand from a growing population, creating a mismatch between the demand for livestock products and what is actually being produced [6]. Background Livestock production in Ghana has stagnated since 2009, and even fell from 32 to 22% in 2013 [14], even though over 40% of households in Ghana are involved in livestock rearing [15]. According to the estimates by the Ministry of Food and Agriculture (MoFA), cattle production in Ghana increased by only 7.6% between 2000 and 2010 [16]. Over the same period, Ghana’s population grew by more than 30% [15]. The consequences of this mismatch in growth include high meat imports, high meat prices, and loss of foreign exchange. The production gap is reflected in the total beef imports into Ghana, that have been exponentially increasing by 1876.4%, from a little over 600 metric tons in the year 2000 to about 12,500 metric tons in 2010 [16]. The global demand for live- stock products is estimated by the Food and Agriculture Organization to further double by 2050 [6]. Therefore, there is the urgent need to identify the causes for loss of livestock and mental health problems in farmers to increase produc- tion and wellbeing in order to inform appropriate interven- tions to address these. Food insecurity in Ghana will increase substantially, if livestock production levels in Ghana are not improved. Identifying the root causes of livestock losses and the drivers of farmers’ mental health are key to meeting the SDGs 1, 2 and 3. Even though mental health problems are one of the recog- nized non-communicable diseases with high burden in both developed and developing countries, it has received less fo- cused attention over the past decades, especially in a rural context [7]. The World Health Organization (WHO) esti- mates that each year, more than 450 million people across the general population suffer from mental illness globally and about 75% of people with mental disorders in developing countries, where farming is the only source of income for many, receive no treatment [8]. Additionally, mental disorder-related deaths occur mainly through suicides, ac- counting for about 1 million deaths a year globally [9]. In Ghana, an estimated 3 million persons were living with men- tal disorders in 2007, with a treatment gap of more than 95% © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 12 Nuvey et al. BMC Public Health (2020) 20:825 Nuvey et al. BMC Public Health (Continued from previous page) Conclusions: Livestock diseases are the leading cause of losses to livestock farmers, whose mental wellbeing is negatively affected by these losses. Although an adaptive strategy by farmers to compensate for poor veterinary services, the arbitrary use of veterinary drugs and sale of diseased cattle pose health risks to the public. Further research to evaluate the performance of veterinary services in Ghana, mental health problems and risk to human health due to potential high-risk meat entering the food chain, is needed. Keywords: Livestock loss, Mental health, Veterinary, Food safety, Food security, Ghana Description of study areas This study was carried out in two representative districts of the Northern and Southern Belts in Ghana. The Page 3 of 12 Page 3 of 12 Nuvey et al. BMC Public Health (2020) 20:825 Bunkpurugu-Yunyoo (BY) District in the Northern re- gion of Ghana and Kwahu Afram Plains South (KAPS) District in the Eastern region were drawn randomly from a list of 14 districts in the Northern Belt and seven in the South, of the highest livestock production districts respectively (Fig. 1). activities including crop production, livestock rearing, and fish farming. The landscape is generally undulating with vast stretches of arable land that lies within the Savannah vegetation zone. The district has the Volta River, which flows in the East into the Gulf of Guinea. The soils are suitable for the cultivation of both food and cash crops. There are two main rainfall seasons; the first rainy season starts from May to June and the sec- ond from September to October. The mean annual rain- fall is between 1150 mm and 1650 mm. Livestock rearing is the second most important agricultural activity in the district after crop farming and is operated mainly on a free-range basis. The main animals reared in the district are cattle, goats, and sheep. The Kwahu Traditional Council, the highest traditional authority in the district, allocates land portions to persons interested in farming [18]. The BY District is an agrarian settlement which lies within the Northern Savannah Agro-ecological zone and has common grass vegetation. The landscape is generally gently rolling and the climatic conditions make the dis- trict conducive for the cultivation of food and cash crops, and the rearing of livestock. More than 94% of households within the district are engaged in agricultural activities. There is only a single rainfall season annually, usually between April and October with a mean annual rainfall ranging between 100 mm to 115 mm. The trad- itional authorities allocate land portions to persons who wish to engage in agriculture. Livestock and poultry pro- duction is mostly done under a free-range system with animals including chickens, goats, sheep, cattle, and pigs reared by farmers [17]. Research design This was a cross-sectional survey using a concurrent mixed-method approach. Multistage sampling was adopted in selecting the livestock farmers for the quanti- tative study. To ensure comparability of the value of The KAPS District is also an agricultural settlement with over 80% of the population engaged in agricultural Fig. 1 Map of Ghana showing the Bunkpurugu-Yunyoo (BY) and Kwahu Afram Plains South (KAPS) Districts. Map created by the authors using the Quantum Geographic Information Systems (QGIS) Software version 3.6 Fig. 1 Map of Ghana showing the Bunkpurugu-Yunyoo (BY) and Kwahu Afram Plains South (KAPS) Districts. Map created by the authors using the Quantum Geographic Information Systems (QGIS) Software version 3.6 Page 4 of 12 Page 4 of 12 Nuvey et al. BMC Public Health (2020) 20:825 Nuvey et al. BMC Public Health (2020) 20:825 Nuvey et al. BMC Public Health (2020) 20:825 losses suffered, only livestock farmers rearing cattle were recruited. Firstly, we identified and stratified cattle farm- ing communities into the Northern Belt (farming in the Northern Savannah zone) and Southern Belt (farming in the Savannah vegetation zone). Accordingly, 55 and 40 cattle farming communities, from the BY and KAPS Dis- tricts respectively, were included from which 12 commu- nities in the BY District and 10 communities in the KAPS District were randomly selected (See Add- itional file 1). From the selected communities, 145 and 142 cattle farmers in the BY and KAPS districts respect- ively were recruited consecutively from a sampling frame provided by community leaders of the farmers. For the qualitative approach, 19 farmers’ leaders in the commu- nities were selected purposively; ten (10) from BY and nine (9) from KAPS. In addition, all five (5) veterinary officers (two (2) from BY and three (3) from KAPS), were recruited into the qualitative study. Overall, 22 communities were sampled from the two districts. A total of 287 cattle farmers were selected for interviews with a structured questionnaire, while nineteen (19) cat- tle farmers’ leaders and five (5) veterinary officers took part in in-depth interviews. Factors that were assessed were based on literature review of previous studies. farmers, the coping strategies adopted, and the sup- port available to farmers to deal with these events. The discussions with the veterinary officers captured their perspectives on the nature of services provided to the livestock farmers, common challenges faced in their work, and the utilization of veterinary services by the farmers. Research design The quantitative data collected was coded and ana- lyzed using STATA software (version 15.1) [21]. De- scriptive analysis of the data was expressed as frequencies and proportions for categorical data, and means with standard deviations for continuous data. The DASS – 21 Likert scale scores ranging between 0 and 3, 0 meaning non-experience of the negative emotion and 3 as frequent negative emotion experi- ence, were reversed so that higher scores denote bet- ter mental health; 0 = 3, 1 = 2, 2 = 1 and 3 = 0. The reversed scores were summed under each of the three subscales (depression, stress, and anxiety), and multi- plied by 2 to generate depression, stress and anxiety scores. The mean of these three scores was computed to generate the mental health score. The mental health scores were categorized into two; scores above and below the mean mental health score depicting better and worse mental health respectively. Inferen- tial analysis was done to identify the factors that ex- plained variations in the mental health categories using Chi-square test. To determine the most efficient linear model of mental health and predictors, a back- ward elimination model selection was done using like- lihood ratio tests, starting with the most plausibly complex model [22]. Six (6) stepwise regression models were run, starting with 13 independent vari- ables in the first model. Likelihood ratio tests were used to eliminate the independent variables that were not statistically significant to obtain the most efficient predictor model with eight independent variables. In the multivariate linear analysis, the coefficient of de- termination (R2), F statistic, coefficients of the mul- tiple linear regressions of each independent variable with their respective standard errors, were presented. Diagnostic analysis of the residuals of each regression model was performed. Farmers characteristics and causes of livestock losses Farmers characteristics and causes of livestock losses The mean age (SD) of the cattle farmers was 46.9 ± 11.7 years (range = 19 to 78 years). Almost all the respondents were male (93%) and had some basic education (46%). The average pastoral household had 10 members and al- most all the respondents were married (92%). The ma- jority of the farmers (70%) had experience with cattle rearing and raising livestock in general since their child- hood. Household cattle holdings ranged between 11 and 400 herds (median = 31). The cattle farmers in the Kwahu Afram Plains South (KAPS) District had signifi- cantly larger herd sizes, with about 40% (51/142) having more than 50 cattle herds, compared with 21% (31/145) for those in the Bunkpurugu-Yunyoo (BY) District. More than 90% (261/287) of the cattle farmers grew crops in- cluding cereals (230/261), legumes (114/261), vegetables (49/261) and root tubers (39/261). One-third (90/287) of the respondents reported experiencing some illness at the time of the study. They reported symptoms includ- ing myalgia, asthma, skin lesions, eye problems and high blood pressure. Close to 50% (41/90) of the farmers re- ported musculoskeletal symptoms. About 80% (225/287) of the farmers were emotionally attached to their cattle. Farmers with larger than 50 cattle herds were more emotionally attached to their herds than those with smaller herds (less than 50), p = 0.033. The findings from the qualitative enquiry indicate; the main diseases resulting in losses are Contagious Bovine Pleuropneumonia (CBPP), and Foot and Mouth Disease (FMD). CBPP is reported as the most severe cause of losses. These diseases were widely reported by both vet- erinary officers and the leaders of farmers in the study districts. As this farmer and veterinary officer remark: “For that heart disease (CBPP), I do not know. If it just enters your kraal, it takes time before it goes. It is not easy, not one year or two years. If you don’t take care, you will lose a lot. I know a farmer who had about 300 cattle, when this disease affected him. Before he realized, only 100 remained. Some- times they go out to graze, and before you know it, five of them have died from that sickness; too much!” (Male livestock farmer, 38 years, BY). “ … CBPP kills them a lot. Data collection and analysis The research team comprising the principal investigator and three research assistants visited the cattle farmers and veterinary officers in their homes and/or workplaces to administer the questionnaires between 1st July and 29th September 2018. Two questionnaires; structured and unstructured, were administered to the respondents face-to-face, in one of four (4) languages, English, Ewe, Akan or Bimoba, depending on their language prefer- ence, to obtain quantitative and qualitative information respectively. The Depression, Anxiety and Stress Scale – 21 (DASS-21) was used to assess the mental health of the cattle farmers. It was not possible to do a full-scale validation of the DASS-21 for our study population [19]. Thus, two experts each in the three local languages car- ried out translation of the tools using the back transla- tion approach [20]. Other quantitative data collected included sociodemographic characteristics, sources of losses and number of cattle lost, emotional attachment to livestock and support available to the farmers. To complement this quantitative data, 19 leaders from the sampled communities and 5 veterinary officers were interviewed as key informants. The interview recordings from discussions with the leaders of livestock farmers and veterinary officers were transcribed. The transcripts were coded deductively using NVivo software (version 12) [23], to generate themes and sub-themes from the emerging patterns in the data relevant to the research questions. Triangula- tion of the findings was done to provide depth to the meaning of the quantitative results obtained. The find- ings were expressed as narratives supported by verbatim quotations (see Additional file 2 for the word cloud gen- erated from qualitative analysis of the data). The in-depth interviews were conducted using sep- arate interview guides for the leaders of cattle farmers and veterinary officers in both study districts. The farmers’ leaders interviewed shared their experiences on what livestock rearing meant to the farmers, the key adverse events leading to livestock losses to the Page 5 of 12 Nuvey et al. BMC Public Health (2020) 20:825 Page 5 of 12 Nuvey et al. BMC Public Health (2020) 20:825 Results adverse events accounting for the losses to 91% (218/ 240) of farmers were animal diseases. The other main risk factors for loss were from cattle theft (50%), pasture shortages (27%) and conflicts with other land users (22%) (Fig. 2). These losses have economic implications, costing each pastoralist on average about USD 1500, USD 600 and USD 300, to animal diseases, theft and pasture shortages respectively, per year (average value per head of cattle in Ghana = USD 300). Farmers characteristics and causes of livestock losses Foot and mouth disease is not much of a problem because only the young ones die from it, because they are unable to suck with the sore in the mouth; for the CBPP, it is ser- ious” (Veterinary Field officer, 39 years, KAPS). Around 85% (240/287) of the cattle farmers lost cattle over a one-year period. The median number of cattle lost per year per farmer was six (interquartile range = 3 to 10). Proportionately, the cattle farmers lost on average 15% of their total herd size within 1 year. The main Fig. 2 Root causes of livestock losses to farmers in Ghana (n = 240) Fig. 2 Root causes of livestock losses to farmers in Ghana (n = 240) Nuvey et al. BMC Public Health (2020) 20:825 Nuvey et al. BMC Public Health (2020) 20:825 Page 6 of 12 Nuvey et al. BMC Public Health (2020) 20:825 check on our animals. It is virtually nil” (Male live- stock farmer, 72 years, BY). The majority of livestock farmers (170/287) received support from veterinary services to deal with adverse event occurrences. On average, the support interventions received by farmers per year come from two (interquar- tile range = 0 to 4) different of the following four main sources: veterinary services, friends and family or pastor- alist association. The farmers with smaller herds (less than 50 cattle), reported receiving a significantly (p < 0.001) higher number of supporting interventions (me- dian = 2, interquartile range = 0 to 4) compared to those with large cattle herds (median = 1, interquartile range = 0 to 2) (see Table 1). The farmers in the BY District also reported significantly higher average level of support available (median = 3, interquartile range = 1 to 4) com- pared to the farmers in the KAPS District (median = 1, interquartile range = 0 to 2) (p < 0.001). “The challenge has to do with personnel. The area is so vast and we are seriously understaffed. The farmers live in areas that are so far apart, so at times, hmmm, it is difficult to get to all of them...We know there are some quacks going round, injecting the animals, but because we are under-staffed, I don’t fight with them” (Doctor of Veterinary Medi- cine, 51 years, KAPS). “Sometimes the farmers do not even know how to ad- minister the drugs properly. Farmers characteristics and causes of livestock losses Everyone just does what they think is right. Some farmers buy medicine in a red and yellow bottle and mix with “Akpeteshie” be- fore injecting the animals. So sometimes, I think we the farmers ourselves kill the animals because the in- jections are not given properly and we do not even know the right quantity to give. If the drugs are given in excess, the animal may die and people just smoke the meat and send to the market” (Male livestock farmer, 47 years, KAPS). The qualitative findings however, show that veterinary support to farmers is insufficient. The veterinary officers attribute this challenge to inadequate logistics (human and physical resources) and unwillingness of the cattle farmers to utilize vaccination services. Owing to this in- adequate service delivery, the farmers resort to buying veterinary drugs from mainly unlicensed dealers and treat cattle diseases themselves. As these farmers’ leaders and veterinary officers indicate: As the last farmer indicated, selling of diseased cattle for meat on the market to recover losses is one of the adaptive strategies adopted by the livestock farmers to cope with the adverse events they suffer. The veterinary services are unable to regulate the drugs used by farmers in treating animals and to ensure that only wholesome livestock products enter the food chain: “ … you see especially Bunkpurugu here, the farmers don’t want to vaccinate their animals. Because of that, we are facing many problems like PPR, CBPP, and anthrax … we see CBPP and PPR always. That one is not going at all” (Veterinary Field officer, 27 years, BY). “For most of the farmers, they will sell the dead ones; they will not throw it away. If the animal dies, some of them will smoke the meat nicely and send it to market as bush meat. That is what they do” (Veter- inary Field Officer, 39 years, KAPS). “For most of the farmers, they will sell the dead ones; they will not throw it away. If the animal dies, some of them will smoke the meat nicely and send it to market as bush meat. That is what they do” (Veter- inary Field Officer, 39 years, KAPS). “Our animals die because of lack of attention from the veterinary officers. Veterinary services are very, very poor. You go to them and they say there is no medicine. Farmers characteristics and causes of livestock losses So the veterinary services itself is not responding well to the livestock farmer. I cannot tell you the number of times that they even come to “These diseases that affect our animals, pose a health threat to those in the cities. If the meat is unwholesome and the farmer doesn’t discard it, it will be sent there” (Male livestock farmer, 47 years, KAPS). Table 1 Sources of support available to livestock farmers to deal with adverse events (N = 287) Source of support available Small herd (< 50 cattle) Large herd (≥50 cattle) Total frequency (n)* Veterinary services 119 51 170 Family 104 47 151 Friends 76 37 113 Pastoralist association 61 36 97 Insurance 4 4 8 Government compensation 1 1 2 Frequencies denote multiple responses of each respondent for type of support interventions received in a year Table 1 Sources of support available to livestock farmers to deal with adverse events (N = 287) “We do not eat the cattle if it dies of diseases. So we have to sell at very low prices, sometimes 700 Ghan- aian Cedis (USD 128). You lose a lot due to this” (Male livestock farmer, 38 years, BY). Despite the immense contribution of a lack of suffi- cient veterinary staff to decreased livestock farmers’ ac- cess to veterinary services, some of the impediments are purely financial. The veterinary officers report having to pre-finance the purchase of veterinary drugs and Frequencies denote multiple responses of each respondent for type of support interventions received in a year Page 7 of 12 Page 7 of 12 Nuvey et al. BMC Public Health (2020) 20:825 controlling for the farmers’ marital status, number of support received, district of farming, growing of crops and farmers’ experience with raising livestock, the cattle farmers’ mental wellbeing was negatively influenced by the number of adverse events experienced, report of ill health by the farmers, farmers’ emotional attachment to livestock, and proportion of cattle lost to diseases, theft and conflict. Whereas a unit increase in a farmers’ herd size and losses suffered from pasture shortages, were as- sociated with an improvement in their mental wellbeing [F (8, 278) = 14.18, p < 0.001, R2 = 0.29] (Table 3). Farmers characteristics and causes of livestock losses It is also important to note that elimination of other variables in the subsequent models did not result in significant changes in the strength of the relationship (slope) and the statistical significance of the individual predictor var- iables compared to Model 1. We also present simple general linear models demonstrating the effects of the proportions of livestock lost to the major factors includ- ing animal diseases, theft, pasture shortages, conflicts and the total proportions of herd lost to all causes on mental health of livestock farmers in additional informa- tion 4. consumables they use in treating livestock and thereafter charge the livestock farmers for the services delivered. consumables they use in treating livestock and thereafter charge the livestock farmers for the services delivered. “Sometimes when you call the doctor, he will say his fuel, car, etc. cost him, and then charges you a higher amount that I cannot afford. That is how they are. Sometimes it is just a problem with one animal. For this, you will waste all that money?” (Male livestock farmer, 49 years, BY). “ … somebody (farmer) will call you to come and treat the animals. Then you go to these places to vaccinate or treat and he will not give you the money. Another day, he will not call you. He will call another veterinarian, even though he owes you, he will just change the veterinarian … So the next time you call me, I will not go” (Veterinary Field Of- ficer, 27 years, BY). “ … somebody (farmer) will call you to come and treat the animals. Then you go to these places to vaccinate or treat and he will not give you the money. Another day, he will not call you. He will call another veterinarian, even though he owes you, he will just change the veterinarian … So the next time you call me, I will not go” (Veterinary Field Of- ficer, 27 years, BY). “Sometimes they (farmers) agree with you to admin- ister the drugs on credit. Thereafter, the payment be- comes a problem...So, next time if you call me, I won’t bother to go and do the work for him, because it will affect me. I have taken money from my small income to buy the drugs to work for you and you do not want to pay me the money back?” (Veterinary Field Officer, 39 years, KAPS). Discussion Agriculture and livestock production particularly con- tribute significantly to the growth and development of many developing countries including Ghana, and serve as a livelihood source to a vast majority of the poor in society. Despite these benefits, it is also one of the most stressful occupations. Due to negative effects of climate change, adverse events including droughts and conflict have increased in occurrence, leading to livestock losses to farmers and thereby affecting their productivity. Due to this decline in production of livestock, the local de- mand for livestock and livestock products is not ad- equately met, leading to high meat imports, high food prices and loss of foreign exchange. The loss of livestock may also lead to negative livelihood and mental health consequences for livestock farmers. Therefore, identify- ing the drivers of low mental health of livestock farmers and sources of livestock losses is key to inform interven- tions and being able to meet the Sustainable Develop- ment Goals (SDGs) 1, 2 and 3, which aim to reduce poverty, reduce hunger and ensure good health and wellbeing. The study found that the majority of the cattle farmers (60%) have poor mental health. Of those, 72% were de- pressed, 66% anxious and 59% stressed. The mean men- tal health score was 45.7 (SD = 5.7) out of a possible 63. On average, the farmers scored 42.7 (SD = 7.4) on the stress scale, 47.5 (SD = 6.4) on the anxiety scale and 46.8 (SD = 6.6) on the depression scale out of a possible 56 for each of the sub-scales. The livestock farmers per- formed better on the anxiety sub-scale of the mental health assessment. Even though the average mental health levels were not statistically different between the study districts, the livestock farmers in the KAPS District had relatively better mental health than the livestock farmers in the BY District (see additional file 3). Causes of livestock farmers’ poor mental health Causes of livestock farmers’ poor mental health The mental health of the cattle farmers is influenced sig- nificantly by the age of the livestock farmer, the farmers’ emotional attachment to their livestock, the number of stressful events experienced and the proportion of live- stock lost as well as the farmers’ health status (Table 2). Table 3 below illustrates the model selection iteration to determine the most efficient linear model predicting the mental health of cattle farmers. The best model had eight independent variables and accounted for about 30% of the variance in mental health (Model 6). After The mental health of the cattle farmers is influenced sig- nificantly by the age of the livestock farmer, the farmers’ emotional attachment to their livestock, the number of stressful events experienced and the proportion of live- stock lost as well as the farmers’ health status (Table 2). Our study provides evidence, that the root causes of livestock losses are from livestock disease outbreaks, cat- tle theft, pasture shortages and conflicts. The extent to which climate change affected the prevalence of these adverse events, though previously documented [3, 24, 25], could not be ascertained in this study. Livestock dis- eases were the leading cause of losses suffered by farmers because of mainly inadequate access to veterin- ary services and a lack of clarity on the responsibility of Table 3 below illustrates the model selection iteration to determine the most efficient linear model predicting the mental health of cattle farmers. The best model had eight independent variables and accounted for about 30% of the variance in mental health (Model 6). After Nuvey et al. Causes of livestock farmers’ poor mental health BMC Public Health (2020) 20:825 Page 8 of 12 Table 2 Factors influencing the mental health of livestock farmers Factor Category Poor (n = 171) Good (n = 116) Percent (%) Statistical significance Chi-square P-value Age of farmer Less than 50 years 90 81 59.6 8.487 0.004 50 years and above 81 35 40.4 Sex Female 15 4 6.6 3.169 0.092a Male 156 112 93.4 Highest level of education No formal education 56 40 33.4 1.782 0.642a Basic 81 51 46.0 Secondary 24 21 15.7 Tertiary 10 4 4.9 District Kwahu Afram Plains 80 62 49.5 1.228 0.268 South 91 54 50.5 Bunkpurugu-Yunyoo Marital status Not married 12 10 7.7 0.251 0.616 Married 159 106 92.3 Number in household 10 or fewer members 93 73 57.8 2.070 0.150 More than 10 members 78 43 42.2 Number of cattle in herd 50 or fewer cattle 117 88 71.4 1.875 0.171 More than 50 cattle 54 28 28.6 Cultivation of crops No 15 11 9.1 0.042 0.837 Yes 156 105 90.9 Raised with farm animals No 45 42 30.3 3.201 0.074 Yes 126 74 69.7 Support availability No support (≤2 sources) 109 70 62.4 0.340 0.560 Support (> 2 sources) 62 46 37.6 Level of attachment Not attached 24 38 21.6 14.307 < 0.001 Attached 147 78 78.4 Self-reported illness status Not ill 103 94 68.6 13.892 < 0.001 Ill 68 22 31.4 Level of livestock loss No loss (0%) 16 31 16.4 19.556 < 0.001 Low loss (< 10%) 49 32 28.2 Moderate loss (10–25%) 68 42 38.3 Severe loss (> 25%) 38 11 17.1 Numbers (n) of farmers falling into each category of ‘good’ and ‘poor’ mental health, percentage (%) denotes the proportion of farmers with poor mental health within each influencing factor category and their Chi-square and p-value. adenotes Fisher’s exact test probabilities for observations less than 5 persons in each mental health category. Poor and good mental health categories denotes scores of farmers less than and above the study average of 45.7 respectively Table 2 Factors influencing the mental health of livestock farmers Numbers (n) of farmers falling into each category of ‘good’ and ‘poor’ mental health, percentage (%) denotes the proportion of farmers with poor mental health within each influencing factor category and their Chi-square and p-value. adenotes Fisher’s exact test probabilities for observations less than 5 persons in each mental health category. Causes of livestock farmers’ poor mental health Poor and good mental health categories denotes scores of farmers less than and above the study average of 45.7 respectively Agriculture in collaboration with the Africa Develop- ment Bank [27] is commendable. Cattle rustling in pas- toral communities could be addressed if law enforcement is strengthened in farming communities in Ghana. Some of the farmers in the present study also sell diseased animals on the market to recover some losses. These practices appear to be a coping strategy adopted by the livestock farmers to help them deal with the challenge of inadequate access to veterinary services and thus high losses and is consistent with other studies [28, 29]. Agriculture in collaboration with the Africa Develop- ment Bank [27] is commendable. Cattle rustling in pas- toral communities could be addressed if law enforcement is strengthened in farming communities in Ghana. Some of the farmers in the present study also sell diseased animals on the market to recover some losses. These practices appear to be a coping strategy adopted by the livestock farmers to help them deal with the challenge of inadequate access to veterinary services and thus high losses and is consistent with other studies [28, 29]. farmers, resulting in them treating animal diseases them- selves using drugs freely available on the market, arbi- trarily without veterinary prescription. These findings agree with a previous study conducted among livestock farmers in the Northern Region of Ghana [26]. Periodic education provided by veterinary extension workers to livestock farmers could address the problem of pasture shortages. The establishment of a fodder bank for stor- age of feeds against drought periods and provision of other veterinary services in Wawase in the Kwahu Afram Plains South District by the Ministry of Food and Nuvey et al. BMC Public Health (2020) 20:825 Page 9 of 12 Table 3 Backward elimination regression analysis for factors predicting farmers’ mental health (n = 287) Model 1 Model 2 Model 3 Model 4 Model 5 Model 6 Variable Coef. (SE β) Coef. (SE β) Coef. (SE β) Coef. (SE β) Coef. (SE β) Coef. Causes of livestock farmers’ poor mental health R2 is the coefficient of determination for each of the multivariate linear regression models The loss of livestock was found to negatively affect the livelihood and reported physical and mental health of the livestock farmers. The farmers in this study lost an average of USD 1500 per year due to livestock disease outbreaks. This affected their ability to provide for the basic needs of their households. As expected, and as found in previous studies in the United Kingdom [30] and Australia [31], following the outbreak of Foot and Mouth Disease (FMD), livestock losses negatively affect the mental health of farmers. The proportion of the live- stock farmers with psychological distress (60%) is rela- tively high, compared to the previously reported prevalence of psychological distress of 20% among the general Ghanaian population [11]. This finding is not uncommon, as other comparative studies in Europe, America and Australia showed agricultural workers to be highly stressed compared to other occupations [32– 34]. The main diseases leading to the losses in Ghana in- clude FMD and Contagious Bovine Pleuropneumonia (CBPP). The occurrence of disease outbreaks of CBPP and FMD were widespread in both study districts. This is despite the availability of vaccination against FMD. It is surprising though that the losses suffered by the farmers to pasture shortages appear to be positively re- lated to mental health with each loss. This may be pos- sible if the farmers attribute losses to normal seasonality of the weather and may be expectant of better condi- tions during different times of the year. exploring. While the livestock farmers complained of a delayed or absent response by the veterinary officers to requests for veterinary services, the veterinary officers on the other hand, while acknowledging staff challenges, report that the livestock farmers do not patronize pre- ventive services, especially their vaccination services when organized. The lack of and/or poor uptake of vet- erinary services appears to be mostly financial in nature. As findings showed, the veterinary personnel often have to pre-finance the drugs and the consumables used in administering them. Thus, they often have to balance their quest to recuperate their investments with the live- stock farmers’ need for veterinary services. These facts point to a lack of clearly defined roles and responsibil- ities for livestock farmers, veterinary officers and the government in the livestock production system. Causes of livestock farmers’ poor mental health (SE β) Self-reported ill health status −2.91 (0.68)d −2.92 (0.68)d −2.93 (0.66)d −2.93 (0.66)d −2.92 (0.66)d −2.96 (0.66)d Number of adverse events −0.76 (0.21)d −0.76 (0.21)d −0.76 (0.21)d −0.76 (0.21)d −0.81 (0.18)d −0.83 (0.18)d Proportion of herd lost −0.07 (0.02)c −0.07 (0.02)c −0.07 (0.02)c −0.07 (0.02)c −0.07 (0.02)c −0.07 (0.02)c Emotional attachment to cattle −1.86 (0.77)b −1.86 (0.77)b −1.82 (0.77)b −1.82 (0.77)b −1.80 (0.76)b −1.82 (0.76)b Herd size 0.02 (0.01)a 0.02 (0.01)a 0.02 (0.01)b 0.02 (0.01)b 0.02 (0.01)b 0.01 (0.01)b Experience with livestock −0.72 (0.67) −0.72 (0.66) −0.72 (0.66) −0.72 (0.66) −0.73 (0.66) District (Bunkpurugu) −0.48 (0.76) −0.48 (0.73) −0.37 (0.70) −0.37 (0.70) Cultivator of crops 0.61 (1.11) 0.62 (1.10) 0.61 (1.10) Household size 0.01 (0.06) 0.01 (0.06) Number of support sources 0.01 (0.21) Slope 54.52 54.52 54.58 54.99 54.70 54.44 R2 0.24 0.24 0.24 0.24 0.22 0.23 F-ratio 8.68d 9.68d 10.92d 12.47d 14.54d 17.19d “Coef” denotes coefficients that are unstandardized regression slopes with standard errors (SE β) in parentheses. a, b, c, d, denote 10, 5, 1 and 0.1% significance level respectively. “Variable” = Variables included as predictors of the mental health of livestock farmers in Ghana. Categorical variables and their levels, added in the regression include self-reported ill health status (0 = Not ill/1 = Ill), Emotional attachment to cattle (0 = Not attached/1 = Attached), has experience raising livestock since childhood (0 = No/1 = Yes), District (0 = Kwahu Afram Plains South/1 = Bunkpurugu-Yunyoo), cultivator of crops (0 = No/ 1 = Yes). All ‘0’ denote the reference category. R2 is the coefficient of determination for each of the multivariate linear regression models mination regression analysis for factors predicting farmers’ mental health (n = 287) “Coef” denotes coefficients that are unstandardized regression slopes with standard errors (SE β) in parentheses. a, b, c, d, denote 10, 5, 1 and 0.1% significance level respectively. “Variable” = Variables included as predictors of the mental health of livestock farmers in Ghana. Categorical variables and their levels, added in the regression include self-reported ill health status (0 = Not ill/1 = Ill), Emotional attachment to cattle (0 = Not attached/1 = Attached), has experience raising livestock since childhood (0 = No/1 = Yes), District (0 = Kwahu Afram Plains South/1 = Bunkpurugu-Yunyoo), cultivator of crops (0 = No/ 1 = Yes). All ‘0’ denote the reference category. Causes of livestock farmers’ poor mental health The livestock farmers’ that reported some physical ill health were more likely to have psychological problems compared to those without physical ailments. Improved access to health services can therefore greatly influence the general health of livestock farmers. Consequently, the livestock losses to animal diseases have been enormous with economic and health conse- quences. The livestock farmers in Ghana would benefit immensely from awareness campaigns on preventive ser- vices like vaccination of livestock, to reduce economic loss, grief from cattle losses and improve cattle product- ivity. A similar intervention study among smallholder cattle farmers in Cambodia showed incremental gains in cattle weights, improved farmer knowledge levels and had a positive impact on household income over time for smallholder farmers [35]. While the economic influ- ence of livestock losses in this study was evident, many of the livestock farmers have also developed a special bond with their livestock. Livestock rearing, therefore, is more than just a source of livelihood to the farmers. These findings are consistent with qualitative inquiries conducted in Maasai communities in Eastern Africa, in Australia and the Alps of Switzerland which found deep emotional bonds between livestock farmers and their cattle [36–38]. This emotional attachment could eman- ate from the close contact the livestock farmers have with the cattle during milking, feeding, and cleaning of pens among others, which are practices similar to the earlier studies and this present one. The livestock farmers manifest their bond to cattle by talking to the cattle, giving names to cattle and caring for their furs. The emotional attachment to livestock tended to amplify the negative effect of loss on the mental health of farmers, as our findings revealed. The stronger bond of farmers with larger herds is surprising, but may explain why more farmers with large herds tend to seek veterin- ary care for their sick animals compared to those with smaller herds; apart from the obvious fact that farmers with larger herds are more likely to have resources to pay for veterinary services Nevertheless, the ability of the livestock farmers to adapt easily to the lack of veterinary services by learning to treat diseases themselves and selling of diseased meat can be described as a resilience strategy. This is one of many other adaptive strategies of livestock farmers to adverse events found in other studies conducted in East Africa [39, 40]. Causes of livestock farmers’ poor mental health Farmers had taken up duties of veterinarians by administering drugs including intravenous medications, while veterin- ary officers have become private entrepreneurs in a pub- lic service sector. What is clear though is the insufficient level of veterinary services support to livestock farmers. In addition, as qualitative findings revealed, most live- stock farmers do not utilize vaccination services orga- nized, but prefer treating the diseases instead. However, some farmers also report a lack of vaccines at veterinary offices when required. In the instances where vaccina- tions are organized, the veterinary officers operate with an expectation that farmers would pay them back. This arrangement is problematic, as it may result in neglect of farmers by veterinary officers who do not have ad- equate finances of their own, when the Ministry of Food The contrasting accounts given by the livestock farmers and veterinary officers alike on the reasons for the high prevalence of livestock diseases is worth Nuvey et al. BMC Public Health (2020) 20:825 Page 10 of 12 Nuvey et al. BMC Public Health (2020) 20:825 Page 10 of 12 and Agriculture fail to own this crucial aspect of veterin- ary service delivery. The inadequate number of veterin- ary personnel and other logistical constraints undermine the efforts of the ministry to improve livestock production. live in areas isolated from human settlements in order to gain access to pasture as well as avoid confrontations with other land users, limit their access to health ser- vices that are not readily available in rural areas in devel- oping countries. The Mental Health Authority in Ghana would be instrumental in devising strategies to make mental health services more accessible to farmers. The state of a farmers’ physical health also affected their mental health. The livestock farmers’ that reported some physical ill health were more likely to have psychological problems compared to those without physical ailments. Improved access to health services can therefore greatly influence the general health of livestock farmers. live in areas isolated from human settlements in order to gain access to pasture as well as avoid confrontations with other land users, limit their access to health ser- vices that are not readily available in rural areas in devel- oping countries. The Mental Health Authority in Ghana would be instrumental in devising strategies to make mental health services more accessible to farmers. The state of a farmers’ physical health also affected their mental health. Conclusions The livestock farmers in Ghana face multiple stressful events, such as animal disease outbreaks, cattle theft and pasture shortages as well as conflicts with other land users, all of which lead to livestock losses. The loss of livestock, mainly due to a lack of sufficient veterinary support to farmers, negatively affects the productivity of the farmers, with livelihood and mental health conse- quences. The livestock farmers have adapted to this shortfall in veterinary support; treat animal diseases themselves and sell diseased meat in the markets, posing health risks to the public. The mismatch between the farmers’ needs and the veterinary services provision has to be addressed to improve mental health, productivity and reduce the threats to food security and safety. Consent for publication Not applicable. Funding This study was conducted within the framework of the DELTAS Africa Initiative [Afrique One-ASPIRE/DEL-15-008]. Afrique One-African Science Part- nership for Intervention Research Excellence (ASPIRE) is funded by a consor- tium of donors including the African Academy of Sciences (AAS), Alliance for Accelerating Excellence in Science in Africa (AESA), the New Partnership for Africa’s Development Planning and Coordinating (NEPAD) Agency, the Well- come Trust [107753/A/15/Z] and the UK government. The funders had no role in the study. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. All data generated or analyzed during this study are included in this published article and its supplementary information files. Acknowledgments We acknowledge support received from Afrique One ASPIRE. In addition, we would like to thank the livestock farmers and veterinary officers who took the time to participate in this study, as well as the staff and Cohort 11 residents of the Ghana Field Epidemiology and Laboratory Program, in the School of Public Health, University of Ghana Legon. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08949-2. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08949-2. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08949-2. Authors’ contributions d d d d Conceived and designed the study: FSN KK BS GF DC BB. Conducted the investigation: FSN. Analyzed the data: FSN. Secured funding for conduct of project: KA BB. Contributed materials/analysis tools: KK PN AAL BS DKA EK SS EA. Wrote the paper: FSN KK KA DC PN AAL. All authors read and approved the final manuscript. Causes of livestock farmers’ poor mental health BMC Public Health (2020) 20:825 Nuvey et al. BMC Public Health (2020) 20:825 Nuvey et al. BMC Public Health (2020) 20:825 Page 11 of 12 of vaccines supplied by the government, and forestall the negative practices. It is also essential that an evaluation of the performance of veterinary services in Ghana is conducted. Considering the potential of transferring re- sistant bacterial strains from food to humans, there is need for an intensified surveillance by veterinary personnel at livestock markets and slaughter areas to reduce the sale of veterinary drugs and diseased meat. We propose a future study, which would assess for resistant pathogens and drug residues in retail livestock products and explore any links with resistant pathogens which are isolated from hu- man patients presenting with foodborne illnesses in Ghana though the utilization of the One Health approach to explore ways of maximizing the benefits accruable from the implementation of these policies. Additional file 2. Word cloud showing key issues of concern to study participants. This is a figure generated from transcripts of interviews of key informants using NVivo software, showing the key concerns raised by the livestock farmers. Additional file 3. Mental health scores of the livestock farmers by study district. This is a figure (box plot) of mental health scores of the study respondents by district of farming. Additional file 4. Summary of simple regression analyses the effect of loss factors on livestock farmers’ mental health (N = 287). This is a table showing the individual effect of each loss factor on farmers’ mental health. Additional file 4. Summary of simple regression analyses the effect of loss factors on livestock farmers’ mental health (N = 287). This is a table showing the individual effect of each loss factor on farmers’ mental health. Abbreviations AMR A b AMR: Antimicrobial resistance; BY: Bunkpurugu-Yunyoo; CBPP: Contagious Bovine Pleuropneumonia; DASS-21: Depression, Anxiety and Stress Scale – 21; FMD: Foot and Mouth Disease; ILO: International Labour Organization; KAPS: Kwahu Afram Plains South; SDGs: Sustainable Development Goals; WHO: World Health Organization AMR: Antimicrobial resistance; BY: Bunkpurugu-Yunyoo; CBPP: Contagious Bovine Pleuropneumonia; DASS-21: Depression, Anxiety and Stress Scale – 21; FMD: Foot and Mouth Disease; ILO: International Labour Organization; KAPS: Kwahu Afram Plains South; SDGs: Sustainable Development Goals; WHO: World Health Organization Our study revealed concerning findings, by showing the complexity of the challenges faced by livestock farmers in Ghana. Inadequate veterinary services support together with non-adoption of preventive measures such as vacci- nations, increases the frequency of adverse events result- ing in livestock loss, and poor mental health. The prevalence of psychological problems among farmers is higher than observed in the general population in Ghana. What is more alarming, the coping strategies employed by farmers include arbitrary drug use for treating animal dis- eases and the sale of unsafe meat at local markets. These findings raise concerns about the low quality of life of live- stock farmers and threats to public health through high- risk meat entering the food chain. Competing interests Competing interests DKA and EK declare that they are editorial board members of BMC Public Health. All other authors have no competing interests. DKA and EK declare that they are editorial board members of BMC Public Health. All other authors have no competing interests. Ethics approval and consent to participate The study was reviewed and approved by the Noguchi Memorial Institute of Medical Research Institutional Review board (approval number 054/17–18). Within the study districts, permission was obtained from all the relevant authorities within the study districts prior to data collection. The respondents provided written informed consent and data is kept as confidential records. Causes of livestock farmers’ poor mental health This presents an opportunity that can be harnessed by the appropriate government agencies as farmers show a willingness to do everything possible to keep their herd healthy. The timely provision of support services to farmers can therefore contribute to increased productivity and attaining food security and general growth and development. The recently launched pro- gram, “rearing for food and jobs”, by the Ministry of Food and Agriculture in Ghana, will come to nothing if the shortfalls in veterinary service delivery are left unaddressed. More so, the arbitrary use of freely available veterinary drugs to treat diseases and the sale of diseased livestock for meat on the markets by livestock farmers, presents potential food safety concerns. The insufficient number of veterinary personnel would mean that not all meat presented at the markets would have been cleared to be wholesome for the consumption of the public. This is also a concern for the emergence of antimicrobial resist- ance (AMR) in retailed food. The identification of AMR bacteria including: Campylobacter, Enterococcus, Sal- monella, Escherichia coli, Listeria, and Vibrio spp., in meat have been attributed to the indiscriminate use of veterinary drugs by farmers to treat animal diseases [41– 43]. Hence, the formulation of the AMR policy by the government of Ghana through the Ministries of Food and Agriculture, Health, and Environment, Science and Technology is commendable. The policy is very well for- mulated, but requires concerted efforts to implement the strategies outlined. The state of mental health of livestock farmers in Ghana is worrying. While we found in this study that about two-thirds of livestock farmers had psychological problems, previous studies in Ghana showed a preva- lence of psychological distress in less than one-third of the general population [11]. This finding is similar to previous research by the International Labour Organization which identified agriculture as one of the most stressful occupations [32]. The high levels of stress livestock farmers are confronted with from multiple adverse events they suffer could be contributory to the higher than normal prevalence of psychological prob- lems among farmers compared to the general popula- tion. Additionally, the tendency of livestock farmers to We have demonstrated how the health of humans, ani- mals and the environment depend on each other. 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https://openalex.org/W3201631671	https://europepmc.org/articles/pmc8459460?pdf=render	English	null	Changes in serum biomarkers of inflammation in bovine besnoitiosis	Parasites & vectors	2,021	cc-by	9,449	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Acute and chronic besnoitiosis in extensive natural-service herds can have relevant effects in the health of bulls and negative consequences in their productive performance. Recent progress has been made in order to elucidate the pathogenesis of this disease. In this context, the study of biomarkers of inflammation in serum would contribute to gaining knowledge about the physiopathology of bovine besnoitiosis. Serological biomarkers could help in early diagnosis and prognosis, as seropositive bulls may have mild or severe testicular lesions. Methods: Herein, we have investigated the diagnostic and/or prognostic value of a panel of serum (serological) bio‑ markers related to inflammation, including total protein, globulin and albumin, haptoglobin (Hp), adenosine deami‑ nase (ADA) paraoxonase-1 (PON-1) and acetylcholinesterase (AChE) in naturally and experimentally B. besnoiti-infected males classified according to different clinical phases of the disease (acute, chronic and subclinical besnoitiosis). Results: Results showed a similar response pattern in these biomarkers for naturally and experimentally infected cattle, with a few relevant variations. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during the chronic infection. Haptoglobin, albumin, PON-1 and ADA were identified as the biomarkers that showed changes of higher magnitude in the acute phase of the infection, whereas high total protein and globulin values were found in chronically infected cattle. We have described the changes of a panel of inflammatory biomarkers of acute and chronic bovine besnoitiosis. Conclusions: In summary, several biomarkers with promising diagnostic value have been identified. The biomarkers associated with acute infection are related to previously reported molecular biomarkers in testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement results from specific immunoglob‑ ulin M (IgM) detection. Keywords: Bovine besnoitiosis, Serological biomarkers, Acute-phase response, Haptoglobin, Albumin, Paraoxonase-1, Adenosine deaminase, Acetylcholinesterase cattle disease in sub-Saharan countries [1, 2] and has been spreading throughout different countries in Europe for the last two decades [3, 4] in the absence of vaccines and therapeutic tools. The disease progresses in two sequen- tial steps. First, acute bovine besnoitiosis is characterised by fever, depression and anorexia followed by generalised oedema, ocular and nasal discharge, and orchitis [5, 6]. Next, chronic besnoitiosis is characterised by skin lesions such as hyperkeratosis, skin folding and alopecia that occur because of the development of tissue cysts in the Parasites & Vectors Parasites & Vectors González‑Barrio et al. Parasites Vectors (2021) 14:488 https://doi.org/10.1186/s13071-021-04991-0 Changes in serum biomarkers of inflammation in bovine besnoitiosis David González‑Barrio1* , Ana Huertas‑López2, Carlos Diezma‑Díaz1, Ignacio Ferre1, José Joaquín Cerón2, Luis Miguel Ortega‑Mora1 and Gema Álvarez‑García1 Background Bovine besnoitiosis caused by the protist cyst-form- ing coccidian parasite Besnoitia besnoiti is a chronic and debilitating disease. It is a well-known endemic Correspondence: davigo20@ucm.es; dgonzalezbarrio@gmail.com 1 Animal Health Department, Faculty of Veterinary Sciences, SALUVET, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain Full list of author information is available at the end of the article Correspondence: davigo20@ucm.es; dgonzalezbarrio@gmail.com 1 Animal Health Department, Faculty of Veterinary Sciences, SALUVET, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain Full list of author information is available at the end of the article 28040 Madrid, Spain Full list of author information is available at the end of the article , p ull list of author information is available at the end of the article Correspondence: davigo20@ucm.es; dgonzalezbarrio@gmail.com 1 Animal Health Department, Faculty of Veterinary Sciences, SALUVET, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain Full list of author information is available at the end of the article Sera from experimentally infected calves p y Sera were obtained from experimentally infected calves that developed a subclinical chronic infection [17]. Twelve healthy 3-month-old calves were randomly allo- cated into four different groups composed of three ani- mals each (G1, G2, G3 and G4) (Table 1). The inoculum consisted of 106 bradyzoites diluted in 2 ml of phosphate- buffered saline (PBS) administered through three differ- ent inoculation routes: intravenous inoculation by single jugular venepuncture (G1), subcutaneous inoculation in the left prescapular area (G2) and intradermal inocula- tion in the thigh area (G3). A non-infected control group was intravenously inoculated with 2 ml of PBS (G4). Daily clinical monitoring was carried out in all inocu- lated animals. Blood samples were collected on the day of inoculation, twice a week for the first month post-infec- tion and once a week until the end of the assay at 70 days post-infection, with a total of 16 blood samplings for each animal. Five millilitres of peripheral blood in Vacutainer tubes (Becton, Dickinson and Company, Plymouth, UK) without anticoagulant was obtained by jugular venepunc- ture. Vacutainers without anticoagulant were centrifuged (1,200×g for 1 min) to obtain serum samples. A total of 64 serum samples were collected [17]. A relevant role of interstitial and recruited mac- rophages in the testicular parenchyma was recently sug- gested to contribute to the pathogenesis of infection at the testicular level, and different molecular markers were identified as prognostic indicators of sterility. In sterile bulls, the acute phase was mainly characterised by the upregulation of interleukin-1 alpha (IL-1α), IL-6 and matrix metalloproteinase inhibitor-1 (TIMP1), whereas in the chronic phase, the upregulation of intercellular adhesion molecules (ICAM) and the downregulation of metalloproteinase 13 (MMP13), tissue-type plasmino- gen activator (PLAT) and IL-1α were observed in the testicular parenchyma [13]. Since monitoring of disease progression in testicular parenchyma by measuring tis- sue markers may be difficult to implement as a routine diagnostic procedure, identification of markers in serum samples may be a more straightforward approach.l Acute or chronic systemic inflammation involves, among other events, an innate immune response through neutrophils or inflammatory cytokines produced by mac- rophages that enter the circulation and induce an oxida- tive response [14] and an increase in positive acute-phase proteins (APPs) and a decrease in negative APPs [15]. Bovine serum panel and study design Bovine serum panel and study design Sera from experimentally (Table 1) and naturally (Tables 2 and 3) B. besnoiti-infected cattle were ana- lysed. All our experimental procedures were approved by the Animal Welfare Committee of the Community of Madrid, Spain following procedures described in Span- ish and EU legislation (PROEX 92/14, Law 32/2007, R.D. 53/2013), and Council Directive 2010/63/EU. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. González‑Barrio et al. Parasites Vectors (2021) 14:488 Page 2 of 13 connective tissues. Pathognomonic tissue cysts are vis- ible in the ocular conjunctiva, mucous membranes of the upper respiratory tract and vestibulum vaginae. However, only a small proportion of animals show severe clinical signs and develop characteristic lesions, and most ani- mals remain subclinically infected [7]. globulins, total protein, and adenosine deaminase (ADA) and acetylcholinesterase (AChE), which are also related to the immune response and can be considered markers of inflammation [16]. Sera from experimentally infected calves Since profiles involving various biomarkers rather than individual tests are recommended [15], we have investi- gated the diagnostic and/or prognostic value of a panel of biomarkers of inflammation in B. besnoiti-infected cattle grouped according to different clinical phases of the disease (acute, chronic and subclinical besnoitiosis). Haptoglobin (Hp), representative of positive APPs, albu- min, representative of negative APPs, and paraoxonase-1 (PON-1), representative of oxidative stress markers, were measured. In addition, we determined the levels of Methods Bull infertility or even sterility with testicular degen- eration and azoospermia is one of the most relevant economic consequences of B. besnoiti infection, particu- larly in extensive natural-service herds [8–11]. Bulls may already develop sterility during the acute phase as a con- sequence of vascular injury in the pampiniform plexus and scrotal skin lesions that hamper testicular ther- moregulation [6]. In chronically infected bulls, numer- ous cysts are present in the scrotal skin, epididymis and ampullae and in the walls of blood vessels in the pam- piniform plexus that may contribute to thermoregula- tion failure favoured by intense fibrosis and thickening of scrotal skin that interfere with normal spermatogenesis [8, 10, 12]. Sera from naturally infected animals l l fi d d ff Animals were classified into different categories [bulls with (i) early acute infection, (ii) late acute infection, (iii) chronic infection, (iv) subclinical infection and (v) non-infected bulls] according to clinical signs and sero- logical results. Molecular results as well as macroscopic and microscopic lesions were available and are shown in Table 2 for acutely infected bulls and Table 3 for chroni- cally and subclinically infected bulls. Twelve acutely infected bulls were analysed. All ani- mals showed clinical signs characteristic of the acute phase, such as fever and orchitis. The acutely infected animals were divided into two subgroups as described Page 3 of 13 González‑Barrio et al. Parasites Vectors (2021) 14:488 Table 1 Summary of the most relevant results regarding clinical signs, macroscopic and microscopic lesions, serological results and parasite detection in experimentally infected calves [17] and naturally infected bulls [6] Pos positive, Neg negative, nd not determined. More detailed data on experimental calf infection were previously published by Diezma-Diaz et al. [43] Group No. animals Systemic clinical signs/ lesions Lesions in testicles Parasite DNA detection Immune response Macroscopic lesions Microscopic findings Humoral Cellular (IFN) IgM IgG WB IgG Avidity Innate Adaptive G1 intravenous inoculation 3 Fever (1 day), lymphadenopathy, congestive ocular sclera, cough, nasal discharge and ocular tissue cysts Not detected Inflammatory infil‑ tration, thrombus in epididymis and scrotum Yes Seroconversion at 12 dpi Seroconversion at 19 dpi Pos Low Peak at 12 dpi Peak at 19 dpi G2 subcutaneous inoculation 3 Fever (7 days), lymphadenopathy, congestive ocular sclera, Cough, nasal discharge ocular tissue cysts Not detected Inflammatory infil‑ tration, oedema and thrombus in the scrotum Yes Seroconversion at 19 dpi Seroconversion at 22 dpi Pos Low Peak at 20 dpi Peak at 12 dpi G3 intradermal inoculation 3 Fever (8 days), lymphadenopathy, congestive ocular sclera, cough and nasal discharge, large number of ocular tissue cysts and skin lesions Not detected Inflammatory infil‑ tration, oedema and thrombus in the scrotum and epididymis hyperkeratosis in scrotum and tissue cysts (aver‑ age diameter of tissue cysts in G3 = 143.8 μm) Yes Seroconversion at 19 dpi Seroconversion at 25 dpi Pos Low Peak at 22 dpi Peak at 19 dpi G4 non-infected control group 3 No clinical signs and/or lesions No lesions No lesions No Seronegative Seronegative Neg nd Neg Neg González‑Barrio et al. Sera from naturally infected animals l l fi d d ff The second ve of a late acute infection, with all bulls showing IgG seroconversion nd not determined, Pos positive, Neg negative aBulls (A1–A6) were sampled twice at a 3-week interval as previously described in Diezma-Diaz et al. [44]. The second sampling was representative of a late acute infection, with all bulls showing IgG seroconversion Thirty-four serum samples from subclinically infected bulls were also analysed. These included seropositive fer- tile bulls without clinical signs or lesions (Table 3). in Table 2: (i) sera from 11 bulls that presented high levels of IgM without the detection of anti-B. besnoiti IgG (early acute infection [EA]) and (ii) sera from seven bulls with IgM and IgG levels (later acute infection [LA]). All animals came from extensive natural-service herds. Sera from naturally infected animals l l fi d d ff The second sampling was representative of a late acute infection, with all bulls showing IgG seroconversion Group Bull number Systemic clinical signs/ lesions Lesions in testicles Parasite DNA detection Immune response Macroscopic lesions Microscopic findings Humoral IgM IgG WB IgG avidity Early acute infection A1a Fever Orchitis nd nd Pos Neg Neg nd A2 Fever Orchitis nd nd Pos Neg Neg nd A3 Fever Orchitis nd nd Pos Neg Neg nd A4 Fever Orchitis nd nd Pos Neg Neg nd A5 Fever Orchitis nd nd Pos Neg Neg nd A6 Fever, pneumonia, oedema in limbs Orchitis, petechiae, haemorrhages and hydrocele Vascular dam‑ age, inflammatory infiltrate, skin lesions and aspermia Yes Pos Neg Neg nd A7 Fever Orchitis nd nd Pos Neg Neg nd A8 Fever Orchitis nd nd Pos Neg Neg nd A9 Fever, pneumonia, oedema in limbs Orchitis, petechiae, haemorrhages and hydrocele Vascular damage, inflammatory infil‑ trate and skin lesions Yes Pos Neg Neg nd A10 Fever, pneumonia, oedema in limbs Orchitis, petechiae, haemorrhages and hydrocele Vascular dam‑ age, inflammatory infiltrate, skin lesions and aspermia Yes Pos Neg Neg nd A11 Fever, pneumonia, oedema in limbs Orchitis, petechiae, haemorrhages and hydrocele Vascular dam‑ age, inflammatory infiltrate, skin lesions and aspermia Yes Pos Neg Neg nd Late acute infection A1b Fever Orchitis nd nd Pos Pos Pos Low A2 Fever Orchitis nd nd Pos Pos Pos Low A3 Fever Orchitis nd nd Pos Pos Pos Low A4 Fever Orchitis nd nd Pos Pos Pos Low A5 Fever Orchitis nd Yes Pos Pos Pos Low A6 Fever, pneumonia, oedema in limbs Orchitis, petechiae, haemorrhages and hydrocele Vascular dam‑ age, inflammatory infiltrate, skin lesions and aspermia Yes Pos Pos Pos Intermediate A12 Congestive scleral conjunctiva Orchitis, hyperkera‑ tosis, acanthosis and ulcers Vascular damage, inflammatory infil‑ trate, skin lesions and tissue cysts (average diameter of tissue cysts = 56.6 μm) Yes Pos Pos Pos Intermediate nd not determined, Pos positive, Neg negative aBulls (A1–A6) were sampled twice at a 3-week interval as previously described in Diezma- sampling was representative of a late acute infection, with all bulls showing IgG seroconversion sitive, Neg negative aBulls (A1–A6) were sampled twice at a 3-week interval as previously described in Diezma-Diaz et al. [44]. Sera from naturally infected animals l l fi d d ff Parasites Vectors (2021) 14:488 Page 4 of 13 Table 2 Summary of the most relevant results regarding clinical signs and serological results in acutely infected bulls [6, 13] nd not determined, Pos positive, Neg negative aBulls (A1–A6) were sampled twice at a 3-week interval as previously described in Diezma-Diaz et al. [44]. Sera from non‑infected bulls Nine serum samples from seropositive chronically infected bulls were analysed. These animals showed clinical signs and lesions compatible with chronic bes- noitiosis, such as scrotal skin hyperkeratosis and testis atrophy with azoospermia (Table 3). Forty serum samples from bulls that were seronegative for B. besnoiti and came from herds without a previous history of bovine besnoitiosis were included in this study (Table 3). González‑Barrio et al. Parasites Vectors (2021) 14:488 Page 5 of 13 Table 3 Summary of the most relevant results regarding clinical signs and serological results in naturally infected bulls (chronically and subclinically infected bulls) Pos positive, Neg negative, nd not determined. aBulls with subclinical infection were IgG-seropositive in several consecutive samplings Group No. animals Systemic clinical signs/ lesions Lesions in testicles Parasite DNA detection Serology Macroscopic lesions Microscopic findings Humoral IgM IgG WB IgG avidity Chronic infection 9 Folds and hyperkera‑ tosis skin of perineum, carpus and tarsus, presence of cysts in scleral conjunctiva Folds and hyperkera‑ tosis in scrotum, skin of perineum, carpus and tarsus Inflammatory infil‑ trate, skin lesions and tissue cysts (average diameter of tissue cysts = 191.0 μm) Yes Pos Pos Pos High Subclinical infection 34 No clinical signs or lesions No lesions Samples not available nd nd Pos Pos nda Non-infected bulls 40 No clinical signs or lesions No lesions No lesions nd Neg Neg Neg nd Blood samples were preserved at 4 °C until arrival at the laboratory and then centrifuged at 3000×g for 10 min, and serum was preserved at − 80 °C until further analysis.h Statistical analysish The levels of APPs (Hp and albumin), an oxidative stress marker (PON-1) and three additional markers of inflammation (globulin, AChE, ADA) were analysed by repeated-measures two-way analysis of variance (ANOVA) and Tukey’s post hoc tests in experimentally infected calves. The non-parametric Kruskal–Wallis test was used to compare naturally infected cattle groups, and Dunn’s post-test was used for multiple comparisons, as the data were not normally distributed. Two different analyses were performed in naturally infected bulls: (i) acutely (bulls A6, A10 and A11) and chronically infected bulls with testicular degeneration and subclinically fertile bulls were compared, and (ii) bulls classified in the dif- ferent categories according to clinical signs and serologi- cal results [bulls with (i) early acute infection (n = 11), (ii) late acute infection (n = 7), (iii) chronic infection (n = 9), (iv) subclinical infection (n = 34) and (v) non-infected bulls (n = 40)] were included in the analyses. Statistical significance for the analysis was established with P < 0.05 using GraphPad Prism 6.01 software (San Diego, CA, USA). The biomarkers measured were Hp, globulin, albumin, total protein, PON-1, ADA and AChE. Analytical methods Analysis of the concentration of Hp, total proteins, albu- min, globulin, serum PON-1 and AChE was performed in an automated analyser (Olympus AU 600, Beckman Coulter), following previously described methods [18– 21]. In brief, the commercial Tridelta Phase range serum Hp kit (Tridelta Development Limited, Ireland) was used to determine Hp. This assay is based on the principle that the peroxidase activity of haemoglobin against inactiva- tion is preserved when haemoglobin-Hp complexes are formed at low pH [18]. Total proteins and albumin were measured using commercially available reagents follow- ing the manufacturer’s instructions. The globulin concen- tration was calculated by subtracting the concentration of albumin from the total protein concentration. Serum PON-1 activity was determined by the analysis of the hydrolysis of p-nitrophenyl acetate to p-nitrophenol [19]. ADA activity was measured using an automated spec- trophotometric method (Adenosine Deaminase Assay Kit, Diazyme Laboratories). This method determines the enzymatic deamination of adenosine to inosine, which after several reactions is transformed to N-ethyl-N-(2- hydroxy-3-sulfopropyl)-3-methylaniline and 4-aminoan- tipyrine. These molecules, in the presence of peroxidase, generate quinine dye, which is kinetically monitored at a 550-nm wavelength [20]. Finally, the AChE activity was analysed by measuring the hydrolyzation of butyrylthi- ocholine iodide (BTCI, Sigma) using 5,5′-dithiobis-2-ni- trobenzoic acid (DTNB, Sigma) as chromophore [21]. Acute‑phase and oxidative stress responses in naturally infected cattlehf Total protein levels were similar among the three inoc- ulated groups with the exception of a few relevant find- ings when compared to the control group: higher levels at 54 dpi (ANOVA followed by Tukey’s post hoc multi- ple comparison tests, q = 3.83, df = 144, P < 0.05) in the subcutaneously inoculated group; higher levels at days 33, 47, 54 and 75 dpi (ANOVA, F(15, 135) = 5.94, P < 0.05) in the intravenously inoculated group; and a significant decrease at 22 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 5.05, df = 144, P < 0.001), followed by an increase at 33 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 3.13, df = 144, P < 0.05) and maintenance of higher levels at 47 and 75 dpi (ANOVA, F(15, 135) = 5.94, P < 0.05) in the intradermally inoculated group.i The serological results of the different markers are shown in Fig. 2. Similar results were obtained regardless of the criteria employed to classify the animals in the differ- ent categories (panel a: sterile acutely and chronically infected bulls versus fertile subclinically infected bulls; panel b: bulls with acute, chronic or subclinical infection based on clinical signs and serological results). However, differences were more evident in panel b when animals were classified according to serological results and clini- cal signs compared to a more restrictive criterion where only sterile and fertile bulls were considered. Non-significant variations in Hp, AChE and PON-1 values were observed among the groups studied (Fig. 2). The remaining markers showed significant differences between some of the groups, as mentioned below. Globulin levels showed a significant increase com- pared to the control group from 22 dpi in the subcu- taneously inoculated group (ANOVA, F(15, 135) = 7.66, P < 0.05) and from 29 dpi in the intravenously (ANOVA, F(15, 135) = 7.66, P < 0.01) and intradermally (ANOVA, F(15, 135) = 7.66, P < 0.01) inoculated groups and thereafter. Total protein levels were higher in chronically infected bulls than in non-infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 28.66, df = 2, P < 0.05) and acutely infected bulls (Kruskal–Wal- lis H-test followed by Dunn’s multiple comparison test: χ2 = 15.51, df = 2, P < 0.05) as shown in Fig. 2a. These values were similar in those groups with clinical signs (Fig. Acute‑phase and oxidative stress responses in experimentally infected calveshf The serological results of the different markers are shown in Fig. 1. The levels of albumin were lower in all inoculated groups than in the control group. This decrease was sig- nificant in the subcutaneously (ANOVA, F(15, 135) = 6.53, P < 0.0001) and intravenously (ANOVA, F(15, 135) = 6.53, P < 0.01) inoculated groups from 8 dpi onwards and in the intradermally inoculated group from 12 dpi onwards (ANOVA, F(15, 135) = 6.53, P < 0.01).i Haptoglobin concentrations were increased from 8 to 12 days post-infection (dpi) in all infected groups. This increase was significant in the subcutaneously inoculated group at 8 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 5.88, df = 144, P < 0.001) and 12 dpi (ANOVA followed by Tukey’s post hoc mul- tiple comparison tests, q = 10.02, df = 144, P < 0.0001), and concentrations decreased thereafter. In both the intravenously and intradermally inoculated groups, this increase was not significant at 8 dpi, and then the lev- els decreased, followed by a significant increase at 22 dpi in the intravenously inoculated group (ANOVA fol- lowed by Tukey’s post hoc multiple comparison tests, q = 4.318, df = 144, P < 0.05) and at 29 dpi (ANOVA fol- lowed by Tukey’s post hoc multiple comparison tests, q = 4.55, df = 144, P < 0.01) and 33 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 5.82, df = 144, P < 0.001) in the intradermally inoculated group. Next, Hp levels decreased until the end of the study. ( ) Significantly lower PON-1 levels were observed at 15 (ANOVA, F(15, 135) = 7.25, P < 0.01) and 19 dpi (ANOVA, F(15, 135) = 7.25, P < 0.01) in the subcutaneously infected group and at 22, 26, 29 and 33 dpi (ANOVA, F(15, 135) = 7.25, P < 0.05) in the intradermally infected group compared to the control group. ( ) Significantly lower PON-1 levels were observed at 15 (ANOVA, F(15, 135) = 7.25, P < 0.01) and 19 dpi (ANOVA, F(15, 135) = 7.25, P < 0.01) in the subcutaneously infected group and at 22, 26, 29 and 33 dpi (ANOVA, F(15, 135) = 7.25, P < 0.05) in the intradermally infected group compared to the control group. ADA levels were generally higher in all infected groups. Acute‑phase and oxidative stress responses in experimentally infected calveshf The most relevant differences corresponded to 19 and 33 dpi in the subcutaneously infected group (ANOVA, F(15, 135) = 3.33, P < 0.05) and to 75 dpi in the intradermally infected group (ANOVA, F(15, 135) = 3.33, P < 0.05). Results Serological results showed a similar response pattern for naturally and experimentally infected cattle, with a few relevant variations. In both naturally and experimentally infected cattle, globulins, albumin and AChE showed sig- nificant variations. Haptoglobin was one of the most rel- evant markers in experimentally infected cattle, whereas ADA showed significant variations in naturally infected cattle. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during chronic infec- tion. The levels of each biomarker at 0 dpi in experimen- tally infected calves and in non-infected field cattle were Page 6 of 13 González‑Barrio et al. Parasites Vectors (2021) 14:488 very similar except for PON-1, which was lower in field animals than in experimentally infected calves. showing lower levels than the control group (ANOVA, F(15, 135) = 15.78, P < 0.05) until the end of the study, and in the intravenously infected group at 64 dpi (ANOVA, F(15, 135) = 15.78, P < 0.01) and 75 dpi (ANOVA, F(15, 135) = 15.78, P < 0.0001). The values increased similarly in both the intradermally infected group and the non- infected group. showing lower levels than the control group (ANOVA, F(15, 135) = 15.78, P < 0.05) until the end of the study, and in the intravenously infected group at 64 dpi (ANOVA, F(15, 135) = 15.78, P < 0.01) and 75 dpi (ANOVA, F(15, 135) = 15.78, P < 0.0001). The values increased similarly in both the intradermally infected group and the non- infected group. Acute‑phase and oxidative stress responses in naturally infected cattlehf 2b) in which levels of total protein were higher in chronically infected and subclinically infected bulls ) AChE values increased from 25 dpi. In general terms, in all infected groups, the levels were similar to or lower than those detected in the control group. The most relevant differences corresponded to the subcu- taneously and intravenously infected group at 47 dpi, González‑Barrio et al. Parasites Vectors (2021) 14:488 Page 7 of 13 than in non-infected bulls (Kruskal–Wallis H-test fol- lowed by Dunn’s multiple comparison test: χ2 = 32.93, df = 2, P < 0.05 and χ2 = 18.37, df = 2, P < 0.07, respec- tively) and early acutely infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 51.28, df = 2, P < 0.001 and χ2 = 36.72, df = 2, P < 0.01, respectively). Intravenous inoculation group (G1) Subcutaneos inoculation group (G2) Intradermal inoculation group (G3) Negative control group (G4) Fig. 1 Serum concentrations of acute-phase response biomarkers in experimentally infected calves with 106 bradyzoites inoculated by intravenous, subcutaneous and intradermal routes and in the control group. P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001 Intravenous inoculation group (G1) Subcutaneos inoculation group (G2) Intradermal inoculation group (G3) Negative control group (G4) rimentally infected calves with 106 bradyzoites inoculated by intravenous, * P < 0.01, * P < 0.001, ** P < 0.0001 Fig. 1 Serum concentrations of acute-phase response biomarkers in experimentally infected calves with 106 bradyzoites inoculated by intravenous, subcutaneous and intradermal routes and in the control group. * P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001 Fig. 1 Serum concentrations of acute-phase response biomarkers in experimentally infected calves with 106 bradyzoites i subcutaneous and intradermal routes and in the control group. * P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001 H-test followed by Dunn’s multiple comparison test: χ2 = 51.28, df = 2, P < 0.001 and χ2 = 36.72, df = 2, P < 0.01, respectively). Acute‑phase and oxidative stress responses in naturally infected cattlehf than in non-infected bulls (Kruskal–Wallis H-test fol- lowed by Dunn’s multiple comparison test: χ2 = 32.93, df = 2, P < 0.05 and χ2 = 18.37, df = 2, P < 0.07, respec- tively) and early acutely infected bulls (Kruskal–Wallis than in non-infected bulls (Kruskal–Wallis H-test fol- lowed by Dunn’s multiple comparison test: χ2 = 32.93, df = 2, P < 0.05 and χ2 = 18.37, df = 2, P < 0.07, respec- tively) and early acutely infected bulls (Kruskal–Wallis Page 8 of 13 González‑Barrio et al. Parasites Vectors (2021) 14:488 González‑Barrio et al. Parasites Vectors (2021) 14:488 besnoitiosis (acute, chronic and subclinical infection). In the present study, several biomarkers showed significant variations in infected animals with a slightly different profile for naturally and experimentally infected cattle. Globulins levels were significantly higher in chroni- cally and subclinically infected bulls than in non-infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 32.78, df = 2, P < 0.01 and χ2 = 19.26. df = 2, P < 0.01, respectively) (Fig. 2a). The similar is found in bulls with clinical signs (Fig. 2b), where signifi- cantly higher levels in were chronically and subclinically infected bulls than in non-infected bulls (Kruskal–Wal- lis H-test followed by Dunn’s multiple comparison test: χ2 = 38.15, df = 2, P < 0.01 and χ2 = 22.44, df = 2, P < 0.05, respectively) and early acutely infected bulls (Kruskal– Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 48.12, df = 2, P < 0.01 and χ2 = 32.40, df = 2, P < 0.05, respectively).i i The search for serum biomarkers can help with early diagnosis and elucidate the clinical and molecular patho- genesis puzzle of bovine besnoitiosis [6, 22]. Previous attempts have focused on haematological and biochemi- cal parameters [23, 24]. However, changes in a few bio- markers were observed when animals with well-defined acute, subacute and chronic besnoitiosis were studied compared to another study where only seropositive cattle were compared with seronegative cattle [23, 24]. There- fore, a well-characterised panel of clinically affected animals is more convenient in the search for biomark- ers, and acute-phase response markers could be of value in bovine besnoitiosis, where a systemic inflammatory reaction is especially intense during the acute phase and focused at the testicular level during acute and chronic besnoitiosis. Acute‑phase and oxidative stress responses in naturally infected cattlehf Moreover, biomarkers with prognostic value at the reproductive level could be helpful to discern ster- ile, subfertile and fertile bulls, since ultrasound analysis is presently the only tool available to determine the extent of genital lesions. However, mild testicular fibrosis is a common genital lesion not necessarily associated with altered semen quality [10]. In this context, the usefulness of acute-phase response biomarkers should be clarified, taking into account their diagnostic value in numerous inflammatory diseases in cattle [25]. Higher values of AChE but not significant were observed in late acute and chronic infections (Fig. 2). Albumin values were lower in all infected groups than in the non-infected group. These differences were significant in acutely infected bulls [acute infection: Kruskal–Wallis H-test followed by Dunn’s multiple com- parison test: χ2 = 45.53, df = 2, P < 0.05 (Fig. 2a); early acute infection: χ2 = 4.15, df = 2, P < 0.001 (Fig. 2b); late acute infection: χ2 = 45.43, df = 2, P < 0.01 (Fig. 2b)] and in subclinically infected bulls (χ2 = 17.17, df = 2, P < 0.05 in Fig. 2a, and χ2 = 18.79, df = 2, P < 0.06 in Fig. 2b) com- pared to non-infected bulls.i Non-significant variations in PON-1 values were observed among the groups studied (Fig. 2a, b). The high- est levels corresponded to bulls with chronic infection, followed by bulls with acute infection and subclinically infected bulls (Fig. 2a). The lowest levels corresponded to bulls with early acute infection (Fig. 2b). l We have reported relevant differences among differ- ent naturally infected bull groups. These animals were grouped following a restrictive criterion based on a com- bination of clinical, histopathological, molecular and serological findings to compare homogeneous groups according to the different clinical phases of B. besnoiti infection. However, in the present study, sample limita- tion was unavoidable, since the time post-infection in field animals was unknown. In fact, the tissue cyst size vari- ability observed in some animals evidenced asynchrony in the chronobiology of the infection, as previously noted for acutely infected bulls [6]. Another limitation was the small sample size (only three acutely infected bulls with testicular degeneration were evaluated), which may have also contributed to the fact that no markers of steril- ity were identified. Acute‑phase and oxidative stress responses in naturally infected cattlehf Nevertheless, this study offers robust Finally, the highest ADA levels corresponded to bulls with an early acute infection, followed by bulls with a late acute infection, chronically infected bulls and finally subclini- cally infected cattle. Significant differences were observed between bulls with an early acute infection and subclinically infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 29.55, df = 2, P < 0.05) and the negative control (χ2 = 33.66, df = 2, P < 0.01). (See figure on next page.) Fig. 2 Serum concentrations of acute-phase response biomarkers in naturally infected and non-infected bulls. a Sterile bulls with acute or chronic infection and testicular degeneration and fertile subclinically infected bulls (seropositive without macroscopic lesions or clinical signs). b All bulls were classified according to clinical signs and serological results. Early acute infection: IgM+, IgG−, clinical signs compatible with an acute infection. Chronic infection: IgM+, IgG+, high IgG avidity index, clinical signs and lesions compatible with a chronic infection. Subclinical infection: IgM+, IgG+, high IgG avidity index, absence of clinical signs and lesions compatible with a chronic infection. #P = 0.07; &P = 0.06; P < 0.05, P < 0.01, P < 0.001, **P < 0.0001 Discussion p The Hp biomarker in experimentally infected cattle appeared to be a good predictor biomarker of the infec- tion, as the first peak of Hp levels at 10 dpi appeared simultaneously in all infected groups and coincided with the incubation period and enlarged lymph nodules just prior to fever development (between 7 and 24 dpi) [17], except for the subcutaneously inoculated group, which presented fever at 7 dpi. This peak was also detected prior to innate immune response interferon (IFN) lev- els (12–22 dpi) and seroconversion (19–25 dpi). A sec- ond increase in Hp levels was detected at approximately 22 and 35 dpi in the intravenously and intradermally inoculated groups, respectively, which may be related to a delay in seroconversion in both groups and the higher severity of clinical signs observed in the intradermally infected group. According to the concentration of Hp, both the subcutaneously and intradermally infected groups presented higher Hp concentrations of 1–2 g/l, while Hp levels were lower in the intravenously infected group (between 0.2–1 g/l), probably related to the sever- ity of the infection, as stated by others [25, 26]. In fact, there are several examples of the diagnostic and prognos- tic value of serum Hp in mastitis, enteritis, peritonitis, pneumonia, endocarditis and endometritis in cattle [25, 26]. However, Hp values did not significantly vary in nat- urally infected animals despite the severity of the infec- tion, particularly in bulls with early acute infection [6]. In our study, Hp levels were in the range of 0.1–0.15 g/l in all naturally infected bulls, similar to the low Hp serum concentrations (< 20 mg/l) reported in healthy cattle [25]. These results might be explained by the narrow diag- nostic window of Hp levels observed in experimentally infected cattle, where the first peak was observed prior to fever development. Bulls with early acute infection had already developed fever a few days prior to sampling. Another feasible explanation could be that the concentra- tion of Hp may decrease dramatically during accelerated haemolysis [27]. However, our serum samples were not apparently haemolysed. ADA is an important enzyme that participates in neu- romodulation, apoptosis, necrosis and proliferation of lymphocytes during cellular response [30]. According to Franco et al. [31], ADA acts during inflammation in injured tissue, i.e., regulates the concentration of extra- cellular adenosine, an important molecule with anti- inflammatory properties, since this enzyme converts adenosine to inosine. Discussion We have studied for the first time a panel of serum bio- markers representative of either APPs or oxidative stress response in the different clinical scenarios of bovine Page 9 of 13 González‑Barrio et al. Parasites Vectors (2021) 14:488 Haptoglobin Total proteins Globulin AChE Albumin PON-1 ADA a b Fig. 2 (See legend on previous page.) ADA Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) González‑Barrio et al. Parasites Vectors (2021) 14:488 González‑Barrio et al. Parasites Vectors (2021) 14:488 Page 10 of 13 Page 10 of 13 Albumin and PON-1 peak detection varied among groups at 15 dpi in the subcutaneously inoculated group and 25 dpi in the intradermally and intravenously inocu- lated groups around humoral (seroconversion; 19–25 dpi) and cellular adaptive immune responses (adaptive immune response IFN levels; 12–19 dpi). A significant decrease in albumin concentrations was also observed in acutely infected bulls that agrees with the decrease in serum albumin levels also reported in naturally infected bulls with acute and subacute B. besnoiti infection by Langenmayer et al. [23]. These findings indicate that the concentrations of albumin may reflect the magnitude of changes and predict adverse clinical outcomes. Vincent et al. [28] suggested that low concentrations of albumin in the blood serum serve as an important prognostic indicator that is associated with increased mortality and morbidity. In fact, three naturally infected bulls died dur- ing the acute phase with respiratory clinical signs, fever and orchitis [6]. On the other hand, it is possible that the reduction in PON-1 levels in acutely infected animals coincides with endothelial dysfunction, as stated by oth- ers [29]. Indeed, severe vascular lesions were found in the pampiniform plexus of acutely infected bulls [6]. data and complements a previous study that identified a few molecular markers related to disease progression in testicular tissues [13]. Moreover, the results obtained in experimentally infected cattle offer valuable data about the kinetics of the acute-phase response during acute and chronic infection, as discussed below. Haptoglobin, albumin, PON-1 and ADA were identified as the most promising biomarkers for the acute phase of the infection. Haptoglobin was increased, whereas albu- min and PON-1 showed a decrease in experimentally infected cattle, and peaks were detected during the first month post-infection. In naturally infected cattle, the lowest values of albumin and PON-1 and the highest val- ues of ADA corresponded to bulls with acute infection. Discussion In our study, ADA levels from acutely infected bulls were higher than in the other groups of bulls (significant differences with non-infected and subclinically infected bulls). This increase in ADA activity has been described in the course of diseases that induce a cellular immune response, as for example in neosporosis [32] and toxo- plasmosis [33]. Thus, increased ADA activity leads to decreased adenosine levels, which might regulate to the inflammatory process and tissue damage induced by B. besnoiti infection. In general terms, apart from a suggested correla- tion of APP levels with acute besnoitiosis severity, this acute response appears to be related to a few upregu- lated molecular markers (IL-6, IL-1α, PLAT and TIMP1) detected in the testicular parenchyma of acutely infected bulls versus a downregulation of PLAT, IL-1α, IL-6, IL-8 and IL-10 in chronically and subclinically infected bulls [13]. APPs are predominantly glycoproteins synthesised by hepatocytes in response to IL-6, tumour necrosis fac- tor alpha (TNF-α), and other proinflammatory cytokines such as IL-1α [34]. This relationship between overexpres- sion of pro-inflammatory cytokines and high levels of González‑Barrio et al. Parasites Vectors (2021) 14:488 Page 11 of 13 Page 11 of 13 APPs has been described in experimental Trypanosoma evansi infections in rats [35]. promising diagnostic value. The biomarkers associ- ated with an acute infection (Hp, albumin, PON-1 and ADA) are related to previously reported molecular biomarkers in the testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement IgM detection. High total protein and globulin values were character- istic of chronically infected cattle, in agreement with the observations made by Langenmayer et al. [23, 36], who reported high levels of serum total proteins and globulins in cows with chronic B. besnoiti infection. In addition, higher antibody levels have been associated with severely chronically affected animals [37]. Moreover, high values of globulins have been described in cows with chronic diseases [38], high values of total proteins in cows diag- nosed with endometritis [39] and chronic lameness [40]. Globulin concentrations above 50 g/l are an indicator of chronic inflammation [41]. In our study, animals with chronic infection, in addition to having values of 70 g/l (Fig. Acknowledgements We are grateful to Professor Javier Blanco who contributed to sample col‑ lection. We would like to thank the veterinarian practitioners Javier Carvajal, Fernando Criado and Javier Brieva, the slaughterhouses and veterinarian of Encinar de Humienta (Cáceres), Cárnica Colmenar (Madrid) and Carnes Sierra Madrid (Madrid). Availability of data and materials Data supporting the conclusions of this article are included within the article. Authors’ contributions GA-G, LMO-M, JJC and DG-B contributed to the design of the study. GA-G and JJC provided project supervision and coordination. CD-D, IF and DG-B collected samples. DG-B, CD-D and AH-L performed the laboratory work. DG-B and GA-G contributed to the interpretation of data and writing of the first draft of the manuscript. All authors edited the manuscript. All authors read and approved the final manuscript. Abbreviations AChE A l h AChE: Acetylcholinesterase; ADA: Adenosine deaminase; APPs: Acute-phase proteins; Hp: Haptoglobin; ICAM: Intercellular adhesion molecule; IFN: Inter‑ feron; IL-1α: Interleukin-1 alpha; IL-6: Interleukin-6; IL-8: Interleukin-8; IL-10: Interleukin-10; MMP13: Metalloproteinase 13; PLAT: Tissue-type plasminogen activator; PON-1: Paraoxonase-1; TIMP1: Matrix metalloproteinase inhibitor-1; TNF: Tumour necrosis factor. i Markers of sterility were not identified, since acutely and chronically infected sterile bulls did not show a simi- lar pattern of acute-phase response compared to non- infected and subclinically infected animals. The most relevant finding was that AChE values were higher in late acutely infected bulls, and significantly higher levels of AChE were detected in chronically infected animals than in subclinically infected animals. An increase in AChE activity may lead to enhanced degradation of acetylcho- line, a molecule that has an anti-inflammatory effect [42]. Therefore, the presence of an intense inflammatory infil- trate observed in natural infection [6] in both acute and chronic besnoitiosis may be associated with an increase in AChE activity. Remarkably, AChE values were higher in calves (≈ 0.5 μmol/ml·min) than in bulls (≈ 0.3 μmol/ ml·min), even though both groups were classified as subclinically infected cattle. A more severe infection in experimentally infected cattle could explain this find- ing, since microscopic lesions (e.g., thrombus, oedema, inflammation, hyperkeratosis and dilated sweat glands) were detected in testes from infected calves [43].hf Discussion 2), also presented an intense inflammatory infiltrate with the presence of lymphocytes, some macrophages, abundant young granulation tissue and an abundance of fibroblasts in the testicular parenchyma [13].i On the other hand, the value of AChE associated with elevated globulin and total protein levels as indicative of testicular injury remains to be elucidated. The kinet- ics of the different biomarkers should be corroborated in future longitudinal field studies, since biomarkers could aid in decisions regarding the use of seropositive bulls for natural mating. Conclusions We have described the pattern of the acute-phase response during acute and chronic bovine besnoitio- sis. The results have identified several biomarkers with Competing interests The authors declare that they have no competing interests. Funding This study was financed by the Spanish Ministry of Economy and Competi‑ tiveness (AGL-2016-75202-R), the Spanish Ministry of Science and Innovation (PID2019-103960RB-I00) and by the Community of Madrid (PLATESA P2018/ BAA-4370). DGB was funded by the Spanish Ministry of Science through a Juan de la Cierva postdoctoral fellowship (FJCI-2016-27875). CD-D was finan‑ cially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-069839). AH-L was supported by a pre-doctoral grant from University of Murcia (R-1207/2017). IF, LMO-M and GA-G are part of the TOXOSOURCES consortium, supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement no. 773830: One Health European Joint Programme. 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Risk factors associated with excessive negative energy balance in commercial United Kingdom dairy herds. Vet J. 2019;250:15–23. 42. Schwertz CI, Do Carmo GM, Bottari NB, Da Silva ES, Gabriel ME, Lucca NJ, et al. Relationship between pathological findings and cholinesterase activity and nitric oxide levels in cattle infected naturally by Eurytrema coelomaticum. J Comp Pathol. 2016;154:150–6. 43. Diezma-Díaz C, Tabanera E, Ferre I, Pizarro-Díaz M, González-Huecas M, Blanco-Murcia J, et al. Histological findings in experimentally infected male calves with chronic besnoitiosis. Vet Parasitol. 2020;281:109120. 40. Herzberg D, Strobel P, Müller H, Meneses C, Werner M, Bustamante H. Proteomic profiling of proteins in the dorsal horn of the spinal cord in dairy cows with chronic lameness. PLoS ONE. 2020;15:e0228134. Publisher’s Note 42. Schwertz CI, Do Carmo GM, Bottari NB, Da Silva ES, Gabriel ME, Lucca NJ, et al. Relationship between pathological findings and cholinesterase activity and nitric oxide levels in cattle infected naturally by Eurytrema coelomaticum. J Comp Pathol. 2016;154:150–6. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 43. Diezma-Díaz C, Tabanera E, Ferre I, Pizarro-Díaz M, González-Huecas M, Blanco-Murcia J, et al. Histological findings in experimentally infected male calves with chronic besnoitiosis. Vet Parasitol. 2020;281:109120. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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https://openalex.org/W4206646077	https://bmcwomenshealth.biomedcentral.com/track/pdf/10.1186/s12905-020-01042-1	English	null	Breast self-examination and associated factors among women in Wolaita sodo city, Ethiopia: Community based cross sectional study	Research Square (Research Square)	2,020	cc-by	7,582	Breast self-examination and associated factors among women in Wolaita Sodo, Ethiopia: a community-based cross- sectional study Breast self-examination and associated factors among women in Wolaita Sodo, Ethiopia: a community-based cross- sectional study Temesgen Lera1* , Aman Beyene2, Befekadu Bekele1 and Solomon Abreha1 Abstract Background: The early detection of breast cancer plays an important role in decreasing morbidity and mortality of breast cancer. Breast self-examination (BSE) is one screening method used for the early detection of breast cancer. BSE involves the woman looking at and feeling each breast for possible lumps, distortions, or swellings. BSE is a simple exercise that can potentially save women’s lives, but BSE receives relatively little attention and no study has yet addressed BSE at the community level. Here we assessed BSE and associated factors among women aged 20– 65 years in Wolaita Sodo city, Ethiopia. Methods: This was a community-based, cross-sectional study. Systematic random sampling was used to select 626 women aged 20–65 years old. Data were collected using a pre-tested and structured questionnaire. Data were recorded using EpiData version 3.5.1 and exported to SPSS version 21 for cleaning and statistical analysis. Bivariable analysis was performed, and variables with a p-value < 0.25 were used in multiple logistic regression analysis. Multiple logistic regression was employed, and variables with p-values < 0.05 were considered statically significant. Results: A total of 629 women aged between 20 and 65 years were included in the study. Over half (60.9%) of participants were aged between 20 and 29 years, and 8.2% were < 50 years old. Women who mentioned BSE as a method for the early detection of breast problems were 6.36-times (95% CI: 3.72, 10.71) more likely to perform BSE than those who reported that they did not know of any method. Those who had breast fed for 13–24 months were 2.43 times (95% CI: 1.28, 4.59) more likely to examine their breasts than those who breast fed for different durations or used other methods. Employed study participants were 3.13-times (95% CI: 1.14, 8.58) more likely to practice BSE than those who were not employed. Likewise, students were 3.73-times (95% CI: 1.19, 11.73) more likely to perform BSE. Conclusions: In our sample, women’s practice of BSE was relatively low. Knowledge of BSE, breastfeeding up to 24 months, being employed, and being a student were factors affecting performing BSE. Educating girls and increasing awareness, including through electronic media, are important to encourage BSE and improve breast cancer outcomes. Keywords: Breast self-examination (BSE), Breast cancer, Wolaita Sodo, Ethiopia * Correspondence: temesgenlera@yahoo.com 1School of Public Health, Wolaita Sodo University, Wolaita, Ethiopia Full list of author information is available at the end of the article Lera et al. BMC Women's Health (2020) 20:167 https://doi.org/10.1186/s12905-020-01042-1 Lera et al. BMC Women's Health (2020) 20:167 https://doi.org/10.1186/s12905-020-01042-1 Study population Breast self-examination (BSE) is a breast cancer screening method that involves the woman looking at and feeling her own breasts for possible lumps, distor- tions, or swellings. BSE is a simple exercise that can po- tentially save women’s lives. BSE is recommended for every woman from the age of 20 years onwards, and BSE is recommended to be performed for 20 min every month [5]. However, women in developing countries are known not to perform BSE for various reasons [1]. A woman who performs BSE may be more motivated to seek medical attention, including clinical breast examin- ation (CBE) and mammography [6]. Houses in selected kebeles were included by systematic random sampling, and study unit was selected by the simple random sampling technique. Source population All women aged 20-65 years were considered as a source population. Study setting and design The study was carried out in Wolaita Sodo city. The city has a total population of 250,521 (male 79,871 (52%), fe- male 73,650 (48%)), and the city has three sub-cities, 18 kebeles, three health centers, one Ministry of Health- owned hospital, and one private hospital. The city is lo- cated 160 km from the regional city Hawassa and 327 km from Addis Ababa, the capital of Ethiopia [9]. A community-based cross-sectional study design was employed. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 10 Lera et al. BMC Women's Health (2020) 20:167 Page 2 of 10 Inclusion and exclusion criteria Women age 20–60 years were included in the study, and women who were seriously ill during the period of data collection, had known breast cancer, and those not will- ing to participate in the study were excluded. Plain English summary patients ignore their initial symptoms for an average of over 1.5 years [7]. BSE is still recommended as a general approach to increasing breast health awareness and de- tecting any anomalies because it is free, painless, and easy to practice [5]. Furthermore, the American Cancer Society also recommends that women, from age 20, should be educated on the advantages and disadvantages of performing monthly BSE [8]. Detecting breast cancer early is important for decreasing its associated morbidity and mortality. Breast self- examination (BSE) is a screening method used to detect breast cancer early. In this study, respondents were asked via a closed-ended structured questionnaire con- ducted through face-to-face interviews whether or not they practiced breast self-examination regularly. Six hun- dred twenty-nine women participated, with a response rate of 100%. 76% of study participants were married, and 49.8% percent of women were housewives. We found that the prevalence of BSE among this sample of women was 34.5%. Knowledge of BSE, breastfeeding up to 24 months, being employed, and being a student were factors associated with performing BSE. Therefore, edu- cating girls and increasing awareness, including through electronic media, are important to encourage BSE and improve breast cancer outcomes. Breast cancer in low- to middle-income countries tends to present late and has poor clinical outcomes due to several factors such as unequal access to prompt high-quality treatment and a lack of early screening [3]. Despite the fact that breast cancer has recently become a leading cause of mortality in young women, especially those in urban areas, the Ethiopian health system has traditionally concentrated on communicable disease pre- vention as a public health priority [7]. Furthermore, there are very few reports measuring BSE at the popula- tion level. Here we addressed this knowledge gap by assessing BSE and its associated factors in women aged 20–65 years in Wolaita Sodo city, Ethiopia, to identify the need for information on BSE in Ethiopia. Background Breast cancer is an issue of public health importance in both developed and developing nations. Due to its high prevalence, breast cancer places significant pressure on health system resources and incurs significant healthcare costs. Breast cancer is the second leading cause of death among women globally, more than a million new cases detected yearly, accounting for 10.9% of all cancer cases, second to lung cancer [1, 2]. Its occurrence is growing both in developed and developing regions. An estimated 636,000 new cases were identified in high-resource countries, while 514,000 cases were identified in low- and middle-resource countries in 2008. Breast cancer is the most recurrent cause of death among women both in developing and developed counties, with an estimated 269,000 and 189,000 losses, respectively. Seventy percent of all breast cancer cases will be in low- and middle- resource countries by 2020 [3] globally. The occurrence of breast cancer varies across the African region, ranging from 19.3 per 100,000 per year in Eastern Africa to 38.1 per 100,000 per year in Southern Africa [4]. Data collection procedure Data collection procedure Structured, pre-tested, and interviewer-administered questionnaires were used. The questions included sociodemographic characteristics and BSE-related is- sues. The questionnaires were adapted from the Ethi- opian Development and Health Survey (EDHS) and the published literature. Data were collected between 24 November 2018 and 2 December 2018 by trained data collectors. Data were collected through face-to- face interviews, maintaining the pre-determined sam- pling intervals. The data collectors informed the re- spondents about all the details of the research and procedures, what was expected of them, and the po- tential risks and benefits in order to encourage accur- ate and honest responses. When the woman was not available at the first visit, data collectors arranged al- ternative visits. If a woman was still not available on second visits or declined to participate, the household was skipped and the immediate next household in the sampling frame was considered. N ¼ Z 1:96 ð Þ2 P 1 −P ð Þ=d2 with the assumptions z = 1.96 at a 95% confidence level; the P-prevalence of BSE was taken as 53.6% (0.536) from a previous study [10]; and the non-response rate was 10%, confidence levels of 95%, and a 5% margin of error. with the assumptions z = 1.96 at a 95% confidence level; the P-prevalence of BSE was taken as 53.6% (0.536) from a previous study [10]; and the non-response rate was 10%, confidence levels of 95%, and a 5% margin of error. Therefore, the calculated sample size was 572 and, after considering a 10% non-response rate, the final sam- ple size was 629. Therefore, the calculated sample size was 572 and, after considering a 10% non-response rate, the final sam- ple size was 629. Sampling procedure l l Multi-stage sampling was used to select study respondents. First, among the 18 kebeles in the city, 6 kebeles were ran- domly selected by simple random sampling to represent all kebeles. The source population in each selected kebele was identified from Wolaita Sodo Finance Economic Develop- ment Department data [9]. The sample size allocated to the selected kebeles was proportional to the source population in the kebele. The sampling interval was calculated by div- ing the source population by our sample [(N/n) = 15,098/ 629 = 24]. The first household was selected by simple ran- dom sampling from 1 to 24 listed households, and the 10th household was chosen randomly. Sample size and sampling procedure Sample size was calculated with EPidata statistical soft- ware version 3.03 using the single population proportion statistical formula: In Ethiopia, over half of women with breast cancer are aged 50 and younger. It has been shown that 69.6% of Page 3 of 10 Lera et al. BMC Women's Health (2020) 20:167 Age 20 to 65 Age 20 to 65 American Cancer Society-recommended BSE for women aged 20 or older, and mammography for women aged 40 or older [8]. Data quality management Before data collection, the questionnaire was first pre- pared in English and translated into Amharic and back to English to maintain consistency. Data collectors and supervisors received 2 days of training by the principal investigator before data collection. Sampling frame (household)-containing lists of the population in the selected kebeles were obtained, and every 24th house was visited to select the study popula- tion by systematic random sampling until reaching the intended sample size for a given kebele. The respondents from each selected household were taken by simple ran- dom sampling whenever there was more than one eli- gible woman in a selected household (Fig. 1). A pre-test was conducted in Dilbetigil kebele, which was not one of the selected kebeles, and 5% of the total sample size was tested. Based on the pre-test, question- naires were revised, edited, and necessary corrections made. Daily checks of data for completeness and consistency were performed during data collection. Fig. 1 Sampling procedure of the study Page 4 of 10 Lera et al. BMC Women's Health (2020) 20:167 Page 4 of 10 Distribution of spousal/parents support to perform BSE and the need of information on BSE Spousal and other support for BSE was 67.2%. However, 88% of BSE performers were confident in performing self-examination. Almost all study participants (98.6%) knew early detection of breast cancer improved the chances of survival. 91% of respondents wanted more in- formation about BSE. Within the year before this study, among performers of BSE, 149 participants performed it more than six times and 26 participants did it four to six times (Table 4). Sociodemographic characteristics of the subjects Six hundred and twenty-nine women were interviewed and subjected to analysis. The participants were aged be- tween 20 and 65 years. 60.9% of participants were aged 20–29 years, and 8.2% were aged over 50 years. Eighty- seven percent of participants were Wolaita in ethnicity, and 70.6% were protestant. Three hundred and thirty- eight respondents had completed secondary education, and 478 study participants were married (Table 1). Knowledge of the right age to perform BSE and the reasons given to perform BSE Ethical clearance was obtained from Wolaita Sodo Uni- versity Institutional Review Board (IRB). Written permis- sion was obtained from Sodo City Health Department. During data collection, all respondents were asked for permission, and informed consent was obtained from each study participant. Those performing BSE had different perspectives on the correct age to commence BSE, which should be between 10 and 30 years of age (mean age 18.41 ± 2.8 SD). Of these, 63 recommended BSE at 20 years and 144 responded “I don’t know”. Of those who had performed BSE, 107 respondents reported no specific time/any time they could remember, and 133 reported practicing it on a regular basis (Table 3). Operational definitions Breast self-examination (BSE) The self-examination of the breasts to identify any changes in the breasts [11]. Data analysis procedures sources of information. Forty five respondents reported receiving information on breast cancer from other sources like journals, books, and non-governmental or- ganizations (Fig. 2). Data were entered into EpiData version 3.1 and cleaned to check for accuracy, consistency, completeness, and values, and any identified error was corrected. Data were exported into SPSS version 21 (IBM Statis- tics, Chicago, IL) for analysis. Descriptive statistics were performed. Bivariable analyses were computed, and vari- ables with p-values < 0.25 were considered candidates for multiple logistic regression analysis. Multiple logistic regression analysis was performed, and variables with p- values ≤0.05 were considered statistically significant. Ad- justed odds ratios (AOR) with 95% confidence intervals (CI) were calculated. The reported methods of breast cancer screening were clinical breast examination (45.3%) and BSE (18%) and 36.5% women responded that they did not know of any method of breast cancer screening. Of those who had heard about breast cancer, 46% had also heard about BSE and, among study participants who had received in- formation on BSE, 79.8% had performed BSE and 71.6% reported they regularly performed it. Among those who had ever heard of breast cancer, 92% knew (heard) their family history of breast cancer (Table 2). Breast self-examination performed Of respondents who had ever heard of BSE, 45 believed that they had some kind of barrier to practicing BSE. However, over half of performers (54.8%) claimed that there was no obstacle to performing BSE (Fig. 3). Breast self examination performed If the woman performed BSE at least once in the last 12 months. Factors associated with BSE T different educational level and/or differences in the that 46% of women had lower than in several oth countries: 78.4% in the M danian women [14], 47% teacher training college Chinese immigrants [1], a area of western Turkey [1 in our study was higher t showed that only 41.5% [17]. These differences are in socioeconomic and dem study populations. The relatively low know BSE i h h Table 1 Sociodemographic characteristics of the women in Wolaita Sodo, 2019 (n = 629) Variables/characteristics Frequency (%) Age distribution of the women 20–29 years 383 (60.9) 30–39 years 139 (22.1) v40–49 years 55 (8.7) v ≥50 years 52 (8.3) Marital status Never married 113 (18) Married 478 (76) Divorce 17 (2.7) Widowed 21 (3.3) Participant’s education No education 73 (11.6) Primary 218 (34.8) Secondary 179 (28.4) Higher education 159 (25.3) Husband’s education No education 20 (4.2) Primary 131 (27.4) Secondary 148 (31) Higher education 179 (37.4) Religion Protestant 444 (70.6) Orthodox 131 (20.8) Muslim 24 (3.8) Catholic 16 (2.5) Others 14 (2.2) Ethnicity Wolaita 549 (87.3) Gamo 32 (5.1) Garage 18 (2.9) Amhara 16 (2.5) vOthers 14 (2.2) Occupational status of the women House wife 312 (49.6) Employee 133 (21.1) Merchant 74 (11.8) Student 54 (8.6) Other 56 (8.9) Age at which first pregnancy occurred 15–24 years 397 (76.9) 25–34 years 111 (21.5) 35–44 years 2 (0.4) Table 1 Sociodemographic c Wolaita Sodo, 2019 (n = 629) Variables/characteristics Duration of breastfeeding Birth-12 months 13–24 months 25–34 months Number of children None One Two Three or more Table 1 Sociodemographic characteristics of the women in Wolaita Sodo, 2019 (n = 629) (Continued) Variables/characteristics Frequency (%) Duration of breastfeeding Birth-12 months 77 (15.4) 13–24 months 280 (56.6) 25–34 months 141 (28) Number of children None 23 (4.7) One 104 (21.4) Two 115 (23.7) Three or more 244 (50.2) previously heard of BSE were significantly associated with performing BSE (p < 0.05). Women who had men- tioned BSE as a method for the early detection of breast problems were 6.36-times (95% CI: 3.72, 10.71) more likely to perform BSE than those reported not knowing any method. Those who had breast fed for 13–24 months were 2.43-times (95% CI: 1.28, 4.59) more likely to examine their breasts than those who mentioned dif- ferent categories/duration of breast feeding. Employed study participants were 3.13-times more likely (95% CI: 1.14, 8.58) to practice BSE than those who were un- employed. Factors associated with BSE Likewise, students were 3.73-times (95% CI: 1.19, 11.73) more likely to perform BSE than others (Table 5). Factors associated with BSE Bivariate binary logistic regression analysis revealed that occupational status, duration of breastfeeding, female education, husband’s education, early detection method for breast cancer, source of information, and knowledge of personal history of having a benign breast lump were candidates for multiple logistic regression analysis at p ≤ 0.25. Knowledge and practice of BSE and information sources Among the respondents, 94% knew (heard or read) about breast cancer and their main source of informa- tion was electronic media (62.4%). The contribution of health professionals as a source of breast cancer infor- mation was found to be 14.7%. Electronic media, family/ friends, and health workers were reported as major In the multivariable logistic regression analysis, occu- pational status of women, duration of breastfeeding, and Lera et al. BMC Women's Health (2020) 20:167 Page 5 of 10 Page 5 of 10 Table 1 Sociodemographic characteristics of the women in Wolaita Sodo, 2019 (n = 629) Variables/characteristics Frequency (% Age distribution of the women 20–29 years 383 (60.9) 30–39 years 139 (22.1) v40–49 years 55 (8.7) v ≥50 years 52 (8.3) Marital status Never married 113 (18) Married 478 (76) Divorce 17 (2.7) Widowed 21 (3.3) Participant’s education No education 73 (11.6) Primary 218 (34.8) Secondary 179 (28.4) Higher education 159 (25.3) Husband’s education No education 20 (4.2) Primary 131 (27.4) Secondary 148 (31) Higher education 179 (37.4) Religion Protestant 444 (70.6) Orthodox 131 (20.8) Muslim 24 (3.8) Catholic 16 (2.5) Others 14 (2.2) Ethnicity Wolaita 549 (87.3) Gamo 32 (5.1) Garage 18 (2.9) Amhara 16 (2.5) vOthers 14 (2.2) Occupational status of the women House wife 312 (49.6) Employee 133 (21.1) Merchant 74 (11.8) Student 54 (8.6) Other 56 (8.9) Age at which first pregnancy occurred 15–24 years 397 (76.9) 25–34 years 111 (21.5) 35–44 years 2 (0.4) ≥45 years 6 (1.2) previously heard of BSE with performing BSE (p < tioned BSE as a method f problems were 6.36-time likely to perform BSE tha any method. Those who months were 2.43-times ( to examine their breasts t ferent categories/duration study participants were 3 1.14, 8.58) to practice B employed. Likewise, stud 1.19, 11.73) more likely (Table 5). Discussion This study showed that, i in an Ethiopian city, 94% or read about breast can the 83% reported for wom Ethiopia [10] and lower th students at 99.5% [12]. Discussion A study performed in northern Ethiopia on breast cancer screening methods reported by health extension workers were BSE (14.4%), clinical breast examination (22.3%), and mammography (3%) [23]. These differences may be due to different educational levels and partici- pant groups. In the present study, 98% of breast cancer-informed participants knew that early detection of breast cancer improves chances of survival from the disease. This find- ing is supported by the study of Mekelle town women [10] and higher than a previous in western Ethiopia (74.7%) [19]. Among respondents who reported to have had information on BSE, 79% had at some point per- formed BSE, lower than in a study of Nigerian Nurses in Lagos General Hospital (89%) [4], and higher than stud- ies of women in northern Ethiopia (37.3%) [20], female Malaysian students (25.5%) [12], female traders in Ibadan, Nigeria (18%) [6], women in a rural area of west- ern Turkey 40.9% [16], and female household heads in northern Ethiopia (53.6%) [10]. Our result was similar to that reported for female health professionals in Welega (77%) [19]. Forty-five percent of our study sample per- formed BSE on a regular monthly basis, which is higher than in Jordanian women (only 7%) [14], female Malay- sian students (31.2%) [12], and female undergraduate students in a teacher training college in Cameroon (25.9%) [15]. This could be due to the different periods when these studies were conducted. In this study, 53.6% of women conducting BSE had sup- port from their partners, which was higher than another study reporting that 39.8% of women performing BSE were getting support from their spouses/partners [24]. The major barriers to practicing BSE identified in the present study were pressure of work/being too busy, not having enough privacy to perform BSE, thinking that breast cancer wasn’t possible, forgetfulness, and doubt about its effectiveness in 30 (13.8%), 14 (6.4%), 13 (5.9%), 10 (4.6%), and 11(5%) of respondents, respect- ively. However, over half of women performing BSE (119; 54.8%) could not identify any obstacle to perform- ing BSE. A study conducted in western Ethiopia reported that no current breast problem (12.7%), not feeling com- fortable performing BSE (2.7%), scared of being diag- nosed with breast problem or cancer, not believing it is beneficial (4%), and not knowing how to do it (7.7%) were barriers to not performing BSE [19]. Discussion This study showed that, in a random sample of women in an Ethiopian city, 94% of respondents had ever heard or read about breast cancer. This figure is higher than the 83% reported for women in Mekelle town, northern Ethiopia [10] and lower than a study of female Malaysian students at 99.5% [12]. These differences might reflect different educational levels among the study participants and/or differences in the study settings. We also found that 46% of women had previously heard about BSE, lower than in several other studies conducted in other countries: 78.4% in the Malaysian study [13], 67% in Jor- danian women [14], 47% in undergraduate students in a teacher training college in Cameroon [15], 80.9% in Chinese immigrants [1], and 72.1% in women in a rural area of western Turkey [16]. However, awareness of BSE in our study was higher than in Benghazi, Libya, which showed that only 41.5% of women had heard of BSE [17]. These differences are likely to represent differences in socioeconomic and demographic characteristics of the study populations. The relatively low knowledge of our respondents about BSE might prevent them from performing BSE, which might reduce chances of early detection of the disease. Lera et al. BMC Women's Health (2020) 20:167 Page 6 of 10 Fig. 2 Breast cancer information sources among women in Sodo city, 2019 Fig. 2 Breast cancer information sources among women in Sodo city, 2019 women (60.28% recommended age 20) [21], and women in Kyadondo County, Uganda (40%) [22]. 62% of our participants who had received breast cancer information indicated that their main source of informa- tion was from the media, with colleagues and friends also mentioned as important sources of information about breast cancer. Amazingly, the proportion of re- spondents who mentioned health professionals as a main source of breast cancer information was only 13.8%. This is consistent with findings from a similar study con- ducted in Jordanian females where relatives, friends, and neighbors were found to be the main sources of breast cancer information [14] but different to a study of Iran- ian women, where health professionals were the main source of information (32.4%) [18]. The different breast screening methods recognized by participants in the present study were BSE in 15.4% of women, clinical breast examination in 42.4%, and mam- mography in 0.3%. Canadian women knew about BSE (64.3%), clinical breast examination (45.7%), and mammography (32.7%) [20]. Discussion BMC Women's Health (2020) 20:167 Page 7 of 10 Table 2 Knowledge and practice of BSE and main information sources among women in Wolaita Sodo, 2019 (n = 629) Characteristics/variables Frequency (%) Ever heard of breast cancer Yes 591 (94) No 38 (6) Source of information Electronic media 366 (62) Journal/brochure/leaflet/magazine 4 (0.8) Books 3 (0.5) Educational institution 9 (1.4) Non-governmental organizations 1 (0.2) Health workers 87 (14.7) Family/friend 93 (15.8) Other person 28 (4.7) Previously heard of BSE Yes 272 (46) No 319 (54) Early detection method for breast cancer Breast self-examination (BSE) 107 (18) Clinical breast examination (CBE) 268 (45.3) I don’t know 216 (36.5) Performed breast self-examination Yes 217 (78) No 55 (20.2) Still perform breast self-examination Yes 195 (90) No 22 (10) Knowledge that early detection of breast cancer improves chances of survival Yes 570 (96.7) No 13 (2) I don’t know 8 (1.3) Family history of breast cancer Yes 14 (2.4) No 523 (88.6) I don’t know 54 (9) Personal history of having benign breast lamp Yes 20 (3.3) No 197 (33.3) I don’t know 374 (63.4) Knowledge of someone suffering from breast cancer Yes 116 (20) No 475 (80) Ever nursed a breast cancer patient Yes 4 (1) No 587 (99) Table 2 Knowledge and practice of BSE and main information sources among women in Wolaita Sodo, 2019 (n = 629) (Continued) Characteristics/variables Frequency (%) Had close contact with a person with a benign breast lamp Yes 18 (3) No 573 (97) Knowledge of personal status of other body part cancer Yes 446 (75) No 145 (25) Position of BSE Standing 49 (22.4) Lying 45 (21) Sitting 16 (7.3) Standing and lying 107 (49.3) Technical knowledge of BSE With palm and three middle fingers 35 (16) Simply touch and feel 157 (72.5) I don’t know 25 (11.5) BSE practices applied Inspection 3 (1.4) Palpation 116 (53.6) Inspection and palpation 98 (45) Knowledge about which arm to be used for BSE Right hand for left breast and vice versa 33 (15) The same arm for the same side breast 13 (6) Any (no protocol) 171 (79) northern Ethiopia indicated that the major barriers to practicing BSE were an absence of symptoms and lack of knowledge about its importance [10]. Discussion In another study, the three main reasons for not per- forming BSE were no breast problem (53.2%), not know- ing the BSE technique (30.6%), and not knowing the importance of BSE (21.4%) [23]. In a study of female Debre Birhan University students, the main reasons for not performing BSE were a lack of knowledge on how to conduct BSE and not having any symptoms of breast cancer [25]. A study of female household heads in Twenty-nine percent of our participants knew the cor- rect age at which BSE should be started, slightly greater women in Benghazi, Libya (27.7%) [17] but fewer than those in south east Asia, where 44% of study participants recommended practicing BSE at age 20 [5], Nigerian Lera et al. Discussion Table 3 Distribution of time BSE practiced, and the reasons given to perform or not perform BSE among women in Wolaita Sodo, 2019 (n = 626) Variable/characteristics Frequency (%) Appropriate time of BSE Few days after menses 97 (45) Few days before menses 13 (6) No specific time 107 (49) Frequency of BSE practices Twice per month 48 (22.2) Once every month 98 (45) Once every 6 month 2 (0.9) Once per year 4 (1.9) Any time I observe a change 65 (30) Advantage of regular breast self-examination Detect any abnormality 72 (33) Learn how the breast normally looks and feels 56 (26) Detect breast cancer earlier and promote treatment 89 (41) Reasons for performing BSE Fear of breast cancer 51(23.5) Early detection of breast cancer 128 (59) Breast cancer in the family/friends 13 (6) Previous breast problems 3 (1.4) Heard from media 22 (10) Barriers to BSE I don’t have enough privacy for BSE practice 14 (6.4) Pressure of work/I am too busy 30 (13.8) Doubts about its effectiveness 11 (5) Absence of symptoms/disease 13 (6) I am scared of being diagnosed with breast cancer 7 (3.4) Forgetfulness 10 (4.6) I know I can never have breast cancer 13 (6) No obstacle (barriers) 119 (54.8) Table 4 Distribution of spousal/parental support to perform BSE and the need for further information among women in Wolaita Sodo, 2019 (n = 626) Variables/characteristics Frequency (%) Support on BSE from spouse/partner Yes 146 (67.2) No 71 (32.8) Would like information on how to do BSE Yes 249 (91) No 23 (9) Recognized importance of BSE Very important 207 (95.3) Important 10 (4.7) Self-confidence to perform BSE Yes 191 (88) No 26 (11) Where will you go, if you discover a breast lump Health facility 168 (77.4) Traditional healer 49 (22.6) Table 4 Distribution of spousal/parental support to perform BSE and the need for further information among women in Wolaita Sodo, 2019 (n = 626) Variables/characteristics Frequency (%) Support on BSE from spouse/partner Yes 146 (67.2) No 71 (32.8) Would like information on how to do BSE Yes 249 (91) No 23 (9) Recognized importance of BSE Very important 207 (95.3) Important 10 (4.7) Self-confidence to perform BSE Yes 191 (88) No 26 (11) Where will you go, if you discover a breast lump Health facility 168 (77.4) Traditional healer 49 (22.6) due to these other occupations exposing these women to a wider selection of media, friends, and colleagues to share ideas and experiences and initiating BSE practice. Discussion Being healthy (44.8%) and a lack of knowledge about BSE (26.9%) were the most significant barriers men- tioned for not practicing BSE at Adama Science and Technology University [11]. The current study revealed that women who recog- nized BSE as an early breast cancer detection method were 6.36-times more likely to practice BSE than women who did not know of any methods to detect breast can- cer. This finding is consistent with a study of women in Malaysia, which showed that knowledge that BSE is an early detection method for breast cancer was signifi- cantly associated with BSE [13]. In the current study, being employed in an occupation other than being a housewife was significantly associated with performing BSE, with these women 3.12-times (95% CI: 1.14, 8.58) more likely to practice BSE than other groups. These results are in agreement with find- ings in Nigerian women [26], a study in Benghazi, Libya [17], and a study from southern Ethiopia [24]. This may Lera et al. Discussion BMC Women's Health (2020) 20:167 Page 8 of 10 Table 3 Distribution of time BSE practiced, and the reasons given to perform or not perform BSE among women in Wolaita Sodo, 2019 (n = 626) Variable/characteristics Frequency (%) Appropriate time of BSE Few days after menses 97 (45) Few days before menses 13 (6) No specific time 107 (49) Frequency of BSE practices Twice per month 48 (22.2) Once every month 98 (45) Once every 6 month 2 (0.9) Once per year 4 (1.9) Any time I observe a change 65 (30) Advantage of regular breast self-examination Detect any abnormality 72 (33) Learn how the breast normally looks and feels 56 (26) Detect breast cancer earlier and promote treatment 89 (41) Reasons for performing BSE Fear of breast cancer 51(23.5) Early detection of breast cancer 128 (59) Breast cancer in the family/friends 13 (6) Previous breast problems 3 (1.4) Heard from media 22 (10) Barriers to BSE I don’t have enough privacy for BSE practice 14 (6.4) Pressure of work/I am too busy 30 (13.8) Doubts about its effectiveness 11 (5) Absence of symptoms/disease 13 (6) I am scared of being diagnosed with breast cancer 7 (3.4) Forgetfulness 10 (4.6) I know I can never have breast cancer 13 (6) No obstacle (barriers) 119 (54.8) due to these other occupations exposing these women to a wider selection of media, friends, and colleagues to share ideas and experiences and initiating BSE practice. Women who had breast fed their child for 13–24 months were 2.43-times more likely to examine their breasts than those who mentioned different durations of breast feeding, which may be due to those who optimally breast feed being more conscious of or educated to per- form BSE. Women who used electronic media as a source of in- formation were 1.59-times more likely to practice BSE than women who used other media types. This may be due to its relative accessibility compared to other source of information. Discussion p g p Women who had breast fed their child for 13–24 months were 2.43-times more likely to examine their breasts than those who mentioned different durations of breast feeding, which may be due to those who optimally breast feed being more conscious of or educated to per- form BSE. Women who used electronic media as a source of in- formation were 1.59-times more likely to practice BSE than women who used other media types. This may be due to its relative accessibility compared to other source of information. Fig. 3 Reasons of not performing Breast self-examination Fig. 3 Reasons of not performing Breast self-examination Fig. 3 Reasons of not performing Breast self-examination Lera et al. BMC Women's Health (2020) 20:167 Page 9 of 10 Table 5 Factors associated with breast self-examination among women in Wolaita Sodo, 2019 (n = 626) Variables Perform BSE Odds ratio (95% CI) Yes No COR (95% CI) AOR (95% CI) Participant’s occupation status House wife 96 (15%) 217 (34.4%) 1.00 1.00 Employee 76 (12%) 57 (9%) 2.07 (1.20, 3.59) 3.13 (1.14, 8.58) Merchant 26 (4%) 49 (7.7%) 6.25 (3.42, 11.41) 6.47 (2.31, 18.12) Student 19 (3%) 89 (14%) 2.49 (1.25, 4.94) 3.73(1.19, 11.73) Duration breastfeeding Birth-12 months 39 (7.8%) 39 (7.8%) 1.00 1.00 13–24 months 101 (20%) 181 (36.4%) 2.10 (1.18, 0.74) 2.43 (1.28, 4.59) 25–34 months 46 (9.2%) 92 (18.4%) 1.16 (0.75, 0.78) 1.19 (0.74, 1.92) Early detection method for breast cancer BSE 111 (32%) 9 (33%) 364 7.03 (4.14, 11.95) 6.36 (3.72, 10.71) I don’t know 106 (20%) (59%) 1.00 1.00 Personal history of having benign breast lump Yes 21 (3.5%) 59 (10%) 2.31 (1.20, 4.46) 0.03 (0.08, 1.52) No 196 (33%) 315 (53%) 1.00 1.00 Women’s educational status Primary 82 (28.2%) 209 (71.8%) 1.00 1.00 Secondary 135 (39.9%) 203 (60.1%) 1.70 (1.21, 2.37) 0.81 (0.29, 2.24) Husband’s educational status Primary 51 118 1.00 0.62 (0.26, 1.49) Secondary 134 175 1.80 (1.21, 2.67) Source of information Electronics media 151 (25.5%) 218 (36.8) 1.63 (1.14, 2.32) 1.5 9(1.01, 2.59) Other 66 (11%) 156 (26.3) 1.00 1.00 Adjusted odds ratio (AOR), Significant at p- ≤0.05 Table 5 Factors associated with breast self-examination among women in Wolaita Sodo, 2019 (n = 626) Variables Perform BSE Odds ratio (95% CI) Yes No COR (95% CI) AOR (95% CI) Wolaita Sodo Fana FM, Wolaita Sodo Wogeta FM, and South TV) to advocate performing BSE. Acknowledgments ld l k h g We would like to thank Wolaita Sodo University, College of Health Sciences and Medicine. We also thank Wolaita Zone administrators, the supervisors, respondents, and data collectors. Conclusion In general, a low proportion of participants had previ- ously heard about BSE. Only about half of participants performed BSE regularly, and less than a third of re- spondents correctly recognized the age at which BSE should commence. The use of electronic media as a source of information, occupation, and knowledge about early detection methods for breast cancer were associ- ated with performing BSE. Therefore, we recommend that the Wolaita Sodo administration needs to use elec- tronic media consistently and programmatically (e.g., Abbreviations d d AOR: adjusted odds ratio; BSE: breast self-examination; CBE: clinical breast examination; CI: confidence interval; OR: odds ratio Strengths and limitations of the study The main strength of the study is that previous studies conducted in Ethiopia focused on health professionals, whereas we studied a general urban community. How- ever, this study was limited by being conducted in a sin- gle urban community, which may not be representative of the rural community or other urban communities in Ethiopia. Since this was a cross-sectional study, causal conclusions cannot be drawn. Discussion Weekly or monthly mobile phone messages could be sent to en- courage performing BSE. Video screens could be used in the city center to demonstrate BSE issues. 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W2966506307.txt	https://www.frontiersin.org/articles/10.3389/fcell.2019.00157/pdf	en		The N-Glycosylation Processing Potential of the Mammalian Golgi Apparatus	Frontiers in cell and developmental biology	2,019	cc-by	7,278	ORIGINAL RESEARCH published: 13 August 2019 doi: 10.3389/fcell.2019.00157 The N-Glycosylation Processing Potential of the Mammalian Golgi Apparatus Peter Fisher 1 , Jane Thomas-Oates 2* , A. Jamie Wood 1,3* and Daniel Ungar 1* 1 Department of Biology, University of York, York, United Kingdom, 2 Department of Chemistry and Centre of Excellence in Mass Spectrometry, University of York, York, United Kingdom, 3 Department of Mathematics, University of York, York, United Kingdom Edited by: Kristian Prydz, University of Oslo, Norway Reviewed by: Heike Folsch, Northwestern University, United States Johannes Stadlmann, Institute of Molecular Biotechnology (OAW), Austria *Correspondence: Jane Thomas-Oates jane.thomas-oates@york.ac.uk A. Jamie Wood jamie.wood@york.ac.uk Daniel Ungar dani.ungar@york.ac.uk Specialty section: This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology Received: 28 March 2019 Accepted: 26 July 2019 Published: 13 August 2019 Citation: Fisher P, Thomas-Oates J, Wood AJ and Ungar D (2019) The N-Glycosylation Processing Potential of the Mammalian Golgi Apparatus. Front. Cell Dev. Biol. 7:157. doi: 10.3389/fcell.2019.00157 Heterogeneity is an inherent feature of the glycosylation process. Mammalian cells often produce a variety of glycan structures on separate molecules of the same protein, known as glycoforms. This heterogeneity is not random but is controlled by the organization of the glycosylation machinery in the Golgi cisternae. In this work, we use a computational model of the N-glycosylation process to probe how the organization of the glycosylation machinery into different cisternae drives N-glycan biosynthesis toward differing degrees of heterogeneity. Using this model, we demonstrate the N-glycosylation potential and limits of the mammalian Golgi apparatus, for example how the number of cisternae limits the goal of achieving near homogeneity for N-glycans. The production of specific glycoforms guided by this computational study could pave the way for “glycoform engineering,” which will find uses in the functional investigation of glycans, the modulation of glycan-mediated physiological functions, and in biotechnology. Keywords: computational modeling, Golgi apparatus, glycan biosynthesis, cisternal number, glycan heterogeneity INTRODUCTION N-glycosylation is a process initiated in the endoplasmic reticulum (ER) but specific structural elements such as core fucosylation and branching (Figure 1A) are introduced later in the secretory pathway in the Golgi apparatus. N-glycans are modified by a series of sequentially acting glycosidases and glycosyltransferases (Figure 1B) that modify glycans in the Golgi apparatus and consequently dictate and ultimately determine the glycan profile of the whole cell. However, the structural modification of N-linked glycans is a complex process that results in numerous different glycan structures. In the absence of a “glycan template,” protein glycosylation is inherently heterogenous with a number of factors contributing to the final glycan structure. These variables include the protein structure (Hang et al., 2015; Suga et al., 2018), secretory protein load (JimenezMallebrera et al., 2009), Golgi transport mechanism (Hossler et al., 2007), enzyme protein levels, availability of monosaccharide-nucleotides, and the organization of glycosylation enzymes within the Golgi apparatus (Oka et al., 2004; Zolov and Lupashin, 2005; Fisher and Ungar, 2016). Typically, N-glycan biosynthesis can be characterized as a series of divergent pathways that converge into structural nodes (Figure 1B). Following the initial trimming of mannoses by ManI, the number of possible N-glycan structures generated in the Golgi apparatus increases exponentially with each additional monosaccharide until the capping of antennae with sialic acid (Spahn et al., 2016). Frontiers in Cell and Developmental Biology \| www.frontiersin.org 1 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi N-glycans can play important roles in dictating the properties of glycoproteins. From a biologic’s standpoint, controlling the glycans residing on therapeutic antibodies can tune their pharmaceutical properties. For example, glycans lacking core fucose (Figure 1A) can increase antibody-dependent cellular cytotoxicity (ADCC) (Shinkawa et al., 2003; YamaneOhnuki et al., 2004). Overall it appears that fucosylation and galactosylation are the dominant features of N-glycans that influence Fc-receptor binding (Dekkers et al., 2017), which is linked to ADCC. Furthermore, the presence of sialylated complex type glycans increases the in vivo halflife of therapeutic antibodies compared to oligomannose type glycans (Kanda et al., 2007) further illustrating the attenuating role of N-glycans in IgG properties. Due to the heterogenous nature of glycan biosynthesis the production of homogenous biologics with respect to glycosylation is currently not feasible, giving rise to batch-to-batch variation, and heterogeneity within batches. Glyco-engineering of biologics, for example through the deletion of Mgat1 to eliminate complex and hybrid glycans from CHO cells, reduces glycan heterogeneity, and improves immunological properties (Xu et al., 2016). However, eliminating complex glycans also removes any ability to fine-tune the properties of a therapeutic through presence/absence of other N-glycan features such as those shown in Figure 1A. Therefore, devising novel strategies to reduce glycan heterogeneity and/or enrich the relative abundance of a desired glycan is of great value to the pharmaceutical industry. Glycosylation enzyme levels play a pivotal role in glycan biosynthesis; however, the outcome of enzyme depletion or overexpression can be unexpected. Knockdown of the galactosyltransferase GalT4 alongside overexpression of the branching enzymes Mgat4 and/or 5 in CHO cells substantially increased the abundance of tri- and tetra-antennary N-glycans. Interestingly though, it was the reduction of GalT4 that primarily accounted for the increase in branching (McDonald et al., 2014). Two additional important factors in determining the N-glycans produced by mammalian cells are the distribution of the glycosylation enzymes within the Golgi apparatus (Fisher and Ungar, 2016), and the architecture of the Golgi apparatus itself. For example, disruptions to the conserved oligomeric Golgi (COG) complex, which is involved in tethering and thereby targeting intra-Golgi retrograde vesicles, can lead to glycosylation defects in model cell lines (Shestakova et al., 2006; Bailey Blackburn et al., 2016) and to congenital disorders of glycosylation (reviewed in Wu et al., 2004; Steet and Kornfeld, 2006; Hennet and Cabalzar, 2015). Furthermore, the depletion of the Golgi reassembly stacking proteins (GRASPs) of 55 and 65 kDa results in an acceleration of protein transport, Golgi fragmentation, and impaired glycosylation (Xiang et al., 2013; Bekier et al., 2017). Fragmentation of the Golgi apparatus has also been linked to numerous neurodegenerative disorders (reviewed in Joshi et al., 2015). Alterations to the architecture and the organization of the Golgi apparatus provide a potential route to controlling glycosylation alongside the protein levels of the glycosylation machinery themselves. Other factors such Frontiers in Cell and Developmental Biology \| www.frontiersin.org as the availability of the monosaccharide-nucleotide donors are important and worthy of further investigation but are not considered in this work. In this work we build on previous studies of glycosylation in WT HEK293T cells (Bailey Blackburn et al., 2016) and a stochastic model of N-glycosylation we recently developed and validated (Fisher et al., 2019). We used this model of glycosylation to demonstrate in silico the effects of cisternal number on glycan heterogeneity and complexity. This led us to test computationally what limitations the existing Golgi apparatus architecture places on the degree of glycan homogeneity achievable in mammalian cells. Finally, we used our model to predict strategies to increase the relative abundance of targeted glycan structures. RESULTS AND DISCUSSION It is not the aim of this work to describe in great detail the modeling methodology that has been used (for a more detailed description on the development and validation of the modeling framework used in this work please see Fisher et al., 2019), however, a brief summary of the modeling methodology will give context and a greater understanding of the results of this work. Glycosylation reactions are simulated using a stochastic simulation algorithm (SSA). The SSA incorporates the inherent noise that is present in biological systems and that becomes increasingly relevant when the number of reactants (and enzymes) are small, as is the case for glycosylation in the Golgi. In contrast to models of glycosylation based on ordinary differential equations (ODEs) (Hossler et al., 2007; Krambeck et al., 2009, 2017; Goey et al., 2018), several factors can be included in each parameter, reducing the need for excessive parameterization in our model. As such, we define parameters for each enzyme by using the term “effective enzymatic rate” to encompass the enzyme’s protein level, availability of its nucleotidemonosaccharide substrates and the chemical enzymatic rate. In this work we do not wish to consider changes to intrinsic enzymatic properties such as the inherent chemical enzymatic rate constants, therefore any predicted alterations to the effective enzymatic rate parameter are assumed to be equivalent to alterations in the protein level of the respective enzyme. The effective enzymatic rates, the transit time in the Golgi apparatus, and the composition of glycans entering the Golgi were the parameters that were altered to fit the computed to a target glycan profile. This fitting was done using an approximate Bayesian computation (ABC) algorithm (Marjoram et al., 2003), which samples from parameter distributions to feed the SSA and assess the goodness of fit with a target profile. The shift of the parameter distributions during fitting tells us how the organization (e.g., levels and localizations) of enzymes changes from the starting condition to reach the target glycan profile. We utilize the optimized parameters determined previously for the WT HEK293T cell glycan profile as initial values (Fisher et al., 2019), therefore predicted changes in effective enzymatic rates are 2 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi FIGURE 1 \| N-glycan processing pathways to generate a fully sialylated bi-antennary glycan. (A) Pictorial examples of structural features of complex N-glycans that are referred to in this work. (B) N-glycan biosynthesis pathway for Fuc1 GlcNAc4 Man3 Gal2 NeuAc2 starting with the two oligomannose glycans that enter the Golgi apparatus. The biosynthesis of complex N-glycans is characterized by increasing numbers of reaction paths that ultimately converge on structural nodes often due to the addition of terminating sialic acids. time per cisterna. The reduction in glycan maturity observed in GRASP55/65 double knockout (KO) cells may therefore be a consequence of the fragmented Golgi apparatus (Bekier et al., 2017). Interestingly, by changing the number of cisternal elements the most abundant complex glycans simulated by our model also differed (Figure 2C). For example, and in support of the hypothesis that the organization of a fragmented Golgi apparatus may explain the reduction in glycan maturity in GRASP55/65 KO cells, simulations with more than four cisternal elements generated immature complex glycans in comparison to simulations involving one and two cisternal elements, which generated higher relative abundances of more elaborate glycans containing more antennae, and a higher proportion of sialic acids (Figure 2C). This is possibly a knock-on effect of the reduced efficiency of oligomannose processing as mentioned above, and could possibly be explained with the shorter processing time in each individual cisterna. In the previous simulation, the total time taken to traverse the Golgi apparatus was kept constant while the number of cisternal elements was altered. Next, we were interested in what effect varying the transit time per cisterna had on glycan heterogeneity and complexity while keeping the number of cisternae at four. Indeed, altering the time taken to traverse the Golgi apparatus had a different effect than varying the number of cisternal elements (Figure 2B). The number of different glycan structures predicted when the time was reduced to 10% of the WT time decreased dramatically (Figure 2B). Following a gradual increase and plateauing of glycan heterogeneity with increasing time in the Golgi apparatus, the number of glycan structures begins to decrease again at 250% of WT time. This is due to glycan pathways converging relative to the organization of the glycosylation machinery in WT HEK293T cells. Glycan Heterogeneity and the Golgi Apparatus We set out to evaluate how the level of glycan heterogeneity is affected by different variables in our model of N-glycosylation. An important variable is the number of cisternal elements, which is known to vary between species, cell types (Mironov et al., 2017), and in pathologies (Joshi et al., 2014). In our earlier work four cisternae were found to be required to optimally model HEK293T cell N-glycans (Fisher et al., 2019). While this number does not necessarily reflect the actual number of cisternae in this cell type, deviations from this number during modeling reflect potential changes in cisternal number. In order to investigate the effect of architecture on the glycan processing potential of the Golgi apparatus, the number of cisternal elements in our stochastic model of the Golgi was varied from the four required for HEK293T cells in previous work (Fisher et al., 2019). The total effective rate for each enzyme was kept constant, as was the total time spent in the Golgi apparatus. As a measure of glycan heterogeneity in these simulations, the number of different glycan structures produced in the simulation was recorded (Figure 2A). The trend is of decreasing heterogeneity as the number of cisternal elements is increased from two to ten. In addition to the observed decrease in heterogeneity correlating with increasing cisternal elements, the relative abundance of oligomannose glycans also increases as the trimming of mannose residues becomes less efficient possibly due to the reduced Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi FIGURE 2 \| Effect of cisternal number and transit time on glycan heterogeneity. (A) Total number of glycans (circles) and proportion of oligomannose glycans (bars) produced in silico when starting with parameters fitted for the WT HEK293T cell glycan profile altering the number of cisternal elements (CS) in the system from the four (striped bar) used in the WT HEK293T model. The distribution of enzymes across the model Golgi apparatus, the total effective rates for each enzyme and the total time spent in the Golgi apparatus was kept the same for each simulation. (B) As in “A” but varying the total time spent in the Golgi with four cisternae. Striped bar indicates transit time for the WT HEK293T cell model; the times shown for all other simulations are relative to this value. (C) Relative proportions of the three most abundant complex glycans predicted by the model Golgi apparatus when the number of cisternal elements are altered. For all simulations error bars show standard deviation for n = 3. of the glycosylation enzymes studied (Xiang et al., 2013). Furthermore, our model suggests slower transport through the Golgi apparatus would reduce the oligomannose content, a feature that is not observed in COG subunit depleted cell lines (Bailey Blackburn et al., 2016). on structural nodes with terminating sialic acids. For example, the abundance of the fully sialylated bi-antennary glycan, Fuc1 GlcNAc4 Man3 Gal2 NeuAc2 , increases from 4.5% to close to 10% of the total when the transit time is increased 3.3fold of that of WT. In contrast, this same glycan is absent from the simulated profile when the transit time is reduced 10-fold compared to WT. Interestingly, GRASP55/65 depletion accelerates protein trafficking through the Golgi apparatus (Xiang et al., 2013; Bekier et al., 2017), an effect that our model would predict to decrease the relative abundance of complex N-glycans (Figure 2B). Indeed, published experimental evidence shows less processing for both cell surface and intracellular glycans in these cells (Bekier et al., 2017). In contrast, there was a minor kinetic delay for the delivery of VSV-G to the plasma membrane in Cog3 knockdown HeLa cells (Zolov and Lupashin, 2005), indicating a reduced rate of anterograde transport. This is unlikely to be the direct cause of the glycosylation defect though, as Cog3 depletion results in relocation and partial degradation of Golgi glycosylation enzymes (Shestakova et al., 2006), as opposed to GRASP55/65 depletion that had no effect on the localization and levels Frontiers in Cell and Developmental Biology \| www.frontiersin.org Maximizing the Relative Abundance of Target Glycans The production of single glycoforms is an important goal in the pharmaceutical industry. We therefore used our modeling framework to assess the glycan processing potential of the Golgi apparatus. In this instance, we tested the potential of the Golgi apparatus to produce a single glycoform, within reasonable biological boundaries. Three complex glycans with partial or complete sialylation and two or three antennae (Table 1) were chosen to test this property of the Golgi apparatus. The three complex glycans are referred to as bi-Sia1 , bi-Sia2 , and tri-Sia1 when they are described as the target for glycan engineering. When the same structures appear in the simulation as byproducts though, they are referred to in conventional glycan notation. Starting with the fitted parameters for the effective 4 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi second sialylation reaction to achieve a bi-Sia1 glycan structure. The result is similar when enriching tri-Sia1 , as the dominant predicted by-products are the over-sialylated tri-antennary glycan, Fuc1 GlcNAc5 Man3 Gal3 NeuAc2 , and the under sialylated tri-antennary glycan, Fuc1 GlcNAc5 Man3 Gal3 (Figure 3). The major by-products for bi-Sia2 were the fully sialylated triantennary glycan, Fuc1 GlcNAc5 Man3 Gal3 NeuAc3 suggesting that the modeled sialyation activity is not limiting the production of bi-Sia2 , rather the presence of branching enzymes is diverting flux away from bi-Sia2 . These by-products could of course be eliminated by modeling a cell line in which the branching enzymes are completely removed, but the ABC algorithm has difficulties pushing enzyme levels to complete elimination i.e., zero, as it will always sample from a non-zero distribution. Having started with the WT HEK293T cell parameters (Fisher et al., 2019), relative changes in enzyme distributions between Golgi cisternae that maximize the target glycans in silico can be predicted when using localization dependent fitting (Figure 4). For all target glycans an increase in the Mgat1 effective enzymatic rate within the second cisterna is required, and a similar change is seen for Fut8 (Figure 4). Both of these enzymes act early in the generation of complex N-glycans (Calderon et al., 2016; Fisher et al., 2019). The increases in their levels in an early cisterna presumably ensures that glycan processing can proceed more completely toward the desired products without the accumulation of partially processed intermediates. For the two biantennary glycan targets Mgat2 was required to be redistributed toward the trans side of the Golgi apparatus relative to the simulated WT distribution (Figure 4). Analysis of the flux map revealed that the shift in Mgat2 toward the trans side of the Golgi apparatus changed the dominant substrates for Mgat2 (Figure 5A). For the model of WT N-glycosylation only ∼8% of the Mgat2 substrates were fucosylated. In contrast, more than 60% of the Mgat2 substrates were fucosylated in the model primed to maximize the abundance of bi-Sia1 , and a similar shift in substrate preference is seen for bi-Sia2 (Figure 5B). Unsurprisingly, the effective enzymatic rate of the branching enzyme Mgat5 was predicted to be increased when trying to maximize the abundance of tri-Sia1 ; however, the distribution of Mgat5 was also required to shift more to the trans side of the Golgi apparatus (Figure 4). This shift in Mgat5 coincided with an equivalent shift of GalT into the trans-Golgi, a characteristic that aims to spatially separate the two enzymes within the Golgi apparatus (Figure 4). This is in line with competition between galactosylation and glycan branching, a feature that was suggested to explain the demonstrated role of GalT in determining and controlling antenna number (McDonald et al., 2014; Fisher et al., 2019). If we assume that the intrinsic rates of the glycosylation enzymes are not altered, predicted changes to the effective rates that are made by our model can be rationalized as overexpression or knock down of the relevant protein levels. Such an approach to reducing glycan heterogeneity and enriching the target glycan has indeed been previously applied experimentally for the simple case of core fucosylation (Yamane-Ohnuki et al., 2004). Figure 6 shows the predicted changes in total effective rate for each enzyme necessary to maximize the relevant target glycan. The TABLE 1 \| Target glycans. Glycan Abbreviation % of target glycan % of target % of target structure observed in WT glycan using glycan using HEK293T glycan variable fixed localization profile localization fitting fitting Bi-Sia1 (Fuc1 GlcNAc4 Man3 Gal2 NeuAc1 ) 13.5 70.7 74.6 Bi-Sia2 (Fuc1 GlcNAc4 Man3 Gal2 NeuAc2 ) 4.5 96.3 97.9 Tri-Sia1 (Fuc1 GlcNAc5 Man3 Gal3 NeuAc1 ) 2.9 66.9 63.8 enzymatic rates for WT HEK293T cells, we used ABC fitting to maximize the relative abundance of each target glycan. For this purpose, a hypothetical glycan profile was fitted in which 100% of the complex glycan was set as the desired N-glycan. The parameters that were fitted for these experiments were: effective enzymatic rates, transit time per cisterna and composition of glycans entering the Golgi. In addition, we investigated the maximization of target glycans in two scenarios: One, in which the distribution of enzymes between Golgi cisternae can be varied (variable localization); and another one, in which the distribution of enzymes in the Golgi apparatus is fixed to that observed in WT cells (fixed localization). Table 1 shows the percentage of the target glycan normalized to the total amount of complex glycan observed experimentally in WT HEK293T cell samples (Bailey Blackburn et al., 2016). For all of the target glycans the proportion could be increased above that found in WT HEK293T cells (Table 1). Bi-Sia2 could be produced by the simulation at the highest relative abundance due to the terminating sialic acids on both antennae, which are endpoint structures. In contrast, homogeneous production of partially sialylated glycans is predicted to be much more difficult if not impossible to achieve according to our stochastic model. When comparing the fits to the bi-Sia1 and tri-Sia1 targets we can also say that adding a third antenna makes the drive to homogeneity again more difficult, implying that the inclusion of tri- and/or tetra-antennary glycans in products will inadvertently increase heterogeneity, as was observed in glycan-engineered plant expression systems (Nagels et al., 2012). As expected, the most abundant glycan by-products predicted when maximizing the production of each target glycan were quite different (Figure 3). When maximizing the abundance of bi-Sia1 , the major by-products were the un-sialylated biantennary glycan Fuc1 GlcNAc4 Man3 Gal2 and the fully sialylated Fuc1 GlcNAc4 Man3 Gal2 NeuAc2 . This result suggests a small window in which the glycoprotein must exit the Golgi apparatus following the initial sialylation but prior to the Frontiers in Cell and Developmental Biology \| www.frontiersin.org 5 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi FIGURE 3 \| Glycan by-products of maximizing target complex glycans. Relative abundance of the oligomannose pool and the three most abundant complex glycans that are predicted as by-products when maximising the abundance of the indicated target glycans with the indicated type (fixed or variable enzyme localization) of fitting strategy. FIGURE 4 \| Optimized Golgi distribution of enzymes to maximize the relative abundance of target glycans. Distributions of the effective enzymatic rates for the indicated enzymes throughout the Golgi apparatus following fitting with variable enzyme localization to maximize the abundance of the indicated target glycans. The predicted distribution in WT HEK293T cells (Fisher et al., 2019) is shown for comparison. higher for the fixed localization fitting of tri-Sia1 compared with the variable localization fitting of the same glycan. While simply altering the levels of enzymes could conceptually be much simpler through knock-down or overexpression, their distribution may also be changed through engineering of the intra-Golgi vesicular sorting pathway. For example, manipulation of the interactions of the COG vesicle tethering complex (Miller et al., 2013; Willett et al., 2013), or adjustments in the cytosolic and transmembrane differences in total effective enzymatic rates between the fits using variable and fixed enzyme localizations suggest that the distribution of enzymes is an important factor to be considered when designing genetic manipulations of the glycosylation machinery. When the localization of enzymes is fixed, predicted alterations to the total enzymatic rates are much larger than those required in the variable localization scenario (Figure 6). For example, the increase required for Mgat5 is more than threefold Frontiers in Cell and Developmental Biology \| www.frontiersin.org 6 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi FIGURE 5 \| Flux analysis of Mgat2 substrates in WT, bi-Sia1 , and bi-Sia2 simulations. (A) Proportion of biosynthetic flux carried by Mgat2 from its three dominant substrates in WT HEK293T cells (red) and the simulated profiles maximizing bi-Sia1 (blue) and bi-Sia2 (purple) fitted with variable enzyme localization. (B) The proportion of substrates for Mgat2 that are fucosylated for the simulations described in “A.” FIGURE 6 \| Total enzymatic rate changes to maximize relative abundance of target glycans. Percentage changes in total effective enzymatic rates following fitting to maximize the abundance of bi-Sia1 (blue), bi-Sia2 (red), and tri-Sia1 (green) fitted with variable enzyme localization (solid) and fixed enzyme localization (striped) relative to the parameters predicted for the WT HEK293T cell profile. Total effective enzymatic rates obtained from the different fits were adjusted to ensure that the transit time in each case matched that of the WT HEK293T simulation. domains of particular glycosylation enzymes could both be used to alter enzyme locations (Ferrara et al., 2006; Becker et al., 2018). within the fitting as they are considered highly unlikely relative to the WT values. However, it was possible to devise strategies to increase the output of tetra-antennary glycans from our model (Figure 7A). Manually elevating the effective rates of Mgat4/5 10-fold, while keeping the distribution of the enzymes the same as in WT HEK293T cells, increased the relative abundance of tetra-antennary glycans about sevenfold, to roughly 25% (Figure 7A). However, increasing the effective rates of Mgat4/5 further (100-fold) did not increase the relative abundance of tetra-antennary glycans above that of the 10-fold increased rates, but did increase the abundance of bi-antennary glycans (not shown). In order to investigate why further increasing A Computationally Informed Strategy for Producing Tetra-Antennary Glycans Our ABC fitting strategy was unable to generate parameter values for the effective enzymatic rates that would simulate the production of significant amounts of tetra-antennary glycans. Initial parameter values for this fitting were those of the WT HEK293T cell line and therefore the huge shifts required in the effective rate of Mgat4 and 5 are almost never accepted Frontiers in Cell and Developmental Biology \| www.frontiersin.org 7 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi FIGURE 7 \| Hypothetical engineering of the Golgi apparatus to increase the abundance of tetra-antennary glycans. (A) The relative abundance of all tetra- and tri-antennary glycans produced in silico using effective enzymatic rates for: WT HEK293T (red), WT with the total effective enzymatic rates of Mgat4/5 increased 10-fold (dark blue), WT with the total effective enzymatic rates of Mgat4/5 increased 100-fold (light blue), WT with the distribution of enzymes in the model Golgi apparatus set to separate Mgat4/5 from GalT (yellow), and WT with the total effective enzymatic rates of Mgat4/5 increased 10-fold and the localization of enzymes in the model Golgi apparatus set to separate Mgat4/5 from GalT (yellow and dark blue striped). (B) Flux diagrams for selected N-glycosylation reactions for WT HEK293T, WT with the total effective enzymatic rates of Mgat4/5 increased 10-fold and WT with the total effective enzymatic rates of Mgat4/5 increased 100-fold. (C) Schematic showing the levels and relative localizations of key enzymes for WT HEK293T cells and the computationally engineered scenario predicted to maximize the production of tetra-antennary glycans. the effective rates of Mgat4/5, however, the two strategies were synergistic (Figure 7A). It is important to note that separation of the competing enzymes increased the relative abundance of tetraantennary glycans but these were often not sialylated. Therefore, in this engineered theoretical scenario an additional cisterna to accommodate sialylation reactions may also be required depending on the target glycan. the effective enzymatic rates of Mgat4/5 did not result in a concomitant increase in tetra-antennary glycans we examined the flux diagrams for the three conditions (Figure 7B). Flux analysis revealed that as the effective enzymatic rate of Mgat4 increases it begins to outcompete ManII and Mgat2 in the earlier cisternae resulting in a reduction in flux toward GlcNAc4 Man3 , which is an important intermediate on the path of tetra-antennary glycan biosynthesis (Figure 7B). The enzymatic competition between ManII/Mgat2 and Mgat4 is predicted to limit the production of tetra-antennary glycans alongside the known effect of GalT on controlling the degree of N-glycan branching (Fisher et al., 2019). Elimination of GalT was suggested as an approach to producing a larger proportion of tetra-antennary glycans (McDonald et al., 2014). Spatially separating Mgat4/5 from GalT in the Golgi apparatus and therefore minimizing competition between the enzymes (Figure 7C) could achieve a similar effect for increasing the relative abundance of tetra-antennary glycans (Figure 7A). Indeed, the spatial separation of Mgat5 and GalT that is enforced by the model while enriching tri-Sia1 (Figure 4) demonstrates that cisternal separation allows GalT to evade dominating Mgat5. When applied to the task of increasing the proportion of tetra-antennary glycans the effect was not as large as increasing Frontiers in Cell and Developmental Biology \| www.frontiersin.org CONCLUSION In this work our computational model of mammalian N-glycosylation has been used to probe the glycan processing potential of the Golgi apparatus. While this study was based on computation only, our model’s predictions generate some testable hypothesis, which will be interesting to follow up on using laboratory based experiments. Two aspects of Golgi biology, which are commonly affected in pathologies such as CDGs and neurological disorders, the architecture of the Golgi apparatus and transit time through the Golgi apparatus have been shown to be key determinants of the cellular glycan profile. Golgi architecture and transit time should be considered as important factors in glycosylation disorders that likely 8 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi have an additive effect in disrupting the protein levels of the glycosylation enzymes. The goal of generating homogeneous glycoforms of biologics from a process that is inherently heterogeneous is a difficult one. Our model suggests several strategies that we predict will enrich particular target glycan structures; however, our model could not achieve complete homogeneity of glycoforms, suggesting that full uniformity may not be achievable. In the cases of biSia1 and tri-Sia1 a higher percentage of the target glycan could be achieved if the substrate specificities of the enzymes were treated as a variable and not fixed. This suggests that when partial sialylation (or galactosylation) is required, protein engineering to adjust the substrate specificities of enzymes is a necessary strategy. A combination of engineering glycosylation enzymes’ specificities, controlling effective enzymatic rates and organizing the Golgi apparatus may all be required to attain higher glycan homogeneity, but ultimately the number of cisternae in a mammalian Golgi will likely be a key limiting factor for producing large complex glycans close to homogeneity. TABLE 2 \| Distribution of Mgat2, Mgat4, Mgat5, and GalT in an engineered Golgi apparatus to maximize tetra-antennary glycans. Enzyme % effective % effective % effective % effective enzymatic rate enzymatic rate enzymatic rate enzymatic rate in 1st cisterna in 2nd cisterna in 3rd cisterna in 4th cisterna Mgat2 0 100 0 0 Mgat4 0 0 100 0 Mgat5 0 0 100 0 GalT 0 0 0 100 adjacent cisternae and then rescaling the effective enzymatic rates to equal the total for the fitted WT HEK293T cells. By using this strategy, we ensure any alterations to the simulated glycan profile are not the result of changes in enzyme levels. The number of simulated glycans and the relative abundance of oligomannose glycans was calculated from the average of three simulations for each cisternal number. Maximizing Target Glycans MATERIALS AND METHODS To predict alterations to enzyme levels and localization that can maximize a given target glycan we used the ABC fitting methodology to fit a simulated glycan profile to a hypothetical observed glycan profile. The hypothetical glycan profile was constructed by making the relative abundance of the target glycan to 100% of the combined complex and hybrid glycan abundances. The proportion of oligomannose glycans was kept at the level observed in WT HEK293T cells in all target profiles. ABC fitting methodology was then used to predict alterations in the organization of the Golgi machinery to generate the hypothetical glycan profile. The model was not penalized for what type of byproducts were produced, as such it is reasonable to assume that the predicted by-products would be observed in a true glycan profile of an engineered cell line. Modeling Framework The fitted and validated model of HEK293T cells (Fisher et al., 2019) has been used extensively in this work. This model was presented in detail in Fisher et al. (2019); it is generated using a SSA based on the Gillespie algorithm (Doob, 1945; Gillespie, 1976) that was used to implicitly simulate enzyme competition in the Golgi apparatus. Glycans entered the in silico Golgi apparatus one at a time, hence our model does not account for substrate competition or the potential effects of protein load in the secretory pathway. The processing of 10,000 glycans was simulated in order to generate a simulated glycan profile from which the abundance of individual or structural classes of glycans can be obtained. This profile is compared to a real data set utilizing a Bayesian fitting methodology with priors based upon biological knowledge. In the case of this study the priors were based on the parameters obtained by fitting the WT HEK293T glycan profile. Maximising Tetra-Antennary Glycans WT HEK293T fitted parameters were used as a starting point. The effective enzymatic rates of Mgat4 and Mgat5 were increased 10- or 100-fold and the glycan profile simulated. For the spatial separation of the key enzymes involved in determining tetraantennary glycans, the effective enzymatic rates were condensed into specific cisternae as shown in Table 2. The relative abundance of all tetra-antennary glycans was calculated from the average of three simulations for each different scenario. Transit Time and Cisternal Element Number The enzymatic parameters as well as the proportions of Man9 GlcNAc2 , Man8 GlcNAc2 , and GlcMan9 GlcNAc2 obtained for the fitted WT HEK293T cell line (Fisher et al., 2019) were used. The relative time taken for a glycan to transit through each cisternal element was varied and the glycosylation reactions simulated. The number of simulated glycans and the relative abundance of oligomannose glycans was calculated from the average of three simulations for each transit time. For investigating the effect of cisternal element number on the N-glycosylation process the total effective enzymatic rates for each enzyme were kept constant in addition to the total transit time and proportions of Man9 GlcNAc2 , Man8 GlcNAc2 , and GlcMan9 GlcNAc2 . Extra cisternal elements were added by calculating the average effective enzymatic rate between two Frontiers in Cell and Developmental Biology \| www.frontiersin.org DATA AVAILABILITY The datasets generated for this study are available on request to the corresponding author. AUTHOR CONTRIBUTIONS All authors designed the study, planned the experiments, and wrote the manuscript. PF performed the experiments. 9 August 2019 \| Volume 7 \| Article 157 Fisher et al. Glycan Processing Potential of the Golgi York Centre of Excellence in Mass Spectrometry, which was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1 and EP/M028127/1). FUNDING This work was supported by an Impact Accelerator Award to AW, DU, and JT-O in collaboration with GSK (BB/S506795/1), a BBSRC IB Catalyst grant to DU (BB/M018237/1) and the REFERENCES Kanda, Y., Yamada, T., Mori, K., Okazaki, A., Inoue, M., Kitajima-Miyama, K., et al. (2007). Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the highmannose, hybrid, and complex types. Glycobiology 17, 104–118. doi: 10.1093/ glycob/cwl057 Krambeck, F. J., Bennun, S. V., Andersen, M. R., and Betenbaugh, M. J. (2017). Model-based analysis of N-glycosylation in Chinese hamster ovary cells. 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Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent Frontiers in Cell and Developmental Biology \| www.frontiersin.org Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2019 Fisher, Thomas-Oates, Wood and Ungar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 11 August 2019 \| Volume 7 \| Article 157
https://openalex.org/W4361267476	https://goslasmed.elpub.ru/jour/article/download/801/704	Russian	null	The effectiveness of laser therapy in patients with diabetic foot	Lazernaâ medicina	2,023	cc-by	3,887	Резюме Цель: сравнить эффективность применения лазеротерапии и традиционных методов лечения больных сахарным диабетом с гнойно- некротическим поражением нижних конечностей. Материалы и методы. В работе представлен ретроспективный анализ результатов лечения 76 больных сахарным диабетом с гной- но-некротическим поражением нижних конечностей. Больные были разделены на две группы. Первую (основную) группу составили 34 пациента, у которых традиционное лечение дополнялось внутривенным лазерным облучением крови и местной лазеротерапией, вторую (контрольную) – 42 пациента, которые получали только традиционное лечение. Результаты. Лазеротерапия способствовала более быстрому сокращению площади раневого дефекта (17,4 %) по сравнению с тра- диционными методами лечения (11,1 %), снижала сроки перехода в фазу воспалительно-регенераторных изменений, обеспечивала уменьшение частоты выполнения высокой ампутации нижней конечности с 14 до 6 % и снижение длительности пребывания в стаци- онаре на 11,2 койко-дня. Заключение. Применение лазеротерапии у больных диабетической стопой ускоряет очищение и сокращение площади раны, позволяет в более короткие сроки, чем при традиционном лечении, уменьшить или ликвидировать воспалительный процесс, стимулирует развитие грануляционной ткани и ускоряет процесс эпителизации. Ключевые слова: сахарный диабет, диабетическая стопа, лазеротерапия р д , д , р р Для цитирования: Луценко Ю.Г., Гринцов А.Г., Матийцив А.Б. Эффективность применения лазеротерапии у больных диабетической стопой. Лазерная медицина. 2022; 26(3-4): 26–31. https://doi.org/10.37895/2071-8004-2022-26-3-4-26-31 Контакты: Луценко Ю.Г., e-mail: hirurgiya.ﬁ po@mail.ru Для цитирования: Луценко Ю.Г., Гринцов А.Г., Матийцив А.Б. Эффективность применения лазеротерапии у больных диабетической стопой. Лазерная медицина. 2022; 26(3-4): 26–31. https://doi.org/10.37895/2071-8004-2022-26-3-4-26-31 Контакты: Луценко Ю.Г., e-mail: hirurgiya.ﬁ po@mail.ru Laser medicine. 2022, vol. 26, № 3–4 Laser medicine. 2022, vol. 26, № 3–4 Лазерная медицина. – 2022. – Т. 26, № 3–4 УДК: 617.586-002.44:616.379-008.64+615.849/19 DOI: 10.37895/2071-8004-2022-26-3-4-26-31 Abstract Purpose: to compare the effectiveness of laser therapy and traditional methods for managing diabetic patients with purulent-necrotic lesions in their lower extremities. Material and methods. The article presents a retrospective analysis of outcomes after treatment of 76 patients with diabetes mellitus having purulent-necrotic lesions in their lower extremities. Patients were divided into two groups. The ﬁ rst (main) group consisted of 34 patients in whom traditional treatment was accompanied by intravenous laser blood irradiation and local laser therapy; the second (control) group consisted of 42 patients who received only traditional treatment. Results. Laser therapy contributed to more rapid reduction of wound defect (17.4 %) compared to patients after traditional treatment (11.1 %); it reduced the period of transition to inﬂ ammatory-regenerative changes, promoted a smaller number of high amputations of lower limbs from 14 to 6 % as well as shortened the length of hospital stay by 11.2 days. Conclusion. Laser light therapy in patients with diabetic foot accelerates cleansing and reduction of the wound area, allows to reduce or eliminate inﬂ ammatory process in shorter time than with traditional treatment. Keywords: diabetes mellitus, diabetic foot, laser therapy y , , py For c itations: Lutsenko Yu.G., Grintsov A.G., Matiytsiv A.B. The effectiveness of laser therapy in patients with diabetic foot. Laser Medicine. 2022; 26(3-4): 26–31. [In Russ.]. https://doi.org/10.37895/2071-8004-2022-26-3-4-26-31 Contacts: Lutsenko Yu.G., e-mail: hirurgiya.ﬁ po@mail.ru Lutsenko Yu.G., Grintsov A.G., Matiytsiv A.B. M. Gorky Donetsk National Medical University, Donetsk, Donetsk People’s Republic, Russia M. Gorky Donetsk National Medical University, Donetsk, Donetsk People’s Republic, Russia Ю.Г. Луценко, А.Г. Гринцов, А.Б. Матийцив ГОО ВПО «Донецкий национальный медицинский университет имени М. Горького», Донецк, ДНР, Россия МАТЕРИАЛЫ И МЕТОДЫ Представлены результаты лечения 76 больных са- харным диабетом с гнойно-некротическим поражением нижних конечностей, находившихся на лечении в хирур- гическом отделении № 3 ГКБ № 21 г. Донецка в пери- од с 2019 по 2021 г., из которых мужчины составляли 35,5 % (27 чел.), женщины – 64,5 % (49 чел.). Средний возраст больных – 58,7 ± 18,1 года, средняя продолжи- тельность заболевания сахарным диабетом – 9,3 года. Нейропатическая форма СДС отмечена у 21 (27 %) больного, ишемическая – у 14 (18 %), смешанная – у 41 (55 %). Объем поражения тканей стопы варьировал от II до III степени по классификации Wagner (табл. 1). Для оценки результатов лечения использовали сле- дующие методы. 1. Раневая планиметрия. Осмотр ран при перевяз- ке включал измерение их размеров с последующим вычислением площади. Для определения площа- ди раневой поверхности использовалось приложе- ние LesionMeter на операционной системе Android. Измерения проводились на 1-е, 3-и, 5-е и 7-е сутки, вычислялся процент уменьшения площади раневой поверхности за сутки по отношению к предыдущему результату по формуле: As = ( – ) × 100 × , где S – вели- чина площади раны при предшествующем измерении, Sn – величина площади раны в настоящий момент, t – количество суток между измерениями. ф g ( ) Пациенты были разделены на две группы. В 1-й (основной) группе из 34 больных традиционное ле- чение дополнялось внутривенным лазерным облуче- нием крови (ВЛОК) и местной лазеротерапией, во 2-й (контрольной) группе из 42 пациентов применяли только традиционное лечение (дезинтоксикационная, антибактериальная терапия, коррекция углеводного обмена, иммунитета и нарушений гомеостаза, ней- рометаболические, вазоактивные, антихолинэстераз- ные препараты, перевязки с антисептиками и мазями). При поступлении всем больным проводилась хирур- гическая санация раны с иссечением некротических тканей, дренированием раны, а затем осуществля- лось местное лечение под повязкой с учетом фазы раневого процесса. Площадь раневой поверхности составляла от 32 до 618 см 2. Sn – величина площади раны в настоящий момент, t – количество суток между измерениями. 2. Бактериологическое исследование. Определяли качественный состав патогенной микрофлоры, чувст- вительность к антибиотикам и ее количество в коло- ниеобразующих единицах на 1 г ткани раны (КОЕ/г) на 0-е, 1-е, 3-и, 5-е и 7-е сутки. 3. Цитологическое исследование. Изучали кле- точный состав раневого отделяемого методом маз- ков-отпечатков, окраска гематоксилином и эозином, увеличение ×400. Полученные результаты статистически обработаны с определением средней арифметической величины (М), средней квадратичной (SD). Степень достовер- ности вычисляли по таблице Стьюдента. Различия считали достоверными при р < 0,05. ВВЕДЕНИЕ в 23 раза выше, чем у здорового человека [3, 6]. Около 40–60 % всех выполняемых нетравматических ампу- таций производится у больных сахарным диабетом [2–4]. В настоящее время в клинической практике ис- пользуются различные методы лечения гнойно-некро- тических осложнений СДС, такие как гипербарическая оксигенация, терапия отрицательным давлением (NPWT, VAC-therapy), применение тканевых факто- ров роста, лазеротерапия [1, 7, 8]. Однако процент Согласно прогнозам Международной диабетиче- ской федерации, к 2035 г. количество больных сахар- ным диабетом (СД) в мире увеличится до 592 млн человек. Синдром диабетической стопы (СДС) встречается у 20–80 % пациентов СД в возрасте от 22 до 78 лет [1–6]. Риск ампутации нижних конеч- ностей у больных сахарным диабетом в результате развития гнойно-некротических осложнений СДС 26 Laser medicine. 2022, vol. 26, № 3–4 Лазерная медицина. – 2022. – Т. 26, № 3–4 процедур – 8–10. При местной лазеротерапии выпол- няли ежедневные сеансы во время перевязок с по- мощью аппарата «Лика-терапевт». После механиче- ской обработки краев раны, промывания и санации раневой полости, излучающие головки устанавли- вали по краям раны и проводили 7–8 сеансов воз- действия низкоинтенсивным лазерным излучением. Продолжительность сеанса облучения составляла от 8 до 15 мин и зависела от размеров раны. Длина волны излучения – 940 нм, мощность – от 6 до 8 Вт в импульсном режиме с частотой 1500 Гц, суммарная доза энергии за сеанс – 0,05–1,0 Дж/см 2. высоких ампутаций и летальность у данной группы пациентов остаются значимыми [2, 5, 6, 9]. Цель работы: сравнить эффективность примене- ния лазеротерапии и традиционных методов лечения у больных сахарным диабетом с гнойно-некротиче- ским поражением нижних конечностей. МАТЕРИАЛЫ И МЕТОДЫ Для ВЛОК применяли аппарат «Лика-терапевт» (Россия), мощностью 2 мВт, длиной волны излучения 632,8 нм в непрерывном режиме. Продолжительность одного сеанса составляла 10–20 мин, количество Таблица 1 ц Распределение больных синдромом диабетической стопы по классификации Wagner Распределение больных синдромом диабетической стопы по классификации Wagner Distribution of patients with diabetic foot syndrome by Wagner classiﬁ cation Форма Form Степень Degree Всего Total II III Нейропатическая Neuropathic 12 9 21 Ишемическая Ischemic 8 6 14 Смешанная Mixed 27 14 41 Всего Total 47 29 76 % 62 38 100 Distribution of patients with diabetic foot syndrome by Wagner classiﬁ cation 27 Laser medicine. 2022, vol. 26, № 3–4 Лазерная медицина. – 2022. – Т. 26, № 3–4 РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ реакций, однако процесс очищения протекал с разной скоростью. В первые 3 суток доминировал некротиче- ский (I) тип цитограммы (68 %): большое количество некротических тканей, детрита, превалирование нейтро- филов (92–97 %), большинство из которых находилось в состоянии дегенерации и деструкции; незавершенный и извращенный фагоцитоз (в 80–85 % случаев), отсутст- вие макрофагов, лимфоцитов и полибластов (рис. 1). Планиметрические исследования показали, что у всех пациентов отмечалась положительная ди- намика сокращения раневой поверхности. Однако ис- пользование лазеротерапии способствовало более быстрому сокращению площади раневого дефекта – на 17,4 % на 7-е сутки исследования по сравнению с традиционными методами лечения (табл. 2). р д ц д ( ) Ведущая этиологическая роль в развитии гной- ных заболеваниях мягких тканей принадлежала Staphylococcus aureus – 42 случая (55,3 %). Далее, по убывающей: Proteus mirabilis – 17 (22,4 %), Escherichia coli – 8 (10,5 %), Enterobacter cloacae – 7 (9,2 %), Pseudomonas aeruginosa – 2 случая (2,6 %). До начала лечения наблюдалась высокая бактери- альная обсемененность раны (10 7–8 КОЕ/г ткани). Уменьшение уровня обсемененности ниже критическо- го (10 5 КОЕ/г) при лазеротерапии достигалось в сред- нем к 3-м суткам против 7-х суток при традиционных методах лечения. На 7-е сутки после лазеротерапии пациентов с микробной колонизацией не наблюдалось в обеих группах. Таким образом, при лазеротерапии снижение микробной обсемененности происходило быстрее по сравнению с традиционными методами лечения (табл. 3). В 32 % отмечался дегенеративно-воспалительный (II) тип цитограммы: наличие некротических тканей, детрита и клеточных обломков, преобладание ней- трофильных лейкоцитов (до 90 %), из которых до 50 % клеток находилось в состоянии дегенерации и де- струкции, в 65–80 % незавершенный и извращенный фагоцитоз, с обильной микрофлорой, расположенной внутри- и внеклеточно и почти полным отсутствием макрофагов, лимфоцитов и полибластов. Уменьшение количества некротических тканей, детрита, клеточных обломков, нейтрофильной ин- фильтрации, появление и увеличение числа лимфо- цитов и моноцитов (до 5–10 % клеток в поле зрения), наличие незавершенного и извращенного фагоцитоза (в 65–80 %), появление единичных макрофагов и поли- бластов на 5-е сутки наблюдения свидетельствовали о переходе раневого процесса в фазу воспалитель- ных изменений (III тип цитограммы). При использова- нии лазеротерапии этот тип цитограммы имел место у 8 %, а при традиционном лечении – у 4,2 % пациен- тов (рис. 2). Цитологические исследования мазков-отпечатков показали, что процесс очищения ран в обеих группах больных протекал по общепатологическим законам. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ Во всех группах сохранялись общие фазы клеточных Таблица 2 Таблица 2 Скорость уменьшения площади раны Table 2 Rate of reduction of wound area Сроки наблюдения, сутки Observation period, day Сокращение площади раневого дефекта, % Reduction of wound area, % 1-я группа Group 1 2-я группа Group 2 1-е 6,2 4,0 3-и 11,1* 5,3 5-е 16,3* 8,8 7-е 17,4* 11,1 Примечание: * – различия достоверны, р < 0,05. Note: * – differences are signiﬁ cant, р < 0.05. Таблица 3 Динамика элиминации микробных возбудителей из ткани Table 3 Dynamics of elimination microbial pathogens from tissue Группы больных Groups of patients Сроки наблюдения, сутки Observation period, day 0 1 3 5 7 Степень обсемененности раны (КОЕ/г) Degree of contamination of wound (CFU/g) 1 3,4 × 10 7 3,7 × 10 5 2,2 × 10 4 2,5 × 10 2 – 2 2,7 × 10 7 5,8 × 10 6 1,8 × 10 5 1,4 × 10 5 3,6 × 10 3 Скорость уменьшения площади раны Table 2 Rate of reduction of wound area Сроки наблюдения, сутки Observation period, day Сокращение площади раневого дефекта, % Reduction of wound area, % 1-я группа Group 1 2-я группа Group 2 1-е 6,2 4,0 3-и 11,1* 5,3 5-е 16,3* 8,8 7-е 17,4* 11,1 Примечание: * – различия достоверны, р < 0,05. Note: * – differences are signiﬁ cant, р < 0.05. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ Table 2 Динамика элиминации микробных возбудителей из ткани Динамика элиминации микробных возбудителей из ткани Table 3 Dynamics of elimination microbial pathogens from tissue Группы больных Groups of patients Сроки наблюдения, сутки Observation period, day 0 1 3 5 7 Степень обсемененности раны (КОЕ/г) Degree of contamination of wound (CFU/g) 1 3,4 × 10 7 3,7 × 10 5 2,2 × 10 4 2,5 × 10 2 – 2 2,7 × 10 7 5,8 × 10 6 1,8 × 10 5 1,4 × 10 5 3,6 × 10 3 Динамика элиминации микробных возбудителей из ткани Table 3 Dynamics of elimination microbial pathogens from tissue Группы больных Groups of patients Сроки наблюдения, сутки Observation period, day 0 1 3 5 7 Степень обсемененности раны (КОЕ/г) Degree of contamination of wound (CFU/g) 1 3,4 × 10 7 3,7 × 10 5 2,2 × 10 4 2,5 × 10 2 – 2 2,7 × 10 7 5,8 × 10 6 1,8 × 10 5 1,4 × 10 5 3,6 × 10 3 Dynamics of elimination microbial pathogens from tissue 28 Laser medicine. 2022, vol. 26, № 3–4 Лазерная медицина. – 2022. – Т. 26, № 3–4 Рис. 1. Нейтрофильная инфильтрация сосочкового слоя дер- мы. Окраска гематоксилином и эозином, увеличение ×400 Fig. 1. Neutrophil inﬁ ltration of the papillary dermis layer. Staining with hematoxylin and eosin, magniﬁ cation ×400 Крайне редкое выявление в мазке-отпечатке некротических тканей и детрита, преобладание ней- трофильных лейкоцитов (до 70–80 %), сохранность нейтрофилов (до 70 %), появление недифференци- рованных полибластов, фибробластов и лимфоцитов (до 10–20 %), увеличение до 5 % макрофагов, наличие до 15–20 % нейтрофилов в состоянии дегенерации, повышение до 50–60 % завершенного фагоцитоза на 7-е сутки наблюдения свидетельствовали о пере- ходе раневого процесса в фазу воспалительно-реге- неративных изменений (IV тип цитограммы). Наиболее выраженно этот процесс наблюдался при применении лазеротерапии (рис. 3). Таким образом, результаты проведенных цито- логических исследований показали, что применение лазеротерапии способствовало снижению в мазках- отпечатках гнойных ран содержания клеток, опре- деляющих острую фазу воспаления (нейтрофилов, лимфоцитов, моноцитов) и рост числа клеток, фор- мирующих репаративные процессы (макрофагов, фи- бробластов, полибластов). В основной группе полное закрытие трофической язвы отмечено у 7 больных (21 %), у 12 (35 %) наблюдали значительное улучше- ние (уменьшение размеров язвенного дефекта за счет частичной эпителизации), у 15 (44 %) – незначитель- ное улучшение (исчезновение явлений воспаления, наличие грануляций). РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ В контрольной группе результа- ты были следующие: 10 % – закрытие и значительное улучшение, 90 % – незначительное улучшение. Сроки госпитализации больных основной группы составили 18 ± 2,3, контрольной – 29,6 ± 2,5 койко-дня. Рис. 1. Нейтрофильная инфильтрация сосочкового слоя дер- мы. Окраска гематоксилином и эозином, увеличение ×400 Fig. 1. Neutrophil inﬁ ltration of the papillary dermis layer. Staining with hematoxylin and eosin, magniﬁ cation ×400 Fig. 1. Neutrophil inﬁ ltration of the papillary dermis layer. Staining with hematoxylin and eosin, magniﬁ cation ×400 у 9 больных (75 %). Благоприятные результаты приме- нения лазеротерапии указывают на необходимость ла- зерной предоперационной подготовки больных с ней- ротрофическими язвами. Применение лазеротерапии позволило сберечь жизнеспособность стопы у боль- ных с гнойно-некротическим процессом в 94 % случа- ев, при этом некрэктомия была альтернативой высо- кой ампутации нижней конечности, которая выполнена 2 пациентам. Напротив, у пациентов контрольной груп- пы чаще наблюдались осложнения в виде длительного течения раневого процесса, распространения акраль- ных некрозов, увеличения частоты повторных опера- ций в форме высоких ампутаций нижней конечности. У 6 (14 %) больных этой группы комплексная консерва- тивная терапия была неэффективной, им выполнена Необходимо отметить, что у 12 пациентов после лазеротерапии проведены реконструктивные пла- стические операции с положительным результатом Рис. 3. Рана на 9-е сутки воздействия лазерного излучения. Компактное скопление фибробластов, окруженных тонкими пучками оксифильных коллагеновых волокон. Окраска гема- токсилином и эозином, увеличение ×400 Рис. 2. Рана на 5-е сутки после воздействия лазерного излу- чения. Единичные нейтрофилы и мелкие группы лимфоцитов в сосочковом слое дермы. Окраска гематоксилином и эозином, увеличение ×400 Fig. 2. Wound on day 5 after exposure to laser irradiation. Single neutrophils and small groups of lymphocytes in the papillary layer of the dermis. Staining with hematoxylin and eosin, magniﬁcation ×400 Рис. 2. Рана на 5-е сутки после воздействия лазерного излу- чения. Единичные нейтрофилы и мелкие группы лимфоцитов в сосочковом слое дермы. Окраска гематоксилином и эозином, увеличение ×400 Рис. 3. Рана на 9-е сутки воздействия лазерного излучения. Компактное скопление фибробластов, окруженных тонкими пучками оксифильных коллагеновых волокон. Окраска гема- токсилином и эозином, увеличение ×400 Fig. 2. Wound on day 5 after exposure to laser irradiation. Single neutrophils and small groups of lymphocytes in the papillary layer of the dermis. Staining with hematoxylin and eosin, magniﬁ cation ×400 Fig. 2. Wound on day 5 after exposure to laser irradiation. Single neutrophils and small groups of lymphocytes in the papillary layer of the dermis. ЛИТЕРАТУРА 1. Баранов А.В., Исмаилов Г.И., Дербенев В.А., Раджа- бов А.А. Комбинированное применение фотодинамиче- ской терапии и гидрохирургических технологий в ком- плексном лечении обширных гнойных ран у больных синдромом диабетической стопы. Международный науч- но-практический конгресс «Раны и раневые инфекции», посвященный 140-летию со дня рождения С.С. Гиргола- ва: сборник научных трудов. М.; 2021: 18–21. 3. Dedov I.I., Shestakova M.V., Mayorov A.Yu., et al. Algorithms of specialized medical care for patients with diabetes mel- litus. 9th edition. Diabetes Mellitus. 2019; 22 (1S1): 1–144. [In Russ.]. DOI: 10.14341/DM221S1 4. Derbenev V.A., Radzhabov A.A., Huseynov A.I., Ismailov G.I. An integrated approach to the treatment of purulent-necrotic wounds in diabetic foot syndrome. 4-y mezhdunarodnyy nauchno-prakticheskiy kongress «Rany i ranevye infektsii»: sbornik nauchnykh trudov. Moscow; 2018: 58–60. [In Russ.]. 2. Галстян Г.Р., Викулова О.К., Исаков М.А. и др. Эпиде- миология синдрома диабетической стопы и ампутаций нижних конечностей в Российской Федерации по данным Федерального регистра больных сахарным диабетом (2013–2016 гг.). Сахарный диабет. 2018; 21 (3): 170–177. DOI: 10.14341/DM9688 5. Tsvetkov V.O., Kolovanova O.V., Mikaelyan L.S., et al. Os- teomyelitis of diabetic foot: A balance between radical surgi- cal treatment and prolonged antibiotic therapy from the posi- tion of the surgeon. Consilium Medicum. 2020; 22 (4): 61–65. [In Russ.]. DOI: 10.26442/20751753.2020.4.200143 3. Дедов И.И., Шестакова М.В., Майоров А.Ю. и др. Алго- ритмы специализированной медицинской помощи боль- ным сахарным диабетом. 9-й выпуск. Сахарный диабет. 2019; 22 (1S1): 1–144. DOI: 10.14341/DM221S1 6. Armstrong D.G., Boulton A.J.M., Bus S.A. Diabetic foot ul- cers and their recurrence. N Engl J Med. 2017; 376 (24): 2367–2375. DOI: 10.1056/NEJMra1615439 4. Дербенев В.А., Раджабов А.А., Гусейнов А.И., Исмаи- лов Г.И. Комплексный подход к лечению гнойно-некротиче- ских ран при синдроме диабетической стопы. 4-й междуна- родный научно-практический конгресс «Раны и раневые инфекции»: сборник научных трудов. М.; 2018: 58–60. 7. De Alencar Fonseca Santos J., Campelo M.B.D., de Olivei- ra R.A., et al. Effects of low-power light therapy on the tis- sue repair process of chronic wounds in diabetic feet. Pho- tomed Laser Surg. 2018; 36 (6): 298–304. DOI: 10.1089/ pho.2018.4455 5. Цветков В.О., Колованова О.В., Микаелян Л.С. и др. Остеомиелит диабетической стопы: баланс между ради- кальным хирургическим лечением и пролонгированной антибиотикотерапией с позиции хирурга. Consilium Medi- cum. 2020; 22 (4): 61–65. DOI: 10.26442/20751753.2020.4. 200143 8. Saeedi P., Petersohn I., Salpea P., et al. ЗАКЛЮЧЕНИЕ 9. Milcheski D.A., Portocarrero M.L., Alvarez D.M., et al. Initial experience with negative-pressure wound therapy with instil- lation in complex wounds. Rev Col Bras Cir. 2017; 44 (4): 348–353. DOI: 10.1590/0100-69912017004008 Применение лазерного излучения у больных диа- бетической стопой способствует более быстрому со- кращению площади раневого дефекта (17,4 %) в срав- нении с традиционным лечением (11,1 %) на 7-е сутки. Использование лазеротерапии в комплексном лечении гнойных ран ускоряет переход в фазу воспалительно- регенеративных изменений. Применение лазерного излучения в лечении гнойно-некротических ран ниж- них конечностей у больных сахарным диабетом обес- печивает уменьшение частоты выполнения высокой ампутации нижней конечности с 14 до 6 %, сокраще- ние длительности стационарного лечения на 11,2 кой- ко-дня по сравнению с традиционными методами. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ Staining with hematoxylin and eosin, magniﬁ cation ×400 Fig. 3. Wound on day 9 after exposure to laser irradiation. Compact cluster of ﬁ broblasts surrounded by thin bundles of oxyphilic colla- gen ﬁ bers. Staining with hematoxylin and eosin, magniﬁ cation ×400 29 Laser medicine. 2022, vol. 26, № 3–4 Лазерная медицина. – 2022. – Т. 26, № 3–4 national Diabetes Federation Diabetes Atlas, 9th edition. Di- abetes Res Clin Pract. 2019. 157: 107843. DOI: 10.1016/j. diabres.2019.107843 высокая ампутация нижней конечности в связи с рас- пространением гнойно-некротического процесса. 9. REFERENCES 1. Baranov A.V., Ismailov G.I., Derbenev V.A., Radzhabov A.A. Combined application of photodynamic therapy and hydro- surgical technologies in the complex treatment of extensive purulent wounds in patients with diabetic foot syndrome. Mezhdunarodnyy nauchno-prakticheskiy kongress «Rany i ranevye infektsii», posvyashchennyy 140-letiyu so dnya ro- zhdeniya S.S. Girgolava: sbornik nauchnykh trudov. Moscow; 2021: 18–21. [In Russ.]. 2. Galstyan G.R., Vikulova O.K., Isakov M.A., et al. Epidemiol- ogy of diabetic foot syndrome and lower limb amputations in the Russian Federation according to the Federal Regis- ter of Patients with Diabetes mellitus (2013-2016). Diabetes Mellitus. 2018; 21 (3): 170–177. [In Russ.]. DOI: 10.14341/ DM9688 ЛИТЕРАТУРА Global and re- gional diabetes prevalence estimates for 2019 and pro- jections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res Clin Pract. 2019. 157: 107843. DOI: 10.1016/j.di- abres.2019.107843 6. Armstrong D.G., Boulton A.J.M., Bus S.A. Diabetic foot ul- cers and their recurrence. N Engl J Med. 2017; 376 (24): 2367–2375. DOI: 10.1056/NEJMra1615439 9. Milcheski D.A., Portocarrero M.L., Alvarez D.M., et al. Initial experience with negative-pressure wound therapy with instil- lation in complex wounds. Rev Col Bras Cir. 2017; 44 (4): 348–353. DOI: 10.1590/0100-69912017004008 7. De Alencar Fonseca Santos J., Campelo M.B.D., de Olivei- ra R.A., et al. Effects of low-power light therapy on the tis- sue repair process of chronic wounds in diabetic feet. Pho- tomed Laser Surg. 2018; 36 (6): 298–304. DOI: 10.1089/ pho.2018.4455 Конфликт интересов Конфликт интересов Авторы заявили об отсутствии конфликта интересов. Conﬂ ict of interest The authors declare no conﬂ ict of interest. 8. Saeedi P., Petersohn I., Salpea P., et al. Global and re- gional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the Inter- 30 Laser medicine. 2022, vol. 26, № 3–4 Лазерная медицина. – 2022. – Т. 26, № 3–4 Information about the authors Lutsenko Yuri – Cand. Sc. (Med.), Assistant Professor at the Department of Sur- gery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@mail. ru; ORCID: http://orcid.org/0000-0002-9442-5207 Grintsov Alexander – Dr. Sc. (Med.), Professor, Head of the Department of Sur- gery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@mail. ru; ORCID: https://orcid.org/0000-0003-3936-8323 Matiytsiv Alexander – Cand. Sc. (Med.), Assistant Professor at the Department of Surgery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@ mail.ru; ORCID: https://orcid.org/0000-0001-8813-1766 Сведения об авторах Information about the authors Lutsenko Yuri – Cand. Sc. (Med.), Assistant Professor at the Department of Sur- gery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@mail. ru; ORCID: http://orcid.org/0000-0002-9442-5207 Information about the authors Lutsenko Yuri – Cand. Sc. (Med.), Assistant Professor at the Department of Sur- gery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@mail. ru; ORCID: http://orcid.org/0000-0002-9442-5207 Луценко Юрий Григорьевич – кандидат медицинских наук, доцент кафедры хирургии ФИПО, ГОО ВПО «Донецкий национальный медицинский университет им. М. Горького»; e-mail: hirurgiya.ﬁ po@mail.ru; ORCID: http://orcid.org/0000- 0002-9442-5207 Grintsov Alexander – Dr. Sc. (Med.), Professor, Head of the Department of Sur- gery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@mail. ru; ORCID: https://orcid.org/0000-0003-3936-8323 Grintsov Alexander – Dr. Sc. (Med.), Professor, Head of the Department of Sur- gery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@mail. ru; ORCID: https://orcid.org/0000-0003-3936-8323 Гринцов Александр Григорьевич – доктор медицинских наук, профессор, заведующий кафедрой хирургии ФИПО, ГОО ВПО «Донецкий национальный медицинский университет им. М. Горького»; e-mail: hirurgiya.ﬁ po@mail.ru; ORCID: https://orcid.org/0000-0003-3936-8323 Matiytsiv Alexander – Cand. Sc. (Med.), Assistant Professor at the Department of Surgery, M. Gorky Donetsk National Medical University; e-mail: hirurgiya.ﬁ po@ mail.ru; ORCID: https://orcid.org/0000-0001-8813-1766 Матийцив Александр Богданович – кандидат медицинских наук, доцент кафедры хирургии ФИПО, ГОО ВПО «Донецкий национальный медицинский университет им. М. Горького»; e-mail: hirurgiya.ﬁ po@mail.ru; ORCID: https:// orcid.org/0000-0001-8813-1766 Матийцив Александр Богданович – кандидат медицинских наук, доцент кафедры хирургии ФИПО, ГОО ВПО «Донецкий национальный медицинский университет им. М. Горького»; e-mail: hirurgiya.ﬁ po@mail.ru; ORCID: https:// orcid.org/0000-0001-8813-1766 31 31
https://openalex.org/W4324058093	https://pasteur.hal.science/pasteur-04079251/document	English	null	Identifying the Most Probable Mammal Reservoir Hosts for Monkeypox Virus Based on Ecological Niche Comparisons	Viruses	2,023	cc-by	12,463	To cite this version: Manon Curaudeau, Camille Besombes, Emmanuel Nakouné, Arnaud Fontanet, Antoine Gessain, et al.. Identifying the Most Probable Mammal Reservoir Hosts for Monkeypox Virus Based on Ecological Niche Comparisons. Viruses, 2023, 15 (3), pp.727. 10.3390/v15030727. pasteur-04079251 Distributed under a Creative Commons Attribution 4.0 International License Identifying the Most Probable Mammal Reservoir Hosts for Monkeypox Virus Based on Ecological Niche Comparisons Manon Curaudeau 1,2 , Camille Besombes 3 , Emmanuel Nakouné 4, Arnaud Fontanet 3,5, Antoine Gessain 2 and Alexandre Hassanin 1,* 1 Institut de Systématique, Évolution, Biodiversité (ISYEB), Sorbonne Université, MNHN, CNRS, EPHE, UA, 75005 Paris, France; manon.curaudeau1@mnhn.fr 1 Institut de Systématique, Évolution, Biodiversité (ISYEB), Sorbonne Université, MNHN, CNRS, EPHE, UA, 75005 Paris, France; manon.curaudeau1@mnhn.fr 2 Unité Épidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Université Paris Cité, CNRS UMR 3569, 75015 Paris, France; antoine.gessain@pasteur.fr 3 Unité d’Épidémiologie des Maladies Émergentes, Institut Pasteur, Université Paris Cité, 75015 Paris, France; camille.besombes@pasteur.fr (C.B.); arnaud.fontanet@pasteur.fr (A.F.) 4 Department of Arboviruses, Emerging Viruses and Zoonosis, Institut Pasteur, Bangui BP 923, Central African Republic; emmanuel.nakoune@pasteur-bangui.cf 5 Conservatoire National des Arts et Métiers, Unité PACRI, 75003 Paris, France * Correspondence: alexandre.hassanin@mnhn.fr 2 Unité Épidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Université Paris Cité, CNRS UMR 3569, 75015 Paris, France; antoine.gessain@pasteur.fr p ( ) p ( ) 4 Department of Arboviruses, Emerging Viruses and Zoonosis, Institut Pasteur, Bangui BP 923, Central African Republic; emmanuel.nakoune@pasteur-bangui.cf 5 Conservatoire National des Arts et Métiers, Unité PACRI, 75003 Paris, France * Correspondence: alexandre hassanin@mnhn fr p ( ) p ( ) 4 Department of Arboviruses, Emerging Viruses and Zoonosis, Institut Pasteur, Bangui BP 923, Central African Republic; emmanuel.nakoune@pasteur-bangui.cf g p p g 5 Conservatoire National des Arts et Métiers, Unité PACRI, 75003 Paris, France * Correspondence: alexandre.hassanin@mnhn.fr Abstract: Previous human cases or epidemics have suggested that Monkeypox virus (MPXV) can be transmitted through contact with animals of African rainforests. Although MPXV has been identiﬁed in many mammal species, most are likely secondary hosts, and the reservoir host has yet to be discovered. In this study, we provide the full list of African mammal genera (and species) in which MPXV was previously detected, and predict the geographic distributions of all species of these genera based on museum specimens and an ecological niche modelling (ENM) method. Then, we reconstruct the ecological niche of MPXV using georeferenced data on animal MPXV sequences and human index cases, and conduct overlap analyses with the ecological niches inferred for 99 mammal species, in order to identify the most probable animal reservoir. Our results show that the MPXV niche covers three African rainforests: the Congo Basin, and Upper and Lower Guinean forests. Citation: Curaudeau, M.; Besombes, C.; Nakouné, E.; Fontanet, A.; Gessain, A.; Hassanin, A. Identifying the Most Probable Mammal Reservoir Hosts for Monkeypox Virus Based on Ecological Niche Comparisons. Viruses 2023, 15, 727. https://doi.org/10.3390/v15030727 Keywords: Monkeypox; animal reservoir; ecological niche model; tropical Africa; evergreen forests; Sciuridae Academic Editor: Benjamin Liu viruses viruses viruses Identifying the Most Probable Mammal Reservoir Hosts for Monkeypox Virus Based on Ecological Niche Comparisons The four mammal species showing the best niche overlap with MPXV are all arboreal rodents, including three squirrels: Funisciurus anerythrus, Funisciurus pyrropus, Heliosciurus rufobrachium, and Graphiurus lorraineus. We conclude that the most probable MPXV reservoir is F. anerythrus based on two niche overlap metrics, the areas of higher probabilities of occurrence, and available data on MPXV detection. 1. Introduction Received: 25 January 2023 Revised: 7 March 2023 Accepted: 8 March 2023 Published: 11 March 2023 Monkeypox, now called mpox [1,2], is an emerging zoonotic disease caused by the Monkeypox virus (MPXV). Infection in humans manifests as fever, swollen lymph nodes, and fatigue, followed by a rash with macular lesions progressing to papules, vesicles, pustules, and scabs, usually on the face, hands, and feet for two to ﬁve weeks [3]. Taxonomically, MPXV belongs to Poxviridae, a family of large double-stranded DNA viruses (130–375 kbp) represented by 22 genera and 83 species. The family is divided into two subfamilies: Entomopoxvirinae, in which hosts are insects; and Chordopoxvirinae, in which hosts are vertebrates (birds, crocodiles, mammals, and teleost ﬁshes) [4–7]. Within Chordopoxvirinae, all viruses of the genus Orthopoxvirus (OPXV) are related to mammalian hosts, and phylogenetic analyses have supported the existence of an Old World group composed of MPXV and eight other species, such as variola virus (the agent of smallpox), vaccinia virus (the source of modern smallpox vaccines), and cowpox [8]. Mpox was ﬁrst described in 1958 in Asian macaques used for polio vaccine production and research at the HAL Id: pasteur-04079251 https://pasteur.hal.science/pasteur-04079251v1 Submitted on 24 Apr 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License viruses Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/viruses Viruses 2023, 15, 727. https://doi.org/10.3390/v15030727 Viruses 2023, 15, 727 2 of 17 2 of 17 Statens Serum Institut in Copenhagen, Denmark [9]. Although the authors concluded that the monkeys were not infected in Denmark, the origin of mpox infection remained elusive. During the following decade, there were several outbreaks in monkeys in laboratories and zoos all over the world: six outbreaks between 1961 and 1966 in macaques and langurs used in American laboratories; two outbreaks between 1964 and 1965 in orangutans, white- handed gibbons, and common marmosets maintained in captivity in Dutch zoos; and one outbreak in 1968 in chimpanzees used in a French laboratory [10]. The ﬁrst human mpox case was detected in 1970 in a young boy from Bokenda, a remote village in the northwest of the Democratic Republic of Congo (DRC) [11]. In the same year, ﬁve cases were identiﬁed in West Africa: four in two villages of northeastern Liberia, and one in southern Sierra Leone [12]. Several cases were subsequently discovered in 1971 in Côte d’Ivoire [13] and Nigeria [12], in 1979 in Cameroon [14], in 1983 in the Central African Republic (CAR) [15], and in 1987 in Gabon [16,17]. A total of 404 cases were reported between 1970 and 1986 [18]. The vast majority of these cases originated in remote villages in tropical rainforests (89% of the 283 reported between 1970 and 1984) [18], and most of them were from the DRC. p In 1996–1997, the ﬁrst large-scale mpox epidemic occurred, with 511 cases in 54 villages of the Katako-Kombe health zone in central DRC [19]. From the 2000s, the number of reported cases has been steadily increasing: from a few hundred cases each year between 2001 and 2009, to more than two thousand cases per year between 2010 and 2014 [20]. Since September 2017, a mpox outbreak has been ongoing in Nigeria [21]. The apparent rise in cases since 2000 could be explained by enhanced ﬁeld surveillance in African countries [20,22], as well as declining levels of population immunity due to the end of smallpox vaccination, which provided cross-protection against MPXV [23]. Several exports of MPXV have occurred outside the African continent: in 2003 in the United States, in connection with a shipment of wild rodents (Cricetomys sp., Funisciurus sp., and Graphiurus sp.) from Ghana [24,25], in 2018 in Israel [26], in 2018 and 2021 in the United Kingdom [27,28], in 2019 in Singapore [29], and in 2021 in the United States [28], all in connection with travellers to and from Nigeria. The virus has also been exported to South Sudan, a non-endemic African country, in 2005 [30,31]. More recently, more than 80,000 cases have been reported in 2022 worldwide, during an epidemic involving mostly homosexual men [32,33]. g p g y Local investigations in African countries have suggested that several human mpox cases or epidemics originated from contacts with wild mammals, such as chimpanzee (Pan troglodytes), Pennant’s red colobus (Piliocolobus pennanti), or Gambian pouched rat (Cricetomys emini) [34,35]. In agreement with that, MPXV has been isolated or sequenced from several mammal species in Africa, including arboreal species, such as monkeys and squirrels, as well as ground-living species, including rodents and shrews [36–41]. In addition, antibodies against OPXV were detected in many species from four mammalian orders and nine families. Among these mammal species, one or more could be involved as natural reservoir host(s), in which the virus has been circulating for centuries, while others could be secondary hosts, occasionally contaminated through contact with the reservoir. y y g Although the MPXV reservoir has not yet been identiﬁed, several lines of evidence point to mammal species endemic to African rainforests of West and Central Africa. First, MPXV belongs to the genus Orthopoxvirus (OPXV), a genus exclusive to mammals [5,6]. Second, most human mpox cases have been reported in rainforests of West and Central Africa, or travellers from these regions [24,26–31,42]. Third, the geographic distribution of MPXV has been inferred with ecological niche modelling (ENM) methods, and the results have shown that the virus could be found in all rainforests of West and Central Africa [31,43–45]. Fourth, phylogenetic studies based on complete MPXV genomes have revealed a strong geographic structure [45–47]: viruses from Central Africa (Gabon, Cameroon, CAR, Republic of the Congo, and DRC; clade I [48]) are divergent from those from West Africa; the latter can be separated into two geographic subgroups, one including viruses from Sierra Leone, Liberia, Côte d’Ivoire, and Ghana (clade IIa [48]), and the other including viruses from Nigeria (clade IIb [48]). These results suggest that the animal reservoir populations have been genetically isolated from each other for many generations in three separate rainforest Viruses 2023, 15, 727 3 of 17 3 of 17 blocks, including the Upper and Lower Guinean forests in West Africa, and the Congo Basin in Central Africa. Therefore, we hypothesized that the MPXV reservoir is represented by one or several rainforest mammal species with a geographic distribution very similar to that of MPXV. In this study, we explore this hypothesis using a four-step approach: (i) we provide the full list of mammal genera and species previously identiﬁed as MPXV natural hosts in Africa; (ii) we predict the geographic distributions (or ecological niche) of all species of these genera based on georeferenced specimens catalogued in museum collection databases and ENM methods; (iii) we reconstruct the ecological niche of MPXV using reliable data on human index cases and MPXV sequences available for georeferenced wild animals; and (iv) we make statistical overlap comparisons between the ecological niches of mammals and MPXV in order to identify the most probable mammalian reservoir(s). 2.1. Mammal Species Identiﬁed as MPXV Hosts Many wild mammal species have been linked with a potential MPXV infection in Africa. Some people reported contact with a wild animal prior to an infection (for instance from hunting or through the consumption of bushmeat), while anti-OPXV antibodies have been found in many species [49–57]. MPXV was also isolated, and its genome fully sequenced, from two species: one rodent, Funisciurus anerythrus (Thomas’s rope squirrel) from Yambuku in the Mongala Province in northern DRC [36–38,45], and one primate, Cercocebus atys (Sooty Mangabey) from Taï National Park in Côte d’Ivoire [39]. In addition, the MPXV genome was recently sequenced from P. troglodytes from Taï National Park in Côte d’Ivoire [40], and from six species sampled in DRC, including one shrew (Crocidura littoralis) and ﬁve rodents, i.e., Cricetomys sp., F. anerythrus, Funisciurus bayonii, Malacomys longipes, and Stochomys longicaudatus [41]. g p y g For this study, we focused on all mammal species for which some biological evidence, such as virus isolation, MPXV DNA sequence, PCR ampliﬁcation, and detection of anti- OPXV antibodies, supports their role as reservoir or secondary hosts. To limit the inﬂuence of taxonomic issues at the species level (i.e., no identiﬁcation, such as Cricetomys sp., or possible misidentiﬁcation), we chose to study every species of all 14 genera of Table 1. Therefore, our taxonomic selection includes 213 African mammal species (full list provided in Table S1). 4 of 17 Viruses 2023, 15, 727 Table 1. Mammal genera and species potentially involved as reservoir or secondary hosts of MPXV. al genera and species potentially involved as reservoir or secondary hosts of MPXV. Table 1. Mammal genera and species potentially involved as reservoir or secondary hosts of MPXV. Order Family Genus Virus Isolation MPXV Fragment Ampliﬁed by PCR (Sequenced in Bold) Anti-OPXV Antibodies Eulipotyphla Erinaceidae Atelerix Atelerix spp. [55] Atelerix spp. [55] Eulipotyphla Soricidae Crocidura C. littoralis [41] Macroscelidea Macroscelididae Petrodromus P. tetradactylus [54,57] Primates Cercopithecidae Allenopithecus A. nigroviridis [52] Primates Cercopithecidae Cercocebus C. atys [39] C. atys [39] C. galeritus [52] Primates Cercopithecidae Cercopithecus C. ascanius [38,51–53] C. mona [52] C. nictitans [52] C. petaurista [49,50] C. pogonias [38,51,52] Primates Cercopithecidae Chlorocebus C. aethiops * [49] Primates Cercopithecidae Piliocolobus P. badius * [50] P. pennanti * [52] Primates Hominidae Pan P. troglodytes [40] Primates Lorisidae Perodicticus P. potto [52] Rodentia Dipodidae Jaculus Jaculus spp. [55] Rodentia Gliridae Graphiurus G. lorraineus [55] Graphiurus spp. [55,56] G. lorraineus [57] Graphiurus spp. 2.1. Mammal Species Identiﬁed as MPXV Hosts [55,56] Rodentia Muridae Malacomys M. longipes (MT724769) Rodentia Muridae Oenomys O. hypoxanthus [57] Rodentia Muridae Stochomys S. longicaudatus [41] Rodentia Nesomyidae Cricetomys Cricetomys sp. [41] Cricetomys spp. [55,56] C. emini [54,57] Cricetomys spp. [55,56] Rodentia Sciuridae Funisciurus F. anerythrus [37] F. anerythrus [37,41,55,56,58] F. bayonii [41] F. carruthersi [58] F. congicus [58] F. lemniscatus [58] F. pyrropus [58] Funisciurus. spp. [55,56] F. anerythrus [38,51,52,54] F. isabella [52] F. lemniscatus [52] Funisciurus spp. [52,53,56,57,59] Rodentia Sciuridae Heliosciurus H. rufobrachium [51–54] H. gambianus [52,56] Heliosciurus spp. [39,53,57] Rodentia Sciuridae Xerus Xerus sp. [56] *: current species names provided by the IUCN [60]. Xerus sp. [56] Xerus sp. [56] Viruses 2023, 15, 727 5 of 17 5 of 17 2.3. Occurrence Records of MPXV Cases To reconstruct the ecological niche of MPXV, we need occurrence records of MPXV cases. We included the six occurrence records previously published for mammals from which the virus was isolated and/or sequenced, including one F. anerythrus from Yambuku in the Mongala Province in northern DRC, several P. troglodytes from the Taï National Park in Côte d’Ivoire, and two primate sanctuaries in Cameroon [36–38,40,71]. We also included human index cases, which were presumably infected from an animal source and conﬁrmed by PCR, DNA sequencing, or MPXV isolation. However, the GPS coordinates of mpox cases were not provided in previous ENM studies [31,43–45]. All of the 103 human records used in our study are index cases of known village origin [11,12,14–17,21,34,35,45–47,54,59,72–90], for which the GPS coordinates were recovered using Google Maps (https://www.google. fr/maps, accessed on 22 June 2022), OpenStreetMap (http://www.openstreetmap.org, accessed on 22 June 2022), and Joint Operational Graphic (JOG) topographic maps. Most index cases reported in previous mpox epidemics in Central Africa were young boys living in remote villages surrounded by forests [90,91]. Therefore, we assumed that most human outbreaks began in the forest around the village after direct or indirect contact with an animal infected with MPXV. Since the ecological niche of MPXV was inferred using a cell size of 2.5 min (of latitude) × 2.5 min (of longitude) (see Section 2.4), each of the 103 selected villages was considered to be in the same cell as the nearby forest where the ﬁrst human infection occurred. We were able to gather 109 occurrence records for MPXV (Table S2), including 95 in Central Africa and 14 in West Africa. Occurrence records lower than 10 km apart were ﬁltered out using spThin [68], leaving a total of 96 occurrence records, with 84 in Central Africa and 12 in West Africa. 2.2. Occurrence Records of Selected Mammal Species 2.2. Occurrence Records of Selected Mammal Species To reconstruct the ecological niche of mammal species, we obtained species occurrence data from multiple databases, in an effort to reduce sampling biases [61–63]: the Global Bio- diversity Information Facility (GBIF; www.gbif.org, accessed on 22 June 2022), Integrated Digitized Biocollections (IdIgBio), VertNet, and INaturalist, using the R package spocc [64]. All of these databases carry occurrence records for mammal species, but all have their speciﬁcity: GBIF is the largest database of species occurrence records; IdIgBio is the United States digitised collection of specimens [65]; VertNet is a publicly accessible database on vertebrate specimen records from natural history collections around the world [66]; ﬁnally, iNaturalist is a citizen-science database with research-grade observations of multiple taxa. Both IdIgBio, VertNet, and Inaturalist communicate part of their data to GBIF, and some records can be found in multiple databases. Occurrence records that were duplicated, erroneous (e.g., in the ocean or in capital cities), or more than 30 km away from the IUCN distribution were ﬁltered out using CoordinateCleaner [67]. Finally, occurrence records lower than 10 km apart were ﬁltered using spThin [68], as spatial ﬁltering is recommended to improve the performance of ecological niche models and reduce sampling biases [69,70]. 2.4. Ecological Niche Modelling Ecological niche modelling (ENM) correlates occurrence records to environmental variables in order to predict the probability of occurrence for both sampled and non- sampled localities. ENM has been used to infer the distribution of species in the past [92] or future [93], in the context of migration [94], and also to deduce the distribution of invasive species [95] or viruses [96]. Our goal was to reconstruct the ecological niches for MPXV and the 213 selected mammal species. However, the minimum number of occurrence records needed to infer an ecological niche depends on the species prevalence, deﬁned as the fraction of the study area occupied by a species [97]. Here, the species prevalence was calculated as the percentage occupied by the geographic distribution of the species provided by the IUCN [60] on the whole modelling space, i.e., Africa (including Madagascar and other islands). According to Viruses 2023, 15, 727 6 of 17 van Proodij [98], the minimum number of occurrence records required to build an ecological niche in Africa is 14 for narrow-ranged (with a species prevalence below 0.2), and 25 for widespread species (with a species prevalence above 0.2). van Proodij [98], the minimum number of occurrence records required to build an ecological niche in Africa is 14 for narrow-ranged (with a species prevalence below 0.2), and 25 for widespread species (with a species prevalence above 0.2). p p p p The ecological niches were reconstructed using occurrence records and variables available in the WorldClim 2.1 [99,100] and ENVIREM [101] datasets at 2.5 min resolution (approximately 4.5 × 4.5 km gridcell). The WorldClim dataset includes 19 bioclimatic variables derived from monthly temperatures and rainfall values; it is the most employed dataset for ENM studies. The ENVIREM (ENVIronmental Rasters for Ecological Modeling) dataset consists of 18 biologically relevant climatic and topographic variables derived from monthly temperature and precipitation data, and monthly extra-terrestrial solar radiation, which are intended to supplement the 19 bioclimatic WorldClim variables [101]. Since one ENVIREM variable (monthCountByTemp10) is categorical, it was not included in the analyses. We added the elevation to these 36 variables and studied them for an area corresponding to a 300 km radius around the selected points, and the caret R package [102] was used to determine and select the least correlated variables (\|r\| < 0.7) [103]. 2.4. Ecological Niche Modelling For MPXV and each mammal species, ecological niche modelling was performed with the MaxEnt (Maximum Entropy) algorithm [104,105], a machine learning method, with ENMTools in R [106] using 70% of the dataset points as training. To account for the slight differences in results due to the use of a machine learning algorithm [107], the ecological niche modelling was repeated 10 times. The MaxEnt approach was chosen over presence-absence models (Generalized Linear Models (GLM) or BIOCLIM) because it is based on presence-only data, and can produce reliable results even with a limited number of GPS records [108]. The area under the curve (AUC) was used as the measure of model accuracy, with a value of 0.5 indicating model accuracy not better than random, and a value of 1 indicating perfect model ﬁt [109]. To determine if the ecological niche of each mammal species ﬁts well with that of MPXV, niche overlap comparisons were performed with ENMTools in R [106], using Schoener’s D [110] and Hellinger’s I [111] metrics. The D and I metrics were obtained by comparing the estimated habitat suitability for each grid cell of the ecological niche models. The closer the D and I values are to 1, the more the niches overlap; in contrary, a value of 0 indicates no overlap between niches [111]. Species were then ranked using their D and I values, and re-ranked according to the average of their ranks. 3.2. Ecological Niches of Mammal Species and Overlap with the MPXV Niche As indicated in Table 1, the 14 genera currently detected as MPXV hosts belong to four mammalian orders, and represent 11 families: Eulipotyphla (Erinaceidae and Soricidae), Macroscelidea (Macroscelididae), Primates (Cercopithecidae, Hominidae, and Lorisidae), and Rodentia (Dipodidae, Gliridae, Muridae, Nesomyidae, and Sciuridae). p y In Africa, the 14 genera are represented by 213 species (Table S1), for which we gath- ered occurrence records. In the international databases, we found less than 14 occurrence records for 112 species (52.6%) classiﬁed as narrow-ranged, and only 17 occurrence records for one species classiﬁed as widespread, i.e., Crocidura viara. It was therefore impossible to predict the ecological niche of these 113 species. In total, 100 mammal species had enough occurrence data available, after both cleaning and spatial thinning. However, we obtained the ecological niches for only 99 of these 100 species (Figures 1 and 2; electronic Supplemen- tary Materials, Figure S1). It was indeed impossible to infer the niche of Piliocolobus kirkii because most occurrence records were found on the edge of environmental variables, and were therefore interpreted as missing data. All of the 99 niches inferred for mammals have an AUC > 0.697, and 79% have an AUC > 0.9 (Table S1), indicating an excellent performance of the model. Overall, the variability between the 10 replicates of each niche is very low (Figure S2). Based on niche overlap analyses between MPXV and mammal species, the ﬁrst ten ranked species are Funisciurus anerythrus (Figure 1C), Graphiurus lorraineus (Figure 1D), Funisciurus pyrropus (Figure 1E), Heliosciurus rufobrachium (Figure 1F), Stochomys longicau- datus (Figure 2A), Malacomys longipes (Figure 2B), Pan troglodytes (Figure 2C), Oenomys hypoxanthus (Figure 2D), Crocidura olivieri (Figure 2E), and Crocidura theresae (Figure 2F). The niche overlap with MPXV encompasses both Central and West Africa for all of these species, except M. longipes and O. hypoxanthus, which are mainly present in Central Africa, and C. theresae, which is almost only present in West Africa (Figure 2F). In general, the ecological niches predicted for mammal species differ from the geo- graphic distributions provided by the IUCN (red lines in Figures 1 and 2; see also electronic Supplementary Materials, Figure S1), either by encompassing more regions (e.g., the niche of F. anerythrus also contains a large area covering eastern Liberia, Côte d’Ivoire, Ghana, and Togo; Figure 1C) or fewer regions (e.g., the niche of C. 3.1. Ecological Niche of MPXV Ecological niches of Monkeypox virus (A) and the four mammal species showing the best overlap with it: Funisciurus anerythrus (C), Graphiurus lorraineus (D), Funisciurus pyrropus (E), and Viruses 2023, 15, 727 8 of 17 8 of 17 Heliosciurus rufobrachium (F). Black circles indicate localities used to build the distribution model. The probabilities of occurrence (p) are highlighted using different colours: blue grey for probabilities < 0.5; turquoise green for 0.5 < p < 0.75; yellowish green for 0.75 < p < 0.9; and yellow for p > 0.9. The red line is the IUCN distribution of the species [60]. Indicated at the left of the maps are the number of occurrence records (n) used to infer the ecological niche, and the Schoener’s D and Hellinger’s I values summarizing niche overlap between mammal species and MPXV. For convenience, we have included a map (B) showing the major biogeographic barriers, such as the Dahomey gap, rivers, and Cameroon volcanic line (CVL), and African rainforests (B), including the Upper Guinean forests (UGF) and Lower Guinean forests (LGF) in West Africa, the Atlantic Equatorial coastal forests (AECF) and Congolian lowland forests (CLF) in Central Africa, and the Eastern African coastal forests (EACF) in East Africa. 3.1. Ecological Niche of MPXV The ecological niche of MPXV was predicted using 96 occurrence records with an AUC of 0.956 (0.946–0.964; SD: 0.005). The best probabilities of occurrence (≥0.25) are indicated by different colours, from blue to yellow, passing through green, in Figure 1A. They delineate a large geographic area that encompasses rainforests of West and Central Africa. In addition, three isolated areas receive some support in East Africa, including one region in Ethiopia, another region to the east of Lake Victoria in Kenya, and another area in the Eastern African coastal forest of Tanzania, around Dar es Salam. In West Africa, the geographic distribution is discontinuous, with a gap in southwestern Togo and Benin. In Central Africa, the niche covers all of the Congo Basin, except a large area in eastern Equatorial Africa, including eastern and southern Gabon, and southern Republic of the Congo. The highest probabilities of occurrence (greater than 0.75, in yellowish green to yellow; Figure 1A) are found in four main regions: (i) eastern Sierra Leone, southern Guinea, and Liberia; (ii) southern Nigeria and adjacent western provinces of Cameroon; (iii) the coasts of southern Cameroon and Equatorial Guinea; and (iv) most parts of the Congo Basin in eastern Republic of the Congo, southern CAR, and DRC. 7 of 17 of 18 Viruses 2023, 15, 727 Viruses 2023 15 x FO Figure 1. Ecological niches of Monkeypox virus (A) and the four mammal species showin overlap with it: Funisciurus anerythrus (C), Graphiurus lorraineus (D), Funisciurus pyrropu Heliosciurus rufobrachium (F). Black circles indicate localities used to build the distributi The probabilities of occurrence (p) are highlighted using different colours: blue grey for pr Figure 1. Ecological niches of Monkeypox virus (A) and the four mammal species show overlap with it: Funisciurus anerythrus (C), Graphiurus lorraineus (D), Funisciurus pyrro M k i (A) d h f l i h Figure 1. Ecological niches of Monkeypox virus (A) and the four mammal species showing the best overlap with it: Funisciurus anerythrus (C), Graphiurus lorraineus (D), Funisciurus pyrropus (E), and Heliosciurus rufobrachium (F). Black circles indicate localities used to build the distribution model. The probabilities of occurrence (p) are highlighted using different colours: blue grey for probabilities Figure 1. 4. Discussion 4.1. Ecological Niche of MPXV Fragmented into Three Rainforests 4.1. Ecological Niche of MPXV Fragmented into Three Rainforests 4.1. Ecological Niche of MPXV Fragmented into Three Rainforests The MPXV niche was previously reconstructed in four studies [31,43–45], based on different criteria for the selection of occurrence records: Levine et al. [43] used 156 geo- referenced villages in which at least one human case was detected; Lash et al. [44] used 231 occurrence points corresponding to the 404 cases identiﬁed by the World Health Or- ganization between 1970 and 1986; Nakazawa et al. [31] completed the dataset with four occurrences from the South Sudan outbreak; and Nakazawa et al. [45] divided the same dataset into 25 subsets of about 41 to 43 localities at least 50 km apart (to account for the higher number of occurrences in DRC). While recent mpox cases were added to our dataset, only 96 occurrence points were selected to infer our niche, as, unlike previous studies [31,43–45], we chose to retain only localities of animal cases (6) and human index cases (90). This approach was adopted to focus on human cases with the highest probability of MPXV infection from an animal, and to avoid any bias due to human-to-human trans- mission. Although based on a different occurrence dataset, our results are quite similar to previous ones, since the ﬁve MPXV niches cover, more or less, all rainforests of West and Central Africa. However, it is important to note that our MPXV niche has a fragmented distribution, with at least three (and possibly four) separate rainforest regions (Figure 1B), including in West Africa (i) the Upper Guinean forests (UGF; from Guinea and Sierra Leone through Liberia, Côte d’Ivoire and Ghana to western Togo) and (ii) Lower Guinean forests (LGF; from southeastern Benin through Nigeria to western Cameroon), and in Central Africa (iii) the northern part of Atlantic Equatorial coastal forests (AECF) (from the Sanaga River in southwestern Cameroon to Equatorial Guinea), possibly separated (large area with p < 0.5) from (iv) the Congolian lowland forests (CLF; from southeastern Cameroon and northeastern Gabon through northern Republic of the Congo and CAR to DRC). Such a fragmentation of the niche ﬁts well with previous results [31], but contrasts with several niches showing a division into only two areas, corresponding to UGF, and LGF, AECF, and CLF [43–45]. 3.2. Ecological Niches of Mammal Species and Overlap with the MPXV Niche olivieri does not cover the Sahelian savannah zone; Figure 2E), or a combination of both (e.g., the niche of G. lorraineus also includes the area from Togo to southwestern Nigeria, but does not extend to southeastern DRC; Figure 1D). The niches can also be less fragmented (e.g., the niche of F. pyrropus is not divided into the four IUCN areas, but rather shows a continuum from Guinea to Uganda and southwestern Kenya; Figure 1E) or more fragmented (e.g., the niche of O. hypoxanthus shows an Ethiopian patch isolated from the rest of its distribution in Central Africa; Figure 2D). 9 of 17 of 18 Viruses 2023, 15, 727 Viruses 2023 15 x Figure 2. Ecological niches of mammal species ranked between the 5th and 10th positio overlap with the MPXV niche. Stochomys longicaudatus (A), Malacomys longipes (B), Pan (C), Oenomys hypoxanthus (D), Crocidura olivieri (E), and Crocidura theresae (F). See legend for more details. Figure 2. Ecological niches of mammal species ranked between the 5th and 10th posi overlap with the MPXV niche. Stochomys longicaudatus (A), Malacomys longipes (B), Pan Oenomys hypoxanthus (D), Crocidura olivieri (E), and Crocidura theresae (F). See legend more details. l h f l k d b h h d h Figure 2. Ecological niches of mammal species ranked between the 5th and 10th positions for their overlap with the MPXV niche. Stochomys longicaudatus (A), Malacomys longipes (B), Pan troglodytes (C), Oenomys hypoxanthus (D), Crocidura olivieri (E), and Crocidura theresae (F). See legend of Figure 1 for more details. Figure 2. Ecological niches of mammal species ranked between the 5th and 10th positions for their overlap with the MPXV niche. Stochomys longicaudatus (A), Malacomys longipes (B), Pan troglodytes (C), Oenomys hypoxanthus (D), Crocidura olivieri (E), and Crocidura theresae (F). See legend of Figure 1 for more details. Viruses 2023, 15, 727 10 of 17 10 of 17 4.2. Which Mammal Species Are the Most Probable Reservoir Hosts? Of the 213 species belonging to the 14 mammalian genera potentially involved as reservoir or secondary hosts of MPXV, 113 did not have sufﬁcient occurrences to reconstruct an ecological niche (< 14 for narrow-ranged species and < 25 for widespread species). Among these 113 species, none are distributed in both West and Central Africa, indicating that they cannot be considered as likely reservoir host species for MPXV. Among the 99 mammal species for which it was possible to reconstruct a niche, 49 were only distributed in either West African rainforests (28) or Central African rainforests (21), and 22 were found in both West and Central African rainforests (Table S1). Finally, only seven mammalian niches cover, with high probabilities (greater than 0.75, highlighted in yellowish green to yellow in Figures 1 and 2), the three rainforests corresponding to the Upper Guinean forests (UGF), Lower Guinean forests (LGF), and the Congo Basin, and they were logically found among the top ten classiﬁed mammalian niches for their overlap with the MPXV niche. They are represented by three squirrels (family Sciuridae) of the tribe Protoxerini, i.e., Funisciurus anerythrus (Thomas’s rope squirrel; rank one; Figure 1C), Funisciurus pyrropus (red-legged rope squirrel; rank three; Figure 1E), and Heliosciurus rufobrachium (red-legged sun squirrel; rank four; Figure 1F), one arboreal rodent of the family Gliridae, Graphiurus lorraineus (Lorrain dormouse; rank two; Figure 1D), one ground-living rodent of the family Muridae, Stochomys longicaudatus (target rat; rank ﬁve; Figure 2A), Pan troglodytes (chimpanzee; family Hominidae; rank seven; Figure 2C), and Crocidura olivieri (African giant shrew; family Soricidae; rank nine; Figure 2E). MPXV was detected directly (PCR and DNA sequencing) and/or indirectly (anti-OPXV antibodies) in all of these species, except C. olivieri (Table 1). All seven niches, except that of H. rufobrachium, show signiﬁcant differences with the IUCN range maps. First, some large IUCN areas are missing in several niches, such as northeastern CAR in the niche of F. anerythrus, Gabon and northern Angola in the niche of F. pyrropus, and the Sahelian savannah zone in the niche of C. olivieri. Since the gaps are always in peripheral distribution, the ﬁrst hypothesis is to consider that the species is not present, or rarely, in these areas. As an alternative, we can propose that the IUCN range maps were drawn using some specimens misidentiﬁed at the species level. 4. Discussion diehli), and that from the Middle Pleistocene until today, the Volta River and Da- homey Gap have isolated the western chimpanzee (P. t. verus) from the Nigeria-Cameroon chimpanzee (P. t. ellioti). 4.2. Which Mammal Species Are the Most Probable Reservoir Hosts? 4. Discussion In agreement with our MPXV niche divided into three or four rainforests, phylo- geographic studies on MPXV [45–47] have provided strong support for a sister-group relationship between UGF and LGF viruses, a lineage uniting the viruses from Cameroon and Gabon (AECF), and its grouping with several CLF virus lineages. Taken together, these results suggest that important biogeographic barriers have prevented or limited the dispersal of the MPXV mammalian reservoir in only two regions: (i) between UGF and LGF, where two different barriers may have been involved, the Volta River in eastern Ghana and the Dahomey Gap, a savannah corridor that extends from eastern Ghana through Togo and Benin [112]; and (ii) between LGF and the Congo Basin, where the barrier is obviously the Sanaga River, the largest river in Cameroon. Interestingly, these natural barriers are the distribution limits for a wide diversity of arboreal mammal species [60], including several primates, squirrels, and genets: the Volta River is the eastern limit for the green monkey (Chlorocebus sabaeus), two squirrels (Heliosciurus punctatus and Protoxerus aubinnii), and the pardine genet (Genetta pardina), and the western limit for the large-spotted genet (Genetta maculata); the Dahomey Gap is the eastern limit for the spot-nosed monkey (Cerco- pithecus petaurista) and G. pardina; the Sanaga River is the northern limit for ﬁve primates (Arctocebus aureus, Colobus satanas, Euoticus elegantulus, Mandrillus sphinx, Sciurocheirus gabonensis), the ribboned rope squirrel (Funisciurus lemniscatus), and the servaline genet (Genetta servalina); and the Sanaga River is the southern limit for ﬁve primates (Arctocebus calabarensis, Cercopithecus erythrotis, Cercopithecus mona, Euoticus pallidus, Sciurocheirus al- leni) and the crested genet (Genetta cristata). Several phylogeographic studies, based on DNA sequences, have shown that these barriers can also constitute boundaries between mammal subspecies [113–115]. The most convincing study concerns anthropoid apes, as whole genome data analyses [113] have suggested that from the Middle/Late Pleistocene to present, the Sanaga River has separated two subspecies of chimpanzee (Pan troglodytes troglodytes and P. t. ellioti) and two subspecies of lowland gorilla (Gorilla gorilla gorilla and Viruses 2023, 15, 727 11 of 17 11 of 17 G. g. diehli), and that from the Middle Pleistocene until today, the Volta River and Da- homey Gap have isolated the western chimpanzee (P. t. verus) from the Nigeria-Cameroon chimpanzee (P. t. ellioti). G. g. 4.2. Which Mammal Species Are the Most Probable Reservoir Hosts? This hypothesis can hold for species of the genera Crocidura, Funisciurus, and Graphiurus, as they contain several closely related species that are phenotypically very similar and are therefore concerned by taxonomic issues [60,116]. Second, several niches show important extensions by comparison with the IUCN range maps, either eastward (e.g., P. troglodytes: in Uganda, southwestern Kenya, and northwestern Tanzania), westward (e.g., F. anerythrus and S. longicaudatus: from Liberia through Côte d’Ivoire and Ghana to Togo), or linking separate geographical areas (e.g., F. pyrropus: the three northern IUCN areas form a contin- uum supported by high probabilities). Although high probabilities of occurrence indicate favourable environmental conditions for these species, they can be effectively absent in these suitable areas due to signiﬁcant biogeographic barriers, such as large rivers and savannah corridors, or disadvantageous competition with one or more previously estab- lished species. In agreement with this view, we hypothesize that the current absence of P. troglodytes in East Africa (Uganda, Kenya, Tanzania, and also Ethiopia) is the consequence of competition with other anthropoid species during the Pliocene and Pleistocene epochs. This is corroborated by the discovery of many hominid fossils in East Africa [117], and by the recent disappearance of chimpanzee from southwestern Kenya, where they were still present in the Middle Pleistocene [118]. For poorly known taxa, such as many rodents and shrews, high probabilities of occurrence may indicate that these species are effectively present in these suitable areas, but they have not been considered present by the IUCN due to species misidentiﬁcations. This hypothesis can be advanced for the two similar species Viruses 2023, 15, 727 12 of 17 12 of 17 of Funisciurus, F. anerythrus and F. pyrropus, for which the niches were found to be very different from the IUCN maps. In particular, the niche predicted for F. anerythrus suggests that it could be present in the UGF, whereas its IUCN range map shows that F. anerythrus does not occur west of the Dahomey gap (Figure 1C). Taxonomically, F. anerythrus has often been confused with F. pyrropus: F. anerythrus was ﬁrst described as a subspecies of F. pyrropus [116], and one subspecies of F. pyrropus from Gambia in West Africa, i.e., F. p. mandingo, was placed in F. anerythrus by some taxonomists [119]. Another species, Funisciu- rus substriatus from northeastern Ghana, southeastern Burkina Faso, Togo, and Benin, may be also conspeciﬁc with F. anerythrus [116]. 5. Conclusions Our MPXV niche and recent phylogeographic studies on MPXV [45–47] suggest that two biogeographic barriers have isolated mammalian populations of the MPXV reservoir host: on the one hand, the Volta River and Dahomey Gap between UGF and LGF, and on the other hand, the Sanaga River between LGF and the Congo Basin. Our analyses revealed that the niche of F. anerythrus shows the best overlap (using both D and I metrics) with that of MPXV, suggesting that the Thomas’s rope squirrel could be the main MPXV reservoir. Interestingly, F. anerythrus is the only species from which MPXV was isolated [36,37] and fully sequenced by two independent teams in different provinces of northern DRC [41,45]. In addition, two small fragments of MPXV were ampliﬁed in museum specimens from ﬁve Funisciurus species, all collected in rainforests of the Congo Basin: F. anerythrus (45 out of 362 specimens tested; 12,4%), F. carruthersi (3 of out 109 specimens tested; 2.8%), F. congicus (32 out of 239 specimens tested; 13.4%), F. lemniscatus (5 of out 82 specimens tested; 6.1%), and F. pyrropus (8 of out 201 specimens tested; 4.0%) [58]. These data suggest that the MPXV reservoir could contain not just one species, but several species of Funisciurus. In the Congo Basin, however, F. anerythrus is sympatric with the four other species, including F. pyrropus and the three species endemic to Central Africa, F. carruthersi, F. congicus, and F. lemniscatus. As a consequence, it can be hypothesized that F. anerythrus is indeed the reservoir host species, which can frequently contaminate other arboreal species, such as the squirrels and monkeys listed in Table 1 (secondary hosts), due to regular contacts (direct or indirect) in forest trees. Two complementary studies need to be conducted to further investigate this hypothesis: (i) Funisciurus squirrels caught in future ﬁeld surveys in African forests and those currently housed in museum mammal collections should be systematically tested for the presence of MPXV; and (ii) they should be sequenced for mitochondrial and nuclear genes to compare the phylogeography of F. anerythrus with that already available for MPXV. 4.2. Which Mammal Species Are the Most Probable Reservoir Hosts? To address these taxonomic issues, Funisciurus specimens from museum mammal collections should be examined for morphology (e.g., pelage colour, pattern of stripes, body measurements, skull characteristics) and sequenced for mitochondrial and nuclear genes. Funding: This project was supported by the French Agence Nationale de Recherche (ANR 2019 CE-35) and SCOR Corporate Foundation for Science. References A Human Infection Caused by Monkeypox Virus in Basankusu Territory, Democratic Republic of the Congo. Bull. World Health Organ. 1972, 46, 593–597. [PubMed] 12. Foster, S.O.; Brink, E.W.; Hutchins, D.L.; Pifer, J.M.; Lourie, B.; Moser, C.R.; Cummings, E.C.; Kuteyi, O.E.K.; Eke, R.E.A.; Titus, J.B.; et al. Human Monkeypox. Bull. World Health Organ. 1972, 46, 569–576. [PubMed] 13. Arita, I.; Henderson, D.A. Monkeypox and Whitepox Viruses in West and Central Africa. Bull. World Health Organ. 1976, 53, 347–353. [PubMed] yp p g 347–353. 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Bass, J.; Tack, D.M.; McCollum, A.M.; Kabamba, J.; Pakuta, E.; Malekani, J.; Nguete, B.; Monroe, B.P.; Doty, J.B.; Karhemere, S.; et al. 5. Conclusions Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v15030727/s1, Table S1: Classiﬁcation, habitat preference, and ecological niche data for all selected mammal species; Table S2: Geographic coordinates of the 109 occurrence records used for the MPXV niche; Figure S1: Ecological niches of mammal species ranked between the 11th and 99th positions for their overlap with the MPXV niche; Figure S2: Standard deviation calculated for the ecological niche of MPXV and that of the 10 mammal species showing the best overlap with the MPXV niche. Author Contributions: Conceptualization, A.H.; methodology, A.H. and M.C.; software, M.C.; validation, A.H. and M.C.; formal analysis, M.C.; investigation, A.H. and M.C.; data curation, M.C.; writing—original draft preparation, A.G., A.H. and M.C.; writing—review and editing, A.F., A.G., A.H., C.B., E.N. and M.C.; visualization, A.H. and M.C.; supervision, A.H.; project administration, A.H.; funding acquisition, A.F., A.G., A.H., C.B. and E.N. All authors have read and agreed to the published version of the manuscript. 13 of 17 Viruses 2023, 15, 727 Data Availability Statement: The data presented in this study are available in Table S2. Acknowledgments: We would like to acknowledge Caroline Baillergeau and Mélinée Deretz for administrative support. Conﬂicts of Interest: The authors declare no conﬂict of interest. Data Availability Statement: The data presented in this study are available in Table S2. 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The Rodents of West Africa; Trustees of the British Museum (Natural History): Londo Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W2138897119	https://link.springer.com/content/pdf/10.1007/s10886-012-0097-7.pdf	English	null	Pyrethrins Protect Pyrethrum Leaves Against Attack by Western Flower Thrips, Frankliniella occidentalis	Journal of chemical ecology	2,012	cc-by	6,414	Pyrethrins Protect Pyrethrum Leaves Against Attack by Western Flower Thrips, Frankliniella occidentalis Ting Yang & Geert Stoopen & Gerrie Wiegers & Jing Mao & Caiyun Wang & Marcel Dicke & Maarten A. Jongsma Received: 27 October 2011 /Revised: 2 March 2012 /Accepted: 9 March 2012 /Published online: 29 March 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Pyrethrins are active ingredients extracted from py- rethrum flowers (Tanacetum cinerariifolium), and are the most widely used botanical insecticide. However, several thrips spe- cies are commonly found on pyrethrum flowers in the field, and are the dominant insects found inside the flowers. Up to 80 % of western flower thrips (WFT, Frankliniella occidentalis) adults died within 3 days of initiating feeding on leaves of pyrethrum, leading us to evaluate the role of pyrethrins in the defense of pyrethrum leaves against WFT. The effects of pyr- ethrins on WFT survival, feeding behavior, and reproduction were measured both in vitro and in planta (infiltrated leaves). The lethal concentration value (LC50) for pyrethrins against WFTadults was 12.9 mg/ml, and pyrethrins at 0.1 % (w/v) and 1 % (w/v) had significantly negative effects on feeding, embryo development, and oviposition. About 20-70 % of WFT were killed within 2 days when they were fed chrysanthemum leaves containing 0.01-1 % pyrethrins. Chrysanthemum leaves con- taining 0.1 % or 1 % pyrethrins were significantly deterrent to WFT. In a no-choice assay, the reproduction of WFT was reduced significantly when the insects were fed leaves contain- ing 0.1 % pyrethrins, and no eggs were found in leaves con- taining 1 % pyrethrins. Our results suggest that the natural concentrations of pyrethrins in the leaves may be responsible for the observed high mortality of WFT on pyrethrum. concentrations of pyrethrins in the leaves may be responsible for the observed high mortality of WFT on pyrethrum. Keywords Pyrethrum . Pyrethrins . Western flower thrips . Frankliniella occidentalis . Natural pesticide . toxicity. Tanacetum cinerariifolium . Crop pest J Chem Ecol (2012) 38:370–377 DOI 10.1007/s10886-012-0097-7 J Chem Ecol (2012) 38:370–377 DOI 10.1007/s10886-012-0097-7 T. Yang: G. Stoopen: G. Wiegers: M. A. Jongsma () Plant Research International, Wageningen UR, P.O. Box 619, 6700 AP Wageningen, The Netherlands e-mail: maarten.jongsma@wur.nl Introduction Western flower thrips (WFT), Frankliniella occidentalis, is a highly polyphagous insect that causes both direct and indirect effects on plant development and health. The adults and larvae feed on epidermal and subepidermal cells of both meristematic and mature leaf and flower tissues, inhibiting plant growth and development and causing necrotic or light-reflective blotches on the tissue. Furthermore, they indirectly damage plants by transmitting tospoviruses such as tomato spotted wilt virus (Reitz, 2009). As a result, WFT has become the most serious pest in several vegetable and flower crops world-wide (Daughtrey et al., 1997; Reitz, 2009). The widespread use of chemical insecticides to control WFT has led to increasing resistance against the major classes of synthetic insecticides (Broadbent and Pree, 1997; Flanders et al., 2000; Broughton and Herron, 2009). The growing awareness and demand for insecticides that are not environmentally hazardous has stimu- lated the study of plant-derived compounds for pest control (Boeke et al., 2004). Such compounds could be used as natural pesticides, and in theory, genes responsible for the biosynthesis of those compounds could be isolated and transferred to crops to improve plant defense against WFT (Annadana et al., 2002; Outchkourov et al., 2004). T. Yang: G. Stoopen: G. Wiegers: M. A. Jongsma () Plant Research International, Wageningen UR, P.O. Box 619, 6700 AP Wageningen, The Netherlands e-mail: maarten.jongsma@wur.nl T. Yang: M. Dicke Laboratory of Entomology, Wageningen UR, P.O. Box 8031, 6700 EH Wageningen, The Netherlands J. Mao: C. Wang Key Laboratory for Biology of Horticultural Plants, Ministry of Education, College of Horticulture & Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China Among the sources of botanical pesticides, pyrethrins from pyrethrum plants (Tanacetum cinerariifolium) represent the economically most important class of compounds with broad 371 J Chem Ecol (2012) 38:370–377 usage both in homes and organic agriculture (Casida, 1973). Pyrethrins are neurotoxins that bind to voltage-gated sodium channels of neuronal cells, causing the channels to remain open (Davies et al., 2007). Pyrethrins comprise a group of six closely related esters, named pyrethrin I and II, cinerin I and II, and jasmolin I and II. They are found in all aboveground parts of the pyrethrum plants, but predominantly in the ovaries of the flower heads (Brewer, 1973). On average, the concentra- tion of pyrethrins is about 0.1 % (dry weight) in leaves and 1- 2 % (dry weight) in flowers (Baldwin et al., 1993). Assuming a water content of 90 %, pyrethrins account for around 0.01 % of the fresh weight of leaves and 0.1-0.2 % of the fresh weight of flowers. Pyrethrins are effective against a broad spectrum of insects, while their toxicity for mammals is very low, allowing their use as a preharvest spray (Casida and Quistad, 1995; Schoenig, 1995). Western flower thrips are sensitive to syn- thetic pyrethroids (Thalavaisundaram et al., 2008), but there is no report on the effect of natural pyrethrins against WFT. Pyrethrins might provide pyrethrum with a broad range pro- tection against many different insect pests, but the role of pyrethrins in pyrethrum defense has not been studied. flowering chrysanthemum (Chrysanthemum morifolium Ramat.) cv. Sunny Casa in a greenhouse under a photoperi- od of L16:D8 at 25±2°C. In this study, only adult female thrips were used. The chrysanthemum plants used for bio- assays were from the same cultivar, but were grown in an insect-free compartment of the greenhouse under the same light and temperature conditions. All bioassays were con- ducted in a climate room at 20–22°C with a L16:D8 photo regime. flowering chrysanthemum (Chrysanthemum morifolium Ramat.) cv. Sunny Casa in a greenhouse under a photoperi- od of L16:D8 at 25±2°C. In this study, only adult female thrips were used. The chrysanthemum plants used for bio- assays were from the same cultivar, but were grown in an insect-free compartment of the greenhouse under the same light and temperature conditions. All bioassays were con- ducted in a climate room at 20–22°C with a L16:D8 photo regime. Insecticide Pyrethrum oil (70 % w/w) had been extracted from dried and ground pyrethrum flower heads with liquid CO2 leaving no solvent residue (Honghe Senju Biological Co. Ltd., Yunnan, China). Butylated hydroxytoluene (BHT) had been added to the oil (1 %) to prevent oxidation. We confirmed the concentration and composition of the oil by Gas chromatography–mass spectrometry comparison to a pyrethrin standard (Nguyen et al., 1998). Since the major insecticidal compounds in pyrethrum have long been known as pyrethrins (Casida, 1973), the effect of pyrethrum oil was considered to be the effect of pyrethrins. When calculating the concentrations of pyrethrins in different solutions, the percentage of pyrethrins in the oil (70 %) was taken into account. For example, 1 % (w/v) pyrethrins was prepared by dissolving 14.3 mg pyrethrum oil in 1 ml solvent. In initial experiments, we observed that WFT adults died within one day when fed pyrethrum leaves, but that they were abundant in open flowers. Here, we tested the hypoth- esis that pyrethrins are responsible for protecting pyrethrum leaves against WFT by assessing adult and embryo toxicity, and by examining feeding and oviposition deterrence both in vitro and in planta. In vitro Bioassays-Toxicity Assays The toxicity of pyrethrins was evaluated by topical application to thrips (Robb et al., 1995). Pyrethrum oil was dissolved in acetone to achieve a concentration range of 1 to 30 mg pyrethrins per ml, and the solutions were applied to the thorax with a 10-μl glass syringe at 1 μl per thrips. The droplet briefly covered the thorax of the insect and also the paper support before evap- orating in a few seconds, leaving a residue both on the insect and the support. Acetone alone was used as control. After treatment, all thrips were transferred to Petri dishes contain- ing a piece of chrysanthemum leaf embedded in an agar substrate. Mortality was assayed after 24 h by counting the number of insects that did not respond to prodding with a fine brush. Six replicates were used for each concentration, and 10 thrips were used per replicate. Percent mortality was corrected for mortality observed in acetone control using Schneider-Orelli’s formula (Schneider-Orelli, 1947). Data were analyzed using probit analysis (Finney, 1977). Insects and Plant Material Used in Laboratory Experiments A population of WFT was mass-reared on Methods and Materials The filter papers were drenched in 300 μl of assay solution (water, 0.2 % Tween-80 or pyrethrins at 0.01, 0.1, or 1 % in 0.2 % Tween-80) so that each paper was fully wetted but had no excess solution. After transferring the eggs, the Petri dishes were closed with lids and sealed with Parafilm. The developmental status of eggs was monitored every day for 6 d. To facilitate the observations, the bottoms of the Petri dishes were marked with lines that could be seen through the filter paper from the top, and the eggs were placed on filter paper along these lines. This facilitated finding the eggs under the microscope, and the viability of hatched larvae was assessed in terms of their ability to move away (>0.5 cm) from the hatch position. Four replicates of 10 eggs were used for each assay. Data were analyzed by a one-way ANOVA and mean separation test was conducted using LSD (α00.05). test leaf disks were sprayed with the pyrethrin solutions using a Potter Precision Laboratory spray tower, which produced a uniform deposit (3 μl/cm2) of solution on the leaf disks. After overnight starvation, WFT were anaesthe- tized on ice. Groups of 10 WFT were positioned between a control and a test leaf disk placed abaxial side up and 2 cm apart on a 1.5 % (w/v) agar-bed in a Petri dish (7 cm diam). After positioning the thrips, the Petri dish was covered by a 120 μm mesh size nylon mesh lid to prevent condensation. The number of WFT on each leaf disk was recorded 0.25, 1, 2, 4, 20, and 28 h after the release of the WFT. Each concentration was replicated with 12 leaf disks. At each time point, a Student's paired t-test was used to assess the significance of the differences in the mean number of WFT between test and control. In vitro Bioassays-Oviposition Assays Oviposition-deterrent effects were assayed with a non-choice method slightly modified from Annadana et al. (2002). The assay was con- ducted in Perspex ring cages (3 cm in length and 3.5 cm diam), which were closed with a nylon mesh at the bottom. Pollen of Scotch pine (Pinus sylvestris L.) was supplied in a small open tube as food source for WFT. Methods and Materials Field Observation A pyrethrum field close to Luxi, Yunnan province, China, was used for surveying thrips populations (24°27'10.34"N-103°32'21.01"E). The field was 0.5 ha in size, and the presence of insect species was monitored during the flowering period of spring 2010, when the flowers were predominantly in developmental stages 2–5 [numbered according to Casida (1973)]. To assess populations of small resident insects including thrips, flowers at each developmen- tal stage were collected in each one of 3 blocks of the field. Each flower was taken by the stem and turned upside down into a jar containing 75 % alcohol. Flowers were fully im- mersed and vigorously stirred. The procedure was repeated until each jar contained the insects from 100 flowers from a single block and at a particular stage. The number of insects of each species for each stage was scored. In the case of thrips, the number of adults and larvae were scored separately. Among all collected thrips, 30 were randomly picked and identified, where possible to the species level. In vitro Bioassays-Choice Assays with Topically Applied Pyrethrins A dual-choice leaf disk assay was used to deter- mine the deterrent effect of pyrethrins on WFT. All leaf disks (diam 1.6 cm) were punched from chrysanthemum leaves of similar leaf age. Pyrethrum oil was dissolved in 0.2 % (v/v) aqueous Tween-80 to achieve 3 concentrations of pyrethrins: 0.01, 0.1, and 1 % (w/v). Control leaf disks were sprayed with solvent solution (0.2 % Tween-80), and Insects and Plant Material Used in Laboratory Experiments A population of WFT was mass-reared on J Chem Ecol (2012) 38:370–377 372 Table 1 Frequencies of small insect species living on pyre- thrum flowers in the field Table 1 Frequencies of small insect species living on pyre- thrum flowers in the field aA total of 1200 insects were collected to count the frequen- cies of different insects. b A total of 30 thrips were used to identify species. N.d., not determined Insectsa Frequency (%) Speciesb Frequency (%) Thripidae (thrips) 98 Thrips tabaci 43 Frankliniella occidentalis 25 Thrips flavus 21 Thrips palmi 3 Other species 6 Nysius sp. 1.9 n.d. 1.9 Chrysoperla/Chrysopa sp. (lacewing larva) 0.05 n.d. 0.05 Table 1 Frequencies of small insect species living on pyre- thrum flowers in the field diam). Methods and Materials Error bars indicate SE (N0120 per treatment) infiltrated with 0.2 % Tween-80 were used as control. The assay and data analysis were conducted as described above for the choice assays with topically applied pyrethrins. The number of WFT on each leaf disk was recorded 0.25, 1, 2, 4, 20, and 28 h after the release of the WFT. In planta Bioassays-Reproduction Assays To test the effects of pyrethrins on oviposition and hatching of larvae, WFT were assayed with chrysanthemum leaf disks as described by De Kogel et al. (1997), with slight modifications. Leaf disks were punched from untreated leaves, from leaves infiltrated with 0.2 % Tween-80, or from leaves containing 0.01, 0.1, or 1 % pyrethrins in Tween solution. WFT were placed on leaf disks (1.2 cm diam, 2 WFT/disk), which were embedded, abaxial side up, on agar in wells of 24-well Greiner plates. Plates were covered with Parafilm, and every well was carefully sealed by pressing the Parafilm on the edge of each well. WFT were allowed to oviposit for 48 h and were then removed, with simultaneous assessment of mortality. Subsequently, half of the leaf disks from each plate were used to determine the number of eggs, and the other half of the leaf disks were used to determine the number of hatched larvae. To determine the number of eggs, the leaf disks were boiled in water for 3 min so that the eggs were clearly visible under a binocular microscope with transmitting light. To determine the number of hatched larvae, the leaf disks were transferred to Petri dishes con- taining water and incubated in a climate chamber (25°C, L16:D8) for 5 d to allow the larvae to hatch. The hatched larvae were counted under a binocular microscope. One plate containing 24 identical leaf disks was used for each treatment. Data were analyzed by a one-way ANOVA and mean separation test was conducted using LSD (α00.05). Fig. 2 Dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % Tween (control) or 0.2 % Tween with 0.01 %, 0.1 % or 1 % pyrethrins. The presence on either leaf disk was visually recorded 0.25, 1, 2, 4, 20 and 28 h after WFT release. The x- axis represents 10log-transformed time data. Asterisks indicate signif- icant differences to the control (: P<0.05; : P<0.01). C, control. Pyr, pyrethrins. Methods and Materials After placing 10 WFT in a cage, the top was sealed with two layers of stretched Parafilm, with 300 μl aqueous solution in between the layers. The solutions used were water, 0.2 % Tween-80, or pyrethrins at 0.01, 0.1, or 1 % dissolved in 0.2 % Tween- 80. WFT were allowed to adapt to the diet (pollen and water) for 3 d, and then every day for 5 d fresh test solution was provided. All eggs were deposited in the solutions, and were counted daily under a binocular microscope. Each solution was replicated 6 times. Data were analyzed by a one-way ANOVA and a mean separation test was conducted using LSD (α00.05). In planta Bioassays-Mortality Assays on Pyrethrum Leaves Mature pyrethrum leaves were harvested in November from a field in the Netherlands when they were still flowering (51°59'22.08"N-5°39'44.75"E, Wageningen). Fig. 1 Distribution of thrips adults and larvae across different devel- opemental stages of pyrethrum flowers in the field. Error bars indicate SE (N0300 per stage). Stage 2, vertical ray florets; stage 3, horizontal ray florets and first row of disk florets open; stage 4, 3 rows of disk florets open; stage 5, all disk florets open In vitro Bioassays-Embryo Development Assays Around 200 WFT were kept in a Perspex ring cage (7 cm in length and 9 cm diam) to allow oviposition in a water solution as described above. Eggs laid on the same day were collected with a fine brush under a binocular microscope and then transferred to 2 layers of filter paper in Petri dishes (3.5 cm Fig. 1 Distribution of thrips adults and larvae across different devel- opemental stages of pyrethrum flowers in the field. Error bars indicate SE (N0300 per stage). Stage 2, vertical ray florets; stage 3, horizontal ray florets and first row of disk florets open; stage 4, 3 rows of disk florets open; stage 5, all disk florets open J Chem Ecol (2012) 38:370–377 373 Fig. 2 Dual choice assays of western flower thrips on chrysanthemum leaf disks sprayed with 0.2 % Tween (control) or 0.2 % Tween with 0.01 %, 0.1 % or 1 % pyrethrins. The presence on either leaf disk was visually recorded 0.25, 1, 2, 4, 20 and 28 h after WFT release. The x- axis represents 10log-transformed time data. Asterisks indicate signif- icant differences to the control (: P<0.05; *: P<0.01). C, control. Pyr, pyrethrins. Methods and Materials Error bars indicate SE (N0120 per treatment) Two or three pieces of leaves were placed, abaxial side up, on 1 % (w/v) agar in a Petri dish (7 cm diam). After transferring 10 WFT to each Petri dish, dishes were covered with lids with gauze. Petri dishes with two leaf disks (1.6 cm diam) of chrysanthemum leaves, with a total mass similar to the mass of the pyrethrum leaf samples, or with only agar were used as controls. Six replicates were carried out for each treatment. The mortality of WFT was recorded daily for 3 d. In planta Bioassays-Choice Assays To test the in planta activity of pyrethrins against WFT, pyrethrins were infiltrat- ed into whole chrysanthemum leaves as described by Ratcliff et al. (2001). Leaf disks (diam 1.6 cm) were punched from the infiltrated leaves, avoiding the infiltration points so that WFT would not contact pyrethrins directly except at the edge of the disk. In the initial experiments, we infiltrated only water into the leaves and determined that on average 29.1 mg (± 2.1 mg) water could be infiltrated into each leaf disk (6 replicates). As the fresh weight of each leaf disk was on average 45.3 mg (± 1.2 mg), we infiltrated 0.025, 0.25, or 2.5 % pyrethrins solution to bring the con- centrations to 0.01, 0.1, or 1 % pyrethrins. Leaf disks Results Natural Distribution of Insects in Pyrethrum Fields Our field survey in China showed that several thrips species were the most abundant (98 %) insects on pyrethrum flowers (Table 1). In addition, a few Nysius species (Heteroptera: Fig. 3 The number of eggs deposited by western flower thrips when supplied with different concentrations of pyrethrins starting on Day 1. Data points with the same letter within days are not significantly different, P<0.05. Pyr, pyrethrins. Error bars indicate SE (N060 per treatment) Fig. 3 The number of eggs deposited by western flower thrips when supplied with different concentrations of pyrethrins starting on Day 1. Data points with the same letter within days are not significantly different, P<0.05. Pyr, pyrethrins. Error bars indicate SE (N060 per treatment) J Chem Ecol (2012) 38:370–377 374 ygaeidae) (1.9 %) and lacewing larvae (Neuroptera) (0.05 %) ere found. A total of 30 individuals were identified to pecies level; the thrips species found were mainly Thrips baci (44 %), Frankliniella occidentalis (western flower In vitro Insecticidal and Deterrent Effects To determine the effects of pyrethrins against WFT, pyrethrins were tested in vitro at different concentrations on adult mortality, feeding, oviposition, and embryo development. g. 4 Percentage of larvae atching from western flower rips eggs during incubation ith different concentrations of yrethrins. Data points with the me letter within days are not gnificantly different, P<0.05. yr, pyrethrins. Error bars dicate SE (N040 per eatment) In vitro Insecticidal and Deterrent Effects To determine the effects of pyrethrins against WFT, pyrethrins were tested in vitro at different concentrations on adult mortality, feeding, oviposition, and embryo development. Fig. 4 Percentage of larvae hatching from western flower thrips eggs during incubation with different concentrations of pyrethrins. Data points with the same letter within days are not significantly different, P<0.05. Pyr, pyrethrins. Error bars indicate SE (N040 per treatment) Lygaeidae) (1.9 %) and lacewing larvae (Neuroptera) (0.05 %) were found. A total of 30 individuals were identified to species level; the thrips species found were mainly Thrips tabaci (44 %), Frankliniella occidentalis (western flower thrips, or WFT, 25 %), and Thrips flavus (22 %). The number of thrips in flowers was dependent on the flower’s develop- mental stage (Fig. 1). The number of thrips increased until stage 3 (the first row of disk florets are open), and then decreased in later stages. The thrips found inside flowers were mainly adults. Fig. 5 Effects of pyrethrins on embryo development of western flower thrips at day 5. (a), larva hatched in solutions of water, 0.2 % Tween or 0.01 % pyrethrins at day 5; (b), abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5; (c) and (d), embryos before treatment Results Larvae accounted for 7-26 % of the total number of thrips per flower, depending on the flower devel- opmental stage (Fig. 1). In vitro Insecticidal and Deterrent Effects To determine the effects of pyrethrins against WFT, pyrethrins were tested in vitro at different concentrations on adult mortality, feeding, oviposition, and embryo development. The mortality of WFT female adults increased with the concentration of topically applied pyrethrins in the range of 1 to 30 mg/ml. Probit analysis showed that the LC50 and LC90 of pyrethrins was 12.9 mg/ml (with 95 % confidence limit of 10.9-14.8 mg/ml) and 39.0 mg/ml (with 95 % confidence limit from 30.7 to 50.4 mg/ml), respectively. Thrips were significantly deterred from feeding by 0.1 % and 1 % pyrethrins (Fig. 2). When given a choice between chrysanthemum leaf disks coated with 0.2 % Tween (con- trol) or 0.1 % added pyrethrins, after 2 h significantly more (61-77 % of thrips) settled on control leaf disks. Pyrethrins at 1 % were more highly deterrent. Within 1 h, 72-90 % of thrips chose control leaf disks. For both concentrations of pyrethrins, the maximum deterrent effect was reached at 4 h. Application of 0.01 % pyrethrins on leaf disks did not show significant deterrent effects except at the 4 h time point (Fig. 2). Effect of Pyrethrum Leaves on Mortality of WFT We assayed the suitability of pyrethrum leaves as a food substrate for WFT. Mortality could be as high as 80 % within 3 days, although the degree of mortality depended on the plant source (data not shown). When only water and agar were provided, with no plant-based food, only 20-30 % WFT died in 3 days. All WFT feeding on control chrysanthemum leaves remained alive dur- ing the 3-day-experiment. This showed that the mortality of WFT was caused by a toxic principle of pyrethrum leaves rather than deterrence or starvation. Pyrethrins negatively affected oviposition by WFT (Fig. 3). The carrier, 0.2 % Tween-80, did not affect the oviposition of thrips compared to water throughout the experiment, but WFT oviposited significantly fewer eggs with increasing pyrethrin concentrations during the 5-day experiment (Fig. 3). The toxic principle of pyrethrum plants against insects has long been known to be a group of 6 pyrethrin esters (Casida, 1973). We were, therefore, interested in specifically testing the effect of pyrethrins against WFT. Fig. Discussion In planta Insecticidal and Deterrent Effects To study in planta activity of pyrethrins against WFT, thrips were assayed with chrysanthemum leaves that had been infiltrated with pyrethrins to contain 0.01, 0.1, or 1 % pyrethrins. In this experiment, the pyrethrins could not be contacted di- rectly by thrips except by feeding, and the source of nutri- tion consisted of leaves instead of pollen. Pyrethrins, well-known natural insecticidal compounds, are found exclusively in and extracted from the composite flowers of pyrethrum (Tanacetum cinerariifolium), which belongs to Anthemideae tribe within the Astaraceae family (Casida and Quistad, 1995). Remarkably, the potential role of pyrethrins in pyrethrum plant defense has not been stud- ied. Here, we report that western flower thrips (WFT) adults thrive on pyrethrum flowers, but die within a few days on pyrethrum leaves. The hypothesis that pyrethrins are respon- sible for protecting pyrethrum leaves against WFT was tested by spraying or infiltrating pyrethrins to leaves of chrysanthemum, a related pyrethrins-free species belonging to the same tribe. We assessed toxicity to the adult and In the reproduction assay, thrips fed with chrysanthemum leaf disks containing pyrethrins exhibited higher mortality and lower reproduction rates compared to those fed with untreated leaf disks or leaf disks containing 0.2 % Tween (Table 2). In the dual-choice assay, chrysanthemum leaves containing 0.1 % and 1 % pyrethrins showed significant deterrent effects Fig. 6 Percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % Tween with or without 0.01 %, 0.1 %, or 1 % pyrethrins. The solutions were infiltrated into chrysanthemum leaves. The choices were recorded 0.25, 1, 2, 4, 20, and 28 h after WFT release. The x- axis represents 10log-transformed time data. Asterisks indicate signif- icant differences to the control (: P<0.05; *: P<0.01). C, control. Pyr, pyrethrins. Error bars indicate SE (N0120 per treatment) Fig. 6 Percentage of western flower thrips settled on the control chrysanthemum leaf disk in dual choice assays of leaf disks containing 0.2 % Tween with or without 0.01 %, 0.1 %, or 1 % pyrethrins. The solutions were infiltrated into chrysanthemum leaves. The choices were recorded 0.25, 1, 2, 4, 20, and 28 h after WFT release. The x- axis represents 10log-transformed time data. Asterisks indicate signif- icant differences to the control (: P<0.05; **: P<0.01). C, control. Pyr, pyrethrins. Results 5 Effects of pyrethrins on embryo development of western flower thrips at day 5. (a), larva hatched in solutions of water, 0.2 % Tween or 0.01 % pyrethrins at day 5; (b), abnormally developed embryos in solutions of 0.1 % and 1 % pyrethrins at day 5; (c) and (d), embryos before treatment J Chem Ecol (2012) 38:370–377 375 Table 2 Mortality, number of eggs and hatched western flower thrips larvae per leaf disk on chrysanthemum leaf disks infiltrated with different concentrations of pyrethrins Treatment of leaf disks Mortality (%) Eggs Hatched larvae Untreated leaf disks 0 a 2.0±0.4 a 1.4±0.3 a Leaf disks containing 0.2 % Tween 0 a 1.7±0.5 a 1.3±0.3 ab Leaf disks containing 0.01 % pyrethrins 25.0±6.7 b 1.3±0.3 ab 0.8±0.2 b Leaf disks containing 0.1 % pyrethrins 29.2±7.9 b 0.7±0.2 bc 0.1±0.1 c Leaf disks containing 1 % pyrethrins 68.8±9.3 c 0 c 0 c Values (mean±SE, N048 per treatment) followed by the same letter within a column are not significantly different (ANOVA: P>0.05) on thrips within 15 min of release (Fig. 6). A total of 74-93 % of the thrips settled on the control leaf disk when the other leaf disk contained 0.1 % pyrethrins, and 85-95 % thrips settled on the control leaf disk when the other leaf disk contained 1 % pyrethrins. Chrysanthemum leaves containing 0.01 % pyreth- rins did not show significant deterrent effects. The development of eggs was inhibited by 0.1 % and 1 % pyrethrins. About 80 % of larvae hatched when the eggs were incubated with water, 0.2 % Tween, or 0.01 % pyr- ethrins, while only 28 % or 6 % of the larvae hatched when the eggs were incubated with 0.1 % or 1 % pyrethrins, respectively (Fig. 4). In the latter two treatments, the em- bryos that did not develop into larvae had severely stunted and abnormal shapes (Fig. 5), and dried out after a few days. Discussion Error bars indicate SE (N0120 per treatment) 376 J Chem Ecol (2012) 38:370–377 of pyrethrins is a third component that contributes to their effect for plant defense against WFT (Figs. 4 and 5). embryo stages of WFT, and negative effects on feeding and oviposition both in vitro and in planta, and found that the natural concentrations of pyrethrins present in leaves have strong negative effects on WFT. We speculate that the thrips found on pyrethrum flowers survive on pollen that is devoid of pyrethrins (T. Yang, unpublished data). Compared to some synthetic insecticides, the toxicity of natural pyrethrins against WFT in the absence of synergists was not high. In previous studies using topical application methods, the LC50 values of insecticides tested against susceptible WFT strains ranged from 10 to 83 μg/ml for pyrethroids, 20 to 960 μg/ml for carbamates, and 49 to 522 μg/ml for organophosphates (Espinosa et al., 2005; Robb et al., 1995). The LC50 value of pyrethrins against WFT by topical application was determined as 12.9 mg/ml, and the action of pyrethrins was, therefore, 10 to 1000-fold weaker than for these synthetic pesticides. On the other hand, pyrethrins did show much stronger negative effects on feeding behavior and reproduction, which may be explained by the action of pyrethrins on the nervous system, resulting in disordered function of excitable (nerve and muscle) cells (Bradberry et al., 2005). At 0.01 % (100 μg/ ml), pyrethrins not only caused mortality of adults and embryos, but also significantly reduced oviposition (Table 2). All these factors together cumulatively affect the life history parameters. As a result WFT damage on pyrethrin-containing leaves may be virtually absent, and virus transmission also may be strongly reduced. We hy- pothesize that if plants such as closely related chrysanthe- mum species, which do not contain any pyrethrins, were genetically engineered to produce pyrethrins, their resis- tance to WFT in leaves could be significantly improved. For many populations of WFT, resistance has been reported for some synthetic insecticides (Espinosa et al., 2005). Furthermore, many synthetic insecticides are harmful for human health and the environment. It is relevant, there- fore, to find natural insecticides effective against WFT. Previously, several other plant-derived compounds were tested for their insecticidal effects against WFT adults. Discussion For example, carvacrol at 1 % and thymol at 0.1 % and 1 % significantly reduced the oviposition rate of WFT when these compounds were sprayed on leaf disks, but neither compound affected the feeding activity of WFT (Sedy and Kosehier, 2003). Salicylaldehyde (0.1 % and 1 %) and methyl salicylate (0.1 % and 1 %) were also tested. Within 24 h of applying 1 % methyl salicylate to bean or cucumber, the feeding and oviposition activities of thrips females were significantly reduced (Koschier et al., 2007). The effect on the insect could be a result of changes in the plant induced by methyl salicylate, since it is a plant hormone involved in induced resistance (Pieterse et al., 2009). A series of com- mercially available plant-derived essential oils tested at rec- ommended concentrations (0.02-0.5 %), including neem oil, rosemary oil, peppermint oil, garlic oil, and cottonseed oil, caused less than 30 % mortality within 7 days (Cloyd et al., 2009). Compared to other plant-derived compounds, pyr- ethrins are highly effective against WFT. Our results showed that 0.1 % and 1 % pyrethrin solutions sprayed on leaf disks significantly deterred WFT at 4 h, and topically applied pyrethrins were toxic to adults at an LC50 value of 12.9 mg/ml (1.3 %). By mimicking the natural site of pyrethrin accumulation by infiltration of leaves, we found that 1 % pyrethrins caused 69 % mortality and completely inhibited oviposition. Furthermore, 0.1 % pyrethrins was strongly deterrent and resulted in abortion of 95 % of the embryos, while as little as 0.01 % pyrethrins caused 25 % mortality in 2 days. We propose, therefore, that the natural concentrations of pyrethrin in pyrethrum leaves, around 0.01 % by fresh weight, accounts for the observed high mortality of thrips adults on this plant. Acknowledgements We thank Greet Steenhuis-Broers and Awang Maharijaya for rearing the insects. This research was supported by Technology Top Institute Green Genetics of the Netherlands (grant no. 1C001RP), and by project of Doctoral Fund of Ministry of Education of China (grant no.20100146110027). Director Tang and Wang Yan of Honghe Senju Biology are thanked for their support in the field experi- ments in Luxi, Yunnan, China. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Insecticides have not been reported previously to affect the development of WFT embryos. WFT eggs are embedded in plant tissues (Childers, 1997), and as a result they are unlikely to be affected by non-systemic chemicals that are applied on the surface of plants. However, pyrethrins natu- rally accumulate inside pyrethrum tissues, stored in what appear to be unstructured intercellular cavities (M.A. Jongsma, unpublished observations). 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DDT, pyrethrins, pyrethroids and insect sodium channels. IUBMB Life 59:151–162. SCHNEIDER-ORELLI, O. 1947. Entomologisches Praktikum: Einfüh- rung in die land- und forstwirtschaftliche Insektenkunde. Sauer- länder Aarau, Germany. DE KOGEL, W. J., VAN DER HOEK, M., and MOLLEMA, C. 1997. Oviposition preference of western flower thrips for cucumber leaves from different positions along the plant stem. Entomol. Exp. Appl. 82:283–288. SCHOENIG, G. P. 1995. Mammalian toxicology of Pyrethrum extract, pp. 249–257, in J. E. Casida and G. B. Quistad (eds.), Pyrethrum Flowers Production, Chemistry, Toxicology, and Uses. Oxford University Press, New York. ESPINOSA, P. J., CONTRERAS, J., QUINTO, V., GRÁVALOS, C., FERNÁNDEZ, E., and BIELZA, P. 2005. Metabolic mechanisms of insecticide resistance in the western flower thrips, Frankliniella occidentalis (Pergande). Pest Manag. Sci. 61:1009–1015. SEDY, K. A. and KOSEHIER, E. H. 2003. Bioactivity of carvacrol and thymol against Frankliniella occidentalis and Thrips tabaci. J. Appl. Entomol. 127:313–316. FINNEY, D. J. 1977. Probit Analysis. Cambridge University Press, Cambridge. References THALAVAISUNDARAM, S., HERRON, G. A., CLIFT, A. D., and ROSE, H. 2008. Pyrethroid resistance in Frankliniella occidentalis (Per- gande) (Thysanoptera: Thripidae) and implications for its man- agement in Australia. Aust. J. Entomol. 47:64–69. FLANDERS, K. L., HEINRICHS, E. A. S., FOSTER, J. E. and RICE, M. E. 2000. Maize Insect Pests in North America. Radcliffe's IPM World Textbook. University of Minnesota (last modified 2nd of December,
https://openalex.org/W3090113170	https://leprosyreview.org/admin/public/article_shell/uploads/article_files/Lepra/LEPROSY/91/3/lr2020020/lr2020020.pdf	English	null	Dapsone-induced optic atrophy: a rare case report	Leprosy review	2,020	cc-by	2,123	Submitted 18 August 2020; Accepted 20 August 2020 Submitted 18 August 2020; Accepted 20 August 2020 Summary Dapsone-induced optic atrophy (OA) is very rare at therapeutic doses. A 43-year-old gentleman on WHO-recommended multibacillary Multi-Drug Therapy (WHO MB-MDT) containing rifampicin, dapsone, and clofazimine, presented with bilateral gradual painless diminution of vision after 3 months of medication. He denied any history of trauma, headache, limb weakness, ataxia, diplopia, temporal pain , or weight loss. There was no history of sudden diminution of vision in family members. Fundus examination showed bilateral pale optic discs with well-defined margins. Glucose-6-phosphate dehydrogenase (G6PD) was normal. He was diagnosed with OA secondary to dapsone. There is therefore a possibility of dapsone induced OA even at the therapeutic dose. Hence, we should perform routine eye check-up before starting dapsone. Keywords: Dapsone, optic atrophy, leprosy Keywords: Dapsone, optic atrophy, leprosy Correspondence to: Dr Suchana Marahatta, Associate Professor, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal (Tel.: +977-9862023236; e-mail: dermasuchana@gmail.com; suchanamarahatta@yahoo.com) Lepr Rev (2020) 91, 291–294 Lepr Rev (2020) 91, 291–294 doi:10.47276/lr.91.3.291 © The author(s). This article is Open Access under CC BY 4.0 S. Shresthaa, S. Chaudharyb, H. Giric & S. Marahattad aJunior Resident, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal bAssistant Professor, Department of Ophthalmology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal cJunior Resident, Department of Ophthalmology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal dAssociate Professor, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal S. Shresthaa, S. Chaudharyb, H. Giric & S. Marahattad aJunior Resident, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal bAssistant Professor, Department of Ophthalmology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal cJunior Resident, Department of Ophthalmology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal dAssociate Professor, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal S. Shresthaa, S. Chaudharyb, H. Giric & S. Marahattad aJunior Resident, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal bAssistant Professor, Department of Ophthalmology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal cJunior Resident, Department of Ophthalmology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal dAssociate Professor, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal Case report A 43-year-old gentleman was diagnosed with Borderline Tuberculoid leprosy with normal baseline eye examination. He was started on WHO recommended multibacillary multi-drug therapy for leprosy containing 100mg dapsone daily along with rifampicin and clofazimine. Before starting treatment, a thorough ophthalmic examination was done. The best corrected visual acuity was 6/6 in both the eyes. Corneal sensation was intact, intraocular pressure was normal (14.2mmHg in the right eye and 14.6mmHg in the left eye) and fundus evaluation was within normal limits. After 3 months of treatment, he presented with bilateral sudden onset, progressive diminution of vision. He had controlled diabetes mellitus (DM) and hypertension (HTN) under medication (500mg Metformin and 10mg Amlodipine once daily) for 6 months. He denied intake of large dose of dapsone. He did not give a history of trauma, headache, limb weakness, ataxia, diplopia, temporal pain or weight loss. There was no history of smoking , alcohol consumption or use of other medications besides the above mentioned medications. Family members did not have any history of sudden diminution of vision. On examination, the patient was well-oriented to time, place and person. There was no pallor, icterus, lymphadenopathy, clubbing, cyanosis or edema. His vitals were stable. On ophthalmic examination, the visual acuity was 1/60 and 3/60 in right and left eye, respectively. The patient could not read all the plates while assessing color vision using the Ishihara pseudo-chromatic chart. The eyes were aligned with no conjunctival congestion, clear corneas, normal anterior chamber depth and clear lenses. Intraocular pressure was comparable to baseline. Fundus examination after pupillary dilatation showed pale optic discs with well- defined margins in both eyes (Figure 1). There were no intraretinal hemorrhages, macular edema or retinal detachment. Blood tests revealed normal complete blood count, liver function tests, blood sugar (fasting and post-prandial), lactate dehydrogenase and glucose-6-phosphate dehydrogenase (G6PD). Blood concentration of dapsone was not done due to unavailability. The patient was informed about the disease, its cause and management. Since the patient had already developed OA, no added intervention was done for this. However, the patient was kept under regular follow-up and ophthalmic check-up. Dapsone was withheld from his regular MB- MDT regimen and he is only on rifampicin and clofazimine for leprosy management since last 6 months.11,12 Until 6 months of follow-up, his vision and optic atrophy remained static. Introduction Dapsone has anti-microbial (anti-mycobacterial and anti-protozoal) and anti-inflammatory effects. It is used in the treatment of leprosy, prophylaxis of Pneumocystic jirovecii and toxoplasmosis in AIDS patients, various chronic inflammatory dermatoses and immune- bullous disorders.1,2 Dapsone has no direct toxic effect on the retina at therapeutic doses, nor in case of overdose. However, there are a few reports of dapsone-induced optic neuritis, optic atrophy (OA) and retinal necrosis. There are six case reports of OA following dapsone overdose3–9 and one following therapeutic dose.10 In cases of dapsone overdose, OA was acute Correspondence to: Dr Suchana Marahatta, Associate Professor, Department of Dermatology and Venereology, B.P.Koirala Institute of Health Sciences, Dharan, Nepal (Tel.: +977-9862023236; e-mail: dermasuchana@gmail.com; suchanamarahatta@yahoo.com) 291 © The author(s). This article is Open Access under CC BY 4.0 292 S. Shrestha et al. in onset, occurring within 1 to 2 weeks. In the single case report of OA following a therapeutic dose of dapsone, the manifestation was reported after one month of therapy. We report an interesting case of OA following administration of a therapeutic dose of dapsone. Discussion Dapsone is a synthetic sulfone that has anti-microbial and anti-inflammatory effects. It is used for various dermatological conditions , including leprosy (in combination with rifampicin and clofazimine in WHO MB-MDT), dermatitis herpetiformis, IgA pemphigus, linear IgA dermatosis, bullous pemphigoid, and various neutrophilic dermatoses. Besides these, dapsone is also used for various other non-dermatological conditions. Dapsone is associated with various side effects, including hemolytic anemia, methemoglobin formation, photo-sensitivity, anorexia, abdominal pain, nausea, vomiting and hypersensitivity reaction.2 There are a few case reports of ophthalmic side effects of dapsone, such as macular damage due to ischemia of the capillaries. 293 Dapsone induced optic atrophy: a rare case report Figure 1 Fundus photography taken from 90 diopter lens under slit lamp showing well-defined optic disc margin (a), pale disc with obliteration of cup disc ratio (b) and attenuated vessels (c). Figure 1 Fundus photography taken from 90 diopter lens under slit lamp showing well-defined optic disc margin (a), pale disc with obliteration of cup disc ratio (b) and attenuated vessels (c). It has been shown that intake of dapsone even at therapeutic doses can result in fragmen- tation of red blood cells (RBCs). It has been hypothesized that as retinal capillaries have a small caliber with considerable length, those fragmented RBCs can block them, resulting in ischemic macular damage. However, the exact mechanism is not yet clear.13,14 After a thorough history and clinical evaluation, dapsone was the most probable explanation for optic atrophy in our case. However, HTN and DM might have added the risk of optic neuropathy.15,16 The fundus examination did not reveal changes pertinent to HTN or DM in our patient. Likewise, simultaneous involvement of both optic nerves is less likely in HTN and DM.17 The patient was under regular follow-up at the interval of 1 month and leprosy mediations were provided from the hospital leprosy clinic in a controlled fashion; so, the chance of dapsone overdose is low. Based on these clinical clues and a few published reports, we made the final diagnosis of ‘Dapsone-induced Optic Atrophy’. However, we could not support it further with fluorescein angiography because of its unavailability at our center.i On extensive literature search, we could find only one cross-sectional study on causes of OA from Nepal. The study concluded that glaucoma was the most common cause. References 1 Wozel VEG. Innovative use of dapsone. Dermatol Clin, 2010; 28: 599–610. 1 Wozel VEG. Innovative use of dapsone. Dermatol Clin, 2010; 28: 599–610. 2 Wozel, G., Blasum, C. Dapsone in dermatology and beyond. Arch Dermatol Res 306, 103–124 ( https://doi.org/10.1007/s00403-013-1409-7 2 Wozel, G., Blasum, C. Dapsone in dermatology and beyond. Arch Dermatol Res 306, 103–124 (2014) https://doi.org/10.1007/s00403-013-1409-7 3 3 Homeida M, Babikr A, Daneshmend TK. Dapsone-induced optic atrophy and motor neuropathy. Br M 1980; 281(6249): 1180, [Internet]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC171 3 Homeida M, Babikr A, Daneshmend TK. Dapsone-induced optic atrophy and motor neuropathy. Br Med J, 1980; 281(6249): 1180, [Internet]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1714486/. 4 Stanfield JP. A case of acute poisoning with dapsone. J Trop Med Hyg, 1963; 66: 292–295. 1980; 281(6249): 1180, [Internet]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1714486/. 4 Stanfield JP. A case of acute poisoning with dapsone. J Trop Med Hyg, 1963; 66: 292–295. 4 Stanfield JP. A case of acute poisoning with dapsone. J Trop Med Hyg, 1963; 66: 292–295. 4 Stanfield JP. A case of acute poisoning with dapsone. J Trop Med Hyg, 1963; 66: 292–295. i p g p 5 Cooke TJL. Dapsone poisoning. Med J Aust, 1970; 1(23): 1158–1159, [Internet]. Available from: https://onlin elibrary.wiley.com/doi/abs/10.5694/j.1326-5377.1970.tb84486.x. i 5 Cooke TJL. Dapsone poisoning. Med J Aust, 1970; 1(23): 1158–1159, [Internet]. Available from: https://onlin elibrary.wiley.com/doi/abs/10.5694/j.1326-5377.1970.tb84486.x. y y j 6 Davies R. Fatal poisoning with udolac (diaminodiphenylsulphone). Lancet, 1950; 255(6611): 905–906, net]. Available from: http://www.thelancet.com/article/S0140673650907367/fulltext. 7 Hye Park K, Kim H, Chongseo Lee C, Chul Cha K, Min Park S, Jin Ji H et al. Dapsone intoxication: clinical course and characteristics. Clin Toxicol, 2010; 48(6): 516–521, [Internet]. Available from: http://www.tandfon line.com/doi/full/10.3109/15563650.2010.490534.f 8 Farunfelder FTGJ. In: Farunfelder FTGJ (ed.), Drug-induced ocular side effects. 4th ed, Baltimore: Williams and Wiklins 1996; pp. 57–58. 9 Chakrabarti M, Suresh PN, Namperumalsamy P. Bilateral macular infarction due to diaminodiphenyl sulfone (4,4’ DDS) toxicity. Retina, 1999; 19(1): 83–84, [Internet]. Available from: http://www.ncbi.nlm.nih.gov/pubm ed/10048382. 10 Chalioulias K, Mayer E, Darvay A, Antcliff R. Anterior ischaemic optic neuropathy associated with dapsone, vol. 20, Eye. Nature Publishing Group 2006; pp. 943–945, [Internet]. Available from: https://www.nature.com /articles/6702050. 11 Biswas SK. Chemotherapy of leprosy. J Indian Med Assoc, 2004; 102: 695–698. 12f py p y , ; 12 Sapkota BR, Shrestha K, Pandey B, Walker SL. A retrospective study of the effect of modified multi-drug therapy in Nepali leprosy patients following the development of adverse effects due to dapsone. Discussion Other less frequent causes were trauma, vascular events, intracranial space occupying lesions, papilledema, optic neuritis, alcohol intoxication, myopia, age related macular degeneration and idiopathic.18 There are only a few case reports of dapsone-induced OA and detailed evidence concerning its mechanism is lacking. One study done in 1982 with 7 patients taking therapeutic doses of dapsone with features of hemolysis , evaluated the optic side effects of dapsone, but there was little evidence of ocular damage caused by therapeutic doses of dapsone.19 However, we found a single published case report on dapsone-induced OA at therapeutic doses. As in our case, this patient had pre-existing diabetes (Non-Insulin-Dependent) with possible ocular compromise. However, unlike our patient, he also had some evidence of hemolytic anemia .2 294 S. Shrestha et al. There is a possibility of OA even at therapeutic doses of dapsone, but the mechanism is not well explained, which is also true in cases of dapsone overdose. The risk of developing OA could be increased by other predisposing factors that cause optic ischemia, including old-age, HTN, DM, dyslipidemia and coagulopathy.8 It is advised to have regular ophthalmic check- ups prior to dapsone therapy; and we should be more vigilant in case of patients with added risk factors. References Lepr Rev, 2008; 79(4): 425–428. 13 Kenner DJ, Holt K, Agnello R, Chester GH. Permanent retinal damage following massive dapsone overdose. Br J Ophthalmol, 1980; 64(10): 741–744, [Internet]. Available from: https://europepmc.org/articles/PMC104380 8. 14 Cream JJ, Scott GL. Anaemia in dermatitis herpetiformis: the role of dapsone-induced haemolysis and malabsorption. Br J Dermatol, 1970; 82(4): 333–342, [Internet]. Available from: https://pubmed.ncbi.nlm.ni h.gov/5441766/. g 15 McCulley TJ, Lam BL, Feuer WJ. A comparison of risk factors for postoperative and spontaneous nonarteritic anterior ischemic optic neuropathy. J. Neurooncol, 2005; 25: 22–24. 16 Tsai RK, Liu YT, Su MY. Risk factors of non-arteritic anterior ischemic optic neuropathy (NAION): ocular or systemic. Kaohsiung J Med Sci, 1998; 14(4): 221–225, Available from: https://pubmed.ncbi.nlm.nih.gov/9589 616/. 17 Hayreh SS. Role of nocturnal arterial hypotension in the development of ocular manifestations of systemic arterial hypertension. Curr Opin Ophthalmol, 1999; 10(6): 474–482, [Internet]. Available from: https://pubme d.ncbi.nlm.nih.gov/10662254/. 18 Bajracharya K, Gautam P, Yadav SK, Shrestha N. Epidemiology and causes of optic atrophy in general outp Department of Lumbini Eye Institute. J Univers Coll Med Sci, 2016; 3(2): 26–29. 19 Leonard JN, Tucker WFG, Fry L, Marsh RJ, Ford S. Dapsone and the retina. Lancet, 1982; 319: 453, [Internet]. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0140673682916580.
https://openalex.org/W2129116220	https://iris.unito.it/bitstream/2318/147648/1/Anx-NASH-Hepatol.pdf	English	null	Endogenous annexin A1 is a novel protective determinant in nonalcoholic steatohepatitis in mice	Hepatology	2,014	cc-by	9,624	J y p This work has been supported by grants from the Fondazione Cariplo, Milan, Italy (grant no.: 2011-0470), The Wellcome Trust (Programme 086867/Z/08/Z) and, in part, The William Harvey Research Foundation (London, UK). I.L. Ph.D. training at the Scuola di Alta Formazione of the University of East Piendmont was supported by the Compagnia di San Paolo (Turin, Italy) and partially by the Fondazione Cariplo (NutriAl Network 2010). These authors share senior authorship. Abbeviations: Abs, antibodies; ALT, alanine aminotransferase; ANOVA, analysis of variance; AnxA1; Annexin A1; CCL2, chemokine (C-C motif) ligand 2; CCR2, C-C chemokine receptor type 2; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; FCM, ﬂow cytometry; FPR2/ALX; formyl pep- tide receptor 2/lipoxin A4 receptor; H&E, hematoxilin and eosin; HFD, high-fat diet; hmf, high-magniﬁcation ﬁeld; HSCs, hepatic stellate cells; IgG, immuno- globulin G; IHC, immunohistochemistry; IL, interleukin; iNOS, inducible nitric oxide synthase; IR, insulin resistance; KO, knockout; MCD; methionine-choline deﬁcient; MetS, metabolic syndrome; MGL-1, macrophage galactose N-acetyl-galactosamine-speciﬁc lectin-1; MMP, matrix metalloproteinase; mRNA, messenger RNA; NAFLD; nonalcoholic fatty liver disease; NASH; nonalcoholic steatohepatitis; NO, nitric oxide; p38MAPK, p38 mitogen-activated protein kinase; RT-PCR, real-time polymerase chain reaction; a-SMA; alpha-smooth muscle actin; TGs, triglycerides; TGF-b1, transforming growth factor beta 1; TIMP-1, tissue inhibitor of metalloproteinase-1; TNF-a, tumor necrosis factor alpha; WT, wild type. From the 1Department of Health Sciences and Interdisciplinary Research Center for Autoimmune Diseases, University “Amedeo Avogadro” of East Piedmont, Novara, Italy; 2Cardiovascular Research Institute Maastricht, Department of Biochemistry, Maastricht University, Maastricht, The Netherlands; 3William Harvey Research Institute, Queen Mary University of London, London, United Kingdom; and Departments of 4Clinical and Biological Sciences and 5Medical Sciences, University of Turin, Turin, Italy. Abbeviations: Abs, antibodies; ALT, alanine aminotransferase; ANOVA, analysis of variance; AnxA1; Annexin A1; CCL2, chemokine (C-C motif) ligand 2; CCR2, C-C chemokine receptor type 2; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; FCM, ﬂow cytometry; FPR2/ALX; formyl pep- tide receptor 2/lipoxin A4 receptor; H&E, hematoxilin and eosin; HFD, high-fat diet; hmf, high-magniﬁcation ﬁeld; HSCs, hepatic stellate cells; IgG, immuno- globulin G; IHC, immunohistochemistry; IL, interleukin; iNOS, inducible nitric oxide synthase; IR, insulin resistance; KO, knockout; MCD; methionine-choline deﬁcient; MetS, metabolic syndrome; MGL-1, macrophage galactose N-acetyl-galactosamine-speciﬁc lectin-1; MMP, matrix metalloproteinase; mRNA, messenger RNA; NAFLD; nonalcoholic fatty liver disease; NASH; nonalcoholic steatohepatitis; NO, nitric oxide; p38MAPK, p38 mitogen-activated protein kinase; RT-PCR, real-time polymerase chain reaction; a-SMA; alpha-smooth muscle actin; TGs, triglycerides; TGF-b1, transforming growth factor beta 1; TIMP-1, tissue inhibitor of metalloproteinase-1; TNF-a, tumor necrosis factor alpha; WT, wild type. From the 1Department of Health Sciences and Interdisciplinary Research Center for Autoimmune Diseases, University “Amedeo Avogadro” of East Piedmont, Novara, Italy; 2Cardiovascular Research Institute Maastricht, Department of Biochemistry, Maastricht University, Maastricht, The Netherlands; 3William Harvey Research Institute, Queen Mary University of London, London, United Kingdom; and Departments of 4Clinical and Biological Sciences and 5Medical Sciences, University of Turin, Turin, Italy. Received January 23, 2014; accepted March 19, 2014. These authors share senior authorship. Endogenous Annexin A1 Is a Novel Protective Determinant in Nonalcoholic Steatohepatitis in Mice Irene Locatelli,1 Salvatore Sutti,1 Aastha Jindal,1 Marco Vacchiano,1 Cristina Bozzola,1 Chris Reutelingsperger,2 Dennis Kusters,2 Stefania Bena,3 Maurizio Parola,4 Claudia Paternostro,4 Elisabetta Bugianesi,5 Simon McArthur,3 Emanuele Albano,1* and Mauro Perretti3* HEPATOLOGY, Month 2014 2 of NAFLD to nonalcoholic steatohepatitis (NASH) may involve lipotoxicity caused by increased circulating free fatty acids, oxidative damage, and endoplasmic reticulum stress.2,3 These factors not only cause hepa- tocyte death, but can also stimulate Kupffer cells to secrete inﬂammatory mediators that, in turn, recruit and activate leucocytes within the liver.2,3 Nonetheless, the mechanisms responsible for persistence of hepatic inﬂammation along with those leading to the evolution of NASH to ﬁbrosis are still incompletely character- ized. It is increasingly evident that a failure in the mechanisms responsible for terminating inﬂammatory responses might result in chronic inﬂammation.4 of NAFLD to nonalcoholic steatohepatitis (NASH) may involve lipotoxicity caused by increased circulating free fatty acids, oxidative damage, and endoplasmic reticulum stress.2,3 These factors not only cause hepa- tocyte death, but can also stimulate Kupffer cells to secrete inﬂammatory mediators that, in turn, recruit and activate leucocytes within the liver.2,3 Nonetheless, the mechanisms responsible for persistence of hepatic inﬂammation along with those leading to the evolution of NASH to ﬁbrosis are still incompletely character- ized. It is increasingly evident that a failure in the mechanisms responsible for terminating inﬂammatory responses might result in chronic inﬂammation.4 have investigated the possible role of AnxA1 in the evolution of experimental NASH induced by feeding mice with a methionine-choline deﬁcient (MCD) diet. Material and Methods Animal and Experimental Protocol. Eight-week- old male AnxA1 knockout (AnxA1 KO) mice on a C57BL/6 background and wild-type (WT) animals were purchased from Charles River Laboratories (Charles River UK Ltd., Margate, UK) and fed for 4 or 8 weeks with either MCD or control diets (Labora- torio Dottori Piccioni, Gessate, Italy). In some experi- ments, mice also received a high-fat (35% w/v) liquid diet for 12 weeks (see Supporting Information for details). Experiments were approved by the Italian Ministry of Health and by the university commission for animal care following the criteria of the Italian National Research Council. Resolution of an acute inﬂammation is orchestrated by a variety of protein and autacoids that down- modulate leukocyte recruitment, promote clearance of tissue leucocytes, and switch macrophage phenotype favoring tissue healing.5 Among these proresolving factors, Annexin A1 (AnxA1), also known as lipocortin-1, is receiving increasing attention. AnxA1 is a 37-kDa calcium-phospholipid–binding protein highly expressed in myeloid cells and regulated by glucocorticoids.6 By interacting with its receptor, formyl peptide receptor 2/lipoxin A4 receptor (FPR2/ ALX), AnxA1 down-regulates the production of proinﬂammatory mediators, such as eicosanoids, nitric oxide, and interleukin (IL)-6, reduces neutro- phil migration to inﬂammatory sites, and promotes the clearance of apoptotic granulocytes.6,7 Further- more, recent works suggest that endogenous AnxA1 may orchestrate epithelial repair8,9 and even counter- act the development of lung ﬁbrosis.10 Human Specimen Collection and Analysis. Liver biopsies from 28 consecutive patients with NAFLD or NASH referring to the Division of Gastro-Hepatology of the University of Turin (Turin, Italy) in the period April–November 2011 were analyzed. Specimens were collected at the time of ﬁrst diagnosis and processed for histophatology and extraction of nucleic acid. Patients were characterized by anthropometric, clinical, and biochemical data, and liver biopsies were evaluated for severity of steatohepatitis ﬁbrosis.13 All subjects gave informed consent to the analysis and the study was planned according to the guidelines of the local ethical committee. The major clinical and biochemical parameters are reported in Supporting Table 1. Address reprint requests to: Prof. Emanuele Albano, M.D., Ph.D., Department of Health Science, University “Amedeo Avogadro” of East Piedmont, Via Solaroli 17, 28100 Novara, Italy. E-mail: emanuele.albano@med.unipmn.it; fax: 139 0321 620421. Copyright V C 2014 The Authors. HEPATOLOGY published by Wiley on behalf of the American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited Endogenous Annexin A1 Is a Novel Protective Determinant in Nonalcoholic Steatohepatitis in Mice Irene Locatelli,1 Salvatore Sutti,1 Aastha Jindal,1 Marco Vacchiano,1 Cristina Bozzola,1 Chris Reutelingsperger,2 Dennis Kusters,2 Stefania Bena,3 Maurizio Parola,4 Claudia Paternostro,4 Elisabetta Bugianesi,5 Simon McArthur,3 Emanuele Albano,1* and Mauro Perretti3* Annexin A1 (AnxA1) is an effector of the resolution of inﬂammation and is highly effec- tive in terminating acute inﬂammatory responses. However, its role in chronic settings is less investigated. Because changes in AnxA1 expression within adipose tissue characterize obesity in mice and humans, we queried a possible role for AnxA1 in the pathogenesis of nonalcoholic steatohepatitis (NASH), a disease commonly associated with obesity. NASH was induced in wild-type (WT) and AnxA1 knockout (AnxA1 KO) C57BL/6 mice by feeding a methionine-choline deﬁcient (MCD) diet up to 8 weeks. In MCD-fed WT mice, hepatic AnxA1 increased in parallel with progression of liver injury. This mediator was also detected in liver biopsies from patients with NASH and its degree of expression inversely correlated with the extent of ﬁbrosis. In both humans and rodents, AnxA1 production was selectively localized in liver macrophages. NASH in AnxA1 KO mice was characterized by enhanced lobular inﬂammation resulting from increased mac- rophage recruitment and exacerbation of the M1 phenotype. Consistently, in vitro addi- tion of recombinant AnxA1 to macrophages isolated from NASH livers down-modulated M1 polarization through stimulation of interleukin-10 production. Furthermore, the degree of hepatic ﬁbrosis was enhanced in MCD-fed AnxA1 KO mice, an effect associ- ated with augmented liver production of the proﬁbrotic lectin, galectin-3. Accordingly, AnxA1 addition to isolated hepatic macrophages reduced galectin-3 expression. Conclu- sions: Macrophage-derived AnxA1 plays a functional role in modulating hepatic inﬂam- mation and ﬁbrogenesis during NASH progression, suggesting the possible use of AnxA1 analogs for therapeutic control of this disease. (HEPATOLOGY 2014;00:000-000) N h N onalcoholic fatty liver disease (NAFLD) is regarded as the hepatic feature of so-called metabolic syndrome (MetS) and is becoming the most common form of liver injury worldwide as a result of the diffusion of overweight and obesity.1 In approximately 10%-20% of patients with NAFLD, the disease evolves with the development of hepatocellular damage and lobular inﬂammation, often leading to hepatic ﬁbrosis and cirrhosis.1 Available evidence indi- cates that the mechanisms responsible for the evolution 1 2 LOCATELLI ET AL. Address reprint requests to: Prof. Emanuele Albano, M.D., Ph.D., Department of Health Science, University “Amedeo Avogadro” of East Piedmont, Via Solaroli 17, 28100 Novara, Italy. E-mail: emanuele.albano@med.unipmn.it; fax: 139 0321 620421. Copyright V C 2014 The Authors. HEPATOLOGY published by Wiley on behalf of the American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27141 Potential conflict of interest: Nothing to report. LOCATELLI ET AL. 3 AnxA1-producing cells were detected using a polyclonal anti-AnxA1 rabbit anti- serum (Millipore, Temecula, CA) and phycoerythrin- conjugated anti-rabbit IgG (Sigma-Aldrich). p p Real-Time Polymerase Chain Reaction Analyses. Liver RNA was retrotranscripted with a High Capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems Italia, Monza, Italy). Real-time polymerase chain reaction (RT-PCR) was performed in a Techne TC-312 termalcycler (TecneInc, Burlington, NJ) using TaqMan Gene Expression Master Mix and TaqMan Gene Expression probes for mouse TNF-a, IL- 12p40, IL-23p19, IL-10, chemokine (C-C motif) ligand 2 (CCL2), C-C chemokine receptor type 2 (CCR2), inducible nitric oxide synthase (iNOS), arginase-1, mac- rophage galactose N-acetyl-galactosamine-speciﬁc lectin-1 (MGL-1), AnxA1, Fpr2/3 (orthologs of human FPR2/ ALX),14 galectin-3, a1-procollagen, transforming growth factor beta 1 (TGF-b1), a-SMA, tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase (MMP)-9, MMP-13, and b-actin (Applied Biosystems Italia, Monza, Italy). All samples were run in duplicate, and the relative gene expression calculated as 22DCt is expressed as fold increase over control samples. Human sample analysis was performed using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA), following the manu- facturer’s instructions. The sequence of primers used was: sense, 50-GCAGG CCTGGTTTATTGAAA-30; reverse, 50-GCTGTG CATTGTTTCGCTTA-30. Values were normalized to those of b-actin and are expressed by using the comparative 2DDCt method. Western Blotting. Liver fragments were homoge- nized in ice-cold lysis buffer, as previously reported,13 and protein extracts (100 mg) were electrophoresed on a 10% SDS-polyacrylamide gel. Nitrocellulose mem- branes probed with monoclonal Abs against mouse AnxA1 and galectin-3 were revealed with Western Lightning Chemiluminescence Reagent Plus (ECL; Perkin-Elmer, Boston, MA) using the VersaDoc 3000 quantitative imaging system and Quantity One soft- ware (Bio-Rad). Statistical Analysis. Statistical analyses were per- formed by SPSS statistical software (SPSS, Inc., Chi- cago IL, USA) using the one-way analysis of variance (ANOVA) test with Tukey’s correction for multiple comparisons or Kruskal-Wallis’ test for nonparametric values. Signiﬁcance was taken at the 5% level. Normal- ity distribution was preliminarily assessed by Kolmogorov-Smirnov’s test. LOCATELLI ET AL. 3 LOCATELLI ET AL. 3 LOCATELLI ET AL. 3 Histology and Immunohistochemistry. Lobular inﬂammation was scored blind, according to Kleiner et al.,13 in hematoxilin and eosin (H&E)-stained sec- tions. Necroinﬂammatory foci and apoptotic cells were counted as reported previously.14 Liver macrophages and activated hepatic stellate cells (HSCs) were evidenced in formalin-ﬁxed sections using, respectively, anti-mouse F4/80 or anti-human CD68 (eBioscience, San Diego CA) and a-smooth muscle actin (a-SMA) polyclonal antibodies (Abs; Labvision; Bio-Optica SpA, Milan, Italy) in combination with peroxidase-linked goat anti-rat immunoglobulin G (IgG) or horseradish peroxidase polymer kit (Biocare Medical, Concord, CA). AnxA1, FPR2/ALX, and galectin-3 were detected using speciﬁc Abs from Zymed Laboratories-Invitrogen (Carlsbad, CA), Santa Cruz Biotechnology (Dallas, TX), and R&D Systems (Minneapolis, MN), respectively. Isolation and Puriﬁcation of Liver Macrophages. Macrophages were puriﬁed from livers of either controls or 4-week MCD-fed mice, as previously described.15 Cell purity was above 80%, as determined by ﬂow cytometry (FCM), after immunostaining for CD45 and F4/80. Cells were incubated overnight with AnxA1 (100 nM) in the presence or absence of the p38 mitogen- activated protein kinase (p38MAPK) inhibitor, SB203880 (10 lM; Sigma-Aldrich, Milan, Italy) and processed for messenger RNA (mRNA) extraction, as outlined above. In some experiments, liver nonparenchy- mal cells were separated on a Ficoll density gradient, stained with ﬂuorochrome-conjugated Abs for CD45, F4/80, and IL-10 (eBiosciences, San Diego CA), and analyzed with a FACScalibur (Becton Dickinson, Frank- lin Lakes, NJ) ﬂow cytometer. Unspeciﬁc immunoglobu- lin binding was blocked by incubation with decomplemented mouse serum. AnxA1-producing cells were detected using a polyclonal anti-AnxA1 rabbit anti- serum (Millipore, Temecula, CA) and phycoerythrin- conjugated anti-rabbit IgG (Sigma-Aldrich). Isolation and Puriﬁcation of Liver Macrophages. Macrophages were puriﬁed from livers of either controls or 4-week MCD-fed mice, as previously described.15 Cell purity was above 80%, as determined by ﬂow cytometry (FCM), after immunostaining for CD45 and F4/80. Cells were incubated overnight with AnxA1 (100 nM) in the presence or absence of the p38 mitogen- activated protein kinase (p38MAPK) inhibitor, SB203880 (10 lM; Sigma-Aldrich, Milan, Italy) and processed for messenger RNA (mRNA) extraction, as outlined above. In some experiments, liver nonparenchy- mal cells were separated on a Ficoll density gradient, stained with ﬂuorochrome-conjugated Abs for CD45, F4/80, and IL-10 (eBiosciences, San Diego CA), and analyzed with a FACScalibur (Becton Dickinson, Frank- lin Lakes, NJ) ﬂow cytometer. Unspeciﬁc immunoglobu- lin binding was blocked by incubation with decomplemented mouse serum. Material and Methods Interest for a possible involvement of AnxA1 in the evolution of NAFLD originates from the observation that plasma AnxA1 levels are decreased in obese sub- jects inversely correlating with body mass index as well as with the inﬂammation marker, C-reactive pro- tein,11 whereas an increased AnxA1 expression was observed in adipose tissue of obese mice.12 Further- more, AnxA1 deﬁciency promotes adiposity and insu- lin resistance (IR) in Balb/c mice on a high-fat diet (HFD).12 Because AnxA1-null mice also display inap- propriate experimental inﬂammatory responses,7 we Biochemical Analysis. Plasma alanine aminotrans- ferase (ALT) and liver triglycerides (TGs) were deter- mined by spectrometric kits supplied by Radim S.p.A. (Pomezia, Italy) and Sigma Diagnostics (Milano, Italy), respectively. Circulating tumor necrosis factor alpha (TNF-a) and liver IL-12 levels were evaluated by com- mercial enzyme-linked immunosorbent assay (ELISA) kits supplied by Peprotech (Milano, Italy) and R&D Systems (Abingdon, UK), respectively. Biochemical Analysis. Plasma alanine aminotrans- ferase (ALT) and liver triglycerides (TGs) were deter- mined by spectrometric kits supplied by Radim S.p.A. (Pomezia, Italy) and Sigma Diagnostics (Milano, Italy), respectively. Circulating tumor necrosis factor alpha (TNF-a) and liver IL-12 levels were evaluated by com- mercial enzyme-linked immunosorbent assay (ELISA) kits supplied by Peprotech (Milano, Italy) and R&D Systems (Abingdon, UK), respectively. HEPATOLOGY, Vol. 00, No. 00, 2014 Results The Progression of NASH Is Associated With a Stimulation in Liver AnxA1. AnxA1 was expressed at low extent in livers of na€ıve mice, whereas both mRNA and protein content increased in a time- dependent manner in livers of animals with NASH induced by feeding the MCD diet (Fig. 1). AnxA1 expression in NASH livers was selectively localized in enlarged vacuolized mononucleated cells that were pos- itive for the macrophage marker, F4/80 (Fig. 1). Dou- ble staining of frozen liver sections with an anti-AnxA1 The Progression of NASH Is Associated With a Stimulation in Liver AnxA1. AnxA1 was expressed at low extent in livers of na€ıve mice, whereas both mRNA and protein content increased in a time- dependent manner in livers of animals with NASH induced by feeding the MCD diet (Fig. 1). AnxA1 expression in NASH livers was selectively localized in enlarged vacuolized mononucleated cells that were pos- itive for the macrophage marker, F4/80 (Fig. 1). Dou- ble staining of frozen liver sections with an anti-AnxA1 AnxA1 Recombinant Protein Puriﬁcation. cDNA of human AnxA1 carrying a cleavable N-terminal poly-His tag was expressed in Escherichia coli and puri- ﬁed as previously reported.9 Purity of recombinant AnxA1, as assesed by sodium dodecyl sulfate polyacryl- amide gel electrophoresis and matrix-assisted laser desorption/ionization dual time-of-ﬂight mass spec- trometry, was >95%. LOCATELLI ET AL. 4 HEPATOLOGY, Month 2014 1. Hepatic AnxA1 expression in mice with NASH. Mice were fed an MCD diet over an 8-week period. (A and B) AnxA1 mRNA and protein as measured by RT-PCR and western blotting analyses, respectively, in liver extracts of mice receiving control or MCD diet. Hepatic mRNA e expressed as fold increase over control values after normalization to the b-actin gene. Data are from 8-12 animals per group; boxes the values within 25th and 75th percentile whereas horizontal bars represent the medians The extremities of the vertical bars (10th Fig. 1. Hepatic AnxA1 expression in mice with NASH. Mice were fed an MCD diet over an 8-week period. (A and B) AnxA1 mRNA and protein levels, as measured by RT-PCR and western blotting analyses, respectively, in liver extracts of mice receiving control or MCD diet. Hepatic mRNA data are expressed as fold increase over control values after normalization to the b-actin gene. Data are from 8-12 animals per group; boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. LOCATELLI ET AL. 5 In particular, AnxA1 mRNA was signiﬁcantly lower in subjects with bridging ﬁbrosis, as compared to those with mild/moderate pericentral or -portal ﬁbrosis or without ﬁbrosis (Fig. 2). mice had higher mRNA for the chemokine, CCL2, and its receptor, CCR2, together with an elevated number of hepatic macrophages, when compared to paired WT animals (Fig. 4). g Characterization of AnxA1 Effect on Hepatic Macrophages Functions. To further characterize the molecular and cellular events modulated by AnxA1, hepatic macrophage functions were investigated. In WT mice, NASH progression is characterized by a biphasic change in liver expression of macrophage M1 activation markers, including iNOS, IL-12p40, and IL-23p19. These were elevated after 4 weeks on the MCD diet and declined thereafter (Fig. 4). In these animals, individual AnxA1 mRNAs inversely correlated with those of iNOS (r 5 20.62; P 5 0.01), IL-12p40 (r 5 20.48; P 5 0.03), and IL-23p19 (r 5 20.62; P 5 0.03), pointing to the possible contribution of endogenous AnxA1 in down-regulating macrophage M1 responses during disease progression. Supporting this view, AnxA1 KO mice receiving the MCD diet for 8 weeks did not show any decline in iNOS, IL- 12p40, and IL-23p19 liver mRNAs: These markers were instead approximately 2-3 fold higher than those measured in WT animals (Fig. 4). In the same vein, hepatic IL-12 protein content was signiﬁcantly enhanced in AnxA1-KO mice (Fig. 4). In line with the results obtained in rodents, liver biopsy sections from subjects with NASH showed an increased prevalence of CD681 macrophages producing AnxA1 (Fig. 2). A further evaluation of AnxA1 mRNA levels in 28 liver biopsies from NAFLD/NASH patients evidenced that AnxA1 expression was not related to the degree of IR or severity of liver injury (not shown), but inversely correlated with extension of ﬁbrosis (r5 20.59; P < 0.003). In particular, AnxA1 mRNA was signiﬁcantly lower in subjects with bridging ﬁbrosis, as compared to those with mild/moderate pericentral or -portal ﬁbrosis or without ﬁbrosis (Fig. 2). Recent reports have linked the proresolving properties of AnxA1 with a stimulation of IL-10 production through a p38MAPK signaling path, together with induction of M2 polarization.16,17 FCM analysis of mac- rophages isolated from livers of WT mice fed for 4 weeks with the MCD diet showed that AnxA1-expressing F4/ 801 cells had a higher IL-10 content than those with AnxA1 negatives (Fig. 5). Results The extremities of the vertical bars (10th- 90th percentile) comprise 80% of the values. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. (C and D) Localization of AnxA1 expression by IHC in liver of MCD-fed animals (magniﬁcation, 4003). (E) Detection of macro- phages positive for F4/80 immunostaining (magniﬁcation, 4003). (F) Colocalization of AnxA1 in macrophages containing lipid vacuoles was evi- denced by double staining of frozen liver sections with the lipid dye, Oil Red O (red), and anti-AnxA1 Ab (green immunoﬂuorescence; magniﬁcation, 4003). Cell nuclei were courterstained with 40,6-diamidino-2-phenylindole. Images are representative of 3-4 distinct samples. Fig. 1. Hepatic AnxA1 expression in mice with NASH. Mice were fed an MCD diet over an 8-week period. (A and B) AnxA1 mRNA and protein levels, as measured by RT-PCR and western blotting analyses, respectively, in liver extracts of mice receiving control or MCD diet. Hepatic mRNA data are expressed as fold increase over control values after normalization to the b-actin gene. Data are from 8-12 animals per group; boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the vertical bars (10th- 90th percentile) comprise 80% of the values. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. (C and D) Localization of AnxA1 expression by IHC in liver of MCD-fed animals (magniﬁcation, 4003). (E) Detection of macro- phages positive for F4/80 immunostaining (magniﬁcation, 4003). (F) Colocalization of AnxA1 in macrophages containing lipid vacuoles was evi- denced by double staining of frozen liver sections with the lipid dye, Oil Red O (red), and anti-AnxA1 Ab (green immunoﬂuorescence; magniﬁcation, 4003). Cell nuclei were courterstained with 40,6-diamidino-2-phenylindole. Images are representative of 3-4 distinct samples. HEPATOLOGY, Vol. 00, No. 00, 2014 LOCATELLI ET AL. 5 LOCATELLI ET AL. 5 5 Ab and the lipid dye Oil Red O conﬁrmed that AnxA1-positive macrophages contained lipid droplets (Fig. 1), likely derived from the scavenging of dying fat- laden hepatocytes. Histological analysis indicated that AnxA1-producing cells were more frequent in livers of animals with advanced NASH (8 weeks on the MCD diet) than in those with less-severe steatohepatitis (4 weeks of treatments; 4.7 6 0.5 vs. 2.86 0.8 cells/high- magniﬁcation ﬁeld [hmf]; P 5 0.03). Animals with advanced NASH also showed an increase in hepatic mRNA content of the AnxA1 receptor, Fpr2/3, and presence of Fpr2/3-expressing cells in liver sections (Supporting Fig. 1). Although AnxA1 up-regulation paralleled the severity of NASH, an increased expression of AnxA1 was already evident along with early signs of inﬂammation in livers with only steatosis induced by feeding mice for 12 weeks with an HFD that caused obesity and IR (Supporting Fig. 2). mice had higher mRNA for the chemokine, CCL2, and its receptor, CCR2, together with an elevated number of hepatic macrophages, when compared to paired WT animals (Fig. 4). Ab and the lipid dye Oil Red O conﬁrmed that AnxA1-positive macrophages contained lipid droplets (Fig. 1), likely derived from the scavenging of dying fat- laden hepatocytes. Histological analysis indicated that AnxA1-producing cells were more frequent in livers of animals with advanced NASH (8 weeks on the MCD diet) than in those with less-severe steatohepatitis (4 weeks of treatments; 4.7 6 0.5 vs. 2.86 0.8 cells/high- magniﬁcation ﬁeld [hmf]; P 5 0.03). Animals with advanced NASH also showed an increase in hepatic mRNA content of the AnxA1 receptor, Fpr2/3, and presence of Fpr2/3-expressing cells in liver sections (Supporting Fig. 1). Although AnxA1 up-regulation paralleled the severity of NASH, an increased expression of AnxA1 was already evident along with early signs of inﬂammation in livers with only steatosis induced by feeding mice for 12 weeks with an HFD that caused obesity and IR (Supporting Fig. 2). In line with the results obtained in rodents, liver biopsy sections from subjects with NASH showed an increased prevalence of CD681 macrophages producing AnxA1 (Fig. 2). A further evaluation of AnxA1 mRNA levels in 28 liver biopsies from NAFLD/NASH patients evidenced that AnxA1 expression was not related to the degree of IR or severity of liver injury (not shown), but inversely correlated with extension of ﬁbrosis (r5 20.59; P < 0.003). LOCATELLI ET AL. 5 Furthermore, incubation with AnxA1 of macrophages isolated from NASH livers halved the expression of iNOS and IL-12p40 without affecting that of the M2 markers, arginase-1 and MGL- 1/CD301 (Fig. 5). In AnxA1-treated macrophages, sup- pression of M1 activation was associated with a 2-fold increase in IL-10 mRNA (Fig. 5). Moreover, addition of the p38MAPK inhibitor, SB203880, reverted both AnxA1-induced IL-10 stimulation and down-modulation of iNOS and IL-12p40 (Fig. 5). Interestingly, macro- phage incubation with AnxA1 also signiﬁcantly lowered CCL2 and CCR2 mRNA levels in a p38MAPK- dependent manner (Fig. 5), suggesting the potential capacity of AnxA1 to inﬂuence hepatic monocytic recruitment through CCL2 and CCR2 signalling. p g AnxA1 Deﬁciency Stimulates Hepatic Inﬂamma- tion in Advanced NASH. To further make insights on the role of AnxA1 in the evolution of NASH, AnxA1 KO mice were administered the MCD diet. Livers of AnxA1 KO mice on a control diet displayed normal histological appearance apart from the sporadic presence of monocyte inﬁltration (Fig. 3). Upon feed- ing the MCD diet for either 4 and 8 weeks, liver TGs, transaminase release, and the prevalence of apoptotic cells (4.2 6 1.7 vs. 3.4 6 0.6; P 5 0.33) and necrotic foci (4.5 6 1.4 vs. 3.8 6 1.9; P 5 0.43) in AnxA1 KO and WT mice were not statistically different (Fig. 3). However, already after 4 weeks on MCD diet, semi- quantitative scores of lobular inﬂammation were higher in AnxA1 KO mice (2.2 6 0.4 vs. 1.4 6 0.5; n 5 12; P 5 0.01) and remained elevated by extending the treatment to 8 weeks (2.4 6 0.6 vs. 1.6 6 0.5; n 5 12; P 5 0.03). At this time point, NASH in AnxA1 KO mice was characterized by diffuse inﬂammatory foci containing mononucleated cells and by marked up- regulation in liver and circulating levels of TNF-a (Fig. 3). Accordingly, 8-week MCD-fed AnxA1 KO AnxA1 Deﬁciency Stimulates the Fibrogenic Evo- lution of NASH. The data obtained in the patients with NAFLD/NASH indicate an inverse association between liver expression of AnxA1 and the extension AnxA1 Deﬁciency Stimulates the Fibrogenic Evo- lution of NASH. The data obtained in the patients with NAFLD/NASH indicate an inverse association between liver expression of AnxA1 and the extension 6 LOCATELLI ET AL. HEPATOLOGY, Month 2014 Fig. 2. AnxA1 expression in human livers with or without NASH. LOCATELLI ET AL. 5 AnxA1 detection by IHC in liver specimens from control individuals (A) a SH patients (B). CD68-positive macrophages (from the same NASH patient; C) (magniﬁcation, 4003). Control liver samples refer to surgic ections for hepatic metastasis of colon carcinoma. (D) AnxA1 mRNA was measured in liver biopsies of 28 NASH patients using RT-PCR a malized to that of b-actin. Boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. T emities of the vertical bars (10th-90th percentile) comprise 80% of the values. The extent of liver ﬁbrosis was scored according to Klein al.12 Fig. 2. AnxA1 expression in human livers with or without NASH. AnxA1 detection by IHC in liver specimens from control individuals (A) and NASH patients (B). CD68-positive macrophages (from the same NASH patient; C) (magniﬁcation, 4003). Control liver samples refer to surgical resections for hepatic metastasis of colon carcinoma. (D) AnxA1 mRNA was measured in liver biopsies of 28 NASH patients using RT-PCR and normalized to that of b-actin. Boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the vertical bars (10th-90th percentile) comprise 80% of the values. The extent of liver ﬁbrosis was scored according to Kleiner et al.12 Fig. 2. AnxA1 expression in human livers with or without NASH. AnxA1 detection by IHC in liver specimens from control individuals (A) and NASH patients (B). CD68-positive macrophages (from the same NASH patient; C) (magniﬁcation, 4003). Control liver samples refer to surgical resections for hepatic metastasis of colon carcinoma. (D) AnxA1 mRNA was measured in liver biopsies of 28 NASH patients using RT-PCR and normalized to that of b-actin. Boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the vertical bars (10th-90th percentile) comprise 80% of the values. The extent of liver ﬁbrosis was scored according to Kleiner et al.12 of hepatic ﬁbrosis. By investigating the effects of AnxA1 deﬁciency on the ﬁbrogenic evolution of exper- imental NASH, we observed that the ﬁbrosis markers, pro-collagen-1a, a-SMA, and TIMP-1, were higher in MCD-fed AnxA1 KO mice than in WT animals (Fig. 6). Hepatic collagen deposition, as evidenced by Sirius Red staining, and a-SMA-positive activated HSCs (4.3 6 0.9 vs. 20.3 6 3.8 cells/hmf; P < 0.005) LOCATELLI ET AL. 7 HEPATOLOGY, Vol. 00, No. 00, 2014 3. AnxA1 deﬁciency promotes steatohepatitis in mice with NASH. LOCATELLI ET AL. 5 WT and AnxA1 KO C57BL/6 mice were fed the MCD diet up to 8 weeks. Liver histology was evaluated in H&E-stained sections from control or MCD-fed animals (magniﬁcation, 2003). (E-G) NASH severity was ed by circulating ALT release, hepatic TG content and liver TNF-a mRNA levels, measured by RT-PCR and expressed as fold increase over values after normalization to the b-actin gene. (H) Circulating TNF-a levels were determined by ELISA. Values refer to 6-8 animals per boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the vertical 10th-90th percentile) comprise 80% of the values. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for e comparisons. Fig. 3. AnxA1 deﬁciency promotes steatohepatitis in mice with NASH. WT and AnxA1 KO C57BL/6 mice were fed the MCD diet up to 8 weeks. (A-D) Liver histology was evaluated in H&E-stained sections from control or MCD-fed animals (magniﬁcation, 2003). (E-G) NASH severity was assessed by circulating ALT release, hepatic TG content and liver TNF-a mRNA levels, measured by RT-PCR and expressed as fold increase over control values after normalization to the b-actin gene. (H) Circulating TNF-a levels were determined by ELISA. Values refer to 6-8 animals per group; boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the vertical bars (10th-90th percentile) comprise 80% of the values. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. LOCATELLI ET AL. HEPATOLOGY, Month 2014 4. AnxA1 deﬁciency promotes liver macrophage recruitment and activation. WT and AnxA1 KO C57BL/6 mice were fed the MCD diet eks. (A) Macrophage counts after immunostaining with anti-F4/80 Ab. (B-F) Liver mRNA levels for CCL2, CCR2, and the macrophage n markers, IL-12p40, IL23p19, and iNOS, as measured by RT-PCR. Data are expressed as fold increase over control values after norm o the b-actin gene. (G) Liver IL-12 protein content as determined in the same animals. In all cases, values refer to 8-12 animals nd boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the v (10th-90th percentile) comprise 80% of the values. Statistical differences were assessed by one-way ANOVA test with Tukey’s correc ple comparisons. Fig. 4. AnxA1 deﬁciency promotes liver macrophage recruitment and activation. LOCATELLI ET AL. 5 WT and AnxA1 KO C57BL/6 mice were fed the MCD diet up to 8 weeks. (A) Macrophage counts after immunostaining with anti-F4/80 Ab. (B-F) Liver mRNA levels for CCL2, CCR2, and the macrophage M1 activation markers, IL-12p40, IL23p19, and iNOS, as measured by RT-PCR. Data are expressed as fold increase over control values after normal- ization to the b-actin gene. (G) Liver IL-12 protein content as determined in the same animals. In all cases, values refer to 8-12 animals per group and boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the verti- cal bars (10th-90th percentile) comprise 80% of the values. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. were also more evident in AnxA1 KO mice with NASH. These latter a-SMA-positive HSCs were often surrounded by cell clusters containing F4/80-positive mononucleated cells (Fig. 6). Stimulation of liver ﬁbrosis in AnxA1-KO animals was unrelated to regula- tion of hepatic TGF-b1 or of MMP-9 and MMP-13 (Supporting Fig. 3), suggesting that additional factors may contribute to the proﬁbrogenic evolution of NASH. The beta-galactoside-binding lectin, galectin-3, can direct myoﬁbroblast activation in ﬁbrotic livers and has been associated with the pathogenesis of NASH.18,19 In our experiments, liver galectin-3 expres- sion was up-regulated in WT mice with advanced LOCATELLI ET AL. 9 HEPATOLOGY, Vol. 00, No. 00, 2014 d h h h h d h d Fig. 5. AnxA1 regulates macrophage functions through stimulation of IL-10 production. Hepatic macrophages were isolated from livers of eithe control or 8-week MCD-fed mice. (A) F4/80-positive cells were analyzed by FCM for the coexpression of AnxA1 and IL-10 or MCD-fed mice (B) Macrophages isolated from livers of MCD-fed mice were incubated in vitro with or without recombinant AnxA1 (100 nM) and the p38MAPK nhibitor, SB203880 (SB; 10 lM), and subsequently analyzed for expression of IL-10 mRNA (B). (C and D) RT-PCR analyses for other markers ncluding M1 (iNOS and IL-12p40) and (F and G) M2 activation markers (arginase-1 and MGL-1/CD301), as well as (E and H) CCL2 or CCR2 Values refer to four to ﬁve different cell preparations and are presented as mean 6 standard deviation. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. Fig. 5. AnxA1 regulates macrophage functions through stimulation of IL-10 production. LOCATELLI ET AL. 5 Hepatic macrophages were isolated from livers of either control or 8-week MCD-fed mice. (A) F4/80-positive cells were analyzed by FCM for the coexpression of AnxA1 and IL-10 or MCD-fed mice. (B) Macrophages isolated from livers of MCD-fed mice were incubated in vitro with or without recombinant AnxA1 (100 nM) and the p38MAPK inhibitor, SB203880 (SB; 10 lM), and subsequently analyzed for expression of IL-10 mRNA (B). (C and D) RT-PCR analyses for other markers, including M1 (iNOS and IL-12p40) and (F and G) M2 activation markers (arginase-1 and MGL-1/CD301), as well as (E and H) CCL2 or CCR2. Values refer to four to ﬁve different cell preparations and are presented as mean 6 standard deviation. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. NASH and even more in AnxA1 KO mice which, at the 8-week time point had a hepatic content of galectin-3 2-fold higher than WT mice (Fig. 7). In line with these ex vivo data, in vitro experiments showed that AnxA1-mediated signals effectively down- modulated galectin-3 expression in macrophages LOCATELLI ET AL. 10 HEPATOLOGY, Month 2014 Fig. 6. AnxA1 deﬁciency promotes hepatic ﬁbrosis in mice with NASH. WT and AnxA1 KO C57BL/6 mice were fed the MCD diet for 8 week A-C) Liver mRNA levels for pro-collagen-1a, a-SMA, and TIMP-1, as measured by RT-PCR, and are expressed as fold increase over control valu fter normalization to the b-actin gene. Values refer to 6-8 animals per group and boxes include the values within 25th and 75th percenti hereas horizontal bars represent the medians. The extremities of the vertical bars (10th-90th percentile) comprise 80% of the values. Statistic fferences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. (D and F) Collagen deposition as detecte y Sirius Red staining in representative liver sections from 8-week MCD diet in WT and AnxA1 KO mice. (E and G) Activated HSCs expressi -SMA (magniﬁcation, 4003 and 2003). Enlargement shows a-SMA-positive HSCs surrounded by collagen ﬁbers forming cell foci with mon Fig. 6. AnxA1 deﬁciency promotes hepatic ﬁbrosis in mice with NASH. WT and AnxA1 KO C57BL/6 mice were fed the MCD diet for 8 weeks. (A-C) Liver mRNA levels for pro-collagen-1a, a-SMA, and TIMP-1, as measured by RT-PCR, and are expressed as fold increase over control values after normalization to the b-actin gene. LOCATELLI ET AL. 5 Values refer to 6-8 animals per group and boxes include the values within 25th and 75th percentile, whereas horizontal bars represent the medians. The extremities of the vertical bars (10th-90th percentile) comprise 80% of the values. Statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. (D and F) Collagen deposition as detected by Sirius Red staining in representative liver sections from 8-week MCD diet in WT and AnxA1 KO mice. (E and G) Activated HSCs expressing a-SMA (magniﬁcation, 4003 and 2003). Enlargement shows a-SMA-positive HSCs surrounded by collagen ﬁbers forming cell foci with mono- nucleated cells. (H) These latter were stained by the macrophage marker, F4/80 (magniﬁcation, 6003). HEPATOLOGY, Vol. 00, No. 00, 2014 LOCATELLI ET AL. 11 7. AnxA1 regulates galectin-3 production in livers of mice with NASH. WT and AnxA1 KO C57BL/6 mice were fed the MCD diet (A and B) Galectin-3 mRNA (boxes and whiskers) and protein levels (mean 6 standard deviation) were measured by RT-PCR and we , respectively. Data are from 6-8 mice per group and statistical differences were assessed by one-way ANOVA test with Tukey’s corr tiple comparisons. (C) Macrophages isolated from livers of control (Cont) or 4-week MCD-fed mice were incubated with or w nant AnxA1 (100 nM) and the p38MAPK inhibitor, SB203880 (SB; 10 lM), for 16 hours before quantiﬁcation of galectin-3 m E) Liver galectin-3 expression by IHC in MCD-fed animals (magniﬁcation, 4003). Enlargement shows cell foci containing galectin-3 es and HSCs. g. 7. AnxA1 regulates galectin-3 production in livers of mice with NASH. WT and AnxA1 KO C57BL/6 mice were fed the MCD diet for ks. (A and B) Galectin-3 mRNA (boxes and whiskers) and protein levels (mean 6 standard deviation) were measured by RT-PCR and weste ing, respectively. Data are from 6-8 mice per group and statistical differences were assessed by one-way ANOVA test with Tukey’s correcti multiple comparisons. (C) Macrophages isolated from livers of control (Cont) or 4-week MCD-fed mice were incubated with or witho mbinant AnxA1 (100 nM) and the p38MAPK inhibitor, SB203880 (SB; 10 lM), for 16 hours before quantiﬁcation of galectin-3 mRN Fig. 7. AnxA1 regulates galectin-3 production in livers of mice with NASH. WT and AnxA1 KO C57BL/6 mice were fed the MCD diet for 8 weeks. Discussion Growing evidence points to the importance of AnxA1 in the modulation of anti-inﬂammatory and proresolving responses in rodent models of acute inﬂammation.6,7 We now identify novel modulatory functions of AnxA1 in livers of mice with chronic stea- tohepatitis induced by feeding with an MCD diet. Coupled to the translational data with human NASH, and a series of mechanistic readouts in human and mouse macrophage, we unveil an AnxA1-centerd path- way engaged by the host for liver protection. The homeostatic functions of AnxA1 are mediated by FPR2/ALX, a G-protein-coupled receptor that is shared with other proresolving lipid mediators, includ- ing lipoxin A4 and resolvin D1,23,24 as well as with the proinﬂammatory protein, serum amyloid A, and cathelicidin LL-37.25,26 We observed that in vivo mac- rophage responses to endogenous AnxA1 become appreciable when FPR2/ALX is overexpressed in advaced NASH. Recently, Cooray et al. unveiled an AnxA1-speciﬁc FRP2/ALX proresolving signal pathway involving p38MAPK, MAPKAPK1/2, and heat shock protein 27, leading to generation of IL-10.17 In our experiments with isolated macrophages from NASH livers, down-modulation of iNOS and IL-12p40 is associated with a 2-fold rise in IL-10 expression; importantly, pharmacological inhibition of p38MAPK affects, in opposite ways, AnxA1-induced IL-10 stimu- lation and suppression of M1 polarization. The importance of IL-10 in mediating AnxA1 action is consistent with the observation that an N-terminal AnxA1-derived peptide (Ac1-25) fails to exert anti-inﬂammatory activity in IL-10-deﬁcient mice.27 Collectively, and complementing a recent study,17 these data indicate that AnxA1 can down-regulate macro- phage M1-responses through an FPR2/ALX dimeriza- tion signal centerd on p38 and ultimately leading to IL-10 generation. These new data provide pathophysio- logical relevance to this novel network of resolution, formed by AnxA1/FPR2 and IL-10, and operative in NASH livers to limit and delay disease progression. y g g y p In experimental models of NAFLD/NASH, up- regulation of AnxA1 is already evident in fatty livers and further increases with disease progression speciﬁ- cally involving macrophages containing intracytoplas- matic lipid droplets. The origin of these macrophages has not been characterized in detail, though our pre- liminary data indicate that these cells express, to a low extent, the monocyte marker, Ly6C,20 suggesting that they might derive from inﬂammatory macrophages that had undergone phenotypic changes after scaveng- ing dying fat-laden hepatocytes. LOCATELLI ET AL. 5 (A and B) Galectin-3 mRNA (boxes and whiskers) and protein levels (mean 6 standard deviation) were measured by RT-PCR and western blotting, respectively. Data are from 6-8 mice per group and statistical differences were assessed by one-way ANOVA test with Tukey’s correction for multiple comparisons. (C) Macrophages isolated from livers of control (Cont) or 4-week MCD-fed mice were incubated with or without recombinant AnxA1 (100 nM) and the p38MAPK inhibitor, SB203880 (SB; 10 lM), for 16 hours before quantiﬁcation of galectin-3 mRNA. (D and E) Liver galectin-3 expression by IHC in MCD-fed animals (magniﬁcation, 4003). Enlargement shows cell foci containing galectin-3 mac- rophages and HSCs. LOCATELLI ET AL. 12 HEPATOLOGY, Month 2014 HEPATOLOGY, Month 2014 isolated from NASH livers (Fig. 7). Conversely, addi- tion of AnxA1 to the same macrophage preparations did not affect TGF-b1 mRNA (not shown). At immu- nohistochemistry (IHC), galectin-3 production was particularly evident in the macrophage foci mentioned above (Fig. 7). These galectin-3-positive foci were more frequent in AnxA1 KO livers (2.8 6 0.8 vs. 4.6 6 0.5 cell foci/hmf; P < 0.03). Moreover, in these latter, the presence of HSCs along with the macro- phages is associated with abundant collagen ﬁbers sur- rounding the foci (Fig. 6). weeks of treatment. The mechanisms behind the inﬂammatory phenotype appear related to the direct action of AnxA1 on the macrophages. Indeed, in livers of WT animals with advanced NASH, higher AnxA1 expression is associated with a down-modulation of the macrophage M1 phenotype. Such an effect is absent in AnxA1 KO mice that display instead elevated expression of iNOS, IL-12p40, and IL-23p19 gene products. Moreover, the in vitro addition of recombi- nant AnxA1 reduced M1 marker expression in macro- phages isolated from NASH livers by more than 50%. Altogether, these results suggest that macrophage- derived AnxA1 represents an autocrine/paracrine loop that suppresses, at least in part, proinﬂammatory M1 responses. This new notion is consistent with data indicating a central role for AnxA1 in the down- modulation of TNF-a and IL-6 production by macro- phages exposed to glucocorticoids.6,22 Discussion Here, we report that (1) AnxA1- expressing macrophages are evident in liver biopsies of patients suffering from NAFLD/NASH and (2) AnxA1 mRNA levels in these patients inversely corre- late with the severity of ﬁbrosis. In line with these clinical data, experimental NASH in AnxA1-KO mice is characterized by increased liver ﬁbrosis, suggesting that AnxA1 can prevent the ﬁbrogenic evolution of NASH. Besides the immune-mediated events discussed above, this effect might involve modulation of galectin-3 expression. Galectin-3 is a member of the galectin family, a group of lectins that participates in the regulation of cell adhesion, proliferation and sur- vival, as well as in the modulation of tissue inﬂamma- tion and ﬁbrosis.32 In acutely injured livers, galectin-3 is mainly produced by macrophages and sustains M1 activation in models of acetaminophen- and concanavalin-A-induced hepatitis.33,34 Conversely, in rodent and human livers with ﬁbrosis, galectin-3 is also expressed by activated a-SMA-positive HSCs and regulates their proﬁbrogenic activity.19,35 Recent stud- ies have implicated galectin-3 in the pathogenesis of NASH, although with controversial results, because galectin-3-deﬁcient mice show either protection18 or NASH is increasingly recognized as an important cause for liver ﬁbrosis, and approximately 15% of NASH patients progress to advanced ﬁbrosis/cirrhosis.1 However, the factors responsible for the large interindi- vidual variability in development of ﬁbrosis are still poorly characterized. Here, we report that (1) AnxA1- expressing macrophages are evident in liver biopsies of patients suffering from NAFLD/NASH and (2) AnxA1 mRNA levels in these patients inversely corre- late with the severity of ﬁbrosis. In line with these clinical data, experimental NASH in AnxA1-KO mice is characterized by increased liver ﬁbrosis, suggesting that AnxA1 can prevent the ﬁbrogenic evolution of NASH. Besides the immune-mediated events discussed above, this effect might involve modulation of galectin-3 expression. Galectin-3 is a member of the galectin family, a group of lectins that participates in the regulation of cell adhesion, proliferation and sur- vival, as well as in the modulation of tissue inﬂamma- tion and ﬁbrosis.32 In acutely injured livers, galectin-3 is mainly produced by macrophages and sustains M1 activation in models of acetaminophen- and concanavalin-A-induced hepatitis.33,34 Conversely, in rodent and human livers with ﬁbrosis, galectin-3 is also expressed by activated a-SMA-positive HSCs and regulates their proﬁbrogenic activity.19,35 Recent stud- ies have implicated galectin-3 in the pathogenesis of NASH, although with controversial results, because galectin-3-deﬁcient mice show either protection18 or Discussion Interestingly, macro- phages are also the predominant source of AnxA1 in adipose tissue of obese mice,12 suggesting that, during MetS, AnxA1 up-regulation might be a common response in macrophages, irrespective of their tissue location. In this study, the increase in liver AnxA1 was observed in both an experimental model of NAFLD based on mice feeding with an HFD as well as in NASH induced by the MCD diet. We are aware that the MCD model does not reproduce some of the key features of human diseases, such as obesity and IR. However, it was preferred for characterizing the role of AnxA1 in NASH evolution because it causes extensive steatohepatitis rapidly progressing to ﬁbrosis.21 In line with the homeostatic properties of AnxA1, we observed that AnxA1 deﬁciency promotes lobular inﬂammation in MCD-fed mice, particularly in ani- mals with more advanced NASH, as observed after 8 In addition to modulation of proinﬂammatory activity of macrophages, AnxA1 might also regulate liver recruitment of monocytes by controlling the expression of the CCL2/CCR2 pair. Indeed, CCR2 is HEPATOLOGY, Vol. 00, No. 00, 2014 LOCATELLI ET AL. 13 increased disease severity36,37 in relation to an impaired glucose metabolism and an increased suscep- tibility to obesity and systemic inﬂammation.38,39 In our hands, an increase in liver galectin-3 characterizes advanced NASH in MCD-fed mice and galectin-3 appears susceptible to modulation by AnxA1. More- over, the worsening of ﬁbrosis quantiﬁed in MCD-fed AnxA1 KO mice associates with a further up- regulation in galectin-3 levels. In these latter, hepatic ﬁbrosis is characterized by the diffuse presence of cell clusters composed of galectin-3-expressing macro- phages and activated HSCs, whereas liver collagen deposition is particularly evident around these clusters, supporting a speciﬁc role of galectin-3 in stimulating proﬁbrogenetic cell-to-cell interactions. Consistently, galectin-3 secretion by macrophages has been impli- cated in the promotion of renal and vascular ﬁbro- sis,40,41 whereas genetic deletion or inhibition of galectin-3 attenuates HSC activation and hepatic colla- gen deposition in CCl4 2 or tioacetamide-treated mice19,42 and ameliorated inﬂammation and ﬁbrosis in experimental NASH.43 one of the genes more extensively down-regulated in human monocytes exposed to Ac1-25 AnxA1-derived peptide.28 However, differently from that reported by Lange et al.,28 in our hands, the p38 inhibitor, SB203880, effectively reverted the effect of AnxA1 on CCR2 regulation in mice macrophages. Discussion Thus far, AnxA1 has been implicated in reducing granulocyte- tissue inﬁltration by blocking neutrophil-endothelial interactions and accelerating neutrophil apoptosis.6,7 Neutrophil inﬁltration is not relevant in the pathoge- nesis of NASH, whereas macrophages and lymphocytes are the predominant inﬂammatory cells.29 Thus, our data point to the possibility that, during chronic inﬂammation, AnxA1 might also control the recruit- ment of monocytes. The capacity of AnxA1 to inter- fere with the CCL2/CCR2 axis might be particularly relevant in relation to the evolution of steatohepatitis, because CCR2 deﬁciency and CCR2 antagonism ameliorate liver injury and ﬁbrosis in experimental NASH.30,31 Possibly unrelated to the IL-10 effect dis- cussed above, AnxA1-mediated CCR2 down-regulation provides further mechanistic support to the “protective” role played by this mediator. one of the genes more extensively down-regulated in human monocytes exposed to Ac1-25 AnxA1-derived peptide.28 However, differently from that reported by Lange et al.,28 in our hands, the p38 inhibitor, SB203880, effectively reverted the effect of AnxA1 on CCR2 regulation in mice macrophages. Thus far, AnxA1 has been implicated in reducing granulocyte- tissue inﬁltration by blocking neutrophil-endothelial interactions and accelerating neutrophil apoptosis.6,7 interactions and accelerating neutrophil apoptosis. Neutrophil inﬁltration is not relevant in the pathoge- nesis of NASH, whereas macrophages and lymphocytes are the predominant inﬂammatory cells.29 Thus, our data point to the possibility that, during chronic inﬂammation, AnxA1 might also control the recruit- ment of monocytes. The capacity of AnxA1 to inter- fere with the CCL2/CCR2 axis might be particularly relevant in relation to the evolution of steatohepatitis, because CCR2 deﬁciency and CCR2 antagonism ameliorate liver injury and ﬁbrosis in experimental NASH.30,31 Possibly unrelated to the IL-10 effect dis- cussed above, AnxA1-mediated CCR2 down-regulation provides further mechanistic support to the “protective” role played by this mediator. Altogether, these results afford a novel functional role for AnxA1 in NASH progression, a property mediated through a control of hepatic inﬂammation and ﬁbrogenesis as well as uneven modulation of galectin-3 and IL-10, leading to a reduced macrophage M1 response. It is plausible that strategies aiming at increasing hepatic AnxA1 expression, or the develop- ment of AnxA1 analogs,44 might have a potential for therapeutic control on NASH evolution. NASH is increasingly recognized as an important cause for liver ﬁbrosis, and approximately 15% of NASH patients progress to advanced ﬁbrosis/cirrhosis.1 However, the factors responsible for the large interindi- vidual variability in development of ﬁbrosis are still poorly characterized. References 1. Lazo M, Clark JM. The epidemiology of nonalcoholic fatty liver dis- ease: a global perspective. Semin Liver Dis 2008;28:339-350. 1. Lazo M, Clark JM. The epidemiology of nonalcoholic fatty liver dis- ease: a global perspective. Semin Liver Dis 2008;28:339-350. 2. Marra F, Gastaldelli A, Svegliati-Baroni G, Tell G, Tiribelli C. Molecu- lar basis and mechanisms of progression of non-alcoholic steatohepati- tis. Trends Mol Med 2008;14:72-81. 2. Marra F, Gastaldelli A, Svegliati-Baroni G, Tell G, Tiribelli C. 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https://openalex.org/W2398821347	https://open.library.ubc.ca/media/download/pdf/52383/1.0379270/3	English	null	How Well Do Seniors Estimate Distance to Food? The Accuracy of Older Adults’ Reported Proximity to Local Grocery Stores	Geriatrics	2,019	cc-by	8,804	Geriatrics 2019, 4, 11; doi:10.3390/geriatrics4010011 geriatrics geriatrics geriatrics How Well Do Seniors Estimate Distance to Food? The Accuracy of Older Adults’ Reported Proximity to Local Grocery Stores Benjamin W. Chrisinger 1,,†, Abby C. King 1,2, Jenna Hua 1, Brian E. Saelens 3, Lawrence D. Frank 4, Terry L. Conway 5, Kelli L. Cain 5 and James F. Sallis 5 Benjamin W. Chrisinger 1,,†, Abby C. King 1,2, Jenna Hua 1, Brian E. Saelens 3, Lawrence D. Frank 4, Terry L. Conway 5, Kelli L. Cain 5 and James F. Sallis 5 1 Stanford Prevention Research Center, Stanford University School of Medicine, 1070 Arastradero Road, Suite 300, Palo Alto, CA 94304, USA; king@stanford.edu (A.C.K.); jhua1224@stanford.edu (J.H.) 1 Stanford Prevention Research Center, Stanford University School of Medicine, 1070 Arastradero Road, Suite 300, Palo Alto, CA 94304, USA; king@stanford.edu (A.C.K.); jhua1224@stanford.edu (J.H.) 2 Department of Health Research & Policy (Epidemiology), Stanford University School of Medicine, Stanford, 2 Department of Health Research & Policy (Epidemiology), Stanford University School of Medicine, Stanford CA 94305, USA 3 Department of Pediatrics, Seattle Children’s Research Institute and University of Washington, Seattle, WA 98121, USA; brian.saelens@seattlechildrens.org 4 4 School of Community and Regional Planning, University of British Columbia, Vancouver, BC V6T 1Z2, Canada; ldfrank@ud4h.com 5 Department of Family & Preventive Medicine, University of California, La Jolla, CA 5 Department of Family & Preventive Medicine, University of California, La Jolla, CA 92093, USA; tlconway@ucsd.edu (T.L.C.); kcain@ucsd.edu (K.L.C.); jsallis@ucsd.edu (J.F.S.) * Correspondence: benjamin.chrisinger@spi.ox.ac.uk; Tel.: +44-(0)1865-280333 † Current address: Department of Social Policy and Intervention, University of Oxford, Oxford OX1 2ER, 1. Introduction Built environment characteristics, such as walkability, safety, and food environment, are understood to affect the adoption and maintenance of diet and physical activity behaviors affecting a wide range of chronic disease outcomes [1,2]. Importantly, these relationships appear to endure across the life course. As individuals age, the built environment around where they live supports or deters their ability to maintain healthy behaviors known to help prevent or reduce frailty and other aging-related morbidities [3–6]. Critical to our interpretation of how the built environment affects the health of older adults are the objective built environments where individuals live, work, and play, as well as the attitudes and perceptions individuals hold about these places. Perceived neighborhood environmental factors have been found to be associated with walking in older adult populations [5]. Individuals’ perceptions of safety and other walkability domains are of particular importance to physical activity outcomes [7–9]. For instance, Lee et al. found that perceived but not objective social and environmental variables were signiﬁcantly related to neighborhood satisfaction among a large study of adults in two metropolitan regions in the United States [10]. Meanwhile, a systematic review of neighborhood effects on physical activity among older adults by Yen and colleagues found “fairly consistent” evidence for the associations between both objective and perceived environments and physical activity, but noted that these constructs are likely to be linked to health outcomes in different ways [4]. With this in mind, questions arise for neighborhood-based public health research and practice concerning the accuracy of perceptions of neighborhood environmental domains that could support greater physical activity or the use of health-promoting resources and amenities or, alternatively, present barriers to health-promoting activities through misperception or unawareness of local environmental factors. p g g p p One domain of particular interest is the neighborhood food environment. Several studies have found that individuals’ perceived healthy food access is signiﬁcantly associated with objective physical distance to supermarket retailers [11,12]. One study by Barnes et al. found that living further from the nearest supermarket was associated with a reduction in perceived healthy food access [12]. Other studies have investigated whether food environment perceptions are related to diet [13], including whether they mediate possible dietary changes related to improved healthy food access [14]. Received: 5 November 2018; Accepted: 8 January 2019; Published: 10 January 2019 Recent experience in walking to the closest supermarket, along with personal Geriatrics 2019, 4, 11; doi:10.3390/geriatrics4010011 www.mdpi.com/journal/geriatrics 2 of 15 Geriatrics 2019, 4, 11 safety, represent potentially modifiable perceived environmental factors that were related to older adults’ accuracy of perceptions of their neighborhood food environment. safety, represent potentially modifiable perceived environmental factors that were related to older adults’ accuracy of perceptions of their neighborhood food environment. Keywords: built environment; older adults; food environment; food access; walkability; perception; GIS; neighborhood environment walkability scale Received: 5 November 2018; Accepted: 8 January 2019; Published: 10 January 2019 Received: 5 November 2018; Accepted: 8 January 2019; Published: 10 January 2019 Abstract: (1) Background: Findings from observational studies of relations between neighborhood environments and health outcomes underscore the importance of both objective and perceived experiences of those environments. A clearer understanding of the factors associated with discrepancies between these two assessment approaches is needed to tailor public health interventions to specific populations. This study examined how individual and neighborhood characteristics affect perceptions of supermarket distance, particularly when perceptions do not match objective measures. (2) Methods: Participants were older adults (n = 880) participating in the Senior Neighborhood Quality of Life Study in the Seattle/King County, WA or Baltimore/Washington, DC regions. Two main analyses were conducted. The primary outcome for Analysis I was participants’ geographic information systems (GIS)-based objective network distance to the closest supermarket. Generalized linear mixed models with block group-level random effects were used to assess associations between objective supermarket distance and individual/neighborhood characteristics. The primary outcome for Analysis II was a categorical “accuracy” variable, based on participants’ perceived distance to the nearest supermarket/grocery store relative to the objective distance, assuming a walking speed of 1.0 m/s. Multivariate log-linear models fit neural networks were used to assess influential covariates. (3) Results: Several significant associations with objective distance to the nearest supermarket were observed, including a negative relationship with body mass index (BMI) (95% CI = −45.56, −0.23), having walked to the supermarket in the last 30 days (−174.86, −59.42), living in a high-walkability neighborhood, and residing in Seattle/King County (−707.69, −353.22). In terms of participants’ distance accuracy, 29% were classified as accurate, 33.9% were “Underestimators”, 24.0% “Overestimators”, and 13.2% responded “Don’t Know”. Compared to Accurate participants, Overestimators were significantly less likely to have walked to the supermarket in the last 30 days, and lived objectively closer to a supermarket; Underestimators perceived significantly higher pedestrian safety and lived objectively further from a supermarket; and Don’t Know were more likely to be women, older, not living independently, and not having recently walked to the supermarket. (4) Conclusions: Both modifiable and nonmodifiable factors influence the accuracy of older adults’ perceptions of their proximity to the nearest supermarket. 1. Introduction Among older adults in a rural setting, Sharkey and colleagues found that both objective (e.g., living closer to a supermarket or produce retailers) and perceived food access (e.g., perceived number of grocery retailers nearby, fruit/vegetable variety availability) were related to a higher intake of fresh and processed fruits and vegetables [15]. Caspi et al. found that among residents of low-income housing, perceptions of healthy food access were signiﬁcantly related to fruit and vegetable consumption, though objective distance to supermarkets was not [13]. Furthermore, participants who lived within a kilometer of a supermarket but did not report a supermarket to be within walking distance of home ate signiﬁcantly fewer fruits and vegetables than those who lived similarly close and reported living close to such outlets [13]. Systematic reviews have also reported that the food environment—including the availability of or access to retailers that carry healthy food items [16]—may be a potential moderator of fruit and vegetable consumption for older adults [17]. However, contradictory ﬁndings do exist, where no relationship has been found between the perceived food environment and fruit and vegetable intake among older adults; instead, other factors, such as mobility and self-rated health, were determined 3 of 15 Geriatrics 2019, 4, 11 to be signiﬁcantly related to such intake [18]. Studies have also identiﬁed potential barriers to food shopping for older adults, such as difﬁculties in carrying groceries or ﬁnding items that ﬁt their budgets and preferences [14,19], as well as perceived proximity, route characteristics from home to food retailers [5,20], and self-rated health [21,22]. Thus, even in communities with objectively high walkability, optimal food environments for older adults may have different characteristics than those for the general population [23]. Among seniors, the gap between built environment perception and reality has been noted in at least one study of older adults’ use of and estimated distance to neighborhood resources [24]. Here, the researcher found that public transit use was signiﬁcantly related to both objective and perceived built environment variables, as well as individual characteristics. Importantly, transit use appeared to increase participants’ perceived distance to the nearest transit stop or station (e.g., seniors who were transit users overestimated how far they lived from a transit stop, while non-users underestimated it) [24]. 1. Introduction Given the potential adverse effects or lost opportunities resulting from inaccurate neighborhood perceptions, as well as the possibly exacerbating effects of limited mobility among older adults, a better understanding of what informs and characterizes the accuracy or inaccuracy of older adults’ neighborhood perceptions is warranted. Study Objectives A combination of objective and perceived data was used to answer two questions about food environment perceptions among older adults participating in the observational Senior Neighborhood Quality of Life Study (SNQLS): (1) Which individual sociodemographic and built environment characteristics were associated with objective proximity to a particular food environment resource?; and (2) Which individual sociodemographic and built environment characteristics predicted the degree to which an individual accurately perceived the distance to this resource? In this study, as in previous research, supermarkets were assessed as the primary point of food access given their size, recognizability, and year-round provisioning of fresh fruits and vegetables [12,13,15]. 2.1. Study Overview Participants in the observational multi-site senior neighborhood quality of life study (SNQLS, n = 883) were ages 66 years and older, able to walk at least 10 ft with or without assistive devices, able to complete study surveys in English, and lived in either the Seattle/King County, WA region or the Baltimore/Washington, DC region of the US. [25]. Between 2005 and 2008, SNQLS investigators purposively sampled from census tracts in Seattle/King County, WA and Baltimore/Washington, DC that would allow comparisons across different levels of neighborhood income and walkability. A neighborhood “quadrant” categorical variable was generated to balance participant recruitment across this research design: Low-walkability, low-income; low-walkability, high-income; high-walkability, low-income; and high-walkability, high-income. Similarly, a categorical variable for site (Seattle/King County, WA or Baltimore/Washington, DC) was also generated. Participant recruitment/consent and SNQLS methods are described in detail elsewhere [25,26]. 2.3. Accuracy of Distance to Supermarket Perception A categorical variable for the accuracy of perceived distance to the nearest supermarket was created in three steps. First, we estimated the time required to walk the actual network distances to the nearest supermarket for each participant, assuming a 1.0 m/s walking pace for older adults [31]. For example, a participant who lived 1.0 km from the nearest supermarket would have been assigned an estimated walk time of 16.7 min. This estimated walk time was then used to assign an objective distance category corresponding to the NEWS-A survey: 1–5 min, 6–10 min, 11–20 min, 21–30 min, and greater than 30 min. Finally, estimated walk times were compared to perceived walk times to assign participants one of three classiﬁcations: “Accurate” if participants’ estimated walk times fell within their selected perceived walk time category, “Overestimate” if their perceived walk time was greater than their estimated walk time, and “Underestimate” if their perceived walk time was less than their estimated walk time. A separate category was assigned to participants who selected “Don’t Know” for their perceived supermarket distance in the NEWS-A. To evaluate how the results might change based on assumptions of walking paces other than the 1.0 m/s pace, sensitivity analyses were conducted by constructing distance accuracy variables with slightly faster (1.2 m/s) and slower (0.8 m/s) assumed walking paces, using the same procedure as described in the Materials and Methods section. A second sensitivity analysis was also performed using a reduced dataset only including participants without any of three potential mobility constraints that could substantially slow a participant’s walking speed (use of a cane or walker, low comfort walking four blocks (self-reported score of <6 on a 10-point scale, low to high comfort), and not living independently). 2.2.1. Survey Measures Individual neighborhood perceptions were assessed by selected items from the abbreviated Neighborhood Environment Walkability Survey (NEWS-A) [27], a validated and abbreviated version of the original 98-question NEWS instrument [28,29], and included domains of aesthetics, trafﬁc safety, infrastructure for walking and bicycling, personal safety, and pedestrian safety [27]. For each of these domains, participants assessed their neighborhood by responding to statements (e.g., “There are trees 4 of 15 Geriatrics 2019, 4, 11 along the streets in my neighborhood”) on a 4-point Likert scale from “Strongly Disagree” to “Strongly Agree”. Participants also reported the walking time to the nearest supermarket by selecting one of six response categories: 1–5 min, 6–10 min, 11–20 min, 21–30 min, more than 30 min, or “Don’t Know”. p g Derived from participant surveys described in King et al. [25], dichotomous variables were created to control for potentially inﬂuential variables: Self-reported gender (female or male), race/ethnicity (non-Hispanic white or not), use of a cane/walker (yes or no), having ﬁnished at least some higher education (yes or no), dog ownership (yes or no), holding a valid driver’s license (yes or no), living independently (i.e., not residing in a group facility, yes or no), and having walked to the perceived nearest supermarket in the past 30 days (yes or no). Continuous variables included participant age, body mass index (BMI), household size, length of residence at current address, number of vehicles available at home, and objective network distance to the nearest supermarket. Ordinal variables included comfort walking (10-point Likert, with 1 = no conﬁdence and 10 = complete conﬁdence) [30], and NEWS-A component scores of aesthetics, pedestrian safety, personal safety, trafﬁc safety, and walking/cycling facilities [27]. 2.2.2. Geographic Information Systems (GIS) Measures Geographic information systems (GIS) were used to calculate objective network distance from participants’ home addresses to a variety of neighborhood business locations contained in a proprietary Dun & Bradstreet, Inc./Hoovers database. Four-digit standard industrial classiﬁcation (SIC) codes were used to group destinations, including grocery stores and supermarkets. 2.4. Statistical Procedures Continuous and ordinal variables were centered and scaled with the caret package in R, which subtracts a variable’s mean from each of its values and divides by the standard deviation [32,33]. Descriptive statistics were generated to summarize participant and neighborhood characteristics both by perceived closeness to a supermarket and accuracy of this perception. For Analysis I (variables related to objective distance to the nearest supermarket), a series of linear mixed models were generated (using the lmer function of the lme4 package) in R (Version 3.5.1) [34]. Four models were ﬁt: (1) A null model with only neighborhood perceptions and block-group random effect; (2–3) two partial models 5 of 15 Geriatrics 2019, 4, 11 with individual, objective, and perceived neighborhood characteristics; and (4) a full model adjusting for all covariates described in the previous section. Akaike information criterion (AIC) values were used to evaluate improvement between models, and p-values were calculated based on Satterthwaite’s approximations [35]. For Analysis II (variables related to the accuracy of participants’ perceptions regarding perceived supermarket access), we compared covariates of participants who accurately reported their access to those of overestimators, under-estimators, and participants who reported not knowing the distance to the nearest supermarket) [36]. A series of four multivariate log-linear models were ﬁt via neural networks (using the multinom function of the nnet package in R): (1) A null model that only included neighborhood perceptions, (2–3) two partial models with individual and objective neighborhood covariates, and (4) a full model adjusting for all potential covariates described above [37,38]. AIC values were calculated to assess improvement between the null and full models. Likelihood ratio tests were used to assess overall variation between accuracy categories within covariates, and z-tests were used identify signiﬁcant covariates within categories [39]. Output tables were generated using the sjPlot and stargazer R packages [40,41]. 3.1. Study Population Most participants reported that the nearest supermarket was within a 5, 10, or 20-min walk (47.0%), while 40.0% perceived living further than a 20-min walk, and 13.2% provided a “Don’t Know” response. Averages and counts of sociodemographic and built environment variables by distance perception category are fully reported in Table 1 and were aggregated for ease of presentation. Table 1. Individual and neighborhood characteristics of participant population, by perceived walking time to closest supermarket retailer. NEWS: Neighborhood environment walkability survey, BMI: Body mass index. Table 1. Individual and neighborhood characteristics of participant population, by perceived walking time to closest supermarket retailer. NEWS: Neighborhood environment walkability survey, BMI: Body mass index. <20 min Walk ≥20 min Walk Don’t Know Counts (%) (n = 413) (n = 351) (n = 116) Gender Male 206 (0.50) 152 (0.43) 27 (0.23) Female 207 (0.50) 199 (0.57) 89 (0.77) Race Not white 119 (0.29) 101 (0.29) 42 (0.37) White (not Hispanic) 292 (0.71) 249 (0.71) 73 (0.63) Lives independently Yes 361 (0.87) 286 (0.81) 69 (0.59) No 52 (0.13) 65 (0.19) 47 (0.41) Has a valid driver’s license Yes 368 (0.89) 310 (0.89) 91 (0.78) No 45 (0.11) 40 (0.11) 25 (0.22) Uses a cane or walker Yes 35 (0.08) 37 (0.11) 34 (0.29) No 377 (0.92) 314 (0.89) 82 (0.71) Quadrant 1. Low-Walkability/Low-Income 62 (0.15) 89 (0.25) 32 (0.28) 2. Low-Walkability/High-Income 67 (0.16) 128 (0.36) 32 (0.28) 3. High-Walkability/Low-Income 137 (0.33) 83 (0.24) 36 (0.31) 4. High-Walkability/High-Income 147 (0.36) 51 (0.15) 16 (0.14) 6 of 15 Geriatrics 2019, 4, 11 Table 1. Cont. 3.1. Study Population <20 min Walk ≥20 min Walk Don’t Know <20 min Walk ≥20 min Walk Don’t Know Site Baltimore/Washington, DC 198 (0.48) 172 (0.49) 57 (0.49) Seattle/King County, WA 215 (0.52) 179 (0.51) 59 (0.51) ≥1 vehicle available Yes 366 (0.89) 310 (0.89) 88 (0.76) No 47 (0.11) 40 (0.11) 28 (0.24) Dog owner Yes 57 (0.14) 48 (0.14) 18 (0.16) No 356 (0.86) 302 (0.86) 98 (0.84) Walked to supermarket in last 30 days Yes 227 (0.55) 307 (0.87) 116 (1.00) No 185 (0.45) 44 (0.13) 0 (0.00) Mean (SD) Age (y) 74.74 (6.78) 75.17 (6.5) 78.02 (7.28) BMI 26.11 (4.31) 26.5 (4.95) 27.6 (5.87) Household size (persons) 1.75 (0.68) 1.74 (0.79) 1.47 (0.75) Can walk 4 blocks (scale 1–10) 8.84 (2.53) 7.93 (3.22) 6.26 (3.9) Time at current address (months) 249.57 (189.81) 274.53 (189.84) 209.16 (191.4) NEWS Aesthetic score 3.19 (0.63) 3.1 (0.7) 3 (0.73) NEWS Trafﬁc safety score 2.76 (0.66) 2.71 (0.7) 2.66 (0.76) NEWS Pedestrian safety score 2.73 (0.42) 2.6 (0.45) 2.5 (0.51) NEWS Personal safety score 3.42 (0.56) 3.36 (0.64) 3.2 (0.77) NEWS Walk/cycle facilities score 2.89 (0.75) 2.7 (0.9) 2.53 (0.87) Objective distance to supermarket (ft) 2696 (1947) 4719 (3019) 4630 (2724) .2. Analysis I: Associations with Objective Supermarket Distance 3.2. Analysis I: Associations with Objective Supermarket Distance Participant characteristics, perceptions, and behaviors were signiﬁcantly related to increasing objective supermarket distance: Lower BMI (β = −22.89, p = 0.048), more favorable self-reported neighborhood aesthetics (β = 39.02, p = 0.005), longer time lived at the current address (β = 35.29, p = 0.015), living independently (β = 181.49, p = 0.046), and having walked to the nearest supermarket within the last 30 days (β = −117.14, p < 0.001). Additionally, the quadrant in which a participant resided was signiﬁcantly related to objective distance to a supermarket (further distance in the low-walkability/high-income quadrant and shorter distance in the high-walkability/low-income and high-walkability/high-income quadrants, compared to the low-walkability, low-income quadrant) and their residence in Seattle/King County, WA versus Baltimore/Washington, DC was negatively related to objective distance (all p < 0.001). Table 2 shows the results of the ﬁnal model, and Figure 1 represents variable coefﬁcients and conﬁdence intervals in a forest plot. Table 2. Fully-adjusted mixed model with block group random effect: Individual and neighborhood correlates of actual distance to nearest grocery store. Table 2. 3.1. Study Population Fully-adjusted mixed model with block group random effect: Individual and neighborhood correlates of actual distance to nearest grocery store. Outcome: Actual Distance to Grocery Store (in Meters) Predictors Beta CI p (Intercept) 1416.86 1180.01–1653.72 <0.001 Age (y) −14.45 −39.04–10.14 0.250 Race/Ethnicity: white, non-Hispanic 22.42 −38.37–83.21 0.470 Gender: Female −43.29 −88.77–2.20 0.063 BMI −22.89 −45.56–−0.23 0.048 Household size −2.67 −25.94–20.60 0.822 Has dog 19.13 −41.45–79.72 0.536 Outcome: Actual Distance to Grocery Store (in Meters) 7 of 15 Geriatrics 2019, 4, 11 Table 2. Cont. Outcome: Actual Distance to Grocery Store (in Meters) Predictors Beta CI p Time at current address (months) 35.29 6.97–63.60 0.015 Uses cane or walker 37.68 −30.85–106.22 0.282 Comfort walking 4 blocks1 2.95 −23.08–28.99 0.824 Has driver’s license −22.26 −103.82–59.31 0.593 Has ≥1 vehicle available 13.41 −76.92–103.74 0.771 Lives independently 181.49 3.12–359.87 0.046 NEWS: Aesthetics 39.02 12.14–65.89 0.005 NEWS: Pedestrian Safety −10.52 −37.42–16.38 0.444 NEWS: Personal Safety 17.57 −9.00–44.15 0.195 NEWS: Trafﬁc Safety 12.29 −12.79–37.36 0.337 NEWS: Walking/Cycling Facilities −15.68 −46.40–15.05 0.318 Walked to nearest grocery, last 30 days −117.14 −174.86–−59.42 <0.001 Quadrant: Low-Walk/High-Inc2 534.04 299.05–769.02 <0.001 Quadrant: High-Walk/Low-Inc2 −409.30 −608.16–−210.45 <0.001 Quadrant: High-Walk/High-Inc2 −489.56 −737.33–−241.78 <0.001 Site: Seattle/King County, WA3 −530.46 −707.69–−353.22 <0.001 Observations 868 R2/Ω02 0.926/0.925 AIC 12600.926 1 Comfort walking 4 blocks (10-pt Likert scale); 2 Compared to Quadrant 1 (Low-Walkability, Low-Income); 3 Compared to Baltimore/Washington DC. AIC: Akaike information criterion. Outcome: Actual Distance to Grocery Store (in Meters) 1 Comfort walking 4 blocks (10-pt Likert scale); 2 Compared to Quadrant 1 (Low-Walkability, Low-Income); 3 Compared to Baltimore/Washington DC. AIC: Akaike information criterion. 1 Comfort walking 4 blocks (10-pt Likert scale); 2 Compared to Quadrant 1 (Low-Walkability, Low-Income); 3 Compared to Baltimore/Washington DC. AIC: Akaike information criterion. Figure 1. Coefﬁcient estimates and conﬁdence intervals for ﬁxed effects predicting an individual’s actual distance to nearest grocery store. Note: Signiﬁcance levels indicated by: * p < 0.001; p < 0.01; * p < 0.05. Figure 1. Coefﬁcient estimates and conﬁdence intervals for ﬁxed effects predicting an individual’s actual distance to nearest grocery store. Note: Signiﬁcance levels indicated by: * p < 0.001; p < 0.01; * p < 0.05. Figure 1. Coefﬁcient estimates and conﬁdence intervals for ﬁxed effects predicting an individual’s actual distance to nearest grocery store. Note: Signiﬁcance levels indicated by: * p < 0.001; p < 0.01; * p < 0.05. Figure 1. 3.1. Study Population Coefﬁcient estimates and conﬁdence intervals for ﬁxed effects predicting an individual’s actual distance to nearest grocery store. Note: Signiﬁcance levels indicated by: * p < 0.001; p < 0.01; * p < 0.05. Geriatrics 2019, 4, 11 8 of 15 3.3. Analysis II: Accuracy of Perceived Supermarket Access in Relation to Objective Access 3.3. Analysis II: Accuracy of Perceived Supermarket Access in Relation to Objective Access Following the investigation of inﬂuential individual and neighborhood factors correlated with actual distance to the nearest supermarket, we investigated further the putative reasons underlying individuals’ misperceptions of supermarket access. Assuming (based on previous literature [31]) that a speed of one meter per second is reasonably representative of an older adult’s walking pace, we compared perceived walk time with objectively-measured distance between participants’ homes and the nearest supermarket. As Figure 2 shows, the majority of the study sample misjudged their physical food access by either underestimating the distance (e.g., perceived living closer to a supermarket than they actually did; 33.9%), overestimating (e.g., perceived living further from a supermarket than they actually did; 24.0%) or responding “Don’t Know” (13.2%). Only 29.0 percent of participants accurately reported the walking time to the nearest supermarket (n = 255). Table 3 provides a summary of signiﬁcant differences in sociodemographic characteristics by these accuracy classiﬁcations. Output tables for partial models from Analyses I and II are available as Supplemental Material (Supplemental Tables S1 and S2, respectively). Geriatrics 2018, 3, x FOR PEER REVIEW 8 of 16 individuals’ misperceptions of supermarket access. Assuming (based on previous literature [31]) that a speed of one meter per second is reasonably representative of an older adult’s walking pace, we compared perceived walk time with objectively-measured distance between participants’ homes and the nearest supermarket. As Figure 2 shows, the majority of the study sample misjudged their physical food access by either underestimating the distance (e.g., perceived living closer to a supermarket than they actually did; 33.9%), overestimating (e.g., perceived living further from a supermarket than they actually did; 24.0%) or responding “Don’t Know” (13.2%). Only 29.0 percent of participants accurately reported the walking time to the nearest supermarket (n = 255). Table 3 provides a summary of significant differences in sociodemographic characteristics by these accuracy classifications. Output tables for partial models from Analyses I and II are available as Supplemental Material (Supplemental Tables S1 and S2, respectively). Figure 2. 3.1. Study Population Boxplot of objective distance to the nearest grocery store and perceived walking time. Note: Distance presented as square root for ease of presentation, and lines with markers demarcate the Figure 2. Boxplot of objective distance to the nearest grocery store and perceived walking time. Note: Distance presented as square root for ease of presentation, and lines with markers demarcate the possible distances traveled by walking for a given time at pace of 1 m/s. Figure 2. Boxplot of objective distance to the nearest grocery store and perceived walking time. Note: Distance presented as square root for ease of presentation and lines with markers demarcate the Figure 2. Boxplot of objective distance to the nearest grocery store and perceived walking time. Note: Distance presented as square root for ease of presentation, and lines with markers demarcate the possible distances traveled by walking for a given time at pace of 1 m/s. Figure 2. Boxplot of objective distance to the nearest grocery store and perceived walking time. Note: Distance presented as square root for ease of presentation and lines with markers demarcate the Figure 2. Boxplot of objective distance to the nearest grocery store and perceived walking time. Note: Distance presented as square root for ease of presentation, and lines with markers demarcate the possible distances traveled by walking for a given time at pace of 1 m/s. possible distances traveled by walking for a given time at pace of 1 m/s. Table 3. Likelihood ratio tests for coefficients in the fully-adjusted multinomial model comparing participants categorized by accuracy of perceptions regarding distance to nearest supermarket. Likelihood Ratio Chi- Square p-Value Age (y) 9.74 0.02* Race/Ethnicity: White, non-Hispanic 4.01 0.26 Gender: Female 8.91 0.03* BMI 3.33 0.34 Household size 1.78 0.62 Has dog 3.21 0.36 The unadjusted model comparing the accuracy of perception to NEWS-A measures found signiﬁcant relationships between inaccurate distance perceptions and perceived personal safety: Lower personal safety scores were associated with both overestimated and “Don’t Know” responses. A higher perceived availability of walking and bicycling facilities was associated with overestimating distance, and underestimation was associated with higher perceived pedestrian safety (see Figure 3). In the fully-adjusted model, compared to participants with accurate perceptions of supermarket access, an “Underestimator” status was signiﬁcantly and positively related to perceived neighborhood pedestrian safety (p = 0.04) and distance to the nearest retailer (p = 0.003) (see Figure 4). 3.1. Study Population Compared to the “Accurate” group, an “Overestimator” status was signiﬁcantly and negatively related to having walked to the supermarket in the last 30 days (p = 0.01), and negatively related to objective distance (p < 0.001), compared to the accurate group. Finally, compared to the accurate group, a “Don’t Know” status was signiﬁcantly and positively associated with age (p = 0.01) and identifying as a woman 9 of 15 Geriatrics 2019, 4, 11 (p = 0.03), and negatively associated with living independently (p = 0.04). Likelihood ratio tests for coefﬁcients in the fully-adjusted multinomial model are reported in Table 3. A complete summary of coefﬁcients, conﬁdence intervals, and p-values is provided in Supplemental Table S2. (p = 0.03), and negatively associated with living independently (p = 0.04). Likelihood ratio tests for coefﬁcients in the fully-adjusted multinomial model are reported in Table 3. A complete summary of coefﬁcients, conﬁdence intervals, and p-values is provided in Supplemental Table S2. Table 3. Likelihood ratio tests for coefﬁcients in the fully-adjusted multinomial model comparing participants categorized by accuracy of perceptions regarding distance to nearest supermarket. Table 3. Likelihood ratio tests for coefﬁcients in the fully-adjusted multinomial model comparing participants categorized by accuracy of perceptions regarding distance to nearest supermarket. Likelihood Ratio Chi-Square p-Value Age (y) 9.74 0.02 * Race/Ethnicity: White, non-Hispanic 4.01 0.26 Gender: Female 8.91 0.03 * BMI 3.33 0.34 Household size 1.78 0.62 Has dog 3.21 0.36 Time at current address (months) 5.09 0.17 Uses cane or walker 4.52 0.21 Comfort walking 4 blocks 3.72 0.29 Has driver license 3.03 0.39 Has ≥1 vehicle available 1.86 0.60 Lives independently 8.58 0.04 * NEWS: Aesthetics 1.21 0.75 NEWS: Pedestrian Safety 5.75 0.12 NEWS: Personal Safety 3.68 0.30 NEWS: Trafﬁc Safety 2.74 0.43 NEWS: Walking/Cycling Facilities 3.04 0.39 Walked to nearest grocery, last 30 days 58.86 <0.001 * Actual distance to nearest grocery 216.13 <0.001 * Quadrant 16.86 0.05 Site: Seattle/King County, WA 6.87 0.08 Note: *** p < 0.001; * p < 0.05. Geriatrics 2018, 3, x FOR PEER REVIEW 10 of 16 Neighborhood environment perceptions by accuracy of supermarket distance perception, unadjusted multinomial model Figure 3. Average accurate, over-, and underestimator perceptions of neighborhood environment measured with the abbreviated NEWS (NEWS-A) instrument. Note: *p < 0.01; p < 0.05 for z-test of coefficients for distance perception categories compared to accurate in unadjusted multinomial log- linear model. 3.1. Study Population Multinomial log-linear model results: Predictor coefficient estimates and standard errors for overestimators underestimators and individuals reporting “Don’t Know” for perceived walking “Accurate” perceptions, fully-adjusted multinomial model -1.5 0 1.5 Age (yrs) Race/Ethnicity: white, non-Hispanic Gender: female BMI Household size Has dog Time at current address (months) Uses cane or walker Comfort walking 4 blocks Has drivers license Has >=1 vehicle available Lives independently NEWS: Aesthetics NEWS: Pedestrian Safety NEWS: Personal Safety NEWS: Traffic Safety NEWS: Walking/Cycling Facilities Walked to supermarket, last 30 days Actual distance to nearest grocery Quadrant: Low-Walk/High-Inc Quadrant: High-Walk/Low-Inc Quadrant: High-Walk/High-Inc Site: Seattle/King County Estimate Overestimate Underestimate Don't Know **2 * * * *1 Age (y) Race/Ethnicity: White, non-Hispanic Gender: Female BMI Household size Has dog Time at current address (months) Uses cane or walker Comfort walking 4 blocks Has drivers license Has ≥1 vehicle available Lives independently NEWS: Aesthetics NEWS: Pedestrian safety NEWS: Personal safety NEWS: Traffic safety NEWS: Walking/cycling facilities Walked to supermarket., last 30 days Actual distance to nearest grocery Quadrant: Low-walkability/High-income Quadrant: High-walkability/low-income Quadrant: High-walkability/low-income Site: Seattle/King County, WA –1.5 “Overestimate” “Underestimate” “Don’t Know” Figure 4. Multinomial log-linear model results: Predictor coefﬁcient estimates and standard errors for overestimators, underestimators, and individuals reporting “Don’t Know” for perceived Estimate Figure 4. Multinomial log-linear model results: Predictor coefficient estimates and standard errors for overestimators, underestimators, and individuals reporting “Don’t Know” for perceived walking distance to the closest grocery store, compared to individuals with accurate perceptions. Note: Significance levels (determined by z-test) indicated by: * p < 0.001; ** p < 0.01; * p < 0.05. 1Actual coefficient estimate not shown: −15.48 (standard error< 0.001). 2Actual coefficient estimate not shown: −1.92 (standard error = 0.18). Figure 4. Multinomial log-linear model results: Predictor coefﬁcient estimates and standard errors for overestimators, underestimators, and individuals reporting “Don’t Know” for perceived walking distance to the closest grocery store, compared to individuals with accurate perceptions. Note: Signiﬁcance levels (determined by z-test) indicated by: * p < 0.001; p < 0.01; * p < 0.05. 1 Actual coefﬁcient estimate not shown: −15.48 (standard error< 0.001). 2 Actual coefﬁcient estimate not shown: −1.92 (standard error = 0.18). 4. 3.1. Study Population 0 1 2 3 4 Aesthetics Pedestrian Safety Personal Safety Traffic Safety Walk/Bike Facilities Average NEWS-A Score NEWS-A Domain Overestimate Accurate Underestimate Don't Know ** * Neighborhood environment perceptions by accuracy of supermarket distance perception, unadjusted multinomial model Figure 3. Average accurate, over-, and underestimator perceptions of neighborhood environment measured with the abbreviated NEWS (NEWS-A) instrument. Note: ** p < 0.01; * p < 0.05 for z-test of coefﬁcients for distance perception categories compared to accurate in unadjusted multinomial log-linear model. 0 1 2 3 4 Aesthetics Pedestrian Safety Personal Safety Traffic Safety Walk/Bike Facilities Average NEWS-A Score NEWS-A Domain Overestimate Accurate Underestimate Don't Know ** * Neighborhood environment perceptions by accuracy of supermarket distance perception, unadjusted multinomial model Figure 3. Average accurate, over-, and underestimator perceptions of neighborhood environment measured with the abbreviated NEWS (NEWS-A) instrument. Note: *p < 0.01; p < 0.05 for z-test of coefficients for distance perception categories compared to accurate in unadjusted multinomial log- linear model. Figure 3. Average accurate, over-, and underestimator perceptions of neighborhood environment measured with the abbreviated NEWS (NEWS-A) instrument. Note: ** p < 0.01; * p < 0.05 for z-test of coefﬁcients for distance perception categories compared to accurate in unadjusted multinomial log-linear model. 10 of 15 16 Geriatrics 2019, 4, 11 Geriatrics 2018, 3, x FO “Overestimate,” “Underestimate,” and “Don’t Know” vs. “Accurate” perceptions, fully-adjusted multinomial model Figure 4. 4. Discussion Although 29.0% of participants had accurate estimates of their proximity to a supermarket, a large remainder either over- or underestimated their level of physical access to a supermarket: 24.0% were underestimators, or had “optimistic” feelings about their neighborhood food access (e.g., they perceived living closer to a supermarket than they actually did), while 33.9% were overestimators, or had “pessimistic” feelings about their neighborhood food access (e.g., they perceived living further from a supermarket than they actually did). These groups were characterized by both perceived and objective neighborhood factors. Compared to accurate participants, underestimators reported signiﬁcantly higher feelings of pedestrian safety in their neighborhoods, while overestimators were signiﬁcantly less likely to have walked to the supermarket in the last 30 days. In terms of the objective food environment, overestimators actually lived signiﬁcantly closer to supermarkets than more accurate participants, while underestimators lived signiﬁcantly farther away, consistent with other studies of perceived distance to neighborhood amenities among children and adults [42]. Of note, approximately 13.2% of participants could not identify the distance to the closest supermarket; compared to the “Accurate” participants, this group was signiﬁcantly older, more likely to be women than men, and not living independently. Additionally, “Don’t Know” respondents were signiﬁcantly less likely to have walked to the supermarket in the last 30 days. Several ﬁndings provide insights into how the relationship between the objective and perceived food environments may be moderated. First, travel on foot to the supermarket (e.g., having a relatively recent experience walking to the supermarket) was strongly and positively related to the ability to provide an accurate estimate (compared to responding “Don’t Know”), and not having recently walked to the supermarket was also a signiﬁcant predictor of perceiving the distance to be much greater than it was. Second, individual factors, such as gender (“Don’t Know” respondents were more likely to be women than accurate respondents), age (“Don’t Know” respondents were signiﬁcantly older), and living independently (“Don’t Know” respondents were less likely to live independently) were related to the accuracy of perceptions. Finally, neighborhood perceptions, such as higher perceived pedestrian safety, were associated with more “optimistic” perceptions of the (shorter) time required to walk compared to those with accurate perceptions. The results of the linear mixed-model examining correlates of objective distance to the nearest supermarket help to place these ﬁndings in a broader context. 3.1. Study Population Discussion Although 29.0% of participants had accurate estimates of their proximity to a supermarket, a large remainder either over- or underestimated their level of physical access to a supermarket: 24.0% Sensitivity analyses using assumed walking speeds of 0.8 and 1.2 m/s (slightly slower/faster than the 1.0 m/s speed used here) are summarized in Supplemental Material Table S3 Overall, while some covariates changed in their level of signiﬁcance under slower/faster speed assumptions, none changed in terms of the direction of association (e.g., positive versus negative relationships). Notably, however, one of the largest changes in magnitude of association was in the areas of individual mobility for 11 of 15 Geriatrics 2019, 4, 11 “Underestimators” versus “Accurate” participants, especially those who used a cane or walker: −1.19, (95% CI −1.99, −0.39) under a 0.8 m/s assumption, and −0.41 (95% CI −1.29, 0.46) under the 1.0 m/s assumption. The second sensitivity analysis (performed with a dataset that excluded participants with possible mobility constraints) yielded similar results, though with reduced statistical signiﬁcance for all of the NEWS neighborhood perception variables (see Supplemental Material Table S4). These ﬁndings raise important considerations that are further elaborated in the Limitations. Limitations This study has several limitations. First, as a secondary analysis of a cross-sectional observational study, we cannot establish causal relationships between variables and the primary outcomes. Second, we focused on supermarkets as the largest and most identiﬁable type of food retailer to ensure better agreement between participants’ perceptions and what was objectively measured, though the food environment includes many other potential sources, such as fruit and vegetable markets and convenience stores and restaurants. Additionally, our translation of walking minutes to distance was based on averages, which will have varying degrees of error for speciﬁc individuals. Individual-level variables, such as physical ability, may also inﬂuence both the perceived environment, including distance to neighborhood resources, and one’s walking speed, potentially introducing risk of misclassifying participants as over- or underestimators based on the average speed. Our sensitivity analyses offer some contextual detail on this point and underscore the interrelated nature of individual characteristics and neighborhood perceptions. Different methodological approaches, such as structural equation modeling, could provide possible future avenues to disentangle these effects, particularly in studies with prospective, as opposed to cross-sectional, designs. Finally, other qualitative and quantitative studies have shown how shoppers select food retailers according to a variety of individual and store-level criteria beyond physical distance to one’s home. While we had rich individual-level data, we were not able to include information related to store features, such as pricing, quality, or cultural tailoring, which have been found to be inﬂuential elsewhere [47–49], nor were we able to test whether or not participants were aware of the “objectively” nearest store. 4. Discussion Only neighborhood aesthetics were signiﬁcantly related to objective supermarket distance, with higher scores correlated with greater objective distance from a supermarket. While in the current sample, aesthetics did not appear to play a role in helping to deﬁne an individual’s sense of distance, perception of pedestrian safety was signiﬁcantly and positively related to underestimating distance. Some of the inﬂuential environmental factors associated with older adults’ food environment perceptions may be modiﬁable (e.g., walking safety programs may improve perceptions of pedestrian safety; installation of new or improved walking/cycling facilities may raise awareness of these resources), though other signiﬁcant variables in this study, such as an individual’s place of residence or ability to live independently, are much less so. If modiﬁable perceived or objective environmental factors are related to older adults’ reports of more “optimistic” or “pessimistic” views of their food environments, future interventions could seek to encourage seniors to more fully engage with the neighborhood food environment available to them. This ﬁnding aligns with the conclusions of Park and colleagues, who suggest that individual values and interests are important and measurable 12 of 15 Geriatrics 2019, 4, 11 factors for behavioral interventions (which, in their study, concerned meeting physical activity recommendations), and with the earlier work of Thorndyke and Hayes-Roth, which compared the effects of actual experiences versus a map study in terms of distance estimation [43]. Speciﬁc to the food environment, relevant interventions that employ direct resident engagement in conducting assessments of neighborhood resources, including different kinds of food retailers, may help to set the stage for modifying individual perceptions of the food environment [44–46]. In turn, lowered perceived barriers about accessing food environment resources, such as walking distance to a retailer or neighborhood safety, could lead to opportunities for other diet-related interventions that rely on changing food shopping behaviors. Additionally, this study highlights speciﬁc individual factors, like age and gender, which should be further investigated in terms of their effects on the accuracy of distance perceptions, which could be important in designing future interventions in this area. References 1. Kärmeniemi, M.; Lankila, T.; Ikäheimo, T.; Koivumaa-Honkanen, H.; Korpelainen, R. The Built Environment as a Determinant of Physical Activity: A Systematic Review of Longitudinal Studies and Natural Experiments. Ann. Behav. Med. 2018, 52, 239–251. [CrossRef] [PubMed] 2. Mayne, S.L.; Auchincloss, A.H.; Michael, Y.L. Impact of policy and built environment changes on obesity-related outcomes: A systematic review of naturally occurring experiments. Obes. Rev. 2015, 16, 362–375. [CrossRef] [PubMed] 3. Hernandez, D.C.; Johnston, C.A. Individual and Environmental Barriers to Successful Aging: The Importance of Considering Environmental Supports. Am. J. Lifestyle Med. 2017, 11, 21–23. [CrossRef] [PubMed] 4. 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Sohi, I.; Bell, B.A.; Liu, J.; Battersby, S.E.; Liese, A.D. 5. Conclusions In this study, we assessed individual and neighborhood-level correlates of older adults’ perceptions of living close to a supermarket across a population that lived in two U.S. regions with varying levels of walkability and income. We also tested these perceptions against objective GIS-based measures of the food environment, and classiﬁed participants as accurate, or over- or underestimators. Our results reveal how perceptions of over- or underestimating one’s level of access to a supermarket include individual characteristics and capabilities, as well as neighborhood pedestrian safety perceptions. Further research is needed to understand how these food environment perceptions form, how they are associated with actual food choice, and whether or not they can be modiﬁed by interventions in order to promote positive health behavior outcomes. Supplementary Materials: The following are available at https://www.mdpi.com/2308-3417/4/1/11/s1, Tables S1: Output from null, partial, and full linear mixed models predicting objective distance to the nearest supermarket with block group random effect, Tables S2: Output from null, partial, and full multivariate log-linear models predicting likelihood of over-estimating, under-estimating, or responding “don’t know”, compared to accurately estimating the distance to the nearest supermarket, Tables S3: Participant counts of distance perception accuracy categories by assumed walking speed, Tables S4: Sensitivity analysis using reduced dataset that excludes participants with possible mobility constraints. 13 of 15 13 of 15 Geriatrics 2019, 4, 11 Author Contributions: Conceptualization, B.W.C., A.C.K., J.H., B.E.S., L.D.F., T.L.C., K.L.C. and J.F.S.; Data curation, L.D.F.; Formal analysis, B.W.C.; Funding acquisition, A.C.K. and J.F.S.; Methodology, B.E.S. and T.L.C.; Supervision, A.C.K.; Writing—original draft, B.W.C.; Writing—review and editing, B.W.C., A.C.K., J.H., B.E.S., L.D.F., T.L.C., K.L.C. and J.F.S. Author Contributions: Conceptualization, B.W.C., A.C.K., J.H., B.E.S., L.D.F., T.L.C., K.L.C. and J.F.S.; Data curation, L.D.F.; Formal analysis, B.W.C.; Funding acquisition, A.C.K. and J.F.S.; Methodology, B.E.S. and T.L.C.; Supervision, A.C.K.; Writing—original draft, B.W.C.; Writing—review and editing, B.W.C., A.C.K., J.H., B.E.S., L.D.F., T.L.C., K.L.C. and J.F.S. Funding: The research was supported by US National Heart, Lung, & Blood Institute grant R01 HL077141. The primary author was supported by a NIH/NHLBI grant T32 HL007034. Funding: The research was supported by US National Heart, Lung, & Blood Institute grant R01 HL077141. The primary author was supported by a NIH/NHLBI grant T32 HL007034. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Older Adults’ Outdoor Walking: Inequalities in Neighbourhood Safety, Pedestrian Infrastructure and Aesthetics. Int. J. Environ. Res. Public Health 2016, 13, 1179. [CrossRef] [PubMed] 20. 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https://openalex.org/W4362640713	https://research.tue.nl/files/305001699/1-s2.0-S0360319923014519-main.pdf	English	null	Development of selective Pd–Ag membranes on porous metal filters	International journal of hydrogen energy	2,023	cc-by	10,051	Document status and date: Published: 30/07/2023 Document status and date: Published: 30/07/2023 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. 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If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: Document license: CC BY Document license: CC BY DOI: 10.1016/j.ijhydene.2023.03.306 Document status and date: Published: 30/07/2023 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There ca important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or vis DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication DOI: 10.1016/j.ijhydene.2023.03.306 Development of selective Pd–Ag membranes on porous metal filters Citation for published version (APA): Agnolin, S., Apostolo, F., Di Felice, L., Melendez Rey, J., Pacheco Tanaka, D., Llosa Tanco, M., & Gallucci, F. (2023). Development of selective Pd–Ag membranes on porous metal filters. International Journal of Hydrogen Energy, 48(65), 25398-25409. https://doi.org/10.1016/j.ijhydene.2023.03.306 Citation for published version (APA): Agnolin, S., Apostolo, F., Di Felice, L., Melendez Rey, J., Pacheco Tanaka, D., Llosa Tanco, M., & Gallucci, F. (2023). Development of selective Pd–Ag membranes on porous metal filters. International Journal of Hydrogen Energy, 48(65), 25398-25409. https://doi.org/10.1016/j.ijhydene.2023.03.306 Citation for published version (APA): Agnolin, S., Apostolo, F., Di Felice, L., Melendez Rey, J., Pacheco Tanaka, D., Llosa Tanco, M., & Gallucci, F. (2023). Development of selective Pd–Ag membranes on porous metal filters. International Journal of Hydrogen Energy, 48(65), 25398-25409. https://doi.org/10.1016/j.ijhydene.2023.03.306 Citation for published version (APA): Agnolin, S., Apostolo, F., Di Felice, L., Melendez Rey, J., Pacheco Tanaka, D., Llosa Tanco, M., & Gallucci, F. (2023). Development of selective Pd–Ag membranes on porous metal filters. International Journal of Hydrogen Energy, 48(65), 25398-25409. https://doi.org/10.1016/j.ijhydene.2023.03.306 Download date: 24. Oct. 2024 www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 Available online at www.sciencedirect.com h i g h l i g h t s g r a p h i c a l a b s t r a c t A method to ﬁll the pores of Has- telloy ﬁlters via aspiration of a- Al2O3 water suspension has been developed. The effect of a-Al2O3 particle size on the surface quality has been studied with a statistical method. The asymmetrical ﬁlling method can reduce the average pore size of the ﬁlters from 1.1 mm up to 200 nm. A boehmite-based layer is used to change the surface quality and as interdiffusion barrier. A highly H2-selective PdeAg membrane (~40 000) has been ob- tained on the asymmetrically ﬁlled support. A highly H2-selective PdeAg membrane (~40 000) has been ob- tained on the asymmetrically ﬁlled support. Development of selective PdeAg membranes on porous metal ﬁlters S. Agnolin a, F. Apostolo a, L. Di Felice a, J. Melendez Rey b, A. Pacheko Tanaka c, M. Llosa Tanco c, F. Gallucci a,d,* a Inorganic Membranes and Membrane Reactors, Sustainable Process Engineering, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, De Rondom 70, Eindhoven, 5612 AP, the Netherlands b H2Site, Bilbao, Spain b H2Site, Bilbao, Spain , , p c TECNALIA, Basque Research and Technology Alliance (BRTA), Mikeletegi Pasealekua 2, 20009, Donostia, San Sebastian, Spain , p d Eindhoven Institute for Renewable Energy Systems (EIRES), Eindhoven University of Technology, PO Box 513, Eindhoven 5600 MB, the Netherlands * Corresponding author. Inorganic Membranes and Membrane Reactors, Sustainable Process Engineering, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, De Rondom 70, Eindhoven, 5612 AP, the Netherlands. E-mail address: f.gallucci@tue.nl (F. Gallucci). https://doi.org/10.1016/j.ijhydene.2023.03.306 0360-3199/© 2023 The Author(s). Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. Inorganic Membranes and Membrane Reactors, Sustainable Process Engineering, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, De Rondom 70, Eindhoven, 5612 AP, the Netherlands. Introduction Pd based membranes are well-known for their unique solution- diffusion H2 transport mechanism, which makes them the heart of membrane reactors (MRs) for in-situ selective hydrogen removal [1e3]. MRs have proven themselves as a promising alternative to conventional methane steam re- formers by integrating both reaction and separation in a single unit and thus avoiding the puriﬁcation steps required by a conventional process [4e6]. The introduction of membranes in the reaction environment promotes in fact the continuous removal of H2, resulting in equilibrium shift towards the H2 production reaction owing to the Le Chatelier's principle. In this way, the process operating temperature is reduced, increasing overall energy efﬁciency and reducing operational costs [7e9]. To solve the intermetallic diffusion issue, an additional barrier is required between the Pd layer and the metallic support [25]. Several interdiffusion barriers have in fact been extensively investigated in literature, mostly ceramic based (Al2O3 [26], ZrO [27,28], CeO [29], SiO2 [30]) or oxidation based (controlled metal oxides growth [31]). In our previous work [24], we proposed a boehmite-based layer with a dual function as interdiffusion barrier/smoothening layer. In fact, this layer, besides preventing intermetallic diffusion, if combined with a suitable polishing treatment is also able to reduce the surface roughness of the selected metallic supports. However, the deposition of this barrier alone proves insufﬁcient to close the large superﬁcial pore mouths of the ﬁlter, resulting in mem- branes with low perm-selectivity [24,26]. For this reason, this work focuses on the recovery of superﬁcial porosity of pol- ished ﬁlters via chemical etching [32], and the subsequential pore ﬂow distribution narrowing via the introduction of a pore ﬁller. In literature, several materials have been in fact intro- duced as ﬁllers in an effort to improve the surface morphology of metallic supports [33e37]. Similarly, in the recent work of Kim et al. a selective membrane obtained via pore ﬁlling method and sol-gel barrier deposition has been tested for ammonia decomposition applications [38]. gy y g p [ ] As core of this intensiﬁcation technology, Pd-based mem- branes have been subjected to optimization studies, mostly both in terms of their H2 perm-selectivity performance and their suitability for integration in the reaction environment [10e12]. In particular, thin Pd ﬁlms (<10 mm) have been suc- cessfully deposited on tubular ceramic supports [13,14]. i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25399 Accepted 20 March 2023 Available online 5 April 2023 Keywords: Surface modiﬁcation Metallic membranes Hydrogen separation Pd membranes Metallic supports etching. A method to ﬁll the large pores of the ﬁlters via aspiration of a-Al2O3 water-powder suspension has been applied and characterized via imaging of the ﬁlled pores, inferential statistics, and capillary ﬂow porometry measurements. The most suitable ﬁller particle size for pore size distribution reduction has been identiﬁed as 18 mm, while a 5 mm ﬁller proved optimal for further pore morphology improvement. The wide pore size distribution of the ﬁlters has thus been reduced up to 200 nm by ﬁlling with a-Al2O3 particles of decreasing size, similarly to the ceramic supports used for thin PdeAg membranes deposition. A boehmite based interdiffusion barrier has been deposited, achieving further surface roughness reduction. A highly H2 selective membrane has been obtained via simultaneous PdeAg plating on the pre-treated ﬁlter. © 2023 The Author(s). Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/). surface roughness, large superﬁcial potholes, and wide pore size distribution, making the preparation of Pd-based mem- branes with high H2 permeation and selectivity a complex task [22,23]. In order to then acquire metallic supports with the desired characteristics, tailor made supplier treatments and extremely low media grades must be requested, clearly increasing their ﬁnal costs [24]. In our work, we focus on operating suitable and cost-effective supports pre-treatments while starting from a large media grade, rough and unreﬁned Hastelloy ﬁlter. The focus is on acquiring a low-cost product and operating suitable pre-treatments to achieve a sufﬁcient surface quality for deposition of a selective PdeAg layer. a b s t r a c t Metallic supports with sufﬁcient surface quality to achieve highly selective thin PdeAg membranes require speciﬁc pre-treatments, are not readily available on the market and are generally very expensive. To reduce costs, rough and large media grade Hastelloy X ﬁlters have been acquired and pre-treated via polishing and chemical etching. The loss in gas permeance given by the polishing treatment proved fully recovered after chemical Article history: Received 11 February 2023 Received in revised form 10 March 2023 s). Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. This is an open access article under the CC BY ns.org/licenses/by/4.0/). i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25400 25400 are prepared in distilled water, incorporating a water-based solution of organic additives, namely 3.5 wt% polyvinyl alcohol (PVA) (MW 130000) and 1 wt% polyethylene glycol (PEG) (MW 400). The deposited layers are dried under rotation in a climate chamber at 40 C and 60% relative humidity for 1 h. The layers are then sintered for 1 h at 550 C in a static air furnace. These parameters yield to a mesoporous g-Al2O3 layer about 540 nm thick [24]. are prepared in distilled water, incorporating a water-based solution of organic additives, namely 3.5 wt% polyvinyl alcohol (PVA) (MW 130000) and 1 wt% polyethylene glycol (PEG) (MW 400). The deposited layers are dried under rotation in a climate chamber at 40 C and 60% relative humidity for 1 h. The layers are then sintered for 1 h at 550 C in a static air furnace. These parameters yield to a mesoporous g-Al2O3 layer about 540 nm thick [24]. preparation parameters. The outcomes in terms of support gas permeance, pore ﬂow distribution, surface roughness and morphology are thoroughly investigated for each pre- treatment step and, ﬁnally, the electroless deposition of a PdeAg layer is performed. The resulting membranes are then characterized in terms of ideal H2/N2 perm-selectivity. Preparation of porous Hastelloy X supports Preparation of porous Hastelloy X supports A layer of PdeAg alloy is deposited onto treated supports via electroless plating technique, reported in a previous work by Tanaka et al. [13,14]. To improve membrane selectivity, a consecutive plating procedure is carried out to achieve thicker PdeAg layers. After each plating step, the membrane is annealed at 550 C in 10 vol% H2 - 90 vol% Ar atmosphere for 4 h. At temperatures below 300 C, only Ar was used to avoid fragilization of the PdeAg layer. Commercial unreﬁned porous Hastelloy X ﬁlters with an outer diameter of 1.2 cm, average surface roughness (Ra) of 6.1 mm, and 0.5 mm nominal media grade (MG) were acquired by Hebei Golden Flame Wire Mesh Co, China. The supports are cut in samples of 10 cm length and welded to dense stainless steel (AISI316L) tubes, in order to achieve a one close end conﬁgu- ration. To preliminarily reduce the surface roughness of the ﬁlters, the sample supports were polished in an industrial surface ﬁnishing machine via wet-polishing mechanism (ERBA EVT-170). The chosen polishing time amounts to 6 h, as it allows for a suitable trade-off between surface roughness reduction and gas permeation preservation, which was determined in our previous work [24]. The polished supports are then etched by perpendicular immersion in aqua regia for 30 s and thoroughly rinsed with deionized water to remove all mordant residuals. The supports are then oxidized for 1 h at 750 C in a furnace in static air atmosphere. Before further treatments, the supports are further rinsed both in ethanol and in deionized water in an ultrasonic bath, to remove all impurities resulting from the preparation pre-treatments and handling. Capillary ﬂow porometry To evaluate pore size distribution (PSD) variations for each support pre-treatment, the capillary ﬂow porometry tech- nique (CFP, or gas-liquid displacement method) is employed. This technique relies on imposing a trans-sample pressure at which a suitable liquid is displaced from the pores of the examined sample. The displacement is detected by registering the permeating ﬂow of a non-reactive gas through the media. In CFP tests, the sample is ﬁlled with a wetting liquid, assuming the ﬁlling of its entire accessible porosity. Pressure is applied to one side, while the other is kept at atmospheric pressure. This trans-sample pressure difference forces the wetting liquid out of the pores resulting in a permeating ﬂow. Increasing the trans-sample pressure will promote pore clearance, increasing the permeating ﬂow until the sample is fully cleared from the wetting liquid. Young-Laplace equation is then used to correlate the capillary pressure in the media with its pore diameter. If the capillary is assumed of cylin- drical shape, Washburn equation can be applied, which is a typical assumption for indirect method CFP measurements [39,40]: Filler introduction The tubular supports are submerged in a powder-water suspension, which is pulled through the superﬁcial pores via vacuum assisted dip-coating. The immersion time is set to 60 s per cycle. Between each cycle the support is gently rinsed with distilled water, and no calcination is required. The selected ﬁller powder is a-Al2O3, which is evaluated in three different particle sizes (AA-1 Sumitomo 1 mm, AA-5 Sumitomo 5 mm, AA-20 Sumitomo 18 mm). The powders are 10 wt% suspended in water with a magnetic stirrer. The suspension is improved by addition of HNO3 (67%vol) drop- wise. First, the supports undergo several immersion cycles in order to assess both the aspiration effect and the ﬁller size effect on support's PSD and surface morphology. In a second study, supports are ﬁlled asymmetrically with a- Al2O3 of decreasing particle size. d ¼ 4g cos w DP where DP is the applied trans-sample pressure, d is the nar- rowest diameter of the capillary, g is the surface tension of the chosen wetting liquid, and w is the contact angle between the wetting liquid and the wet surface. The 10 cm porous tubes are measured via CFP technique in a geometry-speciﬁc setup (Fig. 1), which consists of. Introduction The deposition of Pd on an appropriate support allows in fact to reach outstanding H2 perm-selectivity while ensuring me- chanical stability of the thin ﬁlms [12,15,16]. Asymmetric ceramic tubular supports prove in fact suitable for the elec- troless deposition of a thin and defect-free Pd-based layer thanks to their low surface roughness, narrow pore size dis- tribution (PSD), and low resistance to gas permeation [17,18]. However, while they ensure adequate surface quality for thin layer deposition, ceramic supports prove to be fragile when introduced in the reactor environment. Particularly difﬁcult is their connection and sealing to the steel parts of most reactors structures, making them prone to breakage and/or leaks and thus rendering the scale-up of membrane modules a difﬁcult task [19]. For these reasons, a variety of steel and steel alloys supports have gained rising interest as possible substitutes. They in fact do not require sealing (which can be substituted by a simple weld) and are way less prone to breakage or crack formation thanks to their high mechanical stability. Metallic supported Pd ﬁlms, however, are proved to be inclined to atomic migration within the support structure, a phenomenon known as intermetallic diffusion or Pd-support interaction [20,21]. In addition, steel-based supports display in fact large In our work, we propose asymmetrically layered a-Al2O3 inside the ﬁlter's superﬁcial pore mouths. The sequential ﬁlling is carried out with a-Al2O3 of decreasing particle size via vacuum assisted aspiration. The surface roughness of the support is then lowered by the deposition of the boehmite- based barrier, covering the pores and preventing interdiffu- sion issues. The ﬁlling effect was studied by feeding imaging parameters to a Two-way ANOVA design, introducing an applicable method for statistical evaluation of membrane i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25401 Fig. 1 e Graphical representation of the CFP setup for tubular membrane samples. supports are evaluated with a portable contact proﬁlometer (MarSurf PS 10) on 10 random positions on the tubular sup- port. These parameters are normalized on their values for an untreated support and employed as dependent variables for a two-way ANOVA. Being an inferential statistics method, ANOVA allows to infer on the whole population of support pores, while observing only a representative sample amount. The factorial design allows for full observation of the outcome variable (D, Ra, or Rz) while variating the two selected factors “Filling cy- cles” and “Filler size” combinations, making it especially suitable for a comprehensive observation of the ﬁlling phenomena. The experimental designs are summed up in Table 1 and the full statistical documentation is attached in the supple- mentary material to this manuscript. Fig. 1 e Graphical representation of the CFP setup for tubular membrane samples. Results and discussion I. The controlling software allows the user to set a desired pressure ramp and/or step to be imposed within the permeation box; Polishing and etching The evolution of the ﬁlters surface with the chosen pre- deposition treatments is analyzed with the laser confocal microscopy images in Fig. 2. The untreated 0.5 mm media grade ﬁlters are characterized by the presence of high proﬁle peaks (red) and large surface roughness (Fig. 2a), in agreement with the behavior observed in our previous studies [24]. At this stage, the pore mouths of the support are interconnected, showing a superﬁcial diameter larger than 50 mm. After 6 h of polishing (Fig. 2b), the surface of the support is uniformly smoothened, erasing the presence of the large peaks by plastic deformation. However, the smaller superﬁcial openings are mostly erased. A few large, isolated pore mouths remain, with superﬁcial diameters close to 20 mm. After etching the support for 30s in aqua regia (Fig. 2c) the smoothened surface is broken down into smaller channels interconnecting the metallo- graphic structure underneath: the surface roughness reduc- tion achieved by polishing is preserved, while the support's valleys are uncovered. This promotes an increase in the number of superﬁcial openings, leading to improved gas per- meance after polishing, while preserving the treatment's smoothening effect. The evolution of N2 permeance, mean ﬂow pore and contact roughness parameters (Table 2) further conﬁrms the behavior observed via microscopy. Firstly, the polishing treatment promotes both a gas permeance reduc- tion of 76%, and a roughness decrease of 87%. Following the chemical etching, due to the presence of a larger amount of proﬁle valleys, Ra is re-increased solely with an additional II. The correct feed ﬂow is sent to the permeation box in order to increase the pressure according to the ramp set by the user (1); III. The permeating ﬂow at each timestamp is registered at the permeate side by the ﬂowmeter with the correct ﬂow range, which can be automatically switched via the three way valve (2). Gas permeance C. An automatic backpressure regulator at the retentate side (Bronkorst EL-PRESS- Pe502C); The prepared PdeAg membranes are tested for N2 permeance at 20 C and 1 bar in a permeation box, described in our pre- vious work [24]. If the N2 permeance is lower than 1,109 mol/ m2/s/Pa the membrane is selected for further high tempera- ture short-term investigation. The selected membranes are activated at 400 C with an air ﬂow of 1 l/min for 2 min. They are then tested at 400, 450 and 500 C for single-gas H2 and N2 permeance with an imposed pressure difference of 1, 2, 3 bar. C. An automatic backpressure regulator at the retentate side (Bronkorst EL-PRESS- Pe502C); D. An automatic three way valve, which can switch be- tween a low ﬂow automatic ﬂowmeter (Bronkhorst EL- FLOW Prestige FG-111B, range 0.004 ml/min - 0.2 l/ min) and a high ﬂow automatic ﬂowmeter (Bronkhorst EL-FLOW Prestige FG-111B range 0.2 l/min-10 l/min); E. An external computer with LabVIEW software. E. An external computer with LabVIEW software. The setup is fully automated as it follows. The setup is fully automated as it follows. Interdiffusion layer deposition A. a tubular permeation box, which is able to withstand pressures up to 60 bara; A smoothening interdiffusion barrier is deposited to ﬁnalize the improvement of support's surface roughness and ease PdeAg deposition. Solutions with boehmite loading 0,9 wt% B. An automatic mass ﬂow controller for N2 (Bronkhorst EL-FLOW Select F-221 M), which is chosen as inert displacement gas; Laser confocal microscopy and proﬁlometry: statistical The surface morphology of the treated ﬁlters was studied using a dual optical-3D laser confocal microscope (VKX-3000, Keyence, Osaka, Japan). Said imaging method has been employed to verify and observe the presence of the selected ﬁlling particles into the superﬁcial pore mouths of the sup- ports. The extent of superﬁcial pore ﬁlling is evaluated by imaging a superﬁcial pore mouth and registering its highest point on the metal surface and its lowest point on the embedded ﬁller. For each ﬁller size and cycles combination, 10 random pore mouths have been evaluated. The result is a ﬁlling extent parameter D. Consequentially, a lower D in- dicates a fuller pore mouth. D has been employed for statistical considerations as dependent variable in a two-way ANalysis Of VAriance (ANOVA), in order to guarantee a more quantitative approach to the ﬁlling procedure optimization [41]. The average surface roughness (Ra) and average proﬁle height (Rz) of the treated i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25402 Table 1 e Two-way ANOVA design for each dependent variable D, Ra, Rz. Laser-confocal microscopy Deﬁnition Variable type Name Variable levels Outcome Dependent D [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Average surface roughness (Ra) Deﬁnition Variable type Name Variable levels Outcome Dependent Ra [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Average proﬁle height (Rz) Deﬁnition Variable type Name Variable levels Outcome Dependent Rz [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Table 1 e Two-way ANOVA design for each dependent variable D, Ra, Rz. Laser confocal microscopy and proﬁlometry: statistical Laser-confocal microscopy Deﬁnition Variable type Name Variable levels Outcome Dependent D [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Average surface roughness (Ra) Deﬁnition Variable type Name Variable levels Outcome Dependent Ra [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Average proﬁle height (Rz) Deﬁnition Variable type Name Variable levels Outcome Dependent Rz [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Table 1 e Two-way ANOVA design for each dependent variable D, Ra, Rz. Laser-confocal microscopy Deﬁnition Variable type Name Variable levels Outcome Dependent D [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Average surface roughness (Ra) Deﬁnition Variable type Name Variable levels Outcome Dependent Ra [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Average proﬁle height (Rz) Deﬁnition Variable type Name Variable levels Outcome Dependent Rz [mm] e Factor 1 Independent (A) Filling cycles [] 10, 15, 20 Factor 2 Independent (B) Filler size [mm] 1.5, 5, 18 Table 1 e Two-way ANOVA design for each dependent variable D, Ra, Rz. Fig. 2 e Laser confocal microscopy imaging and height distribution of a 0.5 mm media grade Hastelloy ﬁlter (a)untreated (b) 6 h polished, (c) Etched 30s in aqua regia. Fig. 2 e Laser confocal microscopy imaging and height distribution of a 0.5 mm media grade Hastelloy ﬁlter (a)untreated (b) 6 h polished, (c) Etched 30s in aqua regia. Fig. 2 e Laser confocal microscopy imaging and height distribution of a 0.5 mm media grade Hastelloy ﬁlter (a)untreated (b) 6 h polished, (c) Etched 30s in aqua regia. i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25403 Fig. 3 e Interaction effect plot of dependent variable D against factor “Filling cycles”, grouped by “Filler size”. Laser confocal microscopy and proﬁlometry: statistical The points indicate the average value for each factor levels combination. Table 2 e Ra, N2 permeance, and CFP measured mean ﬂow pore of a sample Hastelloy support for each pre- treating step, compared with the values for an untreated Hastelloy ﬁlter and an a-Al2O3 support by Rauschert. Pre-treatment Ra N2 permeance Mean ﬂow pore (CFP) [] [mm] [mol/s/m2/Pa ,105] [mm] Ceramic, a-Al2O3 0,5 8,0 0,1 Untreated Hastelloy 6,1 5,0 1,8 Wet polishing, 6 h 0,8 1,2 1,9 Chemical etching, 30s 1,1 8,9 1,1 Table 2 e Ra, N2 permeance, and CFP measured mean ﬂow pore of a sample Hastelloy support for each pre- treating step, compared with the values for an untreated Hastelloy ﬁlter and an a-Al2O3 support by Rauschert. 20%, while the gas permeance surpasses the untreated sup- port's original value. This behavior is well in agreement with the work of Xu et al. which carried out similar improving pre- treatments on a disk-shaped stainless steel support, observing the same surface variations via Scanning Electron Microscopy [32]. Fig. 3 e Interaction effect plot of dependent variable D against factor “Filling cycles”, grouped by “Filler size”. The points indicate the average value for each factor levels combination. Fig. 5 e Sole main effect plot of factor “Filler size” on dependent variable Ra. The points indicate average Ra decrease for each level of the sole factor “Filler size”. Fig. 4 e Main independent effects plots of factor “Filling cycles” and factor “Filler size” on dependent variable Rz. The points indicate the average Rz decrease values for each independent factor level. Fig. 3 e Interaction effect plot of dependent variable D against factor “Filling cycles”, grouped by “Filler size”. The points indicate the average value for each factor levels combination. Filler introduction Taking into account the mean ﬂow pore of a ceramic support (Table 2), which is proved to produce supported thin and ultra- thin Pd based membranes without defects [42,43], the size of the ﬁlter's through pores after polishing and etching is still too large. For this reason, the introduction of the a-Al2O3 ﬁller into the superﬁcial openings of the pre-treated support is expected to prove crucial for the reduction of their size, ensuring gas tightness (and therefore H2 selectivity) of the completed membrane. Table 3 shows the results of the Two-way ANOVA performed on the outcome variables listed in Table 1. Fig. 4 e Main independent effects plots of factor “Filling cycles” and factor “Filler size” on dependent variable Rz. The points indicate the average Rz decrease values for each independent factor level. The results of the two-way ANOVA highlight a statistically signiﬁcant contribution to D of the interaction between the number of aspiration cycles and the ﬁller size. When an interaction is statistically signiﬁcant, analyzing solely the main effects can be misleading. Therefore, the interaction effect is observed in Fig. 3. In particular, all ﬁller sizes under- perform with the lowest number of cycles, meaning that more Fig. 4 e Main independent effects plots of factor “Filling cycles” and factor “Filler size” on dependent variable Rz. The points indicate the average Rz decrease values for each independent factor level. Fig. 4 e Main independent effects plots of factor “Filling cycles” and factor “Filler size” on dependent variable Rz. The points indicate the average Rz decrease values for each independent factor level. Table 3 e ANOVA test results on outcome variables Ra, Rz and D. The relevant factor effect is assumed signiﬁcant if p-value<0.05, with a conﬁdence interval of 95%. Laser confocal microscopy, D Factor Factor levels p-value Signiﬁcance Filling cycles [¡] 10/15/20 0,581 e Filler size [mm] 1,5/5/18 2,3,108 * Interaction [-,mm] e 0,002 Proﬁlometry, Ra Factor Factor levels p-value Signiﬁcance Filling cycles [¡] 10/15/20 0,001 Filler size [mm] 1,5/5/18 0,123 e Interaction [-,mm] e 0,512 e Proﬁlometry, Rz Factor Factor levels p-value Signiﬁcance Filling cycles [¡] 10/15/20 0,001 * Filler size [mm] 1,5/5/18 0,025 * Interaction [-,mm] e 0,782 e Table 3 e ANOVA test results on outcome variables Ra, Rz and D. The relevant factor effect is assumed signiﬁcant if p-value<0.05, with a conﬁdence interval of 95%. Fig. Filler introduction 5 e Sole main effect plot of factor “Filler size” on dependent variable Ra. The points indicate average Ra decrease for each level of the sole factor “Filler size”. Laser confocal microscopy, D Fig. 5 e Sole main effect plot of factor “Filler size” on dependent variable Ra. The points indicate average Ra decrease for each level of the sole factor “Filler size”. i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25404 signiﬁcant contribution of both independent factors is observed, while the interaction effect is lost. This can be explained by the nature of the outcome variable Rz, which averages roughness proﬁle extremes along a measuring length, rather than purposefully imaged pore mouths, yielding to a loss of information with respect to microscopy. Nevertheless, in Fig. 4b it is noticeable how a 5 mm ﬁller pro- motes the largest reduction in Rz, independently from the number of cycles performed. However, a large number of than 10 cycles are required for a low D; at 15 cycles, the best performing size is 5 mm, while at 20 cycles the largest size (18 mm) and the smallest size (1.5 mm) also contribute to a D reduction. This result suggests that 5 mm is a ﬁller that most suits the shape of the superﬁcial pore mouths of the support, requiring less cycles to reach a performance plateau with respect to the largest and smaller ﬁller, which will require more cycles. This result is conﬁrmed by the ANOVA per- formed on the roughness parameter Rz. For Rz a statistically Fig. 6 e (a)Mean ﬂow pore diameter precent decrease with increasing ﬁlling cycles, for each ﬁller size, measured via CFP. (b) N2 permeance percentage decrease with increasing ﬁlling cycles, for each ﬁller size. (c) Pore ﬂow distribution measured via CFP of a ﬁlter ﬁlled 20x with 18 mm a-Al2O3, measured via CFP between 10% and 90% of total dry ﬂow. Fig. 6 e (a)Mean ﬂow pore diameter precent decrease with increasing ﬁlling cycles, for each ﬁller size, measured via CFP. (b) N2 permeance percentage decrease with increasing ﬁlling cycles, for each ﬁller size. PdeAg deposition All three analyses conclude that 5 mm reaches a perfor- mance plateau quicker than the other sizes, making it the most suitable size for all surface morphology parameters improvement, preferably in combination with an amount of aspiration cycles greater than 15. However, microscopically, as the number of cycles grows larger, the 1.5 and 18 mm size will contribute to the improving of the pore morphology. In Fig. 10a the surface morphology of a membrane obtained via deposition of PdeAg when carried out on a 0.5 mm media grade ﬁlter solely treated with the boehmite based smooth- ening layer is shown. The boehmite based layer allows for uniform metal deposition on the support's surface. However, the sole presence of this layer is not enough to promote full pore closure and obtain a defect-free Pd membrane. In Fig. 10b the same deposition is carried out on a support in which a 5 mm a-Al2O3 ﬁller has been introduced. The leveling of the pore surface promoted by the ﬁlling of the large support's mouths allows for the PdeAg to fully close the superﬁcial openings, obtaining the desired defect-free dense layer even on the large superﬁcial pore mouths. For this reason, the The evolution of surface morphology parameters is not the only phenomenon to be considered while applying a ﬁller to the selected support. An optimal ﬁller design should in fact guarantee sufﬁcient support's average pore diameter reduc- tion and pore distribution sharpening, while preserving gas permeance through the porous media. From Fig. 6c it is clear how an 18 mm ﬁller sharpens the ﬁlter's pore ﬂow distribution around an average pore diameter of about 200 nm, promoting a reduction of the mean ﬂow pore by 80% (Fig. 6a). This shows the ﬁller ability to place itself in large pore necks with respect to the smaller sizes, which only contribute to morphology improvement but require more packing to inﬂuence the PSD. Similarly, as observed in Fig. 6b, the 18 mm ﬁller is the most inﬂuential in the decrease of per- meance in the sample ﬁlter, meaning it is affecting the pore necks rather than the surface openings. This conclusion is further conﬁrmed by the dual optical-laser confocal imaging in Fig. 7, in which the 18 mm ﬁller is observed to close the largest defects, while the smaller ﬁllers tend to assume a packed conﬁguration, leaving larger defects open. i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25405 25405 cycles is preferred independently from the chosen ﬁller size (Fig. 4b). For Ra, a further loss of information is introduced within the ANOVA results. This loss is attributed to the aver- aging nature of Ra, which does not consider any extremes of the roughness proﬁle, but rather averages all the deviations from the proﬁle's mean line. A statistically signiﬁcant contri- bution is attributed solely to the ﬁller size, highlighting how 5 mm is the best performing in terms of Ra reduction inde- pendently from the number of applied cycles, which, in turn, proves to be insigniﬁcant (Fig. 5). cycles is preferred independently from the chosen ﬁller size (Fig. 4b). For Ra, a further loss of information is introduced within the ANOVA results. This loss is attributed to the aver- aging nature of Ra, which does not consider any extremes of the roughness proﬁle, but rather averages all the deviations from the proﬁle's mean line. A statistically signiﬁcant contri- bution is attributed solely to the ﬁller size, highlighting how 5 mm is the best performing in terms of Ra reduction inde- pendently from the number of applied cycles, which, in turn, proves to be insigniﬁcant (Fig. 5). measured through pores larger than 500 nm. This pore dis- tribution tail depends on both the initial ﬁlters themselves and any leftover large openings after pre-treatment. The control of this tail, although representing the minority of the distribution, proves in fact crucial to minimize membrane's N2 permeation and thus selectivity, ensuring reproducibility of performance. For this reason, further optimization on the ﬁlling procedure is the subject of planned studies. Filler introduction (c) Pore ﬂow distribution measured via CFP of a ﬁlter ﬁlled 20x with 18 mm a-Al2O3, measured via CFP between 10% and 90% of total dry ﬂow. Fig. 7 e Dual optical-laser confocal imaging of a Hastelloy ﬁlter's selected pore mouth ﬁlled with 1.5 mm a-Al2O3, 5 mm a- Al2O3, and 18 mm a-Al2O3. Fig. 7 e Dual optical-laser confocal imaging of a Hastelloy ﬁlter's selected pore mouth ﬁlled with 1.5 mm a-Al2O3, 5 mm a- Al2O3, and 18 mm a-Al2O3. Fig. 7 e Dual optical-laser confocal imaging of a Hastelloy ﬁlter's selected pore mouth ﬁlled with 1.5 mm a-Al2O3, 5 mm a- Al2O3, and 18 mm a-Al2O3. laser confocal imaging of a Hastelloy ﬁlter's selected pore mouth ﬁlled with 1.5 mm a-Al2O3, 5 mm a Al2O3. i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 PdeAg deposition i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25406 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 Fig. 10 e Dual optical-laser confocal imaging and height distribution of a PdeAg layer deposited on a large superﬁcial opening of (a) an unﬁlled Hastelloy ﬁlter, (b) a Hastelloy ﬁlter ﬁlled with a-Al2O3. Both samples are coated with a boehmite- based smoothening layer. Fig. 10 e Dual optical-laser confocal imaging and height distribution of a PdeAg layer deposited on a large superﬁcial opening of (a) an unﬁlled Hastelloy ﬁlter, (b) a Hastelloy ﬁlter ﬁlled with a-Al2O3. Both samples are coated with a boehmite- based smoothening layer. Table 4 e H2/N2 perm-selectivity comparison of Pd-based membranes deposited via electroless plating onto steel-based supports pre-treated with similar methods. Support Filler Interdiffusion barrier H2 permeance H2/N2 Ref Material/media grade/pre- treatment Material/particle size Material [mol/m2/Pa/s] [] Hastelloy X/0.1 mm/pre- treated by Mott Corp. PdeAg deposition This ren- ders the 18 mm ﬁller suitable for a ﬁrst reduction in large support's openings, while ﬁllers of smaller size can be layered to promote a subsequential improvement of the superﬁcial pore morphology. These observations, paired with the results obtained by Chi et al. who observed the same behavior for a- Al2O3 ﬁllers on lower media grade stainless steel tubes, allow to speculate that ﬁllers with about half of the size of the su- perﬁcial pore openings are the most suitable for pore ﬂow distribution modiﬁcation, while ﬁllers of lower dimensions can be used for surface morphology improvement [35]. Fig. 8 e Dual optical-laser confocal imaging of a Hastelloy ﬁlter's selected pore mouth, asymmetrically ﬁlled with a- Al2O3 of decreasing size. Fig. 9 e Cumulative percentage ﬂow distribution through the pores of an asymmetrically ﬁlled sample ﬁlter before and after boehmite layer deposition, measured via CFP. Fig. 8 e Dual optical-laser confocal imaging of a Hastelloy ﬁlter's selected pore mouth, asymmetrically ﬁlled with a- Al2O3 of decreasing size. Fig. 8 e Dual optical-laser confocal imaging of a Hastelloy ﬁlter's selected pore mouth, asymmetrically ﬁlled with a- Al2O3 of decreasing size. For this reason, the 18 mm ﬁller is selected as base for the asymmetric support. To then improve the morphology of the support's surface, a 5 mm and 1.5 mm ﬁller are introduced subsequentially. The microscopy in Fig. 8 clearly highlights the presence of large a-Al2O3 ﬁller underneath the smaller particles, promoting uniform leveling of the pore mouths. Fig. 9 shows the cumulative ﬂow distribution through the pores of a sample ﬁlter ﬁlled with the selected asymmetric design. The mean ﬂow pore is sufﬁciently reduced around 100 nm, a comparable value to the one of the a-Al2O3 supports commonly used in ceramic membranes preparation. At this stage, most of the largest pore necks are reduced in size by the ﬁllers. However, about 10% of the inert gas ﬂow is still Fig. 9 e Cumulative percentage ﬂow distribution through the pores of an asymmetrically ﬁlled sample ﬁlter before and after boehmite layer deposition, measured via CFP. PdeAg deposition Al2O3þYSZ powder/e No additional layer 1,0$106 200 000 [34] Ceramic/e/double skin / e 4,6$106 26 000 [12] Inconel 600// YSZ powder þ boehmite -based sol/e Blow coated YSZ 3,4$106 8050 [38] PSS/0.2 mm/e CeO/1e4 mm No additional layer 1,2$106 inﬁnite [44] PSS/0.5 mm/polished and etched in alkaline sol and HCl / none 5,0$107 5000 [45] Inconel 600/0.5 mm/e YSZ/50 nm Blow coated YSZ 7,4$107 335 [46] PSS// YSZ/50 nm Blow coated YSZ 3,0$106 240 [47] Hastelloy X/0.5 mm/6 h polish / Boehmite 2,0$107 512 [24] Hastelloy X/0.5 mm/6 h polish, 30s aqua regia etch a-Al2O3/300 nm boehmite-based layer 1.2 wt% 1,1$106 ~6300 #MS, This work Hastelloy X/0.5 mm/6 h polish, 30s aqua regia etch a-Al2O3/300 nm boehmite-based layer 1.2 wt% 1,1$106 ~830 #MS2 This work Hastelloy X/0.5 mm/6 h polish, 30s aqua regia etch a-Al2O3/300 nm boehmite-based layer 1.2 wt% 9,2$107 ~3600 #MS3 This work Hastelloy X/0.5 mm/6 h polish, 30s aqua regia etch a-Al2O3/18 mm þ 5 mm þ1.5 mm boehmite-based layer 0.9 wt% 7,0$107 ~43 200 #MA, This work selectivity comparison of Pd-based membranes deposited via electroless plating onto steel-based ith similar methods. Table 4 e H2/N2 perm-selectivity comparison of Pd-based membranes deposited via electroless plat supports pre-treated with similar methods. i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 8 ( 2 0 2 3 ) 2 5 3 9 8 e2 5 4 0 9 25407 Fig. 13 e Ideal H2/N2 selectivity of #MA, measured at 400 C,450 C,500 C with a trans-membrane pressure of 1,2,3 bar. The measurements are carried out right after membrane annealing. choice of a suitable ﬁlling procedure proves essential to impact ﬁnal membrane performance and reproducibility. In Table 4 membranes prepared with different ﬁlling procedures are compared in terms of perm-selectivity at 400 C (see Fig. 11). PdeAg membranes prepared with a small size ﬁller present poor selectivity compared to the membrane prepared with the asymmetric design. In particular, symmetrically ﬁlled mem- brane #MS has been selected due to its high H2/N2 selectivity, and its performance is compared to #MAS, prepared on a support ﬁlled asymmetrically. Both membranes have been characterized in terms of activation energy via linear regres- sion through the Arrhenius plot in Fig. PdeAg deposition 12, where #MS shows an activation energy of ~6 kJ,mol1and #MA of 9,3 kJ mol1. While #MA is well in agreement with the activation energy range for thin Pd layer membranes, #MS has a lower value, possibly explained by the presence of scattered larger defects. This deduction is conﬁrmed by the n-values of each mem- brane, amounting to 0,71 and 0,51, respectively. #MA rate determining step for hydrogen transport is given by the Pd layer, while for #MS the value suggests an inﬂuence of the metallic support [34]. Fig. 13 e Ideal H2/N2 selectivity of #MA, measured at 400 C,450 C,500 C with a trans-membrane pressure of 1,2,3 bar. The measurements are carried out right after membrane annealing. #MA displays an outstanding H2/N2 selectivity (Fig. 13), accounting that this membrane is prepared on a ﬁlter with 0.5 mm media grade and 50 mm large superﬁcial pore mouths, the largest in literature obtaining membranes with selectivity >10000. This result proves that it is indeed possible to achieve high-performing membranes using unreﬁned metallic ﬁlters. PdeAg membranes prepared with the same ﬁller aspiration- barrier deposition technique reported in literature show promising performances on steel-based supports with larger media grades (Table 4). This two-step procedure therefore classiﬁes itself as a promising standard method for composite PSS/ceramic/Pd based membranes on cheaper support options. Fig. 11 e H2 permeating ﬂux vs H2 partial pressure at 400 C for #MA and #MS. Fig. 12 e Linear regression performed on Arrhenius plot to determine membrane's activation energy as slope (DEa, in J,mol¡1) and pre-exponential factor as intercept. Fig. 11 e H2 permeating ﬂux vs H2 partial pressure at 400 C for #MA and #MS. Fig. 11 e H2 permeating ﬂux vs H2 partial pressure at 400 C for #MA and #MS. Fig. 12 e Linear regression performed on Arrhenius plot to determine membrane's activation energy as slope (DEa, in J,mol¡1) and pre-exponential factor as intercept. r e f e r e n c e s [15] Cechetto V, Di Felice L, Medrano JA, MakhlouﬁC, Zuniga J, Gallucci F. H2 production via ammonia decomposition in a catalytic membrane reactor. Fuel Process Technol 2021;216:106772. https://doi.org/10.1016/ j.fuproc.2021.106772. [1] Fernandez E, et al. Palladium based membranes and membrane reactors for hydrogen production and puriﬁcation: an overview of research activities at Tecnalia and TU/e. 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https://openalex.org/W2106401668	https://europepmc.org/articles/pmc3120745?pdf=render	English	null	Echocardiographic predictors of early in-hospital heart failure during first ST-elevation acute myocardial infarction: does myocardial performance index and left atrial volume improve diagnosis over conventional parameters of left ventricular function?	Cardiovascular ultrasound	2,011	cc-by	5,461	© 2011 Souza et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH Open Access Abstract Background: Left ventricular ejection fraction (LVEF) has been considered a major determinant of early outcome in acute myocardial infarction (AMI). Myocardial performance index (MPI) has been associated to early evolution in AMI in a heterogeneous population, including non ST-elevation or previous AMI. Left atrial volume has been related with late evolution after AMI. We evaluated the independent role of clinical and echocardiographic variables including LVEF, MPI and left atrial volume in predicting early in-hospital congestive heart failure (CHF) specifically in patients with a first isolated ST-elevation AMI. Methods: Echocardiography was performed within 30 hours of chest pain in 95 patients with a first ST-elevation AMI followed during the first week of hospitalization. Several clinical and echocardiographic variables were analyzed. CHF was defined as Killip class ≥II. Multivariate regression analysis was used to select independent predictor of in-hospital CHF. Results: Early in-hospital CHF occurred in 29 (31%) of patients. LVEF ≤0.45 was the single independent and highly significant predictor of early CHF among other clinical and echocardiographic variables (odds ratio 17.0; [95% CI 4.1 - 70.8]; p < 0.0001). MPI alone could not predict CHF in first ST-elevation AMI patients. Left atrial volume was not associated with early CHF in such patients. Conclusion: For patients with first, isolated ST-elevation AMI, LVEF assessed by echocardiography still constitutes a strong and accurate independent predictor of early in-hospital CHF, superior to isolated MPI and left atrial volume in this particular subset of patients. Keywords: acute myocardial infarction, echocardiography, myocardial performance index, left atrial volume, ejec- tion fraction Echocardiographic predictors of early in-hospital heart failure during first ST-elevation acute myocardial infarction: does myocardial performance index and left atrial volume improve diagnosis over conventional parameters of left ventricular function? ian P Souza, Orlando Campos, Clovis A Peres, Cristiano V Machado and Antonio C Carvalho ian P Souza, Orlando Campos, Clovis A Peres, Cristiano V Machado and Antonio C Carvalho CARDIOVASCULAR ULTRASOUND CARDIOVASCULAR ULTRASOUND Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 CARDIOVASCULAR ULTRASOUND * Correspondence: ocampos@cardiol.br Cardiology Department, Escola Paulista de Medicina, Federal University of Sao Paulo, UNIFESP, Brazil Echocardiography h block, cardiac rupture and pericardial effusion), not always solely related to the extend of LV dysfunction in the acute phase of AMI. These factors may justify some controversy about the short-term independent prognostic significance of MPI in AMI patients, defended by some [10,11,14] but questioned by others [12,13]. Therefore, the value of MPI in predicting early in-hospital development CHF particu- larly in isolated, first ST-elevation AMI it is not yet fully established. A comprehensive two-dimensional, spectral and color flow Doppler echocardiographic examination was performed in all patients within 24 hours of arrival at our coronary care unit (within 48 hours of chest pain). We used commer- cially available equipment (ATL-HDI 5000; Philips Medi- cal System, Bothell, Wash.) with a P4-2 MHz transducer. Harmonic images and Doppler studies were recorded for further analysis. Indexes of global and segmental systolic function, diastolic function and combined systolic and dia- stolic functions were obtained. LV volumes and ejection fraction (EF) were determined using the modified biplane Simpson’s method by orthogonal apical views (2 and 4- chambers), using mean values of 3 cardiac cycles [19]. Wall motion score index (WMSI) was calculated by using a 17-segment model proposed by the American Heart Association [19]. LA volume was obtained from the apical 4-chamber view at end systole by the method of discs [20], indexed for body surface area. Pulsed Doppler curves of blood flow were assessed by the apical 4-chamber view. Mitral diastolic inflow velocities were obtained at the tip of leaflets; LV outflow systolic flow curves were obtained just below the aortic valve closure plane. Mean values of peak velocities resulted from 5 consecutive cardiac cycles. The following variables were calculated: ratio of mitral E/ A wave diastolic velocities; deceleration time (DT) of early LV diastolic filling; patterns of mitral diastolic filling (nor- mal, abnormal relaxation, pseudonormal and restrictive filling). Myocardial performance index (MPI) was calcu- lated by the method proposed by Tei [6,21], derived from its components: isovolumetric contraction time (IVCT), ejection time (ET) and isovolumetric relaxation time (IVRT), as previously described [10,11]. A single investiga- tor performed the echocardiographic exams. One experi- enced observer, blinded to clinical data, made further interpretation. The left atrial (LA) volume measurement constitutes another new echocardiographic parameter that has been studied in post-AMI patients. Echocardiography h Increased LA volume has been considered an independent predictor of adverse late outcome in patients with AMI and prior myocardial infarction and in patients with non-ST elevation AMI [15,16]. As far as we know, there is no description of the prognostic value of this index during the acute phase of first ST-elevation AMI. We aimed to analyze the role of the MPI and LA volume compared to other conventional parameters of systolic and diastolic LV function in an homogeneous group of patients with a first, isolated ST-elevation AMI, in predicting early CHF during in-hospital evolution. Methods Patients We studied prospectively 95 consecutive patients (58 ± 12 years old, 64 males) admitted to our coronary care unit with a first ST-elevation AMI, defined as characteristic chest pain lasting for more than 20 minutes, typical ST segment elevation > 1 mm in at least two contiguous leads associated with transient rise of creatine kinase MB. Exclu- sion criteria comprised: previous AMI, non-ST elevation AMI, early reinfarction, in-hospital death, previous coron- ary bypass surgery or angioplasty, left bundle branch block, non sinus rhythm, valvular heart disease, dilated cardiomyopathy and echocardiographic images of poor quality. Introduction An increasing number of studies has reported the use of a combined index integrating systolic and diastolic LV function, the overall myocardial performance index (MPI) [9], for predicting short-term adverse outcome in AMI [10-14]. However, some included high-risk patients with multiple myocardial infarction [10,13] and previous history of CHF [13] or with non-ST elevation [10-13], while others studied only anteroseptal AMI [14], that could affect their results. Moreover, some authors [11,13,14] considered diverse in-hospital complications besides CHF (recurrent angina, reinfarction, death, arrhythmias, heart Early detection of patients with acute myocardial infarc- tion (AMI) at risk of development of in-hospital conges- tive heart failure (CHF) is necessary to limit myocardial injury and left ventricular (LV) dysfunction. Non-invasive evaluation of LV function has been assessed by systolic as well diastolic echocardiographic indexes and related to short-term clinical outcome [1-8]. * Correspondence: ocampos@cardiol.br Cardiology Department, Escola Paulista de Medicina, Federal University of Sao Paulo, UNIFESP, Brazil Page 2 of 6 Page 2 of 6 Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Legend: data expressed by mean ± dp or absolute values and frequency (%); CHF: congestive heart failure; PTI: percutaneous transluminal intervention; CK: creatine kinase; angiography: non obstructive: obstruction absent or < 70% of luminal diameter, one vessel disease: obstruction ≥70% of luminal diameter in one or more epicardial coronary arteries, multivessel disease: obstruction ≥70% of luminal diameter in ≥2 epicardial coronary arteries. Clinical data All patients presented a first, isolated ST-elevation AMI with wall motion abnormalities shown on echocardio- graphy. Prevalence of anterior location (57%) on EKG was not significantly different from inferior infarction (43%; p > 0.05). Thrombolysis or primary angioplasty was performed in the majority of patients (80%). Signifi- cant coronary artery disease (≥70% of obstruction) was present in at least one major epicardial vessel in 93% of patients who underwent coronary angiography. Left ventricular end diastolic and systolic volumes, as well as indexed LA volumes were similar in both groups. No differences between the groups were found concern- ing other variables (E/A ratio, DT, IVCT and IVRT). Multivariate analysis T bl 3 i Table 3 summarizes the selected cut-off values of echocar- diographic continuous variables with higher statistical sig- nificance (LVEF, WMSI and MPI), with respective diagnostic indexes and area under the curves. A LVEF ≤ 0.45 had the best diagnostic performance. An analysis of interaction between variables with highest clinical rele- vance was made, involving age, CKMB levels, diastolic restrictive pattern (DT < 140), LVEF, WMSI and MPI. During the first week of hospitalization (mean: 5 days), 29 (31%) patients presented CHF (Killip class II:16; III: 5; IV: 8). Univariate comparison of clinical variables in both groups of patients are expressed in Table 1. Those with early CHF were significantly older, with a higher level of creatine kinase MB release. There was no difference between the groups regarding history of hypertension, diabetes, site of infarction, reperfusion therapy, use of betablockers, ACE inhibi- tors and multivessel disease. The final model selected the variables demonstrated in Tables 4 and 5. LVEF was the single independent variable significantly related to in-hospital CHF in this series of patients. Those with a first ST-elevation AMI and a LVEF ≤0.45 obtained by echocardiography at admission showed a higher and significant chance of developing CHF in the first week of hospitalization (odds ratio [OR] 17.0; 95% confidence interval [CI] 4.1 - 70.8; p < 0.0001). MPI alone failed to predict early CHF (p > 0.05) by logis- tic regression. Only when conditioned to older age ( > 60 years old), a MPI ≥0.57 was associated to in-hospital CHF (OR 13.7; 95% CI 2.7 - 68.6; p: 0.02), after interac- tion analysis. The LA volume and all other echocardio- graphic variables considered in logistic regression (WMSI, LV systolic volume, abnormal diastolic patterns, Statistical Analysis Clinical and echocardiographic variables were expressed by mean values ± 1 standard deviation or proportion of patients with a determined characteristic. Comparison of continuous variables was made by a 2-sample “t” Stu- dent test or Mann-Withney rank sum test. Discrete vari- ables were compared by Chi-square or Fisher’s exact test. Linear regression was used to study the relationship between two continuous variables. A receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off point of some echocardio- graphic variables. We used univariate analysis followed by a complete model of multivariate logistic regression analysis to identify independent predictors of early CHF, including clinical and echocardiographic variables alto- gether, studied with interactions. A “p” value < 0.05 was considered to be significant. Patients were observed during daily in-hospital evolution, after receiving conventional clinical therapy (90% with betablockers and angiotensin converting enzyme inhibi- tors). Reperfusion therapy by thrombolysis or primary per- cutaneous angioplasty was instituted according standard guidelines [17]. Those without primary angioplasty were submitted to elective coronary angiography before hospital discharge for invasive risk stratification. In-hospital primary end-point was defined as the development of new-onset CHF in the first week of hospitalization, based on Killip functional status [18]. According to this classification, two groups of patients were established: without CHF (Killip class = I) and with CHF (Killip class ≥II). The study protocol was approved by the institutional ethical committee of our institution. Informed consent was obtained for each patient. Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Page 3 of 6 associated with early CHF (p:0.06). The mean values of DT were not significantly different in patients with or without CHF. Echocardiographic data The echocardiographic variables of the two groups of patients are shown in Table 2. The variables significantly related to the development of early CHF by univariate analysis were: lower LVEF (p < 0.001), higher values of WMSI (p < 0.001), higher values of MPI (p < 0.01) and its component ET (p < 0.05). There was a tendency of abnormal diastolic patterns to be related to early CHF (p: 0.06). There was a tendency of restrictive pattern (DT < 140 ms), present in only 8 patients, to be Table 1 Clinical variables according to absence or presence of early CHF CHF absent (n = 66) CHF present (n = 29) p Age (years) 54.9 ± 10.2 65.8 ± 14.2 < 0.001 Systemic Hypertension 32 (49%) 18 (62%) 0.22 Diabetes Mellitus 12 (18%) 5 (17%) 0.91 Site of infarction Anterior 35 (53%) 19 (66%) 0.26 Inferior 31 (47%) 10 (34%) Treatment PTI 26 (39%) 10 (35%) Trombolysis 30 (46%) 10 (34.5%) Conservative 10 (15%) 9 (31%) 0.19 CKMB peak (iu/l) 205 ± 138 346 ± 239 0.004 Angiography Non obstructive 5 (8%) 1 (3%) One vessel disease 34 (51%) 10 (35%) Multivessel disease 22 (33%) 15 (52%) Not done 5 (8%) 3 (10%) 0.31 Legend: data expressed by mean ± dp or absolute values and frequency (%); CHF: congestive heart failure; PTI: percutaneous transluminal intervention; CK: creatine kinase; angiography: non obstructive: obstruction absent or < 70% of luminal diameter, one vessel disease: obstruction ≥70% of luminal diameter in one or more epicardial coronary arteries, multivessel disease: obstruction ≥70% of luminal diameter in ≥2 epicardial coronary arteries. Table 1 Clinical variables according to absence or presence of early CHF Table 1 Clinical variables according to absence or presence of early CHF Legend: data expressed by mean ± dp or absolute values and frequency (%); CHF: congestive heart failure; PTI: percutaneous transluminal intervention; CK: creatine kinase; angiography: non obstructive: obstruction absent or < 70% of luminal diameter, one vessel disease: obstruction ≥70% of luminal diameter in one or more epicardial coronary arteries, multivessel disease: obstruction ≥70% of luminal diameter in ≥2 epicardial coronary arteries. Souza et al. Echocardiographic data Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Page 4 of 6 Table 2 Echocardiographic variables according to presence or absence of early CHF Echocardiographic data CHF absent (n = 66) CHF present (n = 29) p LVEF 0.51 ± 0.07 0.44 ± 0.07 < 0.001 WMSI 1.57 ± 0.31 1.91 ± 0.35 < 0.001 LVESVI (mL/m2) 18.4 ± 7.6 20.8 ± 8.7 0.18 LVEDVI (mL/m2) 37.3 ± 12.2 37.0 ± 11.0 0.88 LAVI (mL/m2) 18.7 ± 4.8 20.6 ± 5.7 0.10 E/A ratio 1.02 ± 0.35 0.99 ± 0.50 0.76 DT (ms) 210 ± 61.9 212.7 ± 66.8 0.85 DT ≤140 ms 4 (6.1%) 4 (13.8%) 0.24 Diastolic patterns Normal 28 (42.4%) 5 (17.2%) 0.06 Abnormal relaxation 27 (40.9%) 14 (48.3%) Pseudonormal 7 (10.6%) 6 (20.7%) Restrictive 4 (6.1%) 4 (13.8%) MPI 0.57 ± 0.14 0.65 ± 0.16 0.01 ET (ms) 262.3 ± 22.8 247.5 ± 31.8 0.03 IVCT (ms) 46.5 ± 24.2 49.5 ± 27.3 0.60 IVRT (ms) 102.0 ± 26.6 108.4 ± 29.4 0.29 Legend: data expressed by mean ± dp or frequency (%); CHF: congestive heart failure; LVEF: left ventricular ejection fraction; WMSI: wall motion score index; LVESVI: left ventricular end systolic volume index; LVEDVI: left ventricular end diastolic volume index; LAVI: left atrial volume index; E/A:ratio of mitral E and A wave diastolic velocities; DT:deceleration time; MPI: myocardial performance index; ET: ejection time; IVCT: isovolumetric contraction time; IRVT: isovolumetric relaxation time; ms = miliseconds, ml/m2 = mililiters per square meter. Table 2 Echocardiographic variables according to presence or absence of early CHF E h di hi d t CHF b t ( 66) CHF t Legend: data expressed by mean ± dp or frequency (%); CHF: congestive heart failure; LVEF: left ventricular ejection fraction; WMSI: wall motion score index; LVESVI: left ventricular end systolic volume index; LVEDVI: left ventricular end diastolic volume index; LAVI: left atrial volume index; E/A:ratio of mitral E and A wave diastolic velocities; DT:deceleration time; MPI: myocardial performance index; ET: ejection time; IVCT: isovolumetric contraction time; IRVT: isovolumetric relaxation time; ms = miliseconds, ml/m2 = mililiters per square meter. ET and IVCT), as well as the remaining clinical variables, were excluded by multivariate analysis. ET and IVCT), as well as the remaining clinical variables, were excluded by multivariate analysis. excluded in the multivariate analysis when all clinical and echocardiographic variables were included in the com- plete model of multiple logistic regression. Echocardiographic data Therefore, in first ST-elevation AMI patients, MPI alone was of limited short-term prognostic value in respect to in-hospital CHF. The interaction analysis between variables of our study demonstrated that only in patients older than 60 years or more, a MPI ≥0.57 could be predictive of early CHF in first ST-elevation AMI (p < 0.02). Legend: LVEF: left ventricular ejection fraction; MPI: myocardial performance index, MPIAge: interaction between variables MPI and age. Discussion Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Page 5 of 6 Page 5 of 6 Table 5 Hazard ratio with confidence intervals of independent predictive variables Variable Hazard ratio 95% CI LVEF ≤0.45 17.0 (4.1 - 70.8) Age ≥60 and MPI < 0.57 0.5 (0.1 - 4.1) Age ≥60 and MPI ≥0.57 13.7 (2.7 - 68.6) Legend: CI: confidence interval; LVFE = left ventricular ejection fraction; MPI: myocardial performance index. Table 5 Hazard ratio with confidence intervals of independent predictive variables motion, WMSI could not have independent significance in predicting early CHF in these patients, as observed by others [11,14]. Like other authors [11,14] we did not find any independent prognostic value of conventional diasto- lic function parameters in the early echocardiogram of first ST- elevation AMI patients in predicting early CHF. A restrictive pattern in the exam at admission was pre- sent in only 8 (8.4%) patients in our series, and could be more prevalent in later periods of evolution [6], leading to a more significant prognostic information. one month after AMI. According to them, MPI had a low predictive accuracy with no additional early prognostic value in AMI. In our patients with first ST-elevation AMI, LA volume index was not significantly different between patients with or without CHF in the univariate analysis. This result was expected, since LA remodeling could not occur within 48 hours of initial presentation of a first AMI, because it is not a marker of acute changes in dia- stolic function and/or increased filling pressures, as the instantaneous transmitral diastolic flow parameters are [23]. Other studies described the long-term prognostic value of LA volume in AMI, including a significant pro- portion of patients with prior myocardial infarction, with a longer period of out-hospital follow-up [15,16]. It seems that for patients with isolated first ST-elevation AMI, echocardiographic structural indexes of LA volume are not useful for predicting early in-hospital CHF. On the contrary, other authors demonstrated the inde- pendent prognostic role of MPI in early stages of AMI, superior to LVEF [10,11,14]. Different clinical characteris- tics of their cohorts, like exclusive analysis of anteroseptal AMI [14], broader spectrum of clinical presentation including non-ST elevation AMI [10,11] and multiple infarcts [10] could in part explain their results. Discussion Our study re-emphasizes the power of LVEF, when ana- lyzing other clinical and some more recent echocardio- graphic variables assessed at admission, in predicting early in-hospital CHF in the acute phase of an isolated, first ST-elevation AMI. Particularly in this subset of patients, an LVEF ≤0.45 was the strongest independent and highly significant variable (p < 0.0001) associated to the development of Killip class ≥II. As a marker of myocardial dysfunction in the first week of hospitaliza- tion, it was superior to MPI or LA volume. These results were valid for both anterior and inferior location, irrespective of patient age. In a retrospective study, Lavine [12] found similar results, describing the superiority of LVEF over MPI and diastolic patterns in predicting progressive development of CHF in the first 15 days of admission of a selected subset of patients with first ST-elevation AMI, without any clini- cal evidence of CHF at admission. According to this author, a LVEF < 0,40 was strongly related to CHF, and MPI alone had modest additional prognostic value. Schwammenthal et al. [13] also reported a LVEF ≤0.40 as a powerful and independent predictor of poor outcome Although univariate analysis showed MPI and WMSI to be significantly associated with early CHF, they were Table 3 Cut-off values of echocardiographic continuous variables with statistical significance with respective diagnostic indexes and area under curves Table 3 Cut-off values of echocardiographic continuous variables with statistical significance with respective diagnostic indexes and area under curves Variable Cut- off S (%) E (%) PPV (%) NPV (%) Area under curves ROC LVEF 0.45 87.9 62.1 84.0 69.2 0.77 WMSI 1.8 65.5 75.8 54.3 83.3 0.77 MPI 0.57 79.3 54.6 43.4 85.7 0.67 Legend: S: sensitivity; E: specificity; PPV: positive predictive value; NPV: negative predictive value; ROC: receiver operator characteristics; LVEF: left ventricular ejection fraction; WMSI: wall motion score index; MPI: myocardial performance index. Table 4 Logistic regression model with interaction analysis Table 4 Logistic regression model with interaction analysis Variable Regression Coefficient Standart Error p Constant -3.1329 0.8195 0.1553 LVEF 2.8343 0.7275 0.0001 Age -0.6230 1.0350 0.5472 MPI -0.1780 0.8612 0.8362 MPI Age 3.2431 1.3921 0.0198 Legend: LVEF: left ventricular ejection fraction; MPI: myocardial performance index, MPI*Age: interaction between variables MPI and age. Souza et al. Study limitations l b Our results about the prognostic influence of abnormal diastolic pattern are not conclusive for first ST-elevation AMI, due to the limited number of patients with restric- tive LV filling in the present series. We did not study the E/e’ velocities ratio as an important marker of LV filling pressure, also useful in short-term prognosis after AMI [24], because e’ waves derived from tissue Doppler tra- cings of septal and lateral mitral annulus were not avail- able in all patients, limiting us to obtain this variable. Recent recommendations emphasize the use of averaged values of septal and lateral e’ waves to calculate E/e’ ratio in patients with segmental myocardial dysfunction, as it occurs in AMI [25]. So, this important surrogate of left atrial pressure can not be ruled out as a valuable prog- nostic index of early CHF in this subset of patients with a first ST-elevation AMI. Not only diastolic (E/e’), but also systolic (s’) tissue Doppler-derived indices seems to be useful in early evaluation of patients with AMI and LV dysfunction [26], as well as new markers of myocardial deformation [27]. The prognostic role of these variables should be addressed in further studies in this clinical setting. We did not consider adverse events during in-hospital evolution that could be related to other factors and not to LV dysfunction, like recurrent angina or early malig- nant arrhythmias due to electrical instability that could lead to different results. In the series of Yuasa et al [14] among various other end-points (death, paroxysmal atrial fibrillation, ventricular tachycardia/fibrillation, advanced atrioventricular block, pericardial effusion and cardiac rupture), CHF was present in only 19% of the patients. In our study, LVEF with a cut-off value of 0.45 as defined by a ROC curve presented has having the best diagnostic performance in predicting in-hospital CHF. Contrasting to the prognostic value of global assessment of LV systolic function by EF, our study did not demon- strate additional prognostic information of quantitative regional LV function analysis provided by WMSI. Because of interdependence of both variables (LVEF and WMSI) and some subjectivity when analyzing regional Discussion Non ST- elevation AMI constitutes a particularly heterogeneous subset of patients with different pathophysiologic, prog- nostic and therapeutic features than ST-elevation AMI patients [22], and are difficult to be compared when acute coronary syndromes are analyzed. Multiple infarcts are often associated with LV remodeling and greater systolic and diastolic dysfunction that could be expressed by higher MPI values, as it occurs in dilated cardiomyopathy [21]. MPI, as an integrated index of both systolic and dia- stolic LV function, could be previously elevated in patients with multiple large infarcts, even before the occurrence of additional myocardial injury induced by a novel episode of AMI. In patients with ST-elevation AMI and no previous infarction, without significant LV enlargement like the present series, MPI alone did not show any advantage over conventional LVEF in short-term prognosis. References Oh JK, Ding ZP, Gersh BJ, Bailey KR, Tajik AJ: Restrictive left ventricular diastolic filling identifies patients with heart failure after acute myocardial infarction. J Am Soc Echocardiogr 1992, 5:497-503. 24. Hillis GS, Moller JE, Pellikka PA, Gersh BJ, Wright RS, Ommen SR, Reeder GS, Oh JK: Noninvasive estimation of left ventricular filling pressure by E/e’ is a powerful predictor of survival after acute myocardial infarction. J Am Coll Cardiol 2004, 43:360-7. 6. Nijland F, Kamp O, Karreman AJ, van Eenige MJ, Visser CA: Prognostic implications of restrictive left ventricular filling in acute myocardial infarction: a serial Doppler echocardiographic study. J Am Coll Cardiol 1997, 30:1618-24. 6. Nijland F, Kamp O, Karreman AJ, van Eenige MJ, Visser CA: Prognostic implications of restrictive left ventricular filling in acute myocardial infarction: a serial Doppler echocardiographic study. J Am Coll Cardiol 1997, 30:1618-24. 25. Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, Waggoner AD, Flachskampf FA, Pellikka PA, Evangelista A: Recommendations for the evaluation of left ventricular diastolic function by echocardiography. J Am Soc Echocardiogr 2009, 22:108-133. 7. Poulsen SH, Jensen SE, Egstrup K: Longitudinal changes and prognostic implications of left ventricular diastolic function in first acute myocardial infarction. Am Heart J 1999, 137:910-8. 8. Naqvi TZ, Padmanabhan S, Rafii F, Hyuhn HK, Mirocha J: Comparison of usefulness of left ventricular diastolic versus systolic function as a predictor of outcome following primary percutaneous coronary angioplasty for acute myocardial infarction. Am J Cardiol 2006, 97:160-6. 26. Hameed AK, Gosal T, Fang T, Ahamadie R, Lytwyn M, Barac I, Zieroth S, Hussain F, Jassal DS: Clinical utility of tissue Doppler imaging in patients with acute myocardial infarction complicated by cardiogenic shock. Cardiovascular Ultrasound 2008, 6:11. 9. Tei C: New non-invasive index for combined systolic and diastolic ventricular function. J Cardiol 1995, 26:135-6. 27. Caracciolo G, Eleid MF, Abe H, Bhatia N, Fortuin FD, Wilansky S, Carerj S, Sengupta PP: Non-uniform recovery of left ventricular transmural mechanics in ST-segment elevation myocardial infarction. Cardiovascular Ultrasound 2010, 8:31. 10. Poulsen SH, Jensen SE, Tei C, Seward JB, Egstrup K: Value of the Doppler index of myocardial performance in the early phase of acute myocardial infarction. J Am Soc Echocardiogr 2000, 13:723-30. 11. Abbreviations AMI 15. Moller JE, Egstrup K, Kober L, Poulsen SH, Nyvad O, Top-Pedersen C: Prognostic importance of systolic and diastolic function after myocardial infarction. Am Heart J 2003, 145:147-53. AMI: acute myocardial infarction; LVEF: left ventricular ejection fraction; MPI: myocardial performance index; CHF: congestive heart failure; LA: left atrial; WMSI: wall motion score index; DT: deceleration time; IVRT: isovolumic relaxation time; IVCT: isovolumic contraction time; ET: ejection time 16. Beinart R, Boyko V, Schwammenthal E, Kuperstein R, Sagie A, Hod H, Matetzky S, Behar S, Eldar M, Feinberg MS: Long-Term prognostic significance of left atrial volume in myocardial infarction. J Am Coll Cardiol 2004, 44:327-34. Authors’ contributions LPS: participated in the study design, carried out the echocardiographic examinations and draft the manuscript; OC: conceived the study, participated in its design, performed its coordination and contributed to write the manuscript; CAP: participated in the study design and performed the statistical analysis; CVM: contributed in the study design and reviewed the manuscript; ACC: participated in the study design and in the revision of the manuscript. All authors approved the final version of the manuscript. 17. Antman EM, Anbe DT, Armstrong PW, Bates ER, Green LA, Hand M, Hochman JS, Krumholz HM, Kushner FG, Lamas GA, Mullany CJ, Ornato JP, Pearle DL, Sloan MA, Smith SC Jr, Alpert JS, Anderson JL, Faxon DP, Fuster V, Gibbons RJ, Gregoratos G, Halperin JL, Hiratzka LF, Hunt SA, Jacobs AK, Ornato JP: ACC/AHA guidelines for the management of patients with ST-elevation myocardial infarction; A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 1999 Guidelines for the Management of patients with acute myocardial infarction). J Am Coll Cardiol 2004, 44:E1-E211. Conclusion In this series of patients with a first, isolated ST-eleva- tion AMI without significant LV dilation, LVEF was the single independent predictor or early CHF. MPI alone Page 6 of 6 Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 Souza et al. Cardiovascular Ultrasound 2011, 9:17 http://www.cardiovascularultrasound.com/content/9/1/17 or LA volume failed to predict in-hospital CHF during the first week of evolution in this particular subset of patients. in acute myocardial infarction: comparison to measures of systolic and diastolic left ventricular function. Chest 2003, 124:1645-51. in acute myocardial infarction: comparison to measures of systolic and diastolic left ventricular function. Chest 2003, 124:1645-51. 14. 14. Yuasa T, Otsuji Y, Kuwahara E, Takasaki K, Yoshifuku S, Yuge K, Kisanuki A, Toyonaga K, Lee S, Toda H, Kumanohoso T, Hamasaki S, Matsuoka T, Biro S, Minagoe S, Tei C: Noninvasive prediction of complications with anteroseptal acute myocardial infarction by left ventricular Tei index. J Am Soc Echocardiogr 2005, 18:20-5. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. 18. Killip T, Kimball JT: Treatment of myocardial infarction in coronary care unit. A two-year experience with 250 patients. Am J Cardiol 1967, 20:457-64. Received: 16 January 2011 Accepted: 3 June 2011 Published: 3 June 2011 Received: 16 January 2011 Accepted: 3 June 2011 Published: 3 June 2011 19. Cerqueira MD, Weissman NJ, Dilsizian V, Jacobs AK, Kaul S, Warren K, Laskey WK, Pennell DJ, Rumberger JA, Ryan TMD, Verani MS, American Heart Association Writing Group on Myocardial Segmentation and Registration for Cardiac Imaging: Standardized Myocardial Segmentation and Nomenclature for Tomographic Imaging of the Heart. Circulation 2002, 105:539-542. References 1. Nishimura RA, Tajik AJ, Shub C, Miller FA Jr, Ilstrup DM, Harrison CE: Role of two-dimensional echocardiography in the prediction of in-hospital complications after acute myocardial infarction. J Am Coll Cardiol 1984, 4:1080-7. 2. Pierard LA, Albert A, Chapelle JP, Carlier J, Kulbertus HE: Relative prognostic value of clinical, biochemical, echocardiographic and haemodynamic variables in predicting in-hospital and one-year cardiac mortality after acute myocardial infarction. Eur Heart J 1989, 10:24-31. 2. Pierard LA, Albert A, Chapelle JP, Carlier J, Kulbertus HE: Relative prognostic value of clinical, biochemical, echocardiographic and haemodynamic variables in predicting in-hospital and one-year cardiac mortality after acute myocardial infarction. Eur Heart J 1989, 10:24-31. 20. Lester SJ, Ryan EW, Schiller NB, Foster E: Best method in clinical practice and in research studies to determine left atrial size. Am J Cardiol 1999, 84:829-32. 21. Tei C, Ling LH, Hodge DO, Bailey KR, Oh JK, Rodeheffer RJ, Tajik AJ, Seward JB: New index of combined systolic and diastolic myocardial performance: a simple and reproducible measure of cardiac function-a study in normals and dilated cardiomyopathy. J Cardiol 1995, 26:357-66. 3. Berning J, Launbjerg J, Appleyard M: Echocardiographic algorithms for admission and predischarge prediction of mortality in acute myocardial infarction. Am J Cardiol 1992, 69:1538-44. 3. Berning J, Launbjerg J, Appleyard M: Echocardiographic algorithms for admission and predischarge prediction of mortality in acute myocardial infarction. Am J Cardiol 1992, 69:1538-44. 4. Launbjerg J, Berning J, Fruergaard P, Appleyard M: Sensitivity and specificity of echocardiographic identification of patients eligible for safe early discharge after acute myocardial infarction. Am Heart J 1992, 124:846-53. 4. Launbjerg J, Berning J, Fruergaard P, Appleyard M: Sensitivity and specificity of echocardiographic identification of patients eligible for safe early discharge after acute myocardial infarction. Am Heart J 1992, 124:846-53. 22. Romano S, Dagianti A, Penco M, Varveri A, Biffani E, Fedele F, Dagianti A: Usefulness of echocardiography in the prognostic evaluation of non-Q- wave myocardial infarction. Am J Cardiol 2000, 86:43G-45G. 23. Abhayaratna WP, Seward JB, Appleton CP, Douglas PS, Oh JK, Tajik AJ, Tsang TS: Left atrial size: physiologic determinants and clinical applications. J Am Coll Cardiol 2006, 47:2357-63. 5. Oh JK, Ding ZP, Gersh BJ, Bailey KR, Tajik AJ: Restrictive left ventricular diastolic filling identifies patients with heart failure after acute myocardial infarction. J Am Soc Echocardiogr 1992, 5:497-503. 5. References Ascione L, De Michele M, Accadia M, Rumolo S, Damiano L, D’Andrea A, Guarini P, Tuccillo B: Myocardial global performance index as a predictor of in-hospital cardiac events in patients with first myocardial infarction. J Am Soc Echocardiogr 2003, 16:1019-23. doi:10.1186/1476-7120-9-17 Cite this article as: Souza et al.: Echocardiographic predictors of early in- hospital heart failure during first ST-elevation acute myocardial infarction: does myocardial performance index and left atrial volume improve diagnosis over conventional parameters of left ventricular function? Cardiovascular Ultrasound 2011 9:17. doi:10.1186/1476-7120-9-17 Cite this article as: Souza et al.: Echocardiographic predictors of early in- hospital heart failure during first ST-elevation acute myocardial infarction: does myocardial performance index and left atrial volume improve diagnosis over conventional parameters of left ventricular function? Cardiovascular Ultrasound 2011 9:17. 12. Lavine SJ: Prediction of heart failure post myocardial infarction: comparison of ejection fraction, transmitral filling parameters, and the index of myocardial performance. Echocardiography 2003, 20:691-701. 13. Schwammenthal E, Adler Y, Amichai K, Sagie A, Behar S, Hod H, Feinberg MS: Prognostic value of global myocardial performance indices 13. Schwammenthal E, Adler Y, Amichai K, Sagie A, Behar S, Hod H, Feinberg MS: Prognostic value of global myocardial performance indices
https://openalex.org/W1978624673	https://europepmc.org/articles/pmc3835106?pdf=render	English	null	An Overview of Biomarkers and Molecular Signatures in HCC	Cancers	2,010	cc-by	7,571	Seon-Hee Yim 1 and Yeun-Jun Chung 1,2,* 1 Integrated Research Center for Genome Polymorphism, The Catholic University of Korea School of Medicine, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea; E-Mail: lyra@catholic.ac.kr (S.Y.) 1 Integrated Research Center for Genome Polymorphism, The Catholic University of Korea School of Medicine, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea; E-Mail: lyra@catholic.ac.kr (S.Y.) 2 Department of Microbiology, The Catholic University of Korea School of Medicine, 505 Banpo- dong, Seocho-gu, Seoul 137-701, Korea Integrated Research Center for Genome Polymorphism, The Catholic University of K Medicine, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea; E-Mail: lyra@catholi 2 Department of Microbiology, The Catholic University of Korea School of Medicine, 505 Banpo- dong, Seocho-gu, Seoul 137-701, Korea * Author to whom correspondence should be addressed; E-Mail: yejun@catholic.ac.kr; Tel.: +82-2-2258-7343; Fax: +82-596-8969. * Author to whom correspondence should be addressed; E-Mail: yejun@catholic.ac.kr; Tel.: +82-2-2258-7343; Fax: +82-596-8969. Received: 19 March 2010; in revised form: 30 April 2010 / Accepted: 7 May 2010 / Published: 7 May 2010 Abstract: Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality worldwide. Although most HCCs seem to originate from the accumulation of genetic abnormalities induced by various risk factors, underlying mechanisms of hepatocarcinogenesis remain unclear. Long-term survival of HCC patients is also poor, partly due to HCC recurrence. Although serum alpha-fetoprotein (AFP) level is a useful marker for the detection and monitoring of HCC, AFP levels may remain normal in the patients even with advanced HCC. To identify useful biomarkers for HCC, many studies have been conducted on molecular events such as genetic and epigenetic alterations, and gene expression. This review summarizes recent studies of potential molecular markers for diagnosis and monitoring metastasis or recurrence of HCC. Keywords: hepatocellular carcinoma; biomarker; molecular classification cancers OPEN ACCESS ISSN 2072-6694 www.mdpi.com/journal/cancers Review Cancers 2010, 2, 809-823; doi:10.3390/cancers2020809 Cancers 2010, 2, 809-823; doi:10.3390/cancers2020809 2.1. Serum DNA It is known that some tumors release cells and cell lysis material including DNA into the blood, which could be used as potential biomarkers for detection of cancers. Zhang et al. reported that methylated alleles of p16, p15, and RASSF1A were found in serum DNA of 44%, 24%, and 70% of HCC patients, but detected in 4%, 0%, and 6% of control serum DNA samples, respectively [4]. These methylated alleles could be detected one to nine years before the clinical diagnosis of cancer. The average time to clinical diagnosis of cancer since methylated alleles were detected in serum was 4.3 years for p16, 3.4 years for p15, and 4.4 years for RASSF1A. Since a series of genetic and epigenetic events occur during the long period, these genes seem to be potential epigenetic biomarkers for early detection of HCC or precancerous lesions non-invasively [4]. However, they did not examine the relationship between these genes and various etiologic factors. Wong et al. reported a correlation between p16 hypermethylation in HCC tissues and serum DNA and that p16 hypermethylation was not detected in the serum of patients with other chronic liver diseases such as liver cirrhosis or hepatitis [5]. However, 17% of cirrhotic patients had been reported to have serum DNA with aberrant p16 methylation in another study, which may reflect the lack of standardized processing of blood samples and analytic methods of serum DNA [6]. No studies on the relationship between methylation status of p15 and RASSF1A in serum DNA and cirrhosis have been reported. 1. Introduction Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality worldwide, and liver cirrhosis is the most important predisposing factor for it [1]. Hepatitis B and C viral infection is the most common underlying cause of chronic liver disease leading to liver cirrhosis, and aflatoxin B1 and alcohol are also well-known risk factors. Although most HCCs seem to be originated from the 810 Cancers 2010, 2 Cancers 2010, 2 accumulation of genetic abnormalities induced by various risk factors, underlying mechanisms of hepatocarcinogenesis remain unclear. Long-term survival of HCC patients is poor, partly due to HCC recurrence, which up to 80% of the patients experience even after curative resection [2]. Although serum alpha-fetoprotein (AFP) level is a useful marker for the detection and monitoring of HCC, AFP levels may remain normal in up to 30% of the patients with advanced HCC [3]. Various HCC markers have been suggested, but the overall performance has been unsatisfactory in terms of sensitivity and specificity. To improve HCC prognosis, it is imperative to find useful markers for early diagnosis and monitoring of recurrence of HCC. To identify candidate biomarkers for HCC, many studies have been conducted on molecular events such as genetic and epigenetic alterations, and gene expression. High-throughput omics-based technologies have also enabled the genome-wide interrogation of these changes. This review summarizes recent studies of potential molecular markers for diagnosis and monitoring metastasis or recurrence of HCC. Cancers 2010, 2 Cancers 2010, 2 Cancers 2010, 2 them, 12 genes encode secretory proteins detectable in sera, which may be used as markers for early diagnosis of HCC and as targets for chemoprevention. Misregulation of the genes in this signature is common in patients with HBV and HCV infection, hemochromatosis, and Wilson’s disease, but not very common in patients of other etiologies. This is consistent with the clinical findings that patients with cirrhosis caused by HBV infection, HCV infection, and hemochromatosis have a higher risk of developing HCC compared with those with cirrhosis caused by other factors [8]. Jia et al. identified five genes (GPC3, PEG10, MDK, SERPINI1, and QP-C), which were overexpressed in most of the HCC samples, including those with normal serum AFP and early stage tumors [9]. They showed that a combined use of these five genes could classify noncancerous hepatic tissues (100%) and HCC (71%). To use expression profiles as biomarkers, invasive techniques are required to get the tissues, but markers, which encode extracellular proteins or secretory proteins, can be used to develop non-invasive markers. Among the five genes, GPC3, MDK, and SERPINI1 encode extracellular proteins, which can be detected in serum. They tested serum MDK and found that it can distinguish normal and cirrhotic individuals from HCC patients, including those with normal AFP and small tumors, which could be used as a diagnostic marker. Most of HCC cases from this study were positive for hepatitis B. Budhu et al. reported that livers from metastatic HCC patients have a different gene expression pattern compared with the livers from patients without metastatic HCC, and suggested a 17 gene predictor of HCC venous metastasis [10]. Liver tissues bearing metastatic HCC showed a decrease of pro-inflammatory Th1-like cytokines and an increase of anti-inflammatory Th2-like cytokines. It suggests that the predominant humoral cytokine response in the liver inducing anti- inflammatory/immune-suppressive responses may play a role in promoting HCC venous metastases. The identified predictor is only applicable for operable HCC patients and patients positive for HBV due to the characteristics of the cohort. 2.2. RNA Expression RNA expression profiles are one of the most successful markers that can be used to classify many solid tumors including HCC, based on molecular characteristics. Kim et al. examined the mRNA expression of tissue samples derived from cirrhotic patients of various etiologies using cDNA microarray and reported a molecular signature containing 273 significantly altered genes in HCC [7]. The misregulated genes were clustered into several functional pathways, including metal transport, blood coagulation, and immune response, which can be involved in hepatocarcinogenesis. Among 811 2.3.4. Insulin-like Growth Factor (IGF)-II and Related Factors 2.3.4. Insulin-like Growth Factor (IGF)-II and Related Factors IGF-II is a mitogenic polypeptide related to insulin and thought to serve as a growth factor in various cancers through coexpressing IGF-II and IGF-I receptors, which is a kind of fetal growth factor and highly expressed during hepatocarcinogenesis [22]. It has been suggested that IGF-II may play a role in the neovascularization of HCC by increasing vascular endothelial growth factor (VEGF) [23,24]. The circulating free IGF-II levels were significantly higher in HCC patients than in those with chronic hepatitis or liver cirrhosis. The circulating IGF-II mRNA was positive in 34% of HCC patients, but negative in other liver diseases, extrahepatic tumors, and normal controls. The circulating IGF-II mRNA correlated with the stage of HCC, and was detected in 100% of HCC with extrahepatic metastasis. No significant relationship was found between tumor sizes and circulating IGF-II mRNA. The expression of free IGF-II and IGF-II mRNA may be useful markers for diagnosis of HCC and its extrahepatic metastasis, and monitoring recurrence [12]. Cancers 2010, 2 Cancers 2010, 2 Cancers 2010, 2 2.3.2. Hepatoma-specific Gamma-glutamyl Transferase (GGT) Isoenzyme 2.3.2. Hepatoma-specific Gamma-glutamyl Transferase (GGT) Isoenzym .2. Hepatoma-specific Gamma-glutamyl Transferase (GGT) Isoenzyme GGT is an enzyme that catalyzes the degradation of glutathione and other gamma-glutamyl compounds. It is highest in embryonic livers and decreases to the lowest levels right after birth, but total GGT activities in patients with liver disease and extrahepatic tumors are high [16]. GGT is divided into several subfractions, among which the hepatoma-specific GGT bands (including I', II, and II', HS-GGT) in sera of HCC patients have been used for diagnosis of HCC. HS-GGT can only be found in sera of HCC patients, and its analysis may improve the specificity and sensitivity of HCC diagnosis [17]. Yao et al. showed that the circulating HS-GGT activity was elevated in 86% of the HCC patients, but in less than 3% of patients with other diseases, and that there is an increasing tendency of total RNA concentrations also observed from liver cancer to distal noncancerous tissues [16]. The serum HS-GGT level can be a sensitive tumor marker for diagnosis or differentiation of HCC. 2.3.3. Transforming Growth Factor (TGF)-β1 TGF-β1 is one of TGF-β isoforms, which arrests the cell cycle in the G1 phase and elicits inhibition of cell proliferation and triggering apoptosis [18]. Although it is a growth inhibitor, the overexpression of hepatic TGF-β1 was found in HCC tissues and shown to be correlated with carcinogenesis, progression and prognosis of HCC [19,20]. TGF-β1 expression is also associated with the degree of HCC differentiation and status of HBV replication, but neither to the size nor to number of tumors [21]. However, the sensitivity and specificity of circulating TGF-β1 level as a non-invasive marker have been rarely studied in HCC. 2.3.1. AFP and AFP-mRNA Although total AFP has been a useful marker for diagnosis and monitoring of HCC, it is often difficult to distinguish tumors from benign liver diseases based on the elevated AFP level. Recently, a hepatoma-specific AFP (HS-AFP) subfraction was reported to be superior to total AFP level in both sensitivity and specificity in differentiating benign liver diseases from malignant ones [11,12]. Total AFP can be divided into three glycoforms, AFP-L1, AFP-L2, and AFP-L3. Among them, AFP-L3 is a HS-AFP found in the sera of HCC patients. There was a relationship between AFP-L3 percentage with HCC differentiation, metastasis and relapse, suggesting that the percentage of HS-AFP may be a more specific marker than total AFP for early diagnosis of HCC and its recurrence [13,14]. AFP-mRNA from peripheral blood mononuclear cells by RT-PCR has also been studied in recent years [15]. Although AFP-mRNA is frequently detected in the blood of patients with benign liver diseases or of HCC patients without extrahepatic metastases, it seemed to identify more aggressive tumors in terms of clinical behaviors. If hepatocyte-specific mRNAs are detected in circulating blood, it could reflect the presence of circulating, presumably malignant liver cells and suggest the likelihood of hematogenous metastasis. 812 Cancers 2010, 2 Cancers 2010, 2 reported that patients with inoperable HCC had higher levels of serum HGF than healthy controls, and serum HGF was negatively correlated with survival [27]. They suggested that the elevated HGF levels in HCC patients may reflect the impaired clearance of HGF due to significant liver damage or may be caused by increased HGF production to regenerate hepatocytes [27]. Similarly, Yamagamim et al. found that the serum HGF was significantly higher in patients with HCC than in patients with chronic hepatitis or cirrhosis [28]. In addition, patients with relatively higher HGF concentrations had an ectopic recurrence or a diffuse infiltration of HCC. They followed up the patients after measuring the initial HGF levels and found that the cumulative incidence of HCC in patients with higher initial HGF concentrations are higher than those with lower HGF. They suggested that the elevated serum HGF is probably produced by infiltrating mesenchymal and cancer cells based on their observations; in one patient whose HGF level was below the detection limit at the initial examination, the concentrations increased over the time and exceeded 0.4 ng/mL just before HCC was detected; HGF concentrations were higher in patients with diffuse carcinomas or with multiple cancers than in patients with a single cancer [28]. It may be useful to evaluate the HGF level for the detection and follow-up of HCC. 2.3.6. Heat Shock Proteins (HSPs) HSPs are induced in cells under various stress conditions, including carcinogenesis. Enhancement of intracellular HSP is related to the formation and development of HCC and reported as a vital marker indicating the progression and aggravation of HCC [29]. The HSP gp96 expression was reported to increase as the HBV-induced disease progressed from chronic hepatitis to cirrhosis then HCC, and to be correlated with the degree of tumor differentiation and tumor size, but not with the number of tumors [30]. Sakamoto et al. reported the significant overexpression of HSP70 in early HCC compared with precancerous lesions, and in advanced HCC compared with early HCC. HSP70 has been shown to be negative in other benign nodular lesions, hepatocellular adenoma and focal nodular hyperplasia. Hence, HSP70 might work as a molecular marker to differentiate between benign and malignant liver nodules [31]. 2.3.7. Complement C3a Complement C3a, which is produced from C3 by C3 convertase, has been reported as a potent inflammatory mediator of innate immune response, and to contribute to the early priming stages of hepatocyte regeneration after toxic injury and partial hepatectomy [32]. Lee et al. found that complement C3a was elevated in the sera of patients with chronic hepatitis C and HCV-related HCC, but not in those with HBV-related HCC. It supports that the molecular hepatocarcinogenesis might be different between HBV-related and HCV-related HCC, and therefore the effect on the expression levels of C3a might also be different [33]. 2.3.5. Hepatocyte Growth Factor (HGF) The HGF is the most potent growth factor for hepatocytes and has been reported to be elevated in a number of liver diseases, including acute and chronic hepatitis, liver cirrhosis, HCC, primary biliary cirrhosis, and fulminant hepatic failure [25]. Stellate cells and myofibroblasts are induced to secrete HGF by tumor cell products and HGF stimulates tumor cell invasiveness in turn [26]. Vejchapipat et al. 813 2.3.9. Squamous Cell Carcinoma Antigen (SCCA) 2.3.9. Squamous Cell Carcinoma Antigen (SCCA) SCCA is a member of the family of serine protease inhibitors named serpins and has been reported to be overexpressed in HCC tissues [37]. Giannelli et al. compared HCC and liver cirrhosis and reported that the combination of serum AFP and serum SCCA yielded a correct serologic diagnosis in 90.8% of the HCC patients [37]. Guido et al. reported that SCCA was poorly expressed in regenerative tissue, but strongly expressed in dysplastic nodules, suggesting a role as a potential marker for early detection of HCC [38]. It has been reported that SCCA can react with the IgM class of immunoglobulins to form the immunocomplexes SCCAIC, which is detectable in the serum of HCC patients. They suggested that SCCA-IgM ICs alone or in combination with AFP, can increase the sensitivity for diagnosing HCC significantly [39]. Tretoli et al. reported that serum SCCA levels in cirrhotic patients were significantly lower than in HCC patients, but that there was no significant correlation between tissue and serum levels of SCCA [40]. 2.4. Immunohistochemistry (IHC) Markers IHC plays an important role in the diagnosis of HCC. A number of IHC markers are established including several cytokeratins such as HepPar 1 and pCEA. However, none of these markers are specific enough for the diagnosis of HCC and some of them appear or disappear during tumorigenesis, which make a diagnosis difficult. Therefore, the combination of IHC markers is also used for a precise diagnosis. 2.4.1. Hepatocyte Paraffin 1 (HepPar 1) HepPar 1 has been acknowledged as the most sensitive and specific IHC marker for HCC and reflects hepatocyte differentiation [41,42]. Because it is positive in normal liver and adenomas, it is not useful for distinction of benign versus malignant liver lesions. HepPar 1 is more likely to be negative in poorly differentiated and sclerosing HCC (sensitivity 50% or less). Although most adenocarcinomas are negative for HepPar 1, gastric, esophageal, and lung adenocarcinomas can occasionally show strong positive reactions. Given the relatively higher frequency of metastatic cancer to the liver from these sites, the predictive value of positive HepPar 1 is not high. Rare carcinomas with hepatoid morphology occur in the gastrointestinal tract and pancreas and are positive for HepPar 1 [42]. 2.3.8. Glypican-3 (GPC3) GPC3 was suggested as a possible tumor marker for HCC, since the levels of GPC3 were significantly high in the serum of HCC patients, but undetectable in healthy donors and patients with benign liver diseases [34,35]. Capurro et al. showed that GPC3 stimulates the growth of HCC cells by Cancers 2010, 2 814 Cancers 2010, 2 Cancers 2010, 2 stimulating the Wnt pathway through facilitating the interaction between the Wnts and their signaling receptors [36]. GPC3 was found to be expressed in small tumors, indicating its potential as a diagnostic marker for early stage HCC. The study also showed that the combined use of GPC3 and AFP would significantly increase the sensitivity of the test without compromising specificity. 2.4.5. α-Fetoprotein (AFP) AFP is an oncofetal glycoprotein. Serum levels increase in patients with HCC, yolk sac tumors, hepatitis, and cirrhosis. HCC is immunoreactive for AFP in 17%–68% of paraffin embedded sections and in 45%–50% of cell blocks. Almost all hepatoid adenocarcinomas, both primary and metastatic adenocarcinomas, are reported as AFP positive [42]. 3. Candidate Markers for Prognosis Although the etiological factors and the natural history are well defined, HCC are quite heterogeneous and clinical behaviors are hard to predict. Therefore, it is necessary to establish robust methods capable of evaluating the prognosis of HCC patients. 2.4.3. Glypican 3 (GPC-3) GPC-3 is an oncofetal protein, as it is normally expressed in fetal liver and placenta but not in normal adult liver. GPC-3 is reported to be expressed in 64% to 90% of HCCs, but not in benign masses, which helps to distinguish benign versus malignant lesions [41,44]. GPC-3 is also positive in melanoma and nonseminomatous germ cell tumors such as choriocarcinoma. Its sensitivity is known to be high (~80%), but it needs further validation [41]. 2.4.4. MOC-31 Most HCCs are negative or weakly positive for MOC-31, but it is consistently (80%–100%) expressed in cholangiocarcinoma and metastatic adenocarcinoma from colorectum, pancreas, stomach, lung, breast, and ovary [45,46]. MOC-31 is also expressed in a majority of neuroendocrine tumors, urothelial carcinomas, and renal cell carcinomas [41]. Therefore, it can help distinguish HCC from other tumors. Cancers 2010, 2 Cancers 2010, 2 poorly differentiated one (25%–50%). In several studies, the frequency of biliary canalicular stain decreased with grade; 58%–100% well-differentiated, 25%–80% moderately differentiated, and 0%–73% poorly differentiated HCC expressing the antigen [42]. 2.4.2. Polyclonal Carcinoembryonic Antigen (pCEA) Overall efficiency of pCEA IHC for HCC in study was approximately 90% [42]. Diffuse cytoplasmic expression of pCEA is observed in various adenocarcinomas. HCC shows a canalicular pattern in 60% to 90% of cases [42,43], which is characteristic of HCC but not observed in other adenocarcinomas. Sensitivity is high for well- and moderately differentiated HCCs (~80%), but low in 815 Cancers 2010, 2 816 Cancers 2010, 2 oncogenic potential in HCC. Recurrent loss in 9p24.2–p21.1 and gain in 8q11.21–q24.3 were associated with high tumor grade and microvascular invasion, respectively. Gene enrichment analysis showed that functions such as cytokine receptor binding and defense response to virus pathways are significantly enriched in high grade-related RARs [48]. Saelee et al. observed DNA alterations on chromosomes 5q34, 6p25.2, and 8q12.1 in Thai HCC patients and found that allelic loss on chromosome 5q34 correlated with poor survival in HCC, indicating the potential of this chromosomal region as a prognostic marker [49]. Poon et al. examined 158 HBV-associated HCCs by CGH analysis and classified them into three subgroups using self-organizing tree algorithm [50]. Gains in 1q21–23 and 8q22–24 were identified as genomic events associated with the early development of HCC, and the gain in 3q22–24 was associated with tumor recurrence and poor survival. Although the chromosome aberration data reported in HCC have been relatively rarer than expression or methylation profiles so far, CNA profiles, together with expression, epigenomic and clinical data, will be useful resources as a prognostic marker. 3.1. Chromosomal Alterations 3.1. Chromosomal Alterations Chromosome aberrations are frequently detected in HCC and molecular cytogenetic approaches such as comparative genomic hybridization (CGH) have provided information on copy number alterations (CNAs) in HCC. Patterns of CNAs provide useful information that can be used in heterogeneic HCC to identify genetic events involved in hepatocarcinogenesis and associated with clinical characteristics including prognosis. Among CNAs in HCC, gains of 1q, 8q, and 20q, and losses of 4q, 8p, 13q, 16q, and 17p have been frequently reported, but not necessarily with clinical or prognostic implications [47]. Kim et al. defined recurrently altered chromosomal regions in HCCs using whole-genome array-CGH [48]. They found that the extent of chromosomal alterations correlated with tumor grade, size and microvascular invasion. Among the genes in the recurrently gained regions on 1q, expression of KIF14 and TPM3 was significantly increased, suggesting their 3.3. Gene Expression Profiles The profiling of gene expression patterns have provided new insights into the molecular pathogenesis of various human malignancies, including HCC. However, gene expression profiling is even more important to elucidate the mechanisms behind prognostic diversity and resistance against therapeutic agents. Although there have been many genome-wide gene expression studies, we focused on a couple of them which reported prognostic implications with their data. Ye et al. suggested a molecular signature that can classify metastatic HCC and identified genes relevant to metastasis and survival [61]. Among the identified genes, Osteopontin, which was overexpressed in metastatic HCC, was identified as a lead gene in the signature. They showed that an Osteopontin-specific antibody effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis of HCC cells in nude mice. Therefore, Osteopontin can act as both a diagnostic marker and a potential therapeutic target for metastatic HCC. Several genes belonging to cell adhesion and matrix degradation were also identified to be associated with HCC metastasis, including genes encoding SPP1, α9-integrin, interleukin-2 receptor, serine proteinase inhibitor member-5, matrix metalloproteinase-9, leukocyte immunoglobulin-like receptor subfamily A member-2 and CD37 antigen. The potential of SPP1 as a diagnostic marker was also emphasized because it can be found to be elevated in body fluids in cancer patients and as a target for therapy of HCC patients with metastatic potential. Lee et al. reported that human HCC can be subdivided into two subclasses that are associated with survival based on gene expression data [62]. It is possible that the two subclasses of HCC might represent different cellular origins, but it can be caused by considerable molecular heterogeneity within each HCC subclass. After this, Lee et al. reported that individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis by studying gene expression patterns from human HCC, mouse HCC and rat fetal hepatoblasts and adult hepatocytes [63]. It was hypothesized that HCC of this subtype may arise from hepatic progenitor cells. Analyses of gene networks showed that activation of AP-1 transcription factors might have key roles in development of this subtype. Iikazu et al. used mRNA expression profiles of tissue specimens from patients with HCC using oligonucleotide microarrays representing about 6000 genes and developed a scoring system for predicting early intrahepatic recurrence [64]. They identified 12 genes for the system, which could classify all early-stage and late-stage HCC. Cancers 2010, 2 Cancers 2010, 2 and prognostication of HCC based on DNA methylation profiles using liver biopsy specimens may be useful for close follow-up of patients who are at higher risk of HCC development. 3.2. DNA Methylation Profiles Aberrant DNA methylation is frequently observed in human cancers; global DNA hypomethylation is associated with activation of protooncogenes and genomic instability; hypermethylation on CpG islands in the promoter regions of tumor suppressor genes results in transcriptional silencing and genomic instability [51–54]. It has been shown that multiple tumor-related genes, such as the E-Cadherin and Hypermethylated-in-Cancer (HIC)-18 genes, are silenced by DNA hypermethylation in HCC [55–57]. Since DNA methylation status is altered in a coordinated manner, it is important to have a global picture of methylation changes to interpret the results correctly. However, only a few previous studies have examined genome-wide DNA methylation status in HCC. Calvisi et al. analyzed the global levels of DNA methylation and the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with clinical phenotypes and prognosis of HCC [58]. However, there was no significant association between methylation patterns and any etiologic agents in their study. They identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) by epigenetic silencing of inhibitors of the Ras pathway in all HCC. Selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3, BNIP3L, IGFBP3, and EGLN2) was found to be associated with poor prognosis. Gao et al. used methylated CpG island amplification microarrays to investigate DNA methylation in cancerous and precancerous tissues from HCC [59]. They found aberrant DNA methylation of several important genes, such as DUSP2 and BMP6, which are known to be associated with tumorigenesis. Early detection of aberrant DNA methylation in these genes in precancerous tissues might be useful in risk and prognosis assessment of HCC. Arai et al. reported that the extent of DNA methylation alterations and the degree of DNA methylation alterations increased during hepatocarcinogenesis from precancerous stage to HCC [60]. DNA methylation status was significantly different between HBV- and HCV-positive patients with HCCs in both noncancerous liver tissue and cancerous tissue, suggesting that the HBV-related carcinogenetic pathway may result in distinct DNA methylation profiles. They suggested the types of DNA methylation profiles that were able to discriminate a poor-outcome group from a favorable-outcome group. Therefore, risk estimation 817 Cancers 2010, 2 Cancers 2010, 2 a molecular signature having features of hepatic progenitor cells, whereas EpCAM-negative HCC displayed the molecular features of mature hepatocytes. EpCAM-positive and EpCAM-negative HCC could be further subclassified into four groups with prognostic implications by combining the level of AFP. They found a close correlation between Wnt-h-catenin signaling and EpCAM-positive HCC. Wnt-h-catenin signaling is known to be activated during liver development and in HCC [66–68]. They have recently shown that EpCAM is a direct transcriptional target of the Wnt-h-catenin signaling pathway, further emphasizing the functional link between these two molecular nodes [69]. 4. Conclusions HCC exhibits numerous molecular abnormalities, which may be involved in the process of HCC development and progression. Thus, it is important to identify accurate predictors of prognosis and a reasonable selection criterion that can be applied to patients with HCC, particularly with early stage HCC, for rational treatment decisions remains a challenging task [70]. As reviewed in this article, many potentially useful molecular biomarkers have been reported, but almost none have been incorporated into the conventional TNM staging system yet. Because of the small study sizes used for most of the studies, predictors reviewed in this article are only suggestive and need to be confirmed in larger independent populations for the clinical use. In addition, since most HCC samples were obtained from hepatitis B virus–positive patients, cautions are required to apply the study results to other populations with different clinical characteristics. Given the heterogeneity of etiology and clinical behaviors of HCC, it would be very difficult to find one biochemical or molecular marker that is both specific and sensitive enough. Combination of pathological features and biomarkers with high sensitivity and specificity will be more efficient and practical for early diagnosis and prognostication of HCC. Acknowledgements This work was supported by (FG09-11-06) of the 21C Frontier Functional Human Genome Project from the Ministry of Education, Science and Technology in Korea and a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare &Family Affairs, Republic of Korea (A092258). 3.3. Gene Expression Profiles By combining their system with the TNM staging, they could predict early intrahepatic recurrence or non-recurrence. Among the 12 genes, TNFAIP3 was considerably down-regulated in HCC with venous invasion. HLADRA and TRIM22 were also down-regulated in HCC with early intrahepatic recurrence. Since these genes are immune-related, immunotherapy can be considered to reduce recurrence and invasion of HCC and their scoring system, based on the 12 genes, could be useful when such immunotherapy is being considered for patients with HCC. Yamashita et al. reported that a hepatic stem cell marker, epithelial cell adhesion molecule (EpCAM) expression was significantly elevated in premalignant hepatic tissues and in a subset of HCC and suggested its possibility as an early biomarker of HCC [65]. EpCAM-positive HCC showed 818 4. Zhang, Y.J.; Wu, H.C.; Shen, J.; Ahsan, H.; Tsai, W.Y.; Yang, H.I.; Wang, L.Y.; Chen, S.Y.; Chen, C.J.; Santella, R.M. 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Budhu, A.; Jia, H.L.; Forgues, M.; Liu, C.G.; Goldstein, D.; Lam, A.; Zanetti, K.A.; Ye, Q.H.; Qin, L.X.; Croce, C.M.; Tang, Z.Y.; Wang, X.W. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 2008, 47, 897–907. © 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2010 by the authors; licensee MDPI, Basel, Switzerland. Cancers 2010, 2 This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W4321105716	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/0A4BB2266C8E7E079A9A8ABA1EA2DCB7/S1052150X22000410a.pdf/div-class-title-hiring-algorithms-and-choice-why-interviews-still-matter-div.pdf	English	null	Hiring, Algorithms, and Choice: Why Interviews Still Matter	Business ethics quarterly	2,023	cc-by	17,818	Business Ethics Quarterly 34:2 (April 2024), pp. 201–230. DOI:10.1017/beq.2022.41 Published by Cambridge University Press on behalf of the Society for Business Ethics. © The Author(s), 2023. 1This is not to say that all contemporary HRM scholars necessarily endorse the efficacy of interviews toward their stated ends. Indeed, a number of HRM scholars doubt the effectiveness of interviews toward https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press Hiring, Algorithms, and Choice: Why Interviews Still Matter Indeed, a number of HRM scholars doubt the effectiveness of interviews toward https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 202 BEQ although the costs may be undesirable, they are the price to pay, as it were, to be able to judge whether a candidate will match the needs of the role and the organization.2 In this article, we suggest that the question of why to conduct interviews is a more difficult one than it first seems. The force of this question can be appreciated when juxtaposed against a twofold threat we argue the traditional view of interviewing faces. The first threat, the behavioral threat, holds that a large body of behavioral evidence suggests that we are poor predicters of future performance and bad judges of fit. This is for multiple reasons: the judgments of interviewers are riddled with biases, interviewers overestimate their assessment capacities, and organizations rarely assess the performance of candidates they might have passed on (in relation to the candidates they ultimately selected). As one HRM textbook notes, “tradition- ally, interviews have not been valid predictors of success on the job” (Mondy & Martocchio, 2016: 165). In short, those involved in making hiring decisions are demonstrably bad at predicting future performance and assessing fit. The behavioral threat has brought some management theorists to suggest aban- doning interviews as traditionally conceived (i.e., unstructured interviews) and moving toward structured interviews. Yet structured interviews, too, face problems: they can collapse into unstructured interviews, or alternatively, they start out unstructured either before or after the official start of the interview and, in doing so, increase exposure to the behavioral threat. More fundamentally, the behavioral threat is simply pushed back one step, to the point at which one decides the structure of the interview. Thus, although structured interviews may be an improvement upon unstructured interviews, they, too, do not fare especially well with respect to the behavioral threat. A defender of the traditional view might acknowledge the force of the behavioral threat yet still respond, “We have no better alternative!” But this argumentative maneuver is cut off by the second threat the traditional view faces: the algorithmic threat. Algorithms already have a superior track record to humans, even expert humans, of predicting the performance and fit of candidates in a number of domains. Hiring, Algorithms, and Choice: Why Interviews Still Matter Vikram R. Bhargava George Washington University, USA Pooria Assadi California State University, Sacramento, USA Why do organizations conduct job interviews? The traditional view of interviewing holds that interviews are conducted, despite their steep costs, to predict a candidate’s future performance and fit. This view faces a twofold threat: the behavioral and algorithmic threats. Specifically, an overwhelming body of behavioral research suggests that we are bad at predicting performance and fit; furthermore, algorithms are already better than us at making these predictions in various domains. If the traditional view captures the whole story, then interviews seem to be a costly, archaic human resources procedure sustained by managerial overconfidence. However, building on T. M. Scanlon’s work, we offer the value of choice theory of interviewing and argue that interviews can be vindicated once we recognize that they generate commonly overlooked kinds of noninstrumental value. On our view, interviews should thus not be entirely replaced by algorithms, however sophisticated algorithms ultimately become at predicting performance and fit. Key Words: ethics of interviews, hiring ethics, employment ethics, recruiting automation, algorithmic ethics, algorithmic decision systems W hy do organizations conduct job interviews, despite the enormous costs associ- ated with the interview process? At first blush, this does not seem like an especially challenging question. This is because a natural and seemingly obvious answer immediately comes to mind: interviews are for predicting a candidate’s future performance and fit with respect to the hiring organization’s requirements, values, and culture—that’s why organizations conduct interviews, despite their costs (Cappelli, 2019b; Elfenbein & Sterling, 2018; Muehlemann & Strupler Leiser, 2018; Society for Human Resource Management [SHRM], 2017). This is also the traditional view of interviewing espoused by managers and is how the nature and function of interviews are characterized in human resource management (HRM) textbooks (Dessler, 2020; Mathis, Jackson, Valentine, & Meglich, 2016; Mondy & Martocchio, 2016).1 Thus, W 1This is not to say that all contemporary HRM scholars necessarily endorse the efficacy of interviews toward their stated ends. predicting future performance and fit. The key point is that, even though a number of HRM scholars are skeptical of the efficacy of interviews at predicting future performance and fit, they nevertheless agree that the nature and function of interviews are for predicting future performance and for assessing candidate fit. 2We note that with respect to a range of candidates, especially ones with more experience, the evaluation process is often mutual (i.e., a candidate may be evaluating whether a position at a given firm would satisfy the candidate’s needs). predicting future performance and fit. The key point is that, even though a number of HRM scholars are skeptical of the efficacy of interviews at predicting future performance and fit, they nevertheless agree that the nature and function of interviews are for predicting future performance and for assessing candidate fit. 2We note that with respect to a range of candidates, especially ones with more experience, the evaluation process is often mutual (i.e., a candidate may be evaluating whether a position at a given firm would satisfy the candidate’s needs). Hiring, Algorithms, and Choice: Why Interviews Still Matter Indeed, 67 percent of eighty-eight hundred recruiters and hiring managers globally surveyed by LinkedIn in 2018 noted that they use artificial intelligence (AI) tools to save time in sourcing and screening candidates (Ignatova & Reilly, 2018). So, where does this leave the practice of interviewing? The behavioral and algorithmic threats, taken together, pose what we call the “interview puzzle” for the traditional view of interviewing. If the traditional view is correct about the nature and function of interviews—that interviews are for predict- ing the future performance and fit of a candidate with respect to the role’s and organization’s needs—then it seems as though the justification for the practice is https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press H, A, C: WISM 203 undermined. Not only is interviewing costly (Cappelli, 2020; Muehlemann & Strupler Leiser, 2018; SHRM, 2017) but we also are bad at it, and we may have better alternatives for predicting performance and fit (i.e., algorithms). Continuing to interview, then, if it is only about predicting performance and fit, seems to be at best an anachronistic human resources (HR) practice or at worst blatant wastefulness sustained by irrational managerial overconfidence. For these reasons, we argue that the traditional view of interviewing must be reexamined. If interviews were singularly a means to predicting performance and fit, as the traditional view posits, we maintain that the justification for interviews is under- mined. However, we argue that the antecedent in this conditional is false: interviews are not singularly a means to predicting performance and fit; rather, they are a much richer normative practice. In particular, we argue that interviews offer different kinds of value that have thus far been overlooked and thus the practice can be worth preserving, despite the behavioral and algorithmic threats. Something of normative significance would be lost were we to abandon the practice of interviewing, and this must be accounted for in our understanding of the nature of interviews. In other words, we dissolve the interview puzzle by arguing that although the behavioral and algorithmic threats are indeed concerning, they only threaten to undermine our interview practices if the traditional view of interviewing is the whole story. Hiring, Algorithms, and Choice: Why Interviews Still Matter But we argue that the traditional view of interviewing accounts for only part of its function—the parts it overlooks are the other kinds of value that interviews create, and these other kinds of value do not succumb to the behavioral and algorithmic threats. By reframing how we understand the nature of interviews, we advance a broader, normative conception of interviewing that suggests that our ability to choose whom we relate to in the workplace is an important source of value and that our work lives may be worse off without the practice. We proceed as follows. In section 1, we characterize the traditional view of interviewing and discuss the costs of interviewing that are exhaustively documented in the HRM literature. In section 2, we discuss the behavioral and algorithmic threats and argue that they together undermine the traditional view of interviewing and thus generate the interview puzzle. In section 3, we introduce our value of choice theory of interviewing, grounded in the work of the philosopher T. M. Scanlon (1988, 1998, 2013, 2019). We show how the interview puzzle can be dissolved once we grasp the inadequacy of the traditional view of interviewing: it fails to account for a broader range of contenders for the kinds of value that can be realized through interviewing. If the view we advance is correct, then the current understanding in HRM and management scholarship about the nature and function of interviews must be significantly expanded. In section 4, we offer several clarifications of our account and discuss some potential objections. In section 5, we discuss some new avenues of research that follow from our work. Finally, in section 6, we conclude. 1. THE TRADITIONAL VIEW OF INTERVIEWING The traditional view of interviewing holds that interviews are one class of selection tools (among other tools, such as tests and background checks) that are useful for https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 204 BEQ predicting a candidate’s performance and fit.3 In particular, a selection interview is defined as “a selection procedure designed to predict future job performance based on applicants’ oral responses to oral inquiries” (Dessler, 2020: 207) and is consid- ered a tool for assessing a candidate’s knowledge, skills, abilities, and competencies in relation to what is required for the job (Dessler, 2020; Graves & Karren, 1996; McDaniel, Whetzel, Schmidt, & Maurer, 1994). Interviews are widespread, in part, because of the belief that they are effective in simultaneously assessing candidates’ ability, motivation, personality, aptitude, per- son–job fit, and person–organization fit (Highhouse, 2008). Several common assumptions sustain this belief: that making accurate predictions about candidates’ future job performance is possible (Highhouse, 2008); that experience and intuition are necessary in effective hiring (Gigerenzer, 2007); that human beings (i.e., can- didates) can be effectively evaluated only by equally sensitive complex beings (e.g., hiring managers), rather than by tests or algorithms (Highhouse, 2008); and that oral discussions with candidates can be revealing, as they allow for “reading between the lines” (Highhouse, 2008: 337). Despite the widespread use of interviews, they are recognized to be a costly and time-consuming practice. The United States “fills a staggering 66 million jobs a year. Most of the $20 billion that companies spend on human resources vendors goes to hiring” (Cappelli, 2019b: 50). On average, employers in the United States spend approximately $4,000 per hire to fill non-executive-level positions and about $15,000 per hire to fill executive-level positions (SHRM, 2016, 2017), and a substantial portion of these costs is attributed to interviews. Outside the United States, employers report similar experiences. For example, in Switzerland, on average, employers spend as much as 16 weeks of wage payments to fill a skilled worker vacancy, of which 21 percent involves search costs, and roughly 50 percent of the search costs are direct interview costs (Muehlemann & Strupler Leiser, 2018). In addition, significant opportunity costs are associated with interviews for all parties involved (Muehlemann & Strupler Leiser, 2018). With respect to the time spent on interviews, according to a recent talent acqui- sition benchmarking report, on average per job, US employers spend approximately eight days conducting interviews (SHRM, 2017). 3Two types of fit characterized in a number of HRM textbooks include “person–job fit,” the candidate’s fit in relation to the role (Dessler, 2020; Mathis, Jackson, Valentine, & Meglich, 2016; Mondy & Martocchio, 2016), and “person–organization fit,” the candidate’s fit in relation to the organization (Dessler, 2020; Mondy & Martocchio, 2016). 4Although the focus of our article is on employers, candidates bear significant costs too. For example, candidates must expend resources to sort through job opportunities, schedule commitments, and purchase new professional attire, among other costs. Relatedly, expending effort and time on interviewing could involve intangible short- and long-term opportunity costs that take candidates away from other productive activities. Furthermore, the psychological effects of the interview process can be onerous for the candidates. Although, in our article, we primarily highlight the costs employers bear, we acknowledge that the costs candidates bear ought to be taken seriously in their own right. 1. THE TRADITIONAL VIEW OF INTERVIEWING Employers report similar experi- ences outside the United States. For example, in Switzerland, on average, employers spend approximately 8.5 hours on job interviews per candidate (Muehlemann & Strupler Leiser, 2018). Of course, the costs of hiring and interviewing are not uniform. The costs vary depending on the skill requirements of the job (Muehlemann & Strupler Leiser, 2018) and the degree of labor market tightness (Davis, Faberman, & Haltiwanger, 2012; Pissarides, 2009; Rogerson & Shimer, 2011), among other factors. That said, https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press H, A, C: WISM 205 these costs on average remain substantial and are increasing—employers today spend twice as much time on interviews as they did in 2009 (Cappelli, 2019b).4 As costly and time consuming as interviews are, there are also difficulties associated with verifying whether they are worth these costs. Indeed, “only about a third of US companies report that they monitor whether their hiring practices lead to good employees; few of them do so carefully, and only a minority even track cost per hire and time to hire” (Cappelli, 2019b: 50). Even if it were not so difficult to assess whether interviews are worth the costs with respect to the end posited by the traditional view (i.e., predicting performance and fit), two additional threats remain. 2.1 The Behavioral Threat The traditional conception of interviews—as a means to predict a candidate’s performance and fit in relation to a vacancy—hinges on an important assumption, namely, that performance and fit can be effectively predicted through interviewing. However, a considerable body of knowledge from the social sciences challenges this basic assumption and chronicles the poor track record of predicting performance and fit through interviews (Bishop & Trout, 2005; Bohnet, 2016; Chamorro-Premuzic & Akhtar, 2019; McCarthy, Van Iddekinge, & Campion, 2010; Rivera, 2012). Spe- cifically, although there is empirical evidence that highlights the outsized role interviews have in the hiring process (Billsberry, 2007), interview-based hiring decisions have been found only to account for up to 10 percent of the variation in job performance (Conway, Jako, & Goodman, 1995). Additionally, biases pervade the process of predicting performance and fit through interviews, both in their unstructured and structured formats (Huffcutt, Roth, & McDaniel, 1996; McDaniel et al., 1994). 2.1.1 Unstructured Interviews 2. THE INTERVIEW PUZZLE: THE BEHAVIORAL AND ALGORITHMIC THREATS 2.1 The Behavioral Threat https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 2.1.1 Unstructured Interviews Unstructured interviews do not have a fixed format or a fixed set of questions, nor do they involve a fixed process for assessing the given responses (Schmidt & Hunter, 1998). During unstructured interviews, both the interviewer and the candidate investigate what seems most relevant at the time (Bohnet, 2016). This process often produces an overall rating for each applicant “based on summary impressions and judgments” (Schmidt & Hunter, 1998: 267). Unstructured interviews are often https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 206 BEQ assumed to be effective in concurrently assessing a range of dimensions associated with predicting performance and person–organization fit (Highhouse, 2008). However, recent research shows that unstructured interviews may not in fact aid in hiring decisions. This research maintains that unstructured interviews are riddled with biases and are often swayed by the whims of the interviewers (Chamorro- Premuzic & Akhtar, 2019). Specifically, this research suggests that unstructured interviews are ineffective because interviewers tend to overlook the limits of their knowledge (Kausel, Culbertson, & Madrid, 2016), “decide on the fly” what ques- tions to ask of which candidates and how to interpret responses (Cappelli, 2019b: 50), place disproportionate emphasis on a few pieces of information (Dawes, 2001), and confirm their own existing preferences (Chamorro-Premuzic & Akhtar, 2019). Subsequently, they become increasingly confident in the accuracy of their decisions, even when irrelevant information is introduced (Bohnet, 2016; Dawes, 2001).5 One reason for interviewers’ overconfidence regarding their predictive abilities is that they cannot often ascertain whether, absent interviews, their predictions would turn out to be better or worse, and they would generally lack a large enough sample to deduce any statistically valid inferences (Bishop & Trout, 2005). y y ( p , ) While managers more heavily value a given trait or ability if evaluated by unstructured interviews rather than by alternative methods (e.g., paper-and-pencil tests) (Lievens, Highhouse, & DeCorte, 2005), a long-standing body of empirical evidence shows that unstructured interviews are unhelpful with selection decisions. For example, in the context of medical school applications, DeVaul, Jervey, Chap- pell, Caver, Short, and O’Keefe (1987) compare the students who were initially accepted versus those who were rejected for medical school and find that only 28 percent of the difference between these groups is related to academic and demographic factors and that 72 percent is related to the admissions committee’s preferences developed through interviews. 5Recent research suggests that part of why overconfidence persists, despite its considerable costs, is the status benefits it confers; moreover, these status benefits largely persist, even when the person’s over- confidence is exposed (Anderson, Brion, Moore, & Kennedy, 2012; Kennedy, Anderson, & Moore, 2013). 6See also Oskamp’s (1965) study of the clinical decisions of psychologists which shows that the 6See also Oskamp’s (1965) study of the clinical decisions of psychologists, which shows that the accuracy of their decisions does not increase significantly with additional information from interviews (but confidence in their decision-making steadily increases). https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 5Recent research suggests that part of why overconfidence persists, despite its considerable costs, is the status benefits it confers; moreover, these status benefits largely persist, even when the person’s over- confidence is exposed (Anderson, Brion, Moore, & Kennedy, 2012; Kennedy, Anderson, & Moore, 2013). 6See also Oskamp’s (1965) study of the clinical decisions of psychologists, which shows that the accuracy of their decisions does not increase significantly with additional information from interviews (but confidence in their decision-making steadily increases). 2.1.1 Unstructured Interviews They report that when it comes to attri- tion and clinical performance during medical school and a subsequent year of postgraduate training, there are no significant differences between the accepted and the rejected groups, suggesting that interviews in this context are unhelpful to the decision-making process. In a similar fashion, Milstein, Wilkinson, Burrow, and Kessen (1981: 77) compare the performance of “a group of 24 applicants who were interviewed and accepted at the Yale University School of Medicine but went to other medical schools … with a group of 27 applicants who attended the same schools but had been rejected at Yale following an interview and committee deliberation.” In this context, too, the researchers find no statistically significant relationship between admission decisions and performance, again pointing to the inefficacy of interviews in aiding the achievement of the decision-making ends.6 https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press H, A, C: WISM 207 Medical school admissions decisions are, of course, not hiring decisions, but similar results are seen in hiring contexts. In a study of the hiring practices at elite professional services firms, Rivera (2012) finds that employers often seek candi- dates who enjoy similar leisure pursuits and have shared experiences and self- presentation styles. In doing so, Rivera shows that unstructured interviews may be less about assessing knowledge, skills, and abilities and more about exercising biases through replicating ourselves, including, but not limited to, our culture, gender, and ethnicity, in hiring decisions. Finally, through a meta-analysis, Schmidt and Hunter (1998) conclude that unstructured interviews are ineffective at predicting the performance of future employees. Not only do we know that unstructured interviews are unhelpful in hiring decisions but there is also some empirical evidence that unstructured interviews reliably undermine those decisions (Bishop & Trout, 2005; DeVaul et al., 1987; Eysenck, 1954; Kausel et al., 2016; Milstein et al., 1981; Oskamp, 1965; Wiesner & Cronshaw, 1988). For example, as far back as the middle of the past century, in a large-scale empirical study, Bloom and Brundage (1947) found that the predic- tive gain in adding an interviewer’s assessment of a candidate’s experience, interest, and personality may well be negative. They specifically report that pre- dictions based on test scores and interviewing were 30 percent worse than pre- dictions based on test scores alone. 2.1.1 Unstructured Interviews More recently, Behroozi, Shirolkar, Barik, and Parnin (2020) have shown that even when tests are conducted in interview formats, such as “whiteboard technical interviews” common in software engi- neering, the mechanics and pressure of the interview context reduce the efficacy of the technical tests. This effect is heightened especially among minorities and other underrepresented groups (Munk, 2021). Other recent research reports sim- ilar findings: for example, research on human judgment documents that when decision makers (e.g., hiring managers, admissions officers, parole boards) judge candidates based on a dossier and an unstructured interview, their decisions tend to be worse than decisions based on the dossier alone (Bishop & Trout, 2005). In a similar fashion, Dana, Dawes, and Peterson (2013) show that adding an unstruc- tured interview to diagnostic information when making screening decisions yields less accurate outcomes than not using an unstructured interview at all. In this case, even though the decision makers may sense that they are extracting useful information from unstructured interviews, in reality, that information is not useful (Dana et al., 2013). 2.1.2 Structured Interviews 2.1.2 Structured Interviews Unlike the unstructured version, a structured interview involves a formal process that more systematically considers “rapport building, question sophistication, ques- tion consistency, probing, note taking, use of a panel of interviewers, and standard- ized evaluation” (Roulin, Bourdage, & Wingate, 2019: 37) in hiring decisions. In this interview format, to predict good hires, an expert interviewer systematically and consistently poses the same set of validated questions about past performance to all candidates and immediately scores each answer based on a set of predetermined criteria relevant to the tasks of the job (Cappelli, 2019b). https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 208 BEQ Although structured interviews are available and designed to standardize the hiring process and minimize subjectivity and bias (Bohnet, 2016; Reskin & McBrier, 2000), they are in effect not much more successful than unstructured interviews in aiding hiring decisions for at least three reasons. First, even though structured interviews, in theory, may be less biased7 and a better predictor of future job performance8 than their unstructured counterparts, they are not widely adopted in practice (König, Klehe, Berchtold, & Kleinmann, 2010; Roulin et al., 2019). The resistance to structuring interviews (Lievens et al., 2005; van der Zee, Bakker, & Bakker, 2002) is driven by interviewers’ belief that a candidate’s character is “far too complex to be assessed by scores, ratings, and formulas” (Highhouse, 2008: 339) that are predetermined in a structured format. Second, even in cases when structured interviews are accepted, they are not well implemented for various reasons. For example, structured interviews tend to be more costly to construct (Schmidt & Hunter, 1998) in part because of the difficulties in designing and validating standardized questions and evaluation criteria (Bohnet, 2016; Roulin et al., 2019). Also, in reality, we rarely see structured interviews conducted by trained and experienced interviewers who manage to avoid having their idiosyncratic personalities distort the process (Roulin et al., 2019). Even when structured interviews are conducted by trained and experienced interviewers, the process sometimes deviates to a semistructured or unstructured format. For instance, in conforming to a predeter- mined set of questions, the flow of conversation in a structured interview might feel stilted, awkward, or uncomfortable for both the interviewer and the candidate, thereby inadvertently shifting the interview process to a less structured format (Bohnet, 2016). 8With respect to the predictive power of structured interviews, they “predict performance in job training programs with a validity of about .35” (Schmidt & Hunter, 1998: 267). 7The average validity of the structured interviews (at about 0.51) is greater than the average validity of the unstructured interviews (at about 0.38) and far greater than the average validity of poorly conducted unstructured interviews (Schmidt & Hunter, 1998: 267). 8With respect to the predictive power of structured interviews, they “predict performance in job training programs with a validity of about .35” (Schmidt & Hunter, 1998: 267). https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press ( ) g g y p y unstructured interviews (Schmidt & Hunter, 1998: 267). 8With respect to the predictive power of structured interviews, they “predict performance in job training programs with a validity of about .35” (Schmidt & Hunter, 1998: 267). 7The average validity of the structured interviews (at about 0.51) is greater than the average validity of the nstructured interviews (at about 0.38) and far greater than the average validity of poorly conducted nstructured interviews (Schmidt & Hunter, 1998: 267). 2.2 The Algorithmic Threat Interviews, both in their unstructured and structured formats, if not by design, in practice are ineffective at assessing fit or predicting future performance and create a significant opportunity for bias in hiring decisions (Chamorro-Premuzic & Akhtar, 2019; Rivera, 2012). However, proponents of the traditional view of interviewing might respond that there are no alternatives. But this assertion falls short in the face of the second threat the traditional view faces, that is, the algorithmic threat. That is, algorithms, even simple ones, in a number of domains, already are no worse (and are at times superior) at predicting the performance and fit of candidates than humans, even expert humans (Bishop & Trout, 2005; Cappelli, 2020). Algorithms can be an effective method for predicting future performance and fit primarily because the hiring challenge at its core is a prediction problem, and statis- tical algorithms are designed to take on and address prediction problems (Danieli, Hillis, & Luca, 2016). For example, a simple statistical prediction rule (SPR) in a linear model is designed to predict a desired property P (e.g., future performance) based on a series of cues (e.g., education, experience, and past performance) such that P = w1(c1) þ w2(c2) þ w3(c3) þ … þ wn(cn), where cn and wn reflect the value and weight9 of the nth cue (Bishop & Trout, 2005). Research shows that even this simple statistical algorithm is, at least in overall effect, better than humans in hiring pre- dictions, in part because such a hiring algorithm is more consistent than humans (and cheaper, to boot). And, in practice, this algorithm can be better scaled and automated in a consistent way (Chamorro-Premuzic & Akhtar, 2019). Also, the increasing availability of good data, advances in statistical algorithms, and new capacities to analyze large-scale data have made this algorithmic route even more promising (Cappelli, 2020). Indeed, more advanced statistical hiring algorithms based on machine learning can be better than humans at predicting performance and fit because they are specifically designed to “adaptively use the data to decide how to trade off bias and variance to maximize out-of-sample prediction accuracy” (Chalfin et al., 2016: 124). In this respect, for example, Cowgill (2019) finds that more advanced statis- tical hiring algorithms based on machine learning better predict job performance than humans because they lack some of the biases from which humans suffer. Also, Chalfin et al. 9The weight for each cue reflects its importance and is assigned based on the comparison of any given cue to a large set of data on performance (Bishop & Trout, 2005). H, A, C: WISM H, A, C: WISM Although research shows that interviews can undermine the aims of the hiring process, interviews have remained a popular norm for employee selection for more than a hundred years (Buckley, Norris, & Wiese, 2000; van der Zee et al., 2002). They have remained popular not necessarily because the inefficacy of interviews is unknown. In fact, Rynes, Colbert, and Brown (2002) report that HR professionals appreciate the limitations of interviews. Still, hiring managers remain reluctant to outsource their judgment (Bohnet, 2016). 2.1.2 Structured Interviews y g p Third, even when structured interviews are conducted by trained and experienced interviewers and the process does not deviate to an unstructured format, empirical evidence shows that structured interviews may not be systematic and free of bias because interviewers may used them to confirm their preexisting judgments rather than to evaluate the candidates—that is, a potential self-fulfilling prophecy (Dougherty, Turban, & Callender, 1994). On the candidates’ side, there is also much room for introducing bias. For example, Stevens and Kristof (1995) show that applicants engage in significant impression management, even in structured inter- views, thereby undermining the decision-making process. Furthermore, even when structured interviews are implemented properly, these issues and biases may not be eliminated: they may simply be shifted to the previous step of designing the inter- view and deciding its structure. Therefore not only are structured interviews rare but, even when they are used and properly implemented, they are afflicted with issues that complicate the evaluation of performance and fit. It is not surprising, then, that Cappelli (2019b: 56) argues that a structured interview is the “most difficult tech- nique to get right.” https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 209 H, A, C: WISM https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 2.2 The Algorithmic Threat (2016) find that, compared to the existing rank-ordering police hiring https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 210 BEQ systems, machine learning algorithms that use sociodemographic attributes; prior behavior, including prior arrest records; and polygraph results would yield a 4.8 percent reduction in police shootings and physical and verbal abuse complaints. In addition to the hiring domain, advanced statistical algorithms based on machine learning have been shown to be more effective than humans in a broader set of screening decisions where “a decision-maker must select one or more people from a larger pool on the basis of a prediction of an unknown outcome of interest” (Rambachan, Kleinberg, Ludwig, & Mullainathan, 2020: 91). For example, Klein- berg, Lakkaraju, Leskovec, Ludwig, and Mullainathan (2018) show that machine learning algorithms exhibit better performance than judges in bail decisions because they incorporate fewer irrelevant perceptions of the defendant (e.g., demeanor) into their decisions. Also, Dobbie, Liberman, Paravisini, and Pathania (2018) illustrate that machine learning algorithms minimize bias against certain types of applicants (e.g., immigrants). Other related studies in lending find that machine learning algorithms are better at predicting default (Fuster, Plosser, Schnabl, & Vickery, 2019) and are less discriminatory compared to face-to-face lenders (Bartlett, Morse, Stanton, & Wallace, 2019). Critics of algorithmic decision-making in hiring (and elsewhere) raise at least two objections. The first objection pertains to the seeming ability of humans to pick up on soft, qualitative, or noncodifiable cues during interviews that are difficult to capture in algorithms (Gigerenzer, 2007; Highhouse, 2008). However, this is precisely where the research shows that there is a high likelihood and magnitude of bias clouding human decision-making. Indeed, the “speculation that humans armed with ‘extra’ qualitative evidence can outperform SPRs has been tested and has failed repeatedly” (Bishop & Trout, 2005: 33). Even if we grant that humans are skilled at inferring relevant information from subtle personality and intellect cues, as some research suggests (Gigerenzer, 2007), statistical algorithms often simply pull on the same cues. While many algorithms tend to draw on codifiable cues (rather than bias- prone, noncodifiable cues), in contrast to humans, algorithms are more efficient and consistent, and they need not be managed with respect to their sense of self-esteem or self-importance (Chamorro-Premuzic & Akhtar, 2019). https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 2.2 The Algorithmic Threat The second objection regarding the algorithmic method of predicting future performance and assessing fit concerns fairness (Cappelli, Tambe, & Yakubovich, 2020; Newman, Fast, & Harmon, 2020; Raisch & Krakowski, 2021; Tambe, Cap- pelli, & Yakubovich, 2019). In this respect, although legitimate fairness concerns are associated with algorithmic predictions of human performance, research has shown that algorithms are often no worse than the alternative means of hiring, including using human judgment through interviews. For example, using data on teacher and police characteristics, Chalfin et al. (2016) show that statistical algorithms predict future performance better than humans. Though there are indeed fairness concerns with algorithms, these concerns are prevalent in human decision-making too (Danieli et al., 2016). Specifically, Danieli et al. grant the prevalence of fairness issues in algorithms but also highlight several comparably concerning psychological biases in human judgment. For example, in hiring contexts, humans engage in bracketing (i.e., overemphasizing subsets of choices over the universe of all https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press H, A, C: WISM 211 options), that is, choosing the top candidate who was interviewed on a given day instead of the top candidate interviewed throughout the search process (Danieli et al., 2016).10 In addition, Li (2020) summarizes research that shows how human judg- ment in hiring may discriminate based on race, religion, national origin, sex, sexual orientation, and age. Given this research, Cappelli (2020) warns us not to romanti- cize human judgment and to recognize “how disorganized most of our people management practices are now.” He notes, “At least algorithms treat everyone with the same attributes equally, albeit not necessarily fairly.” Indeed, a significant portion of the algorithmic fairness issues arguably stems from human actions, as well as the lack of diversity in the humans who designed them (Li, 2020) and the types of data with which humans trained them (Cappelli, 2020; De Cremer & De Schutter, 2021). For example, Dastin (2018) reports that Amazon’s recruiting algorithm was biased against women because it was trained to assess candidates by discovering patterns in submitted résumés over a ten-year time frame—most of those résumés were submitted by men (see also Cappelli, 2019a).11 As it turns out, recent research challenges the common assumption that biased data in the training stage of machine learning will lead to undesirable social out- comes. 10What about the possibility of complementing algorithmic predictions with human oversight? In other words, one might be tempted by the thought that a firm should use both algorithms and its own judgment; that is, one should consider the predictions of the algorithms, but vet these predictions against one’s own assessment of the candidate. After all, algorithms will, at least on occasion, offer what seem to be obviously mistaken prescriptions. And if one’s intuition contradicts what the algorithm is prescribing in such a case, one might defect from the algorithmic strategy. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press Although tempting, this strategy faces serious problems. A crucial lesson from the literature on how to benefit from SPRs (including decision assistance algorithms) is that partial or selective compliance with the strategy results in significantly worse overall outcomes (Bishop & Trout, 2005; Dawes, Faust, & Meehl, 1989; Meehl, 1957). This has been confirmed on multiple occasions in the laboratory context and is a problem in contexts as wide-ranging as medical decision systems and criminal recidivism, as well as in interviews (Bishop & Trout, 2005: 46–47, 91; Goldberg, 1968; Leli & Filskov, 1984; Sawyer, 1966). Specifically, when one opts for a selection strategy based on a SPR (such as an algorithm), but then defects from this strategy on a case-by-case basis—because this particular case seems unique—this yields worse overall outcomes (Bishop & Trout, 2005). This is so even if there is a strong sense that the particular circumstance at hand is somehow exceptional (see the literature on the “broken leg problem” [Bishop & Trout, 2005: 45–46; Dawes et al., 1989; Meehl, 1957] when the decision maker “comes to believe she has strong evidence for defecting from the strategy” [Bishop & Trout, 2005: 46]). In other words, to secure the most overall instrumental benefits of an algorithm, its advice generally cannot be taken a la carte. 10What about the possibility of complementing algorithmic predictions with human oversight? In other words, one might be tempted by the thought that a firm should use both algorithms and its own judgment; that is, one should consider the predictions of the algorithms, but vet these predictions against one’s own assessment of the candidate. After all, algorithms will, at least on occasion, offer what seem to be obviously mistaken prescriptions. And if one’s intuition contradicts what the algorithm is prescribing in such a case, one might defect from the algorithmic strategy. Although tempting, this strategy faces serious problems. A crucial lesson from the literature on how to benefit from SPRs (including decision assistance algorithms) is that partial or selective compliance with the strategy results in significantly worse overall outcomes (Bishop & Trout, 2005; Dawes, Faust, & Meehl, 1989; Meehl, 1957). This has been confirmed on multiple occasions in the laboratory context and is a problem in contexts as wide-ranging as medical decision systems and criminal recidivism, as well as in interviews (Bishop & Trout, 2005: 46–47, 91; Goldberg, 1968; Leli & Filskov, 1984; Sawyer, 1966). Specifically, when one opts for a selection strategy based on a SPR (such as an algorithm), but then defects from this strategy on a case-by-case basis—because this particular case seems unique—this yields worse overall outcomes (Bishop & Trout, 2005). This is so even if there is a strong sense that the particular circumstance at hand is somehow exceptional (see the literature on the “broken leg problem” [Bishop & Trout, 2005: 45–46; Dawes et al., 1989; Meehl, 1957] when the decision maker “comes to believe she has strong evidence for defecting from the strategy” [Bishop & Trout, 2005: 46]). In other words, to secure the most overall instrumental benefits of an algorithm, its advice generally cannot be taken a la carte. 11 2.2 The Algorithmic Threat Specifically, Rambachan and Roth (2020) empirically examine the “bias in, bias out” assumption and highlight the conditions under which machine learning may reverse bias and ultimately prioritize groups that humans may have marginal- ized. More specifically, through mathematical modeling and simulation, they show Although tempting, this strategy faces serious problems. A crucial lesson from the literature on how to benefit from SPRs (including decision assistance algorithms) is that partial or selective compliance with the strategy results in significantly worse overall outcomes (Bishop & Trout, 2005; Dawes, Faust, & Meehl, 1989; Meehl, 1957). This has been confirmed on multiple occasions in the laboratory context and is a problem in contexts as wide-ranging as medical decision systems and criminal recidivism, as well as in interviews (Bishop & Trout, 2005: 46–47, 91; Goldberg, 1968; Leli & Filskov, 1984; Sawyer, 1966). Specifically, when one opts for a selection strategy based on a SPR (such as an algorithm), but then defects from this strategy on a case-by-case basis—because this particular case seems unique—this yields worse overall outcomes (Bishop & Trout, 2005). This is so even if there is a strong sense that the particular circumstance at hand is somehow exceptional (see the literature on the “broken leg problem” [Bishop & Trout, 2005: 45–46; Dawes et al., 1989; Meehl, 1957] when the decision maker “comes to believe she has strong evidence for defecting from the strategy” [Bishop & Trout, 2005: 46]). In other words, to secure the most overall instrumental benefits of an algorithm, its advice generally cannot be taken a la carte. 11We recognize that, in some instances, algorithms risk amplifying our biases and can further entrench bad organizational cultures (because firms would use their own past HR decisions as data sets, which can in turn deepen morally untoward hiring practices). In such cases, this is indeed a significant added concern with using algorithms in lieu of humans. This, of course, would undermine the strength of our characterization of the algorithmic threat and, in turn, lessen the force of the puzzle we raise for the traditional view of interviewing, but it does not undermine our ultimate thesis that there are strong grounds for preserving the practice of interviewing—indeed, this would amount to a further independent consideration that supports our thesis. 2.2 The Algorithmic Threat https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 212 BEQ that, unlike the bias generated by measurement errors caused by mislabeled data, the bias generated by sample selection may be flipped by machine learning such that the machine learning outcomes would favor groups that encountered discrimination in the training data.12 Rambachan and Roth argue that the bias reversal occurs because members of groups that are underrepresented in the original training data, for example, women, that make the cut are typically ones that are statistically outstand- ing performers. As such, in subsequent rounds of learning, the algorithm is fed data in which women are overly positively correlated with being outstanding performers. Rambachan and Roth show that this can ultimately reverse the underrepresentation in the data that is due to human decision makers. We have thus far considered two objections to using algorithms instead of interviews, and we’ve suggested that these objections fall short. Yet one might correctly point out that many more objections to algorithms have recently appeared in the algorithmic ethics literature (Birhane, 2021; Hunkenschroer & Luetge, 2022; Martin, 2019; Müller, 2021; Tasioulas, 2019; Tsamados et al., 2022). For example, there are concerns related to algorithms systemically excluding certain individuals (Creel & Hellman, 2022), eliciting organizational monocultures (Kleinberg & Raghavan, 2021), or disproportionately harming marginalized groups (Birhane, 2021); worries related to the legitimacy and trustworthiness of algorithms (Benn & Lazar, 2022; Martin & Waldman, 2022; Tong, Jia, Luo, & Fang, 2021) and the lack of explainability in the case of opaque algorithms (Anthony, 2021; Kim & Routledge, 2022; Lu, Lee, Kim, & Danks, 2020; Rahman, 2021; Rudin, 2019; Selbst & Powles, 2017; Véliz, Prunkl, Phillips-Brown, & Lechterman, 2021; Wachter, Mittelstadt, & Floridi, 2017);13 issues related to whether algorithms preclude us from taking people seriously as individuals (Lippert-Rasmussen, 2011; Susser, 2021); and concerns related to whether automated systems create responsibility or accountability gaps (Bhargava & Velasquez, 2019; Danaher, 2016; Himmelreich, 2019; Nyholm, 2018; Roff, 2013; Simpson & Müller, 2016; Sparrow, 2007; Tigard, 2021), among other concerns (Bedi, 2021; Tasioulas, 2019; Tsamados et al., 2022; Yam & Skorburg, 2021). In short, there’s now a rich literature involving a wide range of concerns related to adopting algorithms in lieu of human decision makers (Hunkenschroer & Luetge, 2022; Martin, 2022; Müller, 2021; Tsamados et al., 2022). 12The algorithm will continue to replicate and exacerbate any bias generated by measurement errors caused by mislabeled data. 13See also the related debate concerning trade-offs between interpretability and accuracy (London, 2019). 14We are grateful to an anonymous reviewer for raising this concern. 2.2 The Algorithmic Threat And the thought might be put more forcefully: insofar as these two afore- mentioned concerns could be objections to using algorithms (and in turn objections to the force of the interview puzzle), many more objections—like the ones articu- lated in the algorithmic ethics literature—may succeed.14 We grant the force of this concern. Taken together, the arguments developed in the algorithmic ethics literature constitute a powerful concern regarding using algo- rithms in lieu of human decision makers. Furthermore, to the extent that these objections to algorithms succeed, it would weaken the strength of the algorithmic https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 213 H, A, C: WISM threat (and, correspondingly, the force of the interview puzzle). However, for our ultimate aims, this does not concern us. This is because our broader project is not to defend algorithms—we do so in the context of the interview puzzle strictly for the sake of argument. Our ultimate aim is instead to argue that even if these wide-ranging objections to the use of algorithms fall short, there nevertheless remain independent moral considerations that tell against abdicating hiring choices to an algorithm. Crucially, the kinds of moral considerations on which we draw do not depend on certain bad outcomes that may arise due to algorithms. This is to say, even if algorithms were not systemically excluding individuals in arbitrary ways (Creel & Hellman, 2022), did not result in an organizational monoculture (Kleinberg & Raghavan, 2021), did not create responsibility gaps (Himmelreich, 2019; Johnson, 2015; Martin, 2019; Matthias, 2004; Roff, 2013; Sparrow, 2007), or did not elicit other morally untoward outcomes, there nevertheless remains an independent moral concern about firms abdicating their choices in the hiring domain to an algorithm. So, the argument we will now provide might be understood as providing further, independent grounds to resist using algorithms (at least in the context of hiring). Moreover, the arguments we offer do not hinge on certain bad outcomes arising due to using algorithms; as such, the force of our arguments remains, even if the bad outcomes associated with algorithms are ultimately engineered away. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 3. THE VALUE OF CHOICE THEORY OF INTERVIEWS The interview puzzle can be dissolved once we recognize that interviews play additional roles beyond predicting performance and fit. For this reason, even if the behavioral and algorithmic threats undermine the plausibility of interviews serving as a means toward the end of securing an employee who fits the role’s and organization’s needs, we need not conclude that the practice of interviewing is unjustified or something that ought to be abandoned: this is because interviews are a source of other kinds of value and are not exclusively a means for predicting performance and fit. To be clear, on the view we develop, we do not challenge the importance of the end posited by the traditional view (i.e., the end of hiring an employee who fits the role’s and organization’s needs); rather, we argue that additional kinds of value are implicated in the practice of interviewing. Thus we offer a pluralistic theory of interviewing and argue that once we recognize the wider range of contenders for the kinds of value generated through interviewing, we can see that abandoning inter- views would risk the loss of certain important kinds of value. To understand the additional kinds of value implicated in the practice of inter- views, we draw on philosopher T. M. Scanlon’s (1988, 1998) account of the value of choice. Scanlon’s (2013: 12) account “begins from the fact that people often have good reason to want what happens in their lives to depend on the choices they make, that is, on how they respond when presented with the alternatives.” His work on the value of choice has been significant for debates and fields of inquiry as wide-ranging as paternalism (Cornell, 2015), bioethics (Walker, 2022), the freedom and moral responsibility debate (Duus-Otterström, 2011; Fischer, 2008), and contract theory (Dagan, 2019). On the value of choice account, at least three different kinds of value can be generated when making a choice: instrumental, representative, and symbolic. The first is the instrumental value of a choice: if I am the one who makes the choice, I might make it more likely that I realize some end than were I not given the opportunity to choose. 2.3 Taking Stock of the Interview Puzzle The behavioral and algorithmic threats present a significant twofold challenge and raise the interview puzzle for proponents of the traditional view of interviewing. To be sure, this does not mean that the traditional view is not, in part, correct. Finding high-performing candidates who fit the job requirements, as the traditional view posits, is plausibly an important end for firms to pursue. However, the behavioral and algorithmic threats, taken in conjunction, challenge whether interviews are a suitable means toward that end. Crucially, if interviews are only about this end, then the interview puzzle remains and threatens to undermine our justification for con- ducting interviews. We will now argue, however, that there is more to be said on behalf of interviews than the traditional view accounts for. Before proceeding, we offer a brief clarification about an assumption we make in the next section: we treat the interview process as equivalent to a hiring process with human decision makers. But, strictly speaking, this assumption is not always correct. Hiring processes with human decision makers can occur without interviews, because interviews are not the only available basis for selection. For example, tests or work samples might instead be used. However, tests and work samples are apt in a much narrower range of positions. Moreover, as HRM textbooks note, “interviews are one of the most common methods used for selection” (Mathis et al., 2016: 259), and “interviews continue to be the primary method companies use to evaluate applicants” (Mondy & Martocchio, 2016: 165). In fact, “while not all employers use tests, it would be very unusual for a manager not to interview a prospective employee” (Dessler, 2020: 192). For these reasons, we use “the interview process” interchangeably with “hiring process conducted by human decision makers.” At the end of section 4, we briefly discuss the implications of relaxing this assumption. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 214 BEQ https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 3. THE VALUE OF CHOICE THEORY OF INTERVIEWS So, for example, if I’m a prospective car buyer and am given the choice over what color I want for my car, my making this choice realizes a certain instrumental value: of making it more likely that the car will satisfy my aesthetic preferences (in contrast to, for example, were the dealership to choose the color of the car on my behalf or were the color to be selected using a random color generator). So, the instrumental value in a choice is realized when it makes it more likely that a desired end of a prospective decision maker is achieved. The second is the representative value of choice: this is the value that is generated when my making the choice alters the meaning of the outcome of the choice— crucially, this value is realized even if my making the choice is instrumentally worse at achieving certain ends than an alternative method of decision-making (e.g., an algorithm, a coin flip, deference to an expert). For example, it’s important that I am the one who chooses a gift for my partner, not because I’m more likely to satisfy their preferences than they are (were they to choose the gift themselves), but rather because there is value in the fact that I was the one who chose it; in choosing the H, A, C: WISM 215 gift, I expressed myself (e.g., my desires, beliefs, and attitudes toward my significant other) through that act. More simply, representative value relates to how the outcome of the choice takes on a different meaning in virtue of who makes the choice. The third is the symbolic value of choice: this is the value associated with certain choices reflecting that one is a competent member of the moral community who has standing that is “normally accorded an adult member of the society” (Scanlon, 1998: 253). For example, if I, as an adult, were not permitted to choose my bedtime, this would be demeaning and infantilizing. This is so even if a sleep specialist choosing my bedtime would result in outcomes better for my circadian rhythm and other physiological markers. My being able to choose reflects the judgment that I am a “competent, independent adult” (Scanlon, 1998: 253). 3. THE VALUE OF CHOICE THEORY OF INTERVIEWS This is the value that is risked when one is denied the opportunity to make certain choices, ones that, in a given social context, are choices that “people are normally expected to make … for themselves” (Scanlon, 1998: 253). These are the three candidates for the value generated through making a choice. The first is instrumental, and the second two are noninstrumental sources of value. This may not exhaust the candidates for the kinds of value generated in making a choice, but it does taxonomize three important kinds of value that are generated in making a choice. Thus, if a choice is abdicated, (at least) these three kinds of value are at risk and are thus potential candidates for the value that would be lost. Returning to the context of interviewing, when firms conduct interviews, they are making choices about whom to employ. So, let’s now turn to how the value of choice account bears on interviewing. We will discuss each sort of value generated through choice—instrumental, representative, and symbolic—in turn. The first is the instrumental value of choice. Securing instrumental value is the chief value with which the traditional view of interviewing is concerned. The thought goes as follows: interviewing realizes the instrumental value to the extent that it helps the firm predict a candidate’s performance and fit. Those who are inclined to preserve interviews, on the basis of the traditional view of interviewing, might expect that the instrumental value of choice realized in interviewing—helping a firm better predict a candidate’s performance and fit—is what both explains why we interview and also what justifies its costs. j Yet the instrumental value of interviewing is precisely what is called into question by the interview puzzle. Interviewing does not excel at generating the purported instrumental value that it is thought to elicit (namely, predicting future performance and fit). So, if the sole kind of value that could be generated through interviewing is instrumental value, then the grounds for the practice are undermined. But as the value of choice account tells us, there is a wider range of contenders for the kinds of value generated in making a choice. The critical oversight of the traditional view is its failure to recognize that the value generated through interviewing is not entirely conditional on the instrumental value of choice, given that there can be noninstru- mental value generated through the choice. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 3. THE VALUE OF CHOICE THEORY OF INTERVIEWS This brings us to the second potential value—and one overlooked by the tradi- tional view—that is realized through interviews: the representative value of choice. As Scanlon (1998, 253) points out, we value and want certain choices to “result from https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 216 BEQ and hence to reflect [our] own taste, imagination, and powers of discrimination and analysis.” In the interview context, we may value the fact that we are the ones choosing with whom we work, and there is value lost (i.e., representative value) when we abdicate that choice, even if our choosing does not as effectively realize the ends of predicting performance and fit as an algorithm. An algorithm might be better at predicting which romantic partner we should date, whom we should befriend, or which university we should attend—while this all might be correct, abdicating these choices and deferring to an algorithm would result in us losing something of value: representative value. Choosing to whom we relate in the workplace is a way “to see features of ourselves manifested in actions and their results” (Scanlon, 1998: 252). The representative value of a choice is the value that arises in virtue of the choice taking on a different meaning: because of both the fact of who makes the choice and the choice representing or expressing the person’s judgments, desires, and attitudes. The third value generated through interviewing, and another oversight of the traditional view of interviewing, is the symbolic value of choice. Scanlon (2019: 4) points out, “If it is generally held in one’s society that it is appropriate for people in one’s position to make certain decisions for themselves, then failing to make such a decision for oneself or being denied the opportunity to make it, can be embarras- sing, or even humiliating.” Thus the symbolic value of choice is what is lost when a person for whom it would be appropriate (in a given social context) to make a certain decision is precluded from making that decision. 16It is worth noting that the term algorithm is often used to refer to multiple different kinds of processes, systems, and technologies (Leavitt, Schabram, Hariharan, & Barnes, 2021). For instance, some algorithms are rule based (or symbolic) systems, whereas others are association-based systems. Within these broad and rough categories are many varieties of algorithms and ways in which they might be combined and used. For the purposes of our argument, we put to one side the details regarding the technical specifications of algorithms while merely noting that the extent to which a value of choice is undermined by abdicating the choice to an algorithm may also depend on the type and nature of the algorithm. 15For a discussion of the downsides of workplace friendships for organizations, see Pillemer and Rothbard (2018). 16 https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 3. THE VALUE OF CHOICE THEORY OF INTERVIEWS For example, to the extent that workplace norms in a given society involve members of an organization typically having a choice in their future colleagues—people with whom they would collaborate but also, in some cases, those whom they would befriend or with whom they would commiserate and form community (Casciaro, 2019; Estlund, 2003; Porter, Woo, Allen, & Keith, 2019)—through interviewing, depriv- ing people of that choice may result in a loss of symbolic value.15 Relatedly, a certain prestige and status are implicated in making certain choices (including selecting future colleagues through interviewing) that figure into the symbolic value of choice; this is especially vivid, for example, when alumni of a university are involved in on-campus recruiting at their alma mater (Binder, Davis, & Bloom, 2015). This prestige and status that are implicated in the symbolic value of choice are also part of what would be lost were firms to forsake interviews. Crucially, substituting interviews with algorithms can result in a loss of symbolic value even if, as a matter of fact, an algorithm may arrive at a better assessment of a candi- date’s expected performance and fit.16 https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press H, A, C: WISM 217 Although the representative value of choice and the symbolic value of choice may seem similar, especially because, as Scanlon (1998: 253) puts it, “representative and symbolic value may be difficult to distinguish in some cases,” they are not the same. Symbolic value concerns how making certain choices reflects one’s standing, whereas representative value concerns how the meaning of a certain outcome depends on who is making the choice that elicited the outcome. Despite these differences, both are kinds of noninstrumental value, and neither depends on the instrumental effectiveness of the choice with respect to some end (Aristotle, 1962; Donaldson, 2021; Donaldson & Walsh, 2015; Gehman, Treviño, & Garud, 2013; Kant, 2012; O’Neill, 1992; Zimmerman & Bradley, 2019). Our interviewing practices can be vindicated once we recognize that the choice involved in the interview process can realize both representative and symbolic value. The key point is that “the reasons people have for wanting outcomes to be dependent on their choices often have to do with the significance that this dependence itself has for them, not merely with its efficacy in promoting outcomes that are desirable on other grounds” (Scanlon, 1998: 253). 3. THE VALUE OF CHOICE THEORY OF INTERVIEWS And the fact that representative and symbolic value are threatened when abdicating the choice involved in interviewing a candi- date—the choice of whom to relate to in the workplace—generates pro tanto moral reason to preserve interviews as an organizational practice. Crucially, the represen- tative and symbolic value undergirding our interview practices is not imperiled by the behavioral or algorithmic threats. In other words, once we recognize the broader range of contenders for the kinds of value generated through interviewing, we can see that the behavioral and algorith- mic threats only undermine part of the potential value in interviewing—its instru- mental value. But we still have pro tanto moral reason to continue the practice of interviewing, given the noninstrumental value—representative and symbolic value —that may be lost were we to abandon the practice. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 4. CLARIFICATIONS AND OBJECTIONS We now turn our attention to a few clarifications and some potential objections. First, it’s worth keeping in mind that even the noninstrumental values in a choice do not always tell in favor of preserving, rather than abdicating, a choice. For example, with respect to representative value, we might prefer, in some circumstances, for our choices not to reflect our judgments, desires, and attitudes. If one’s organization is considering hiring one’s close friend, one might prefer to have the “question of who will get a certain job (whether it will be my friend or some well-qualified stranger) not depend on how I respond when presented with the choice: I want it to be clear that the outcome does not reflect my judgment of their respective merits or my balancing of the competing claims of merit and loyalty” (Scanlon, 1998: 252). In other words, in circumstances that might present a conflict of interest, for example, there might be reasons related to representative value that tell against preserving the choice. Second, the value of choice is not simply about having a greater number of options from which to select. This is to say, the value of choice generates reasons that “count in favor of ‘having a choice,’ but for reasons of all three kinds having more choice 218 BEQ (over a wider range of alternatives) is not always better than less. Being faced with a wider range of alternatives may simply be distracting, and there are some alterna- tives it would be better not to have” (Scanlon, 2019: 4). So, in the context of interviewing, we remain agnostic about how the value of choice is affected by having more candidates from whom to select. Third, one might doubt whether symbolic value would in fact be risked were we to forgo interviews. The point might be pressed as follows: because many (or even most) employees are not involved in hiring decisions, it is not clear that symbolic value would be lost (or that the failure to be involved in the interview process would be demeaning).17 We grant that symbolic value may not be risked in many instances of abdicating a choice. But this clarification points the way to an advantage of our value of choice account: its contextual sensitivity. 17We are grateful to an anonymous reviewer for this point. We also acknowledge that many hiring decisions are made by internal HR divisions. But it is worth noting that even if these members of HR divisions may not ultimately work with the people they are hiring (unless, of course, the interview is for an HR position), the members of these HR divisions are themselves usually employees of the organization too. Moreover, in a number of fields, it is not uncommon in the final rounds of interviews for candidates to be interviewed by individuals who would be their immediate team members and managers if selected for the position. 4. CLARIFICATIONS AND OBJECTIONS As Scanlon (1998: 253) notes, a key point with respect to whether symbolic value is risked in a given situation is whether the situation is one “in which people are normally expected to make choices of a certain sort for themselves.” Ascertaining whether there is such an expectation in place in a given hiring context and, in turn, whether symbolic value would be lost will depend on certain sociological facts pertaining to the expectations in the given workplace and the norms governing that workplace culture, field, or industry.18 This means that there is an important role for empiricists to play in ascertaining the workplace contexts, fields, or industries in which symbolic value is risked to a greater or lesser extent. And in contexts in which the strength of the norms associated with choosing members of one’s organization are weaker, the reasons provided by the symbolic value of choice would be correspondingly weaker. Fourth, one might raise the following question: what about organizations that outsource hiring to an external head-hunting firm? On our view, such an approach would, in effect, be morally akin to abdicating the choice to an algorithm, with respect to the value of choice. That said, there might be other sorts of considerations —for example, the various objections discussed in the algorithmic ethics literature 18Suppose a firm is deciding on candidates as a collective by using some sort of majoritarian procedure that nevertheless results in an outcome that is no individual’s most preferred choice (List & Pettit, 2011; Pettit, 2007). First, does the individual’s choice still matter? Our aim here in this article is not to enter the debate regarding the metaphysics and morality of group agents. That said, we note that the value of choice of the individual still matters, given that it is a key component of fixing the collective’s choice. It is quite unlike cases in which an individual’s choice (arguably) may not matter due to an outcome being causally overde- termined. That an individual’s most preferred choice was not instantiated is a different matter from the value realized through making the choice. Second, such a collective decision procedure seems morally unobjec- tionable—could automating it render it objectionable? It may very well, albeit perhaps not for reasons related to the value of choice. 18Suppose a firm is deciding on candidates as a collective by using some sort of majoritarian procedure that nevertheless results in an outcome that is no individual’s most preferred choice (List & Pettit, 2011; Pettit, 2007). First, does the individual’s choice still matter? Our aim here in this article is not to enter the debate regarding the metaphysics and morality of group agents. That said, we note that the value of choice of the individual still matters, given that it is a key component of fixing the collective’s choice. It is quite unlike cases in which an individual’s choice (arguably) may not matter due to an outcome being causally overde- termined. That an individual’s most preferred choice was not instantiated is a different matter from the value realized through making the choice. Second, such a collective decision procedure seems morally unobjec- tionable—could automating it render it objectionable? It may very well, albeit perhaps not for reasons related to the value of choice. This is because automating a procedure can change its very nature, morally speaking, for reasons of the sort discussed in the algorithmic ethics literature. We are grateful to an anonymous reviewer for these two questions. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 4. CLARIFICATIONS AND OBJECTIONS This is because automating a procedure can change its very nature, morally speaking, for reasons of the sort discussed in the algorithmic ethics literature. We are grateful to an anonymous reviewer for these two questions. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 219 H, A, C: WISM mentioned earlier—that make relying on algorithms morally worse than abdicating the choice to an external head-hunting firm. Still, it is quite right that the value of choice-related considerations would be morally akin. But this need not mean that there is no role for external head-hunting firms at all. This is because the concerns with respect to the value of choice primarily arise insofar as the firm defers to the judgment of the external head-hunting firm. This, however, does not preclude soliciting advice about hiring decisions from HR consultants or head-hunting firms. Notably, in the context of algorithms, deference to the algorithm is much more likely given that many algorithms are opaque. Moreover, failing to defer to the judgments of the algorithm—that is, picking and choosing on a case-by-case basis when to follow its prescriptions—drastically undercuts its overall instrumental benefits (Bishop & Trout, 2005). Fifth, perhaps, all things considered, in some instances the costs of interviewing may be too burdensome and a firm might be forced to forgo the practice. Perhaps, in other instances, the importance of finding the right person is far too weighty—for example, selecting an airline pilot—for a human to make the decision if an algorithm would do so more effectively. But even in these cases, were we to abandon inter- viewing for a different selection method (e.g., an algorithm), it’s worth keeping in mind that there may still be something of normative significance lost, that is, representative or symbolic value.19 How might these trade-offs be managed? One potential approach might be as follows: suppose one regards instrumental value to be of much greater significance in the business realm than the sorts of noninstrumental value to which we’ve drawn attention. In such a case, a hybrid approach might be considered. Such an approach might involve conducting the initial screening with an algorithm and leaving the ultimate decision to a member of the organization. 19Quite apart from the representative or symbolic value that is risked when abdicating a choice to an algorithm are concerns about how doing so might undermine organizational learning (Balasubramanian, Ye, & Xu, 2022). 20Of course, as earlier noted, picking and choosing when to comply with the predictions of the algorithm significantly undercuts the overall instrumental benefits of the algorithm (Bishop & Trout, 2005). Insofar as one pursues such a hybrid approach, it’s worth keeping in mind that the various other moral objections to the use of algorithms discussed in the algorithmic ethics literature would still be relevant. 21Suppose a physician faces two options: interpret medical images herself or rely on a predictive algorithm. Further suppose that the algorithm yields better instrumental results with respect to patient welfare. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 19Quite apart from the representative or symbolic value that is risked when abdicating a choice to an algorithm are concerns about how doing so might undermine organizational learning (Balasubramanian, Ye, & Xu, 2022). 20Of course as earlier noted picking and choosing when to comply with the predictions of the algorithm 4. CLARIFICATIONS AND OBJECTIONS This may allow for reducing the potential trade-offs between the instrumental and the noninstrumental sources of value of choice.20 In other words, our view is not that, in instances when an algorithm is vastly superior at achieving a given end, firms should pursue the drastically less instru- mentally effective approach. As Scanlon (2019: 4) notes, the various reasons for the value of choice “can conflict with reasons of other kinds, particularly with instru- mental reasons.” So, we are not claiming that firms must always conduct interviews, instead of using algorithms. Nor are we claiming that the instrumental considerations are not of moral significance—in some instances, they may very well be of over- riding moral importance.21 Rather, our point is that multiple kinds of value can be https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 220 BEQ generated through the practice of interviewing—including sources of value that may generate conflicting reasons—and that an adequate theory of interviewing should not overlook this fact. If we are to abdicate interviews in a given context, we should do so in full view of the kinds of value that are risked.22 Sixth, it’s now worth revisiting the assumption we articulated at the end of section 2: treating the interview process as equivalent to a hiring process with human decision makers. As we acknowledged, this assumption is not always, strictly speaking, correct. A hiring process—including one in which humans are making the decisions—might not involve interviews at all; perhaps the hiring process involves choosing on the basis of work samples or tests. So, when we relax this assumption, what follows? Our view would still imply that abdicating the hiring process entirely to algorithms would risk the various values of choice. However, our value of choice account does not entail a particular mode of choosing for a human decision maker—whether interviews, work samples, or tests. 23Of course, the various ways in which we are bad at interviewing characterized in the behavioral threat section might tell in favor of choosing by way of these alternative modes of selection (e.g., tests or work samples) when possible. But we hesitate to make this judgment with confidence, given that different kinds of normative concerns may be associated with relying strictly on work samples or tests; for example, it potentially reduces people to a contrived and narrow set of criteria, rather than treating them with respect as individuals and as fellow members of the moral community. For an additional approach to hiring, see Sterling and Merluzzi’s (2019) exploration of “tryouts” and their theoretical and practical potential. Must the physician insist on making the choice herself? Our view does not rule out that the physician should rely on the algorithm here—in other words, there may very well be cases that the good or bad at stake is so weighty that the instrumental value of relying on the algorithm swamps the various values of choice that may be realized in making the choice oneself. We are grateful to an anonymous reviewer for this example. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 22Our argument is neither about the badness of having fewer choices to make nor about the goodness of having more choices to make (nor is it about preserving the status quo number of choices one makes). With respect to the value of choice, that some other choice is made (e.g., to defer to an algorithm) has little bearing on whether, what kind, and the extent to which one of the values of choice would be undermined in abdicating this choice. Adding a choice elsewhere doesn’t somehow replenish the value of choice that is undermined in no longer choosing one’s colleagues. Must the physician insist on making the choice herself? Our view does not rule out that the physician should rely on the algorithm here—in other words, there may very well be cases that the good or bad at stake is so weighty that the instrumental value of relying on the algorithm swamps the various values of choice that may be realized in making the choice oneself. We are grateful to an anonymous reviewer for this example. 22Our argument is neither about the badness of having fewer choices to make nor about the goodness of having more choices to make (nor is it about preserving the status quo number of choices one makes). With respect to the value of choice, that some other choice is made (e.g., to defer to an algorithm) has little bearing on whether, what kind, and the extent to which one of the values of choice would be undermined in abdicating this choice. Adding a choice elsewhere doesn’t somehow replenish the value of choice that is undermined in no longer choosing one’s colleagues. 5. FUTURE AVENUES OF RESEARCH Our value of choice account of interviewing suggests several new avenues of research. First, a significant body of research in employment ethics primarily emphasizes the ethics of how employers ought to treat their employees (Arnold, 2010; Barry, 2007; Bhargava, 2020; Brennan, 2019; McCall, 2003; Werhane, Radin, & Bowie, 2004), but there is much less, apart from discrimination-related issues, surrounding the ethics of what is owed to prospective employees. Our work highlights the significance of a range of understudied issues to explore in this domain. Although some have explored the question of what is owed to former employees of a firm (Kim, 2014), what, if at all, is owed to potential employees, such as candidates who participate in interviews? Other such issues include, for example, the ethics of exploding offers, accepting applications from candidates that will never be considered, and alerting candidates of rejection. On the side of the candidate, issues include the ethics of feigning enthusiasm for an interview, pursuing an interview merely to solicit an external offer for negotiation leverage, and holding on to offers that one is confident one will not accept. Second, our account of interviewing points the way to questions related to what may make employment relationships meaningful (Robertson, O’Reilly, & Hannah, 2020). Some contributors to the future of work scholarly conversation have argued that employers owe it to their employees to provide meaningful work (Bowie, 1998; Kim & Scheller-Wolf, 2019; Michaelson, 2021; Veltman, 2016).24 By attending to the broader range of values associated with interviewing, managers may have the opportunity to make work and employment relationships more meaningful (Bartel, Wrzesniewski, & Wiesenfeld, 2012; Freeman, Harrison, Wicks, Parmar, & De Colle, 2010; Rosso, Dekas, & Wrzesniewski, 2010). So, an important question to address will be how the process of being selected for a position (i.e., through an interview or through selection by way of an algorithm) can contribute to preserving or promoting the meaningfulness of work (Carton, 2018; Grant, 2012; Jiang, 2021; Kim, Sezer, Schroeder, Risen, Gino, & Norton, 2021; Rauch & Ansari, 2022). Third, there is a sense in which using algorithms in hiring decisions deepens the informational asymmetry between candidates and employers (Curchod, Patriotta, Cohen, & Neysen, 2020; Yam & Skorburg, 2021: 614). Switching to algorithms in hiring may prevent candidates from developing a better understanding of their prospective colleagues and the prospective employer’s workplace culture and norms. 24For a comprehensive discussion of the future of the office, specifically the decisions of firms and employees to work remotely, in a hybrid form, or at an office, see Cappelli (2021). 4. CLARIFICATIONS AND OBJECTIONS With respect to the narrow range of professions where work samples or tests can aptly be implemented, our value of choice arguments are neutral between choosing such an approach and interviewing (but of course, the value of choice account is not neutral between either of these routes and abdicating the choice to an algorithm).23 Interviews are a way—the most prominent and common way, and the way most broadly applicable across a range of positions—for us to choose the members of our organizations, but they are indeed not the only way to choose in the hiring process. To summarize, we have offered an account of some heretofore underappreciated normative dimensions of a widespread business practice, namely, interviewing. Our view helps address some of the challenges to which the traditional conception of interviewing succumbs. The traditional view has difficulty explaining why inter- views persist and justifying why we should not abandon them, given their costs, our poor ability to predict performance and fit, and the presence of algorithmic alterna- tives. Our value of choice theory of interviewing both explains why interviews persist and justifies why there are grounds not to abandon the practice: interviews g g g having more choices to make (nor is it about preserving the status quo number of choices one makes). With respect to the value of choice, that some other choice is made (e.g., to defer to an algorithm) has little bearing on whether, what kind, and the extent to which one of the values of choice would be undermined in abdicating this choice. Adding a choice elsewhere doesn’t somehow replenish the value of choice that is undermined in no longer choosing one’s colleagues. 23Of course, the various ways in which we are bad at interviewing characterized in the behavioral threat section might tell in favor of choosing by way of these alternative modes of selection (e.g., tests or work samples) when possible. But we hesitate to make this judgment with confidence, given that different kinds of normative concerns may be associated with relying strictly on work samples or tests; for example, it potentially reduces people to a contrived and narrow set of criteria, rather than treating them with respect as individuals and as fellow members of the moral community. For an additional approach to hiring, see Sterling and Merluzzi’s (2019) exploration of “tryouts” and their theoretical and practical potential. 4. CLARIFICATIONS AND OBJECTIONS H, A, C: WISM 221 play an important normative function by securing noninstrumental sources of value in hiring. https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 6. CONCLUSION The traditional view of interviewing espoused by both practitioners and manage- ment scholars alike holds that interviews are conducted—despite the steep costs associated with the process—to predict a candidate’s performance and fit in relation to a vacancy. We argue that the traditional view faces a twofold threat: the behavioral and the algorithmic threats. The behavioral threat arises in virtue of a large body of behavioral evidence that points to us being poor predictors of future performance and bad judges of fit. The algorithmic threat arises in virtue of algorithms already being superior predictors of performance and fit than us in a number of domains, including the hiring domain. If the traditional view of interviewing captures all there is to interviewing, then the justification for conducting interviews is undermined by the behavioral and algo- rithmic threats. However, we argue that the practice of interviewing can be vindi- cated once we recognize that there are a broader range of contenders for the kinds of value that can be realized through interviewing—crucially, some of these kinds of noninstrumental value that are realized through interviewing remain insulated from the behavioral and algorithmic threats. In short, we argue that even if algorithms are better predictors of performance and fit than us, it does not follow that we ought to abandon our interview practices: this is because important kinds of noninstrumental value are generated through interviewing that could be lost were we to forgo the practice. 5. FUTURE AVENUES OF RESEARCH On the other hand, if an interview was conducted, the candidate might have acquired this sort of valuable information, even if fallibly. Future scholars should explore the public policy implications of forgoing interviews, especially in jurisdic- tions with employment at will. The symmetrical right to exit is sometimes discussed as a potential justification for employment at will (Bhargava & Young, 2022; https://doi.org/10.1017/beq.2022.41 Published online by Cambridge University Press 222 BEQ Hirschman, 1970; Maitland, 1989; Taylor, 2017). But when candidates and employers enter the employment relationship on starkly asymmetric informational grounds (Caulfield, 2021), it’s worth exploring whether the fact of both parties having a right to exit the relationship loses some of its justificatory force with respect to employment at will and considering whether supplementary regulatory con- straints would be in order. 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W1742143363.txt	https://www.ajol.info/index.php/ajtcam/article/download/31230/36816	en		Antihyperglycemic Effects Of Separate And Composite Extract Of Root Of <i>Musa paradisiacal</i> And Leaf Of <i>Coccinia indica</i> In Streptozotocin-Induced Diabetic Male Albino Rat.	African Journal of Traditional Complementary and Alternative Medicines	2,008	cc-by	4,906	Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 Research Paper 362 Afr. J. Traditional, Complementary and Alternative Medicines www.africanethnomedicines.net ISSN 0189-6016©2007 ANTIHYPERGLYCEMIC EFFECTS OF SEPARATE AND COMPOSITE EXTRACT OF ROOT OF MUSA PARADISIACA AND LEAF OF COCCINIA INDICA IN STREPTOZOTOCIN-INDUCED DIABETIC MALE ALBINO RAT. Chhanda Mallick a b, Kausik Chatterjee b, Mehuli GuhaBiswas b and Debidas Ghoshb a Reproductive Endocrinology and Family Welfare Research Unit, Department of Human Physiology with Community Health, bBio-Medical Laboratory Science and Management Vidyasagar University, Midnapore-721 102 West Bengal, India E-mail: debidas_ghosh@yahoo.co.in Abstract We evaluated the antihyperglycaemic properties of aqueous-methanolic (40:60) extract of root of Musa paradisiaca and leaf of Coccinia indica in separate as well as in composite manner by conducting experiment on streptozotocin-induced diabetic rats. We measured food and water intake ability, the fasting blood glucose level, glucose tolerance, activities of important carbohydrate metabolic enzymes like glucose-6-phosphatase, glucose-6phosphate dehydrogenase, hexokinase in liver along with quantification of glycogen in liver and in skeletal muscle and serum insulin level. We noted that after treatment of aqueous methanolic extract of above plant parts in separate as well as in composite manner at a concentration of 80mg/100g body weight/day to streptozotocin-induced diabetic rat resulted in a significant remedial effect on blood glucose level as well as carbohydrate metabolic enzymes and the quantity of liver and skeletal muscle glycogen. Serum insulin level that was diminished in streptozotocininduced diabetic rat recovered significantly after the co-administration of extract of above plant parts. All the above parameters showed a more potent remedial effect after composite extract treatment with respect to separate treatment and none of the extract has any general metabolic toxicity induction. Key words: Diabetes, Glycaemic index, Insulin, Musa paradisiaca, Coccinia indica Introduction Diabetes is recognized as one of the leading causes of mortality and morbidity in the world (Can et al., 2004). It is a heterogeneous group of diseases and at present it is known as syndrome (Zimmet, 1997). The central identifying feature of diabetes mellitus is chronic and substantial elevation in the circulating glucose concentration. The major mode of control of diabetes can be achieved by diet, exercise, insulin replacement therapy and by the use of herbal hypoglycemic agents (Ivorra and Paya, 1989). Diet therapy along with insulin or oral hypoglycemic agent forms an important way of treatment in diabetes (Clark and Lee, 1995) though it has several demerits. The major drawbacks of insulin therapy are the side effects, which include insulin allergy, lipodystrophy and lipoatropy, insulin antibodies, altered metabolic control, placental transfer of insulin antibodies, autoimmunity and other late complications like morphological changes in kidneys and severe vascular complications (Defronzo et al., 1982; Jarvinen and Koivisto, 1984; Jarvinen and Koivisto, 1986). Similarly, the oral hypoglycemic drugs have many side effects such as nausea and vomiting, cholestatic jaundice, agranulocytosis, aplastic and hemolytic anemias, generalized hypersensitivity reactions, dermatological reaction and lactic acidosis (Khan and Shechter, 1991). Plants have been used for the major source of treatment of diabetes mellitus from ancient time in the Indian medicine and in the world. Herbal drugs are mostly out of toxic or side effect than the chemical drug (Rao et al., 2003). From Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 363 Indian medicinal system it is found that composite extract is most effective than the separate one. On that background this work has been designed to find out the most effective extract of Musa paradisiaca and Coccinia indica in separate and in composite manner to correct the streptozotocin-induced diabetic disorders. From trial and error it has been noted that aqueous-methanolic extract of these two plant parts has a promising antidiabetic effect. . M. paradisiaca is a tree like herb with thick stem composed of convoluted leaf sheaths. Leaves are very large and oblong. This plant belong to the Musaceae family and is distributed throughout the India and Malaysia. Roots of M. paradisiaca are antihelmintic. Flowers are astringent (Joshi, 2000). Fruits are mild laxative. It aids in combating diarrhea and dysentery and promotes healing of intestinal lesions in ulcerative colitis (Joshi, 2000). It is useful in celiac disease, constipation and peptic ulcer (Joshi, 2000). Unripe fruit and cooked flower are useful in diabetes. Different parts of the plant have medicinal value (Kirtikar and Basu, 1991; Joshi, 2000). Coccinia indica is a climbing perennial herb of Cucurbitaceae family and it is distributed widely all over India. Its medicinal importance has been observed earlier by others (Chopra, 1958). Root and leaves of this plant have antilipidemic effects (Eshrat, 2003) as well as antioxidative effects (Venkateswaran and Pari, 2003). Materials and Methods Chemicals Streptozotocin was obtained from spectrochem Pvt. Ltd chemical company (India). Insulin enzyme linked immunosorbant assay (ELISA) kit was purchased from Boehringer Mannheim Diagnostic, Mannheim (Germany). Plant materials and preparation The roots of M. paradisiaca and leaves of C. indica were collected from local area in the month of June and the materials were identified by taxonomist of Botany Department, Vidyasagar University, Midnapore. The voucher specimens with HPCH No: 7, 8 of the plants were deposited in the Department of Botany, Vidyasagar University, Midnapore. Fresh leaves of C. indica and small pieces of fresh roots of M. paradisiaca were dried in an incubator for 2 days at 40 oC, crushed separately in an electric grinder and then powdered. Out of this powder, 100 g was suspended in 500 ml of aqueous-methanol (2:3) mixture and kept in incubator at 37 oC for 36 h. The slurry was stirred intermittently for 2 h and left overnight. The mixture was then filtered and filtrate was dried by low pressure and residue was collected. When required the residue was suspended in olive oil in a fixed dose and used for treatment. Selection of animal and animal care Forty matured normoglycemic Wistar strain male albino rats of 3 month of age weighing about 150.0 ± 10.0 g were used for this experiment. Animals were acclimated for a period of 15 days in our laboratory condition prior to the experiment. Rats were housed at an ambient temperature of 25 ± 2 oC with 12 h light: 12 h dark cycle. Rats have free access to standard food and water ad libitum. The principles of laboratory animal care (NIH 1985) and instruction given by our institutional ethical committee were followed through out the experimental period. Induction of diabetes mellitus Fasting rats for 24 h were subjected to single intramuscular injection of streptozotocin (STZ) at the dose of 4 mg /0.1 ml of citrate buffer/100 g body weight/rat that produced type I diabetes (having fasting blood sugar level more than 250 mg/dl after 24 h of STZ injection. Thirty-two rats were made diabetic in this way. Animal treatment Forty rats were divided into five groups equally as follows and duration of experiment was of 14 days. Group I: (Control group): Rats of this group received single intramuscular injection of citrate buffer (0.1 ml/100 g body weight/rat). Group II: (Diabetic group): Rats of this group were made diabetic by single intramuscular injection of streptozotocin at the dose of 4 mg/0.1 ml citrate buffer/100 g body weight/rat. Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 364 Group III: (C. indica co-administered group): The diabetic rats of this group were fed by gavage method with aqueous-methanolic extract of leaf of C. indica at the dose of 80 mg/0.5 ml olive oil/100 g body weight/rat/day after 24 h of STZ injection for 14 days at fasting state. Group IV: (M. paradisiaca co-administered group): Streptozotocin-induced diabetic rats were fed by gavage with aqueous-methanolic extract of root of M. paradisiaca at the dose of 80 mg/0.5 ml olive oil/100 g body weight/rat/day for 14 days. Group V: (M. paradisiaca and C.indica co-administered group): The diabetic rats of this group were fed with aqueous-methanolic extract of seeds of M. paradisiaca and leaf of C. indica in composite manner at the dose of 80 mg (1:1)/ 0.5 ml olive oil/100 g body weight / rat for 14 days by gavage. Co-administration of extract in group III, IV and V was performed early in the morning and at fasting state. Animals of control (group I) and diabetic groups (group II) were subjected to forceful feeding of 0.5 ml of olive oil/100 g body weight /day for 14 days at the time of extract co-administration to the animals of group III, IV and V to keep all the animals in same experimental condition. On 16th day of experiment, all the animals were sacrificed by decapitation after recording the final body weight, blood was collected from dorsal aorta and serum was separated by centrifugation at 3000 g for 5 min for the assay of insulin followed by ELISA technique. Liver and skeletal muscle were dissected out and stored at –20 oC for biochemical analysis of enzyme activities of glucose-6phosphatase, glucose-6-phosphate dehydrogenase, hexokinase in liver as well as glycogen content in liver and skeletal muscle. Testing of fasting blood glucose level Fasting blood glucose (FBG) level was measured from the first day of extract supplementation to diabetic rats. Fasting blood glucose level in all groups was measured by single touch glucometer at the interval of two days. Blood was collected from the tip of the tail vein and FBG level was measured by single touch glucometer. Glucose tolerance test After overnight fasting, on the day the animals were sacrificed, a zero-min blood sample was taken from tip of tail vein of all the rats: control (Group I), diabetic (Group II), diabetic + M. paradisiaca (Group III), diabetic + C. indica(Group IV) and diabetic + M. paradisiaca +C. indica (Group V) . Glucose solution at the dose of 0.5 g /kg body weight/5ml physiological saline was administered intravenously through femoral vein and blood samples were collected at 30th, 60th, 90th and 120th minute for the measurement of glucose levels by single touch glucometer after the administration of glucose (O'Brien et al., 1985). Biochemical assay of glucose-6-phosphatase activity in liver The liver glucose-6-phosphatase activity was measured according to standard protocol (Swanson, 1955). Tissue was homogenized in ice cold of 0.1 M phosphate buffer saline (pH=7.4) at the tissue concentration of 50 mg/ml. In a calibrated centrifuge tube, 0.1ml of 0.1 M glucose-6-phosphate solution and 0.3 ml of 0.5 M maleic acid buffer (pH=6.5) were taken and brought to 37 oC in water bath for 15 min. The reaction was stopped with 1 ml of 10% trichloroacetic acid (TCA) followed by chilling in ice and centrifuged at 3000 × g for 10 min. The optical density was noted at 340 nm. The enzyme activity was expressed as mg of inorganic phosphate liberated per gm of tissue. Biochemical assay of glucose-6- phosphate dehydrogenase in liver The liver glucose-6-phosphate dehydrogenase activity was measured spectrophotometrically (Langdon, 1966). One unit of enzyme activity is defined as that quantity which catalyses the reduction of 1 µM of NADP per minute. Activity of this enzyme was recorded by using glucose-6-phosphate as a substrate and absorbance was measured at 340 nm. Assay of hexokinase in liver The enzyme activity was determined on the basis of reduction of NADPH coupled with hexokinase which was measured spectrophotometrically at 340 nm (Chou and Wilson, 1975). Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 365 Biochemical assay of glycogen content Glycogen levels in liver and skeletal muscle were measured biochemically (Sadasivam and Manickam, 1996). Tissue homogenized in 80% ethanol, and extract was collected by centrifugation using anthrone reagent, and quantity of glycogen was measured in relation to standards, which was expressed in µg of glucose per mg of tissue. Serum insulin level Serum insulin was measured by enzyme linked imunosorbant assay (ELISA) using the kit (Brugi et al., 1988) (Boehringer Mannheim Diagnostic, Mannheim, Germany). The intra-assay variation was 4.9%. As the samples were run at a time, so there is no inter-assay variation. The insulin level in serum was expressed in µIU/ml. Biochemical assay of glutamate oxalate transaminase (GOT) and glutamate pyruvate transaminase (GPT) Liver and kidney GOT and GPT activities were measured using the kit supplied by Crest Biosystems, Alto Santacruz, Bambolim Complex (Goa, India). The activities of these enzymes were expressed as unit per gram of tissue (Henry et al., 1960). Statistical analysis Analysis of Variance (ANOVA) followed by multiple two-tail ‘t’ test was used for statistical analysis of collected data (Sokal and Rohle, 1997). Differences were considered significant at p< 0.05. Results Body weight, water and food intake Body weight, water and food intake of diabetic animals decreased significantly and after composite extract supplementation there was a significant recovery of these parameters towards the control level although separate or individual extract of the M. paradisiaca and C. indica plants were able to protect these parameters partially (Table 1). Glycemic parameters Streptozotocin injection resulted in a significant elevation in fasting blood glucose level throughout the experiment compared to control and the diabetic-induced animals were unable to tolerate extra glucose load. Treatment of composite extract to streptozotocin diabetic animals resulted in a complete recovery of fasting blood glucose level on 16th day of treatment and the animals were able to tolerate the exogenous glucose load and behaved as control. Individual extract treatment in separate manner to diabetic animal was unable to reverse these parameters to the control level but was able to protect the above parameters partially (Tables 2 and 3). Quantity of glycogen in liver and muscle decreased significantly in diabetic group compared with control group. After composite extract coadministration , the levels of liver and muscle glycogen were reestablished to the control level though the individual extract was unable to reset these values to the control level (Figure 1). Streptozotocin-induced diabetic animal resulted in a significant elevation in glucose-6-phosphatase activity along with diminution in glucose-6-phosphate dehydrogenase and hexokinase activities in liver with respect to control group. Though separate extract co-administration to diabetic rats resulted in a partial protection in these parameters but composite extract resulted in a significant protection and the levels of these parameters reversed to control level (Figure 2). Serum insulin level was decreased significantly in diabetic group in respect to control. After composite extract co-administration, the serum insulin level was restored towards the level of matched control group more effectively than the separate extract co-administration (Figure 1). Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 366 Table 1: Effect of aqueous-methanolic extract of root of M. paradisiaca and leaf of C. indica on body growth, water and food intake in diabetic rats. Group I II III IV V Body weight (g) Initial Final 150.1±6.1 151.6± 6.3 151.9± 6.4 152.1± 6.2 151.2± 6.2 172.2 ± 6.9 126.5± 6.8 * 152.4± 6.4 155.6± 6.7 169.8 ± 6.6 Water intake (ml/rat/d) Before experiment During experiment 37.3 ± 1.1 37.7 ± 1.0 38.2 ± 1.2 60.1 ± 0.9 * 36.7 ± 1.6 41.9 ± 1.2 36.9 ± 1.0 42.2 ± 1.2 38.1 ± 1.2 38.2 ± 1.1 Food intake (g/rat/d) Before experiment During experiment 16.1 ± 2.1 15.1 ± 2.0 17.2 ± 2.3 15.3 ± 2.1 17.4 ± 2.0 15.2 ± 2.1 31.1 ± 2.0 * 23.2 ± 2.3 21.3 ± 2.4 16.1 ± 2.1 Data are expressed as Mean ± SEM, n=8. Values ANOVA followed by multiple comparison two tail 't' test. Values with* differ from others significantly at (p < 0.05 ) in each column, *differ from others significantly in each column (p < 0.05) Table 2.: Effect of aqueous-methanolic extract of root of M. paradisiaca and leaf of C. indica on fasting blood glucose level in diabetic rat. Data are expressed as Mean ±SEM, n = 8 Values with or or * in each column differ from others significantly at (p<0.05) Group I 0 day 80.2 ± 4.3 1st day 80.3 ± 4.1 Fasting blood glucose level (mg/dl) 4th days 7th days 10th days 78.2 ± 4.2 79.5 ± 4.1 78.2 ± 4.5 13th days 78.1 ± 4.2 16th days 77.3 ± 4.3 II 78.7 ± 5.1 299.7 ± 4.3* 347.4 ± 4.6* 331.1 ± 4.4* 354.9 ± 5.2* 380.1 ± 4.9* 421.2 ± 5.0* III 79.2 ± 4.5 297.6 ± 4.2* 271.3 ± 4.5 255.4 ± 4.8 175.3 ± 4.4 161.1 ± 5.1 115.3 ± 4.7** IV 80.3 ± 4.3 298.2 ± 4.1* 273.4 ± 5.0 261.6 ± 4.6 179.2 ± 4.6 165.3 ± 5.0 122.4 ± 4.8** V 79.0 ± 4.1 306.3 ± 4.2* 250.3 ± 4.9* 153.2 ± 4.7* 110.5 ± 4.2* 94.3 ± 4.5* 84.2 ± 4.9 Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 367 Table 3: Effect of aqueous-methanolic extract of root of M. paradisiaca and leaf of C. indica on intravenous glucose tolerance in diabetic rats. Blood glucose level (mg/dl) (mg/dl) 30th min 60th min 90th min 350.6 ± 10.4 99.4 ± 8.3 95.2 ± 8.9 470.3 ± 10.2* 240.3 ± 10.1* 250.3 ± 10.1* 440.2 ± 10.1 118.5 ± 8.1 116.6 ± 9.1 420.7 ± 10.2 100.2 ± 8.4 102.4 ± 9.3 371.8 ± 10.8 90.0 ± 9.2 98.6 ± 9.3 Group 0 min 90.2 ± 8.1 217.0 ± 9.1* 120.3 ± 8.9 102.4 ± 8.3 97.2 ± 9.1 I II III IV V 120th min 92.0 ± 9.2 260.1 ± 10.2* 118.5 ± 8.8 100.3 ± 9.2 96.4 ± 8.9 Data are expressed as Mean ±SEM, n=8. ANOVA followed by multiple comparison two tail 't' test Values with * or in each column differ from others significantly at (p<0.05) 60 Liver 20 18 16 40 14 ** * * 20 µIU/ml µ g/mg of tissue 50 30 GroupI GroupII GroupIII GroupIV GroupV Muscle 12 10 8 6 b * 4 10 2 0 0 Glycogen content Serum1insulin Figure 1: Effect of aqueous-methanolic extract of root of M. paradisiaca and leaf of C. indica on serum insulin and glycogen content in liver and muscle. Data are expressed as Mean ±SEM, n=8. ANOVA followed by multiple comparison two tail 't' test. Values with * or ** on bar in case of specific parameter differ from others significantly at (p<0.05) Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 * 45 35 30 Unit/mg of tissue mg of IP/g of tissue 40 25 20 15 10 18 160 16 140 14 12 * 120 µg/ mg of tissue 50 368 10 8 6 0 G lucose-61 phosphatase * 100 80 60 40 4 5 G roupI G roupII G roupIII G roupIV G roupV 2 20 0 0 Hexokinase 1 Glucose-6-phosphate 1 dehydrogenase Figure 2: Effect of aqueous-methanolic extract of root of M. paradisiaca and leaf of C. indica on glucose-6phosphatase and glucose-6-phosphate dehydrogenase and hexokinase activities in liver. Data are expressed as Mean ±SEM, n = 8. ANOVA followed by multiple comparison two tail 't' test. Values with * or ** on each bar in case of specific parameter differ from others significantly at (p<0.05). 3 GOT * GPT GOT 3 2.5 * GPT * GroupI GroupII GroupIII GroupIV GroupV * 2.5 Unit/g of tissue Unit/g of tissue 2 1.5 1 0.5 2 1.5 1 0.5 0 0 1 Liver 2 1 Kidney 2 Figure 3: Effect of separate and composite methanolic extract of root of M. paradisiaca and leaf of C indica on GOT and GPT levels. Data are expressed as Mean ±SEM, n=8. ANOVA followed by multiple comparison two tail 't' test. Values with * or ** on each bar in case of specific parameter differ from others significantly at (p<0.05). Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 369 GOT & GPT activities Streptozotocin induced diabetes resulted in an elevation of GOT and GPT activities in liver and kidney at significant level when compared to control. After the treatment of composite extract, both parameters in liver as well as in kidney were in the levels of control group although the individual extract was able to protect these parameters partially (Figure 3). Discussion Streptozotocin-induced experimental diabetes is a valuable model for induction of type-I diabetes (Junod et al.,1969). The present investigation highlights the antidiabetic efficacy of aqueous-methanol extract of M. paradisiaca and C. indica along with comparative analysis of composite as well as separate extracts of these plant parts. Antihyperglycemic potency of these plant parts has been evaluated here by the measurement of body growth, water and fluid intake, fasting blood glucose level, intravenous glucose tolerance, activities of glucose-6phosphatase, glucose-6- phosphate dehydrogenase, hexokinase in liver, and quantification of liver and muscle glycogen along with serum insulin level. The present study is important in this respect as this is the first biochemical study on the effects of the extracts of these two plants in the management of type-I diabetes mellitus. Regarding the mode of action of the extract of these plants, the exact mechanism can not be deduced from this experiment but the following hypothesis may be proposed. Glucose-6-phosphate dehydrogenase utilize glucose through pentose phosphate pathway and its activity is under insulin (Tian et al., 1998). Induction of diabetes by STZ resulted in significant diminution in the activity of this enzyme in the liver and this is consistent with our previous report (Mallick et al., 2006). Aqueous-methanolic extract of these plants resulted in a significant recovery of these enzymes which may be one of the ways of antidiabetic efficacy of these plants. Elevation in the activity of glucose-6-phosphatase in STZ-induced diabetic group as reported in the previous studies (Pari and Amarnath, 2004). Extract of these plants resulted in a significant recovery in the activity of this enzyme in liver and this may suggest another possible way of its antidiabetogenic efficacy. The quantity of glycogen in both liver and skeletal muscle was observed to decrease significantly in STZ induced-diabetic rat as STZ selectively destroy β cells of Islets of Langerhance (Kavalali et al., 2002; Palmer et al., 1998) and there by lowering the blood level of insulin has been reported by others (Zhang et al., 2004). This is similar to our observation in this experiment. Separate and composite extract treatment of the above plants resulted in a significant recovery with respect to this effect and this may be due to either stimulation in insulin release from β cells noted in this study or due to insulinomimetic activity of some of the components present in this extract or due to combination of both effects. Protective effect of diabetes of the extract of above plants has been supported with the results obtained with regards to food intake and water intake tending towards the control level in diabetic animal after the extract coadministration. Diabetic condition resulted in liver and renal toxicity induction, which has been observed in this study as shown by GOT and GPT assessment and as reported by others (Ghosh and Suryawanshi, 2001). Both separate and composite extract treatment in diabetic animal resulted in a significant recovery in the levels of GOT and GPT activities in liver and in kidney with respect to diabetic group. As GOT and GPT activities are considered as indicators of general toxicity (Akther et al.,1990; Das et al., 2006) so we suggest that these experimental plant parts have no general toxic effect. From the comparative analysis, it has been revealed that the extract of the studied plants when used in composite manner on streptozotocin-induced diabetic state has a more potent antidiabetogenic effect in comparison to the antidiabetogenic activities of the individual extract of the plants. In conclusion, it may be stated that this composite extract contains the active antihyperglycemic agent (s) that can be used to overcome diabetic complication by pancreatic β cell regeneration or stimulation of insulin secretion or in other ways. Acknowledgement This research work was funded by University Grants Commission, New Delhi, India. Mallick et al., Afr. J. Trad. CAM (2007) 4 (3): 362 - 371 370 References 1. Akther, M. H., Mathur, M. and Vhide, N. K. (1990) Skin and liver toxicity in experimental Lantana camara poisoning in albino rats. Ind. J Physiol. Pharmacol. 34: 13-16. 2. Brugi, W., Briner, M., Fraken, N. and Kessler, A.C.H. (1988). 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https://openalex.org/W3022362948	https://www.pure.ed.ac.uk/ws/files/147076188/s42003_020_0942_0.pdf	English	null	Methylation deficiency disrupts biological rhythms from bacteria to humans	Communications biology	2,020	cc-by	14,870	Edinburgh Research Explorer Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Methylation deficiency disrupts biological rhythms from bacteria to humans Citation for published version: Fustin, J-M, Ye, S, Rakers, C, Kaneko, K, Fukumoto, K, Yamano, M, Versteven, M, Grunewald, E, Cargill, S, Tamai, TK, Xu, Y, Jabbur, ML, Kojima, R, Lamberti, ML, Yoshioka Kobayashi, K, Whitmore, D, Tammam, S, Howell, PL, Kageyama, R, Matsuo, T, Stanewsky, R, Golombek, DA, Johnson, CH, Kakeya, H, Van Ooijen, G & Okamura, H 2020, 'Methylation deficiency disrupts biological rhythms from bacteria to humans', Communications Biology, vol. 3, no. 1, 211. https://doi.org/10.1038/s42003-020-0942-0 Citation for published version: Citation for published version: Fustin, J-M, Ye, S, Rakers, C, Kaneko, K, Fukumoto, K, Yamano, M, Versteven, M, Grunewald, E, Cargill, S, Tamai, TK, Xu, Y, Jabbur, ML, Kojima, R, Lamberti, ML, Yoshioka Kobayashi, K, Whitmore, D, Tammam, S, Howell, PL, Kageyama, R, Matsuo, T, Stanewsky, R, Golombek, DA, Johnson, CH, Kakeya, H, Van Ooijen, G & Okamura, H 2020, 'Methylation deficiency disrupts biological rhythms from bacteria to humans', Communications Biology, vol. 3, no. 1, 211. https://doi.org/10.1038/s42003-020-0942-0 Document Version: Publisher's PDF, also known as Version of record Published In: Communications Biology 1 Graduate School of Pharmaceutical Sciences, Laboratory of Molecular Metabology, Kyoto University, Kyoto, Japan. 2 Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. 3 Graduate School of Pharmaceutical Sciences, Department of System Chemotherapy and Molecular Sciences, Kyoto University, Kyoto, Japan. 4 Institute of Neuro- and Behavioral Biology, University of Münster, Münster, Germany. 5 School of Biological Sciences, University of Edinburgh, Edinburgh, UK. 6 Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, Los Angeles, CA, USA. 7 Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA. 8 Karolinska Institute, Stockholm, Sweden. 9 Department of Science and Technology, National University of Quilmes/CONICET, Buenos Aires, Argentina. 10 Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan. 11 Centre for Cell and Molecular Dynamics, Department of Cell and Developmental Biology, University College London, London, UK. 12 Molecular Medicine, Peter Gilgan Centre for Research and Learning (PGCRL), The Hospital for Sick Children, Toronto, ON, Canada. 13 Department of Biochemistry, University of Toronto, Toronto, ON, Canada. 14 Center for Gene Research, Nagoya University, Nagoya, Japan. 15 Graduate School of Pharmaceutical Sciences, Laboratory of Molecular Brain Science, Kyoto University, Kyoto, Japan. 16Present address: The University of Manchester, Faculty of Biology, Medicine and Health, Oxford Road, Manchester M13 9PL, UK. 17Present address: Kyoto University, Graduate School of Medicine, Department of Neuroscience, Division of Physiology and Neurobiology, Yoshida-Konoe-cho, Oxford Road, Sakyo-ku, Kyoto 606-8501, Japan. 18These authors contributed equally: Jean-Michel Fustin, Shiqi Ye. ✉email: jean-michel.fustin@manchester.ac.uk; okamura.hitoshi.4u@kyoto-u.ac.jp Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio Methylation deﬁciency disrupts biological rhythms from bacteria to humans Jean-Michel Fustin 1,16,18✉, Shiqi Ye1,18, Christin Rakers 2, Kensuke Kaneko3, Kazuki Fukumoto1, Mayu Yamano1, Marijke Versteven4, Ellen Grünewald5, Samantha J. Cargill5, T. Katherine Tamai 6, Yao Xu7, Maria Luísa Jabbur7, Rika Kojima8, Melisa L. Lamberti9, Kumiko Yoshioka-Kobayashi10, David Whitmore 11, Stephanie Tammam12, P. Lynne Howell 12,13, Ryoichiro Kageyama10, Takuya Matsuo14, Ralf Stanewsky 4, Diego A. Golombek9, Carl Hirschie Johnson7, Hideaki Kakeya 3, Gerben van Ooijen 5 & Hitoshi Okamura15,17✉ The methyl cycle is a universal metabolic pathway providing methyl groups for the methy- lation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deﬁciencies. 1 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 M Amino acids contributing to the DZnep binding site showed at least 88% identity between all eukaryotic AHCY sequences, and 78% between human and bacterial sequences, indicating that the DZnep binding site is virtually identical across a wide range of organisms (Fig. 1b, c and Supplementary Fig. 2). Moreover, amino acids that were reported as crucial for the activity of rat AHCY (His55, Asp130, Glu155, Lys186, Asp190, and Asn19119) are universal (Supplementary Fig. 1). In line with the above, molecular docking simulations of AHCY with adenosine or DZnep revealed comparable ligand binding conformations and estimated binding free energies for all organisms (Fig. 1d and Supplementary Fig. 2). M ethylation reactions start with the metabolization of methionine into S-adenosylmethionine, or SAM: the universal methyl donor co-substrate in the trans- methylation of nucleic acids, proteins, carbohydrates, phospho- lipids and small molecules. During the methylation process, SAM is converted into adenosylhomocysteine (SAH) that is rapidly hydrolyzed into homocysteine (Hcy) and adenosine by the enzyme adenosylhomocysteinase (AHCY) to prevent competitive inhibition of methyltransferase enzymes by SAH. Methylation deﬁciency disrupts biological rhythms from bacteria to humans The ratio SAM/ SAH is critical and is a measure of the methylation potential: the tendency to methylate biomolecules1–3. Methylation deﬁciencies, either from poor diet or genetic polymorphisms, contribute to the etiology of many pathologies: cancer, atherosclerosis, birth defects, aging, diabetes and pancreatic toxicity, hepatotoxicity and neurological disturbances4. DZnep is sometimes erroneously considered to be a histone methyltransferase EZH2 inhibitor, because of the misinterpreta- tion of a report showing that it inhibits histone methylation sites targeted by EZH2 in cancer cells21. More recent reports showed that DZnep widely inhibits histone methylation, in line with its inhibitory effect via AHCY22,23. Together these observations strongly support the use of DZnep as a valid approach to disrupt the methyl cycle across taxa, as we next demonstrate. An endogenous circadian clock evolved to anticipate the daily cycles of light and darkness has been found in many organisms, from cyanobacteria to humans. Transcription-translation feed- back loops of “clock genes” directly or indirectly regulating their own transcription underlie many functions of the clock, and drive oscillations of output genes controlling physiology and behavior. Some molecular components of the clock are remarkably con- served in Metazoa, notably the genes Clock and Period, coding for transcription factors, with Clock activating the transcription of Per and Per inhibiting its own transcription5. In 2013, we reported that inhibition of the methyl cycle by AHCY inhibitors strongly affected the circadian clock in mouse and human cells6. We now show that the link between methylation and the circa- dian clock we uncovered in mammals has been conserved during more than 2.5 billion years of evolution, and that circadian rhythm perturbations can be used as a quantitative gauge for the physiological consequences of methylation deﬁciency. Methylation deﬁciency disrupts the metazoan circadian clock. To test the effects of DZnep on the mammalian clock, we exposed a human osteosarcoma cell line (U-2 OS) and PER2::LUC mouse embryonic ﬁbroblasts (MEFs), expressing bioluminescent clock reporters24,25, to DZnep concentrations. In human (Fig. 2a, b) and mouse (Fig. 2c, d) cells, DZnep potently and dose- dependently increased the period of circadian oscillations, visualized by bioluminescent rhythms of Bmal1 or Per2 expres- sion, respectively. Methylation deﬁciency disrupts biological rhythms from bacteria to humans We next tested DZnep on the zebraﬁsh embryonic PAC2 cell line expressing a luminescent reporter of the Per1b gene, a non- mammalian cell type commonly used for circadian studies, and revealed, as observed in mammalian cells, a clear effect of the drug on the circadian period (Fig. 2e, f). An effect on the entrainment to the light/dark cycles (see methods) was also observed: The Per1b gene normally peaks at dawn but was severely blunted and delayed in DZnep-treated cells. Bacterial species exist that lack AHCY but instead express an ancient SAH nucleosidase that hydrolyses SAH into S- ribosylhomocysteine and adenine7,8. Surprisingly, partial rewir- ing of the mammalian methyl cycle by ectopically expressing the E. coli SAH nucleosidase completely protected the methyl cycle from AHCY inhibition, and allowed normal circadian rhythms, even in the presence of a saturating concentration of inhibitor. These observations demonstrate the importance of methyl metabolism in the regulation of biological rhythms from cyano- bacteria to humans and suggest a therapeutic application of methyl cycle reprogramming to alleviate the detrimental impact of methylation deﬁciencies. To test the effects of methylation deﬁciency on circadian rhythms in invertebrates, we exposed Drosophila melanogaster haltere cultures from transgenic luciferase reporter ptim TIM- LUC ﬂies to DZnep. As observed in vertebrates, DZnep caused dose-dependent period lengthening (Fig. 2g, h). In addition, an effect of 100 μM DZnep on the luminescent rhythms reporting the expression of the sur-5 gene was also observed in freely moving C. elegans (Supplementary Fig. 3), a relatively new model in circadian biology26. Results AHCY is a highly conserved enzyme in the methyl cycle. Methylation deﬁciency can be induced by carbocyclic adenosine analogs identiﬁed as AHCY inhibitors more than 30 years ago, such as 3-Deazaneplanocin A (DZnep)9–11. This pharmacological inhibition mimics the pathological symptoms caused by AHCY deﬁciency12, such as high plasma methionine, SAM and SAH; all indicators of methyl cycle aberrations. AHCY catalyzes the clea- vage of SAH to adenosine and L-homocysteine, and DZnep inhibits this reaction by occupying the adenosine binding site of AHCY. The crystal structures of human13, mouse14 and yellow lupin (Lupinus luteus)15 AHCY complexed with adenosine or analogs have been described, and insights into its catalytic activity have been obtained16–19. AHCY is one of the most evolutionarily conserved proteins20, but experimentally determined structures of AHCY complexed with DZnep remain to be described for most organisms. A full-length multiple sequence alignment (Supple- mentary Fig. 1) and homology modeling of AHCY from humans to cyanobacteria (Fig. 1a and Supplementary Movie 1) revealed high conservation of sequence and predicted tertiary structure. Methylation deﬁciency also disrupts the somite segmentation clock. While the metazoan circadian clock is an oscillator with a period near 24 h, the somite segmentation clock in mammalian embryos cycles much faster and orchestrates the appearance of new segments or “somites” from the paraxial mesoderm (Sup- plementary Fig. 4a). The underlying molecular oscillator is cen- tered on the transcription factor Hairy & Enhancer Of Split 7 (Hes7), the expression of which oscillates with a period close to 2 h in mouse27. Like circadian clock genes, Hes7 oscillatory expression can be monitored in real-time from transgenic embryos expressing highly destabilized luciferase under the control of the Hes7 promoter. Testing increasing concentrations of DZnep on these transgenic embryos revealed clear similarity to the effects of DZnep on circadian clock markers, resulting in the lengthening of the somite segmentation clock period (Supple- mentary Fig. 4b–d, Supplementary Movie 2). Together, these data demonstrate the importance of the methyl cycle in the regulation COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio 2 ARTICLE nosylhomocysteinase is a highly conserved protein. a Structural superposition of AHCY from the 9 organisms investigated here, using hu use (5AXA) or lupin (3OND) crystal structures as templates. The blue loop is speciﬁc to plants and green algae; DZnep is shown in yel lack. See also Supplementary Movie 1. Results b Docking simulation of human AHCY with DZnep, based on the 1LI4 crystal structure of human A with Neplanocin A, an analog of DZnep. The amino acids involved in DZnep binding are indicated with their position. See Supplementary F simulations of DZnep to AHCY from other organisms. Red and green arrows are hydrogen bonds, yellow spheres are hydrophobic effects ree energy of binding for depicted DZnep docking conformation was −9.87 kcal/mol. c Discontinuous alignment of amino acids contributin of DZnep, using the human sequence as a reference and with sequence identities shown on the right. When amino acids are identical to hu own in the alignment. The sequence logo on top is a graphical representation of the conservation of amino acids, with the consensus sym fully conserved residue, : = conservation of strongly similar properties, . = conservation of weakly similar properties). The positions of sele amino acids are gi en for the h man se ence on top of the alignment d Molec lar docking sim lations of AHCY ith adenosine and DZ ICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 ARTIC COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 Fig. 1 Adenosylhomocysteinase is a highly conserved protein. a Structural superposition of AHCY from the 9 organis Fig. 1 Adenosylhomocysteinase is a highly conserved protein. a Structural superposition of AHCY from the 9 organisms investigated here, using human (1LI4), mouse (5AXA) or lupin (3OND) crystal structures as templates. The blue loop is speciﬁc to plants and green algae; DZnep is shown in yellow, NAD+ in black. See also Supplementary Movie 1. b Docking simulation of human AHCY with DZnep, based on the 1LI4 crystal structure of human AHCY complexed with Neplanocin A, an analog of DZnep. The amino acids involved in DZnep binding are indicated with their position. See Supplementary Fig. 2 for docking simulations of DZnep to AHCY from other organisms. Red and green arrows are hydrogen bonds, yellow spheres are hydrophobic effects. The estimated free energy of binding for depicted DZnep docking conformation was −9.87 kcal/mol. c Discontinuous alignment of amino acids contributing to the binding of DZnep, using the human sequence as a reference and with sequence identities shown on the right. When amino acids are identical to human, a dot is shown in the alignment. Results The sequence logo on top is a graphical representation of the conservation of amino acids, with the consensus symbols below (* = fully conserved residue, : = conservation of strongly similar properties, . = conservation of weakly similar properties). The positions of selected conserved amino acids are given for the human sequence on top of the alignment. d Molecular docking simulations of AHCY with adenosine and DZnep showing comparable binding free energies in all organisms. Colors represent full sequence identities, relative to human. homocysteinase is a highly conserved protein. a Structural superposition of AHCY from the 9 organisms investigated Fig. 1 Adenosylhomocysteinase is a highly conserved protein. a Structural superposition of AHCY from the 9 organisms investigated here, using human (1LI4), mouse (5AXA) or lupin (3OND) crystal structures as templates. The blue loop is speciﬁc to plants and green algae; DZnep is shown in yellow, NAD+ in black. See also Supplementary Movie 1. b Docking simulation of human AHCY with DZnep, based on the 1LI4 crystal structure of human AHCY complexed with Neplanocin A, an analog of DZnep. The amino acids involved in DZnep binding are indicated with their position. See Supplementary Fig. 2 for docking simulations of DZnep to AHCY from other organisms. Red and green arrows are hydrogen bonds, yellow spheres are hydrophobic effects. The estimated free energy of binding for depicted DZnep docking conformation was −9.87 kcal/mol. c Discontinuous alignment of amino acids contributing to the binding of DZnep, using the human sequence as a reference and with sequence identities shown on the right. When amino acids are identical to human, a dot is shown in the alignment. The sequence logo on top is a graphical representation of the conservation of amino acids, with the consensus symbols below (* = fully conserved residue, : = conservation of strongly similar properties, . = conservation of weakly similar properties). The positions of selected conserved amino acids are given for the human sequence on top of the alignment. d Molecular docking simulations of AHCY with adenosine and DZnep showing comparable binding free energies in all organisms. Colors represent full sequence identities, relative to human. COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 ythms are a quantitative gauge for methylation deﬁciency in Metazoa. Results a Mean luminescence ± SEM of human Bmal1-luc U-2 O increasing concentrations of DZnep; b shows mean ± SEM of period, n = 3 dishes. c Mean luminescence ± SEM of PER2::LUC ME p, n = 3 dishes; mean ± SEM of period shown in d. e Mean luminescence ± SEM of Per1b-luciferase PAC-2 zebraﬁsh cells treated w es; mean ± SEM of period shown in f; note the initial 96 h of data performed under light/dark cycles. g Mean luminescence ± SEM ophila halteres treated with DZnep n = 8 independent halteres with only the upper segment of the error bars shown for clarity; mea Fig. 2 Circadian rhythms are a quantitative gauge for methylation deﬁciency in Metazoa. a Mean luminescence ± SEM of human Bmal cells25 treated with increasing concentrations of DZnep; b shows mean ± SEM of period, n = 3 dishes. c Mean luminescence ± SEM of PER treated with DZnep, n = 3 dishes; mean ± SEM of period shown in d. e Mean luminescence ± SEM of Per1b-luciferase PAC-2 zebraﬁsh cel DZnep, n = 4 dishes; mean ± SEM of period shown in f; note the initial 96 h of data performed under light/dark cycles. g Mean luminesc ptim-TIM-LUC Drosophila halteres treated with DZnep, n = 8 independent halteres, with only the upper segment of the error bars shown for c SEM of period shown in h. All bar graphs analyzed by One-Way ANOVA followed by Bonferroni’s test; all indicated comparisons (a vs. b vs at least p < 0.05. 4 COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio Fig. 2 Circadian rhythms are a quantitative gauge for methylation deﬁciency in Metazoa. a Mean luminescence ± SEM of human Bmal1-luc U-2 OS cells25 treated with increasing concentrations of DZnep; b shows mean ± SEM of period, n = 3 dishes. c Mean luminescence ± SEM of PER2::LUC MEF24 treated with DZnep, n = 3 dishes; mean ± SEM of period shown in d. e Mean luminescence ± SEM of Per1b-luciferase PAC-2 zebraﬁsh cells treated with DZnep, n = 4 dishes; mean ± SEM of period shown in f; note the initial 96 h of data performed under light/dark cycles. Results Ostreococcus tauri and Chlamydomonas reinhardtii represent two different classes of unicellular green algae that have successfully been used in circadian studies and constitute great models to investigate cell-autonomous metabolic processes28–30. We tested increasing concentration of DZnep on Arabidopsis (Fig. 3a, b), Ostreococcus tauri (Fig. 3c, d) and Chlamydomonas reinhardtii (Fig. 3f, g), and observed an increase in period length almost identical to the results obtained in vertebrates. A plant- and green algae-speciﬁc domain has been previously described in AHCY31, from amino acids 151 to 191 (Fig. 1 and Supplementary Fig. 1). Despite this insertion, however, the domains for SAH and NAD+ binding are remarkably conserved. We already showed that DZnep analogs increase SAH in mammalian cells6, but to conﬁrm that DZnep does increase SAH also in the green plant clade, we quantiﬁed SAM and SAH in DZnep-treated algae and revealed a dose- dependent increase in SAH and a decrease in methylation potential (Fig. 3e, h and Supplementary Fig. 5). In conclusion, the effects of DZnep treatment on transcriptional rhythms are also conserved in the plant kingdom. These results are especially meaningful given unicellular algae and humans are separated by more than 1 billion years of evolution32. Bacterial SAH nucleosidase rescues mammalian cells from AHCY inhibition. AHCY mutations (R49C, D86G, Y143C, W112X and A89V) in humans lead to greatly elevated levels of S- adenosylhomocysteine (>100-fold), causing early developmental stagnation and severe pathophysiology12,38,39. If the methyl cycle in these patients could be rewired to prevent S- adenosylhomocysteine build-up, it would protect them from AHCY deﬁciency. To test this potentially transformative hypothesis, we ectopically expressed the wild-type (WT) or the inactive D197A mutant MTAN from E. coli8 in mouse and human cells. In itself, expression of the MTAN variants, which was conﬁrmed in mouse and human cells by immunoblotting, did not signiﬁcantly change circadian parameters (Supplementary Fig. 6a, b). Cells transfected with either vectors grew well and did not display any abnormalities, in line with S-ribosylhomocysteine being a meaningless metabolite in mammalian cells. Strikingly, a complete protection from the effects of DZnep was seen in human cells expressing WT MTAN, even at the saturating con- centrations of 10 and 100 μM (Fig. 5a). D197A MTAN did not provide protection, indicating the speciﬁcity of the effect (Fig. 5b). Identical results were observed in mouse cells (Fig. 5c, d). Results g Mean luminescence ± SEM of ptim-TIM-LUC Drosophila halteres treated with DZnep, n = 8 independent halteres, with only the upper segment of the error bars shown for clarity; mean ± SEM of period shown in h. All bar graphs analyzed by One-Way ANOVA followed by Bonferroni’s test; all indicated comparisons (a vs. b vs. c vs. d vs. e), at least p < 0.05. Fig. 2 Circadian rhythms are a quantitative gauge for methylation deﬁciency in Metazoa. a Mean luminescence ± SEM of human Bmal1-luc U-2 OS cells25 treated with increasing concentrations of DZnep; b shows mean ± SEM of period, n = 3 dishes. c Mean luminescence ± SEM of PER2::LUC MEF24 treated with DZnep, n = 3 dishes; mean ± SEM of period shown in d. e Mean luminescence ± SEM of Per1b-luciferase PAC-2 zebraﬁsh cells treated with DZnep, n = 4 dishes; mean ± SEM of period shown in f; note the initial 96 h of data performed under light/dark cycles. g Mean luminescence ± SEM of ptim-TIM-LUC Drosophila halteres treated with DZnep, n = 8 independent halteres, with only the upper segment of the error bars shown for clarity; mean ± SEM of period shown in h. All bar graphs analyzed by One-Way ANOVA followed by Bonferroni’s test; all indicated comparisons (a vs. b vs. c vs. d vs. e), at least p < 0.05. COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio 4 4 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 of transcriptional programs orchestrating biological rhythms in Metazoa. in cyanobacteria to elicit methylation-dependent morphological changes37. Sinefungin was more potent than DZnep, causing a dose-dependent period lengthening of a much greater magnitude (Fig. 4c, d). These data reveal an unexpected role for methylation even in the cyanobacterial circadian clock, but the methyl cycle in these cells appeared more resilient to DZnep- induced deﬁciency, maybe because of an alternative pathway for SAH catabolism that eukaryotes lack (Fig. 4e). Methylation deﬁciency disrupts the clock in plants and algae. The most commonly used model organism to study circadian rhythms in land plants is Arabidopsis thaliana. We thus tested the effects of DZnep on luminescent rhythms reporting the expres- sion of the plant evening gene TOC1 in protoplasts and extended our investigations to aquatic unicellular green algae. Results Quantiﬁcation of period in human and mouse cells clearly revealed this protective effect of WT MTAN (Fig. 5e, f). Mortality and/or cell growth inhibition was observed at high DZnep con- centrations on cells actively growing, but WT MTAN protected the cells against this effect whereas D197A MTAN did not (Supplementary Fig. 6c–f). Quantiﬁcation of SAM and SAH conﬁrmed the DZnep-dependent increase in SAH and resultant collapse of the methylation potential in cells transfected with mutant, but not wild-type MTAN (Fig. 5g, Supplementary Fig. 6g, h). Interestingly, in vehicle-treated cells the expression of the wild-type MTAN was sufﬁcient to increase the methylation potential compared to cells expressing the mutant enzyme (Supplementary Fig. 6g, h). The methyl cycle in cyanobacteria is less sensitive to AHCY inhibition. The circadian oscillator in cyanobacteria is composed of three proteins. Kai-A, -B and -C form a complex that regulates two key activities of KaiC: ATPase and autophosphorylation activity that result in an autonomous and self-sustained phosphorylation- based oscillator ticking with a period close to 24-h. In a cellular context, this non-transcriptional oscillator controls transcriptional outputs that in turn add robustness to the biochemical oscillator via transcription-translation feedback loops33. Despite these pro- nounced differences in clock architecture, the circadian period in cyanobacteria lengthened in response to low concentrations of DZnep (Fig. 4a, b), but with a much lower magnitude compared to that in the eukaryotes tested here. Finally, we sought to determine the effects of DZnep on global lysine and arginine methylation, on RNA and mRNA N6- methyladenosine (m6A), and on DNA 5-methylcytosine (m5C), as well as the rescue of their potential inhibition by MTAN in mouse cells. In all cases, PER2::LUC MEFs were treated with DZnep 0, 5 or 10 μM (to obtain near-maximum period lengthening) for 48-hours. Inhibition of methylations by DZnep depends on the unique activity of AHCY to directly hydrolyze SAH into Hcy and adenosine. In prokaryotes, the enzyme 5’-methylthioadenosine/S- adenosylhomocysteine nucleosidase (MTAN) has a SAH nucleo- sidase activity that cleaves SAH to adenine and S- ribosylhomocysteine34. This pathway for AHCY catabolism, acting as a buffer against SAH accumulation, could explain the weaker response to DZnep compared to eukaryotes. Indeed, MTANs have been identiﬁed in some Synechococcus strains, such as PCC7336 and MED-G6935. The lack of a strong response to DZnep may be due either to the presence of MTAN in S. COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio Results elongatus, to DZnep being unable to bind to AHCY (this may be supported by the lowest sequence identity of AHCY in S. elongatus shown in Fig. 1) or having low bioavailability, or to the lack of methylation-dependent regulation of the cyanobacterial clock. Surprisingly, higher concentrations of the drug caused no signiﬁcant effects on the period (Fig. 4b). Therefore, to independently verify the importance of methylations for cyanobacterial clock, we used the global methylation inhibitor sinefungin. This is a natural analog of SAM that directly binds to and inhibits methyltransferases36; sinefungin was previously used Immunoblotting using an antibody against mono- and di- methylated lysine showed that a few proteins had lower methylated levels under DZnep treatment, but only in cells transfected with the mutant inactive MTAN (Fig. 5h). Probing mono- and di-methylated arginine in the same cells showed somewhat less pronounced methylation inhibition, which was also rescued by WT MTAN (Supplementary Fig. 6i). We also checked the global levels of the speciﬁc methyl marks Histone 4 Arginine 3 symmetric demethylation (transcriptional repression) and Histone H3 Lysine 4 trimethylation (transcrip- tional activation) but little inhibition was seen (Supplementary Fig. 6i). N6-methyladenosine did not signiﬁcantly ﬂuctuate between treatments when measured from total RNA samples, composed mainly (>90%) of 18S and 28S rRNA, each macromolecule 5 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 l i l 6A it (S ppl t Fi 6j) It i p ibl th t l t t t ith DZ p gical rhythms are a quantitative gauge for methylation deﬁciency in plants and algae. a Mean luminescence ± SEM of Arabidopsi C protoplasts treated with different concentration of DZnep, n = 8 wells per treatment. For comparison between different runs, tra ation to the ﬁrst peak; b shows mean ± SEM of period, n = 8 wells. c Mean luminescence ± SEM of CCA1-LUC Ostreococcus tauri ce ws mean ± SEM of period, n = 7 wells. No signiﬁcance was observed between 10, 20 and 30 μM, but the signiﬁcance compared to nger, i.e., p < 0.05, p < 0.001, p < 0.0001, respectively, indicating dose-dependent effects. e Mean SAH concentration ± SEM in O. tau cated concentrations of DZnep, n = 3 wells. f Mean luminescence ± SEM of tufA-lucCP Chlamydomonas reinhardtii CBR cells treate 5 wells per treatment; g shows mean ± SEM of period, n = 5 wells. h Mean SAH concentration ± SEM in C. Results reinhardtii treated with the ns of DZnep, n = 4 wells. All bar graphs analyzed by One-Way ANOVA (all p < 0.0001) followed by Bonferroni’s test; all indicate (a vs. b vs. c vs. d) at least p < 0.05. See also Supplementary Fig. 5. LE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020 Fig. 3 Biological rhythms are a quantitative gauge for methylation deﬁciency in plants and algae. a Mean luminescence ± SEM of Arabidopsis thaliana CCA1pro:LUC protoplasts treated with different concentration of DZnep, n = 8 wells per treatment. For comparison between different runs, traces were aligned in relation to the ﬁrst peak; b shows mean ± SEM of period, n = 8 wells. c Mean luminescence ± SEM of CCA1-LUC Ostreococcus tauri cells, n = 7 wells; d shows mean ± SEM of period, n = 7 wells. No signiﬁcance was observed between 10, 20 and 30 μM, but the signiﬁcance compared to 0 μM became stronger, i.e., p < 0.05, p < 0.001, p < 0.0001, respectively, indicating dose-dependent effects. e Mean SAH concentration ± SEM in O. tauri treated with the indicated concentrations of DZnep, n = 3 wells. f Mean luminescence ± SEM of tufA-lucCP Chlamydomonas reinhardtii CBR cells treated with DZnep, n = 5 wells per treatment; g shows mean ± SEM of period, n = 5 wells. h Mean SAH concentration ± SEM in C. reinhardtii treated with the indicated concentrations of DZnep, n = 4 wells. All bar graphs analyzed by One-Way ANOVA (all p < 0.0001) followed by Bonferroni’s test; all indicated comparisons (a vs. b vs. c vs. d) at least p < 0.05. See also Supplementary Fig. 5. containing only one single m6A site (Supplementary Fig. 6j). When quantiﬁed from mRNA, however, a decrease in m6A was detected under DZnep treatment, but only in cells transfected by the mutant inactive MTAN (Fig. 5i, j, k). In contrast, DNA m5C was not signiﬁcantly affected (Supplemen- tary Fig. 6k). It is possible that a longer treatment with DZnep may have affected more stable methylations such as m6A in rRNA and m5C in DNA or caused a more widespread inhibition of histone methylation, but since the period lengthening effects of DZnep are observable by 24 h we speculate these methylations may not contribute to the period lengthening. Results Promoter-speciﬁc DNA or COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio 6 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 obacteria are less sensitive to AHCY inhibition. a Mean luminescence ± SEM of Synechococcus PCC 7942 kaiBCp::luxAB knock-in strain treate t concentrations of DZnep, n = 3 colonies, with only the upper section of the error bars and higher DZnep concentrations shown on a differen Fig. 4 Cyanobacteria are less sensitive to AHCY inhibition. a Mean luminescence ± SEM of Synechococcus PCC 7942 kaiBCp::luxAB knock-in with different concentrations of DZnep, n = 3 colonies, with only the upper section of the error bars and higher DZnep concentrations shown Fig. 4 Cyanobacteria are less sensitive to AHCY inhibition. a Mean luminescence ± SEM of Synechococcus PCC 7942 kaiBCp::luxAB knock-in strain treated with different concentrations of DZnep, n = 3 colonies, with only the upper section of the error bars and higher DZnep concentrations shown on a different graph for clarity; b shows mean period ± SEM, n = 3 colonies. c Mean luminescence ± SEM of Synechococcus PCC 7942 kaiBCp::luxAB treated with different Sinefungin concentrations as indicated over the graphs; d shows mean period ± SEM, n = 3 colonies. e Organization of the methyl cycle, with the two-steps SAH conversion to homocysteine in bacteria, starting with MTAN. One arrow represents one enzyme, except for methyltransferases that all use SAM as a co-substrate to methylate different targets, generating SAH in the process. The enzyme AHCY, mediating SAH hydrolysis and inhibited by DZnep, is indicated on the picture. All bar graphs analyzed by One-Way ANOVA followed by Bonferroni’s test; all indicated comparisons (a vs. b vs. c vs. d) at least p < 0.05. COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio 7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 Discussion methionine in the cell, the synthesis of SAM and downstream methylations can continue unimpaired even when Hcy is low. Hcy is however also used for the synthesis of glutathione (GSH) via cystathionine and cysteine, a metabolic route called the transsulfuration pathway (Hcy →cystathionine →cysteine → GSH), which could potentially have been inhibited by MTAN expression and DZnep. To investigate potential disruption of this pathway caused by either DZnep or MTAN, we quantiﬁed these metabolites from cell extracts used in Fig. 5g. As expected, expression of WT MTAN caused a decrease in Hcy compared to cells expressing D197A (Supplementary Fig. 7). Overall, however, the transsulfuration pathway appeared more active in cells expressing WT MTAN, especially given the higher concentrations of cysteine and GSH, which were probably due to the increase in SAM observed under the same conditions (Supplementary Fig. 6g), SAM being a powerful allosteric activator of the trans- sulfuration pathway46. A general tendency of DZnep to inhibit the transsulfuration pathway was observed, more pronounced in cells expressing the mutant MTAN (Supplementary Fig. 7). Glutathione disulﬁde (GSSG), generated during enzyme-catalyzed reduction reactions of GSH with peroxides or disulﬁde bonds, tended to decrease under DZnep treatment (Supplementary Fig. 7). The GSH/GSSH ratio, a measure of oxidative stress, was not signiﬁcantly affected by either DZnep or MTAN, indicating that the mild effects of DZnep and MTAN expression on the transsulfuration pathway are unlikely to contribute to the circa- dian period lengthening (Supplementary Fig. 7). These data do show that WT MTAN also rescues the cells from the mild inhi- bition of the transsulfuration pathway by DZnep, however. Solar ultraviolet radiation played a critical role in prebiotic chemistry, but the energy of UV photons can also degrade bio- logically important molecules, preventing abiogenesis. Once autocatalytic pseudo-life forms evolved with more complex chemistry, avoidance mechanisms may have provided increased ﬁtness in such an environment. The origin of the circadian clock on the early Earth is likely to have been a simple sensing of molecules present in the milieu, increasing or decreasing under UV exposure and triggering an appropriate response. Methyl cycle metabolites are thought to have been present in the pre- biotic world, since methionine can be created by a spark discharge and is proposed to be an intermediate in the prebiotic synthesis of Hcy, which was also found among organic molecules synthesized in the 1972 Miller experiment42. Discussion In addition, formaldehyde, an abundant prebiotic molecule increasing during the day under the action of UV on carbon monoxide and water vapor43 could have affected the chemistry of early life-forms by methylation44 and may have worked like a chemical marker of daylight. A conserved link between methyl transfer and the circadian clock may have arisen from such a scenario. In mammalian cells we previously reported that methyl cycle inhibition decreases the methylation of internal adenosines in mRNA (m6A), as well as that of histones6. While speciﬁc mRNA m6A inhibition was sufﬁcient to elicit period elongation, histone methylation likely contributed to the period elongation obtained by DZnep, as well as that of other methylation sites in mRNA, rRNA and tRNA. Due to the considerable heterogeneity in mechanisms regulating gene expression and function in organ- isms tested here, identifying a single mechanism explaining per- iod elongation in all taxa would be a difﬁcult undertaking and is beyond the scope of the present work. Since the oldest methyl- transferases are RNA methyltransferases45, it is tempting to propose that inhibition of RNA methylation may at least partially contribute to the period lengthening in all species. p y y p The somite segmentation clock was strongly affected by inhi- bition of AHCY, which is in line with the importance of 1 carbon metabolism for embryonic development. As can be seen in the Supplementary Movie 3, showing similar results to Supplemen- tary Movie 2 but merged with brightﬁeld images and displaying luminescence as a pseudo-color green, period lengthening of the oscillatory expression of Hes7-luciferase occurred together with a pronounced delay in the growth of the caudal tip of the pre- somitic mesoderm as well as in the appearance of new somites, demonstrating that this molecular clock as well as its output, i.e. the somitogenesis, were affected by DZnep. The mechanisms underlying this period lengthening may be distinct from those involved in the lengthening of the circadian period. Considering the short period of Hes7 oscillations—2 h—, and the importance of 3’-UTR-dependent regulation of Hes7 mRNA turnover for its cyclic expression47, the inhibition of mRNA methylation may at least in part contribute to the results observed. That mammalian cells expressing E. coli MTAN are completely protected from the period-lengthening effects of DZnep demon- strates that these effects are solely dependent upon methyl cycle inhibition and SAH accumulation. ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 Fig. 5 Rewiring the methyl cycle protects mammalian cells from methylation deﬁciency. a, b Mean luminescence ± SEM of human Bmal1-luc U-2 OS cells transfected with WT and mutant MTAN, respectively, and treated with different concentrations of DZnep, n = 3 dishes per treatment. c, d Mean luminescence ± SEM of PER2::LUC MEFs transfected with WT and mutant MTAN, respectively, and treated with different concentrations of DZnep, n = 3 dishes per treatment. e, f Mean period ± SEM of Bmal1-luc U-2 OS cells and PER2::LUC MEFs from a, b and c, d, respectively, analyzed by two-way ANOVA followed by Bonferroni’s test, n = 3 dishes per treatment. The gray bars indicate which comparisons reach signiﬁcance, all p < 0.0001 (***). g Mean SAH concentration ± SEM in DZnep-treated PER2::LUC MEFs, n = 3 dishes per treatment, analyzed by Two-Way ANOVA (all sources of variations p < 0.0001) followed by Bonferroni’s test; all indicated comparisons (a vs. b vs. c) at least p < 0.05. See also Supplementary Fig. 6. h Immunoblots using an antibody against mono- and di- methylated lysine (K1/2me) reveal that the methylation of some proteins is inhibited by DZnep, and rescued by WT but not D197A MTAN. The red > signs indicate such proteins. Actin loading control is shown in Supplementary Fig. 6i. i, j Mean mRNA m6A methylation levels ± SEM in PER2::LUC MEFs transfected with WT or D197A MTAN, then treated with DZnep for 48 h; i, j showing dot blot assay and enzyme-linked immunosorbent assay-based quantiﬁcation (ELISA), respectively. Dot blot and ELISA analyzed separately by Two-Way ANOVA (DZnep treatment, MTAN effect and interactions at least p < 0.05) followed by Bonferroni’s test (p < 0.05; *p < 0.01), n = 4 dishes. k Dot blot membrane quantiﬁed in i, with 150 ng mRNA/ dot, ﬁrst stained with methylene blue (top) as a loading control. assay-based quantiﬁcation (ELISA), respectively. Dot blot and ELISA analyzed separately by Two-Way ANOVA (DZnep treatment, MTAN effect and interactions at least p < 0.05) followed by Bonferroni’s test (p < 0.05; **p < 0.01), n = 4 dishes. k Dot blot membrane quantiﬁed in i, with 150 ng mRNA/ dot ﬁrst stained with methylene blue (top) as a loading control COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 ARTICLE histone methylation, for example in the promoter of clock genes, which would not be detectable when global methylation is quantiﬁed, may also be inhibited by DZnep. In conclusion, here we have revealed the evolutionarily onserved link between methyl metabolism and biological clocks. Moreover, we have demonstrated in mammalian cells that the period lengthening effects of DZnep exclusively depends on the inhibition of AHCY and the increase in SAH, mainly lea protein and mRNA m6A methylation inhibition. We that the partial rewiring of the mammalian methy protecting proteins and nucleic acids from the conseque methyl cycle inhibition, might present a previously unr strategy to treat methylation deﬁciencies, such as homoc mia or the autosomal recessive AHCY deﬁciency12,38–41. COMMUNICATIONS BIOLOGY \| (2020)3 211 \| htt //d i /10 1038/ 42003 020 0942 0 \| t / bi histone methylation, for example in the promoter of clock genes, which would not be detectable when global methylation is quantiﬁed, may also be inhibited by DZnep. inhibition of AHCY and the increase in SAH, mainly leading to protein and mRNA m6A methylation inhibition. We propose that the partial rewiring of the mammalian methyl cycle, protecting proteins and nucleic acids from the consequences of methyl cycle inhibition, might present a previously unreported strategy to treat methylation deﬁciencies, such as homocysteine- mia or the autosomal recessive AHCY deﬁciency12,38–41. y y In conclusion, here we have revealed the evolutionarily conserved link between methyl metabolism and biological clocks. Moreover, we have demonstrated in mammalian cells that the period lengthening effects of DZnep exclusively depends on the In conclusion, here we have revealed the evolutionarily conserved link between methyl metabolism and biological clocks. Moreover, we have demonstrated in mammalian cells that the period lengthening effects of DZnep exclusively depends on the COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio 8 COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio Methods M l l Molecular modeling and sequence analyses. 3D protein structures were obtained from the Protein Data Bank (PDB) for Homo sapiens (PDB 1LI413, S- adenosylhomocysteine hydrolase complexed with neplanocin, resolution 2.01 Å) and Mus musculus (PDB 5AXA14, S-adenosylhomocysteine hydrolase with ade- nosine, chain A, resolution 1.55 Å). All PDB structures underwent preprocessing including the addition of missing amino acid atoms and hydrogens, removal of solvent atoms, and hydrogen-bond network optimization and residue protonation based on predicted pKa values using the Protein Preparation Wizard51 of Maestro Schrödinger Release 2018-1 [Maestro, Schrödinger, LLC, New York, NY, 2018]. Homology models of Danio rerio (template 1LI4, 100% coverage, 86% sequence identity), Drosophila melanogaster (template 1LI4, 100% coverage, 81% sequence identity), Caenorhabditis elegans (template 1LI4, 98% coverage, 77% sequence identity), Arabidopsis thaliana (template 3OND15 chain A, resolution 1.17 Å, 100% coverage, sequence 92% identity), Chlamydomonas reinhardtii (template 3OND chain A, 100% coverage, sequence 80% identity), Ostreococcus tauri (template 3OND chain A, 99% coverage, 79% sequence identity), and Synechococcus elon- gatus (template 1LI4, 98% coverage, 41% sequence identity) were constructed via the SWISS-MODEL server52. Model quality was evaluated based on GMQE (global model quality estimation) ranging from zero to one with high numbers indicating high model reliability, and QMEAN53, a composite and absolute measure for the quality of protein models, indicating good or poor agreement between model and template with values of around 0 or below −4, respectively. GMQE and QMEAN values for the seven homology models were determined to be 0.98 and 0.85 for D. rerio, 0.93 and 0.82 for D. melanogaster, 0.87 and 0.35 for C. elegans, 0.99 and 0.31 for A. thaliana, 0.95 and 0.08 for C. reinhardtii, 0.91 and −0.54 for O. tauri, and 0.75 and −0.59 for S. elongatus, respectively. GROMACS version 201854 was uti- lized for energy minimization of all nine protein structures including oxidized cofactor NAD+ using force ﬁeld AMBERff99SB-ILDN55. Cofactor parameters and topologies were obtained through ACPYPE56 using the AM1-BCC method with net charge q = −1. The systems including TIP3P water were neutralized with counter ions (Cl- and Na+) to 0.1 M and minimized via steepest descent with a maximum force limit of 100 kJ/mol/nm. Molecular docking simulations were performed using the energy-minimized protein-NAD+ complexes as well as co- crystallized adenosine (ADN), neplanocin (NOC), and 3-deazaneplanocin A (DZnep, constructed from co-crystal NOC in 1LI413) using the Lamarckian Genetic Algorithm provided by the AutoDock4.2 suite57. Methods M l l Protein-ligand interac- tions were analyzed in LigandScout version 4.258,59. Protein methylation was measured by standard immunoblotting using the following antibodies and dilutions: Abcam ab23366, 1:500, for mono- and di- methylated lysine, Abcam ab5823, 1:500, for histone 4 Arginine 3 symmetric demethylation, Abcam ab8580, 1:1000, for Histone H3 Lysine 4 trimethylation, Sigma A5441, 1:10000, for actin, and Abcam ab412 clone 7E6, 1:500, for monomethylated/dimethylated arginine. The second antibody was GE Life Science NA934, 1:5000. Transfection of mammalian cells with expression vectors before luminometry. The bacterial expression vector pPROEX HTa containing wild-type MTAN8 was used as a template to amplify a full length MTAN with ﬂanking SalI/NheI restriction sites at the 5′ and 3′ end, respectively. A single 5′ HA tag 5′-gga- tacccatacgacgtcccagactacgct-3′ was also facultatively inserted during polymerase chain reaction (PCR). The SalI/NheI digested, tagged or untagged PCR products were then ligated into a SalI/NheI-digested pSELECT-HYGRO-MCS (Invivogen, San Diego, California, USA) using Ligation High V2 (Toyobo, Osaka, japan). DH5apha E. coli (Takara Bio, Kusatsu, Japan) were transformed with the ligation products and streaked on a selective plate containing 100 microg/ml hygromycin. Colonies were screened by Sanger sequencing. The D197A mutation was then performed, with the wild-type vector as a template, by PCR using the primers 5′- CCATCTCCGCCGTGGCCGATC3′ and 5′-GATCGGCCACGGCGGAGATGG-3′ containing the mutated GCC codon, together with the 5’ and 3’ primers used for the cloning of MTAN. D197A MTAN was cloned into pSELECT-HYGRO-MCS as described above for the wild-type version. Multiple sequence alignment of AHCY was performed by CLUSTALW60 with the multiple sequence viewer from the Maestro module using the following reference sequences: for human, Genbank: NP_000678.1; mouse, Genbank: NP_057870.3; zebraﬁsh, Genbank: NP_954688.1; fruit ﬂy, Genbank: NP_511164.2; C. elegans, Genbank: NP_491955.1; Arabidopsis: NP_193130.1; Chlamydomonas, Genbank: XP_001693339.1; Ostreococcus, Genbank: XP_022839640.1; cyanobacteria, Genbank: WP_011243218.1. For the molecular structure of drugs shown in Fig. 4e, the Open Source web application MolView was used. Wild-type or D197A tagged MTAN vectors were transfected into PER2::LUC MEFs or U-2 OS in 24-well plates at ~90% conﬂuence with Lipofectamine LTX and PLUS reagent (Invitrogen, Thermo Fisher Scientiﬁc) following manufacturer’s protocol, incubating cells 12–24 (MEFs) or 6–8 (U-2 OS) hours with the transfection mixes. After transfection, medium was changed, and cells were incubated for 24–48 hours. Antibiotic/antimycotic was omitted during transfection and in the ﬁrst medium change. Expression of the HA-tagged WT and D197A MTAN was conﬁrmed by immunoblotting using an anti-HA antibody (Roche 11867423001, 1:1000) (Supplementary Fig. Discussion Moreover, we have shown in mouse cells that the functional consequences of methyl cycle inhibition, i.e., a decrease in the methylation of proteins and mRNA, is also rescued by WT MTAN. This conﬁrms that MTAN, despite removing SAH from the cycle in a way that does not normally occur in mammalian cells, allows the methyl cycle to turn and drive cellular methylations when AHCY does not function. MTAN hydrolyses SAH to S-ribosylhomocysteine and adenine, not to Hcy and adenosine as AHCY does. Since Hcy is needed for the regeneration of methionine in the methyl cycle, how could the methyl cycle still maintain cellular methylations active without Hcy? It is likely that, with the abundance of free We have presented here an identical response to methyl cycle inhibition on biological oscillators across a wide variety of species. Only a small number of examples have been reported that identiﬁed aspects of circadian timekeeping shared among COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio 9 9 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 rhythmic life, such as oxidation cycles of peroxiredoxin29,48,49 and regulation by oscillating intracellular magnesium con- centrations50. Building on those early observations, this study establishes methylation as a universal regulator of rhythmicity. Importantly, to our knowledge we provide a ﬁrst potential avenue of translation of fundamental understanding of methylation deﬁciencies to clinical applications. Biopharmaceutics, such as recombinant MTAN or therapies using genetically engineered cells expressing MTAN, could be used to treat methylation deﬁciencies in the short and longer term. We consider this superior to approaches directly targeting deﬁcient AHCY in cases where the deﬁciency arises from errors of metabolism causing accumulation of metabolites that are inhibitors of AHCY12; and even in cases of AHCY polymorphisms, since the endogenous inactive AHCY in patients would compete with the exogenous active AHCY. DZnep (SML0305) was purchased from Sigma-Aldrich. Period was estimated by BioDare261. For zebraﬁsh cell line and bioluminescence assays, the generation of a per1b- luciferase cell line from zebraﬁsh PAC2 cells has been previously described62. Cells were cultured in Leibovitz’s L-15 medium (Gibco, Thermo Fisher Scientiﬁc, Waltham, MA, USA) containing 15% fetal bovine serum (Biochrom AG, Berlin, Germany), 50 U/mL penicillin/streptomycin (Gibco), and 50 μg/mL gentamicin (Gibco). Discussion The membrane was incubated overnight at 4 °C with the anti-m6A antibody (202 003, Synaptic System, Germany) diluted 1:1000. The next day the membrane was washed three times 10 min with tTBS then incubated 1 h at RT with the secondary antibody (NA934, GE Life Science, USA) diluted 1:5000. After three washes of 10 min with tTBS, the membrane was incubated with ECL Prime reagent (GE Life Science) and imaged with a LAS-4000 imaging system (Fujiﬁlm, Japan). Protein methylation was measured by standard immunoblotting using the following antibodies and dilutions: Abcam ab23366, 1:500, for mono- and di- methylated lysine, Abcam ab5823, 1:500, for histone 4 Arginine 3 symmetric demethylation, Abcam ab8580, 1:1000, for Histone H3 Lysine 4 trimethylation, Sigma A5441, 1:10000, for actin, and Abcam ab412 clone 7E6, 1:500, for monomethylated/dimethylated arginine. The second antibody was GE Life Science NA934, 1:5000. Assays for the inhibition of DNA, total RNA, mRNA and protein methylation. Global DNA methylation was quantiﬁed by the Abcam 5-methylcytosine assay kit (ab233486, colorimetric) following manufacturer’s protocol. Discussion Cells were seeded at a density of 50,000-100,000 cells per well in quadruplicate wells of a 96-well plate in medium supplemented with 0.5 mM beetle luciferin (Promega, Madison, Wisconsin, USA), and drugs were prepared in water and added at the concentrations indicated in the ﬁgures and legends. Plates were sealed with clear adhesive TopSeal (Perkin Elmer, Waltham, MA, USA). Since the circadian clock in these cells is directly light-sensitive and can be entrained by light-dark cycles, cells were synchronized to a 12:12 LD cycle for 7 days and then transferred into DD for at least 3 days. Bioluminescence was monitored at 28°C on a Packard TopCount NXT scintillation counter (Perkin Elmer). DZnep (SML0305) was purchased from Sigma-Aldrich. Period was estimated by BioDare261. Assays for the inhibition of DNA, total RNA, mRNA and protein methylation. Global DNA methylation was quantiﬁed by the Abcam 5-methylcytosine assay kit (ab233486, colorimetric) following manufacturer’s protocol. Global total RNA and mRNA m6A was quantiﬁed by dot blot assay as described below, and mRNA m6A was further conﬁrmed by the Abcam m6A RNA Methylation Assay Kit (ab185912, colorimetric) following manufacturer’s protocol. For the dot blot assay, positively charged nylon membrane was ﬁrst bathed in DEPC-treated water for 10 min followed by DEPC-treated 6× SSC buffer for at least 15 min. During that time, RNA samples were denatured at 95 °C for 3 min and directly transferred on ice. Membranes were then laid on top of two ﬂat layers of blotting paper wet but not dripping with 6× DEPC-treated SSC buffer, the remaining SSC on top of the membrane allowed to seep through it. 3 μl of each RNA sample (50 ng/μl for mRNA or 400 ng/μl for totRNA) were then spotted on the membrane and allowed to seep through. RNA was crosslinked to the membrane in a FUNA-UV crosslinker (Funakoshi, Japan) equipped with 254 nm UV lights, set to “optimal crosslink” (120,000 microjoules/cm2). Membrane was rinsed with DEPC-treated water then transferred to 0.02% Methylene Blue in 0.3 M Na acetate for 15 min in order to conﬁrm the presence and consistent RNA amount for each dot. After several washes with DEPC-water, membrane was scanned for record. The membrane was then washed with 0.1%-tTBS (Nacalai Tesque, Japan) for 10 min, then blocked for 1 h in 5% skimmed milk in 0.1%-tTBS. ARTICLE Dishes were once more rocked by hand for 30 s, then the methanol/MilliQ mix (~0.9 ml due to methanol evaporation) was transferred to a 1.5 ml tube. Two 0.4 ml aliquots of the extracts were ﬁltered through VIVASPIN 500 3KDa cut-off ﬁlters (Sartorius AG, Göttingen, Germany) then kept at −80 °C until quantiﬁcation. Dishes con- taining cells plated simultaneously and submitted to the same treatment were used to calculate cell counts and total cell volume for normalization. Assay for effect of the inhibition of the methyl cycle on invertebrate circadian clock. Halteres of two- to seven-day old transgenic ptim-TIM-LUC males63 kept under 12 h : 12 h light:dark cycles (LD) at 25°C were bilaterally dry dissected. Each pair was transferred into one well of a 96 well plate (Topcount, Perkin Elmer) ﬁlled with medium containing 80% Schneider’s medium (Sigma-Aldrich), 20% inacti- vated Fetal Bovine Serum (Capricorn Scientiﬁc, Ebsdorfergrund, Germany) and 1% PenStrep (Sigma-Aldrich). Medium was fortiﬁed with 226 µM Luciferin (Biosynth AG, Staad Switzerland) and supplemented with 10, 100, or 250 µM Dznep A (Sigma-Aldrich SML0305) diluted in PBS. Plates were sealed with clear adhesive covers and transferred to a TopCount plate reader (PerkinElmer). Bioluminescence emanating from each well was measured hourly in LD for two days, followed by 5 days of constant darkness (DD) at 25 °C as previously described64. The ptim- TIM-LUC reporter contains the timeless (tim) promoter sequences driving rhythmic expression of the tim cDNA, which is fused to the ﬁreﬂy luciferase cDNA. Period was estimated by BioDare261. Preparation of O. tauri and C. reinhardtii for SAM and SAH quantiﬁcation. O. tauri or C. reinhardtii were cultivated in their respective culture medium as described in the corresponding sections on methyl cycle inhibition in these cells. Cells were then treated with DZnep at the indicated concentration at late log phase. After 48 h, cells were harvested (~3 × 10E7 C. reinhardtii and ~10E9 O. tauri) by centrifugation. Cell pellets were then resuspended with 5% mannitol (Naca- lai) for C. reinhardtii or 1M sorbitol (Sigma-Aldrich) for O. tauri, which was then thoroughly removed after centrifugation; this wash was performed twice. Cell pellets were frozen in liquid nitrogen and kept at −80°C before analysis. Cell pellets were resuspended in 0.4 ml methanol (Nacalai), then 0.275 ml of MilliQ containing 125 ng/ml BIS-TRIS (Sigma-Aldrich) was added to the methanol. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 178.90 for GSH (CE –14 V), and m/z 355.10 for GSSG (CE –24 V) from the precursor ions of [M + H]+ (m/z 136 for Hcy, m/z 223 for cystathionine, m/z 122 for cysteine, m/z 308 for GSH, and m/z 613 for GSSG). Since a divalent ion of [M + 2 H]2+ of GSSG (m/z 307) was also abundant as well as an ion of [M + H]+ GSSG (m/z 613) at full ion scan mode, the divalent ion was also chosen as precursor ion to obtain target product ion of m/z 84.10 (CE –26 V). were treated with dexamethasone 200 nM for 2 h for synchronization and, after a medium change (with 1 mM luciferin and various concentrations of DZnep), transferred to a luminometer/incubator (CL24A-LIC, Churitsu Electric Corp., Nagoya, Japan) set at 35 °C, 5% CO2. Photons were counted in bins of 10 s at a frequency of 10 min. p g p The calibration curves prepared were y = 17,580 (±153.7) × for Hcy (P-value <0.0001), y = 6,444 (±50.6) × for cystathionine (P-value <0.0001), y = 2,673 (±33.0) × for cysteine (P-value <0.0001), y = 72,710 (±538.8) × for GSH (P-value <0.0001), y = 8,206 (±133.0) × for [M + H]+ of GSSG (P-value <0.0001), and y = 14,100 (±211.0) × for [M + 2 H]2+ of GSSG (P-value <0.0001). The concentrations of analytes (3 μL injection) were quantiﬁed using the calibration curves. All standards and samples were analyzed in triplicates. Preparation of PER2::LUC cells for SAM and SAH quantiﬁcation. Cells plated in 10 cm culture dishes at 90% conﬂuence were transfected with wild-type or D197A MTAN vectors using lipofectamine LTX with PLUS reagent following manu- facturer’s protocol (Invitrogen). After 12–24 h of incubation with the transfection mixes, medium was changed once, omitting antibiotic/antimycotic, and cells were incubated for one day. Cells were then treated with different concentrations of DZnep and incubated for 48 h. Cells were washed once with 10 ml, then once with 2 ml 5% mannitol (Nacalai). Mannitol was thoroughly removed, and 0.8 ml methanol was added to the dish, rocking vigorously for 30 s for the methanol to homogenously cover the cells. Dishes were tipped and 0.55 ml milliQ containing 125 ng/ml BIS-TRIS (Sigma-Aldrich) was added directly to the methanol. ARTICLE Stocks were maintained on plates with nematode growth medium (NGM) seeded with HB101 Escherichia coli strain, under 12-h/12-h LD/CW cycle (400/0 lx and CW (18.5/20 °C, Δ = 1.5 ± 0.125 °C) environmental cycles. Transgenic animals were generated by microinjection of a Psur-5::luc::gfp construct at 50 or 100 ng/μL with the pRF4 marker (100 ng/μL)26,65. For bioluminescence recordings, transgenic Psur-5::luc::gfp nematode populations were synchronized to the same developmental stage by the chlorine method66. The harvested eggs were cultured overnight in a 50-mL Erlenmeyer ﬂask with 3.5 mL of M9 buffer, 1× antibiotic-antimycotic (Thermo Fisher Scientiﬁc), and 10 µg/mL of tobramycin (Tobrabiotic, Denver Farma S.A., Buenos Aires, Argentina) at 110 rpm with a Vicking M23 shaker, in LD/CW (400/0 lx; 18.5/20 ° C, Δ = 1.5 °C ± 0.125 °C) conditions. The following day, L1 larvae were transferred to NGM plates at ZT1 and were grown for 48 h to the L4 stage under the same LD/CW cycle. Starting at ZT1 (1 h post lights on), the most ﬂuorescent nematodes were selected manually under a SMZ100 stereomicroscope equipped with an epi-ﬂuorescence attachment (Nikon, Minato, Tokyo) with a cool Multi- TK-LED light source (Tolket S.R.L., Buenos Aires, Argentina) to avoid warming the plate. Picking was performed in a room kept at a constant temperature (18 ° C). L4 larvae were selected after 48 h of light-dark/temperature entrainment and placed in a luminescence medium containing Leibovitz’s L-15 medium without phenol red (Thermo Fisher Scientiﬁc) supplemented with 1× antibiotic–antimycotic (Thermo Fisher Scientiﬁc), 40 μM of 5-ﬂuoro-2′- deoxyuridine (FUdR) to avoid new eclosions, 5 mg/mL cholesterol, 10 µg/mL tobramycin (Denver Farma S.A.), 1 mM D-luciferin (Gold Biotechnology, St Louis MO, USA), and 0.05% Triton X-100 to increase cuticle permeabilization. All chemical compounds were purchased from Sigma-Aldrich unless otherwise speciﬁed. Population nematode bioluminescence was recorded in 35- mm plate dishes (CELLSTAR, Greiner Bio-One, Kremsmünster, Austria) with 1 mL of the luminescence medium, by means of an AB-2550 Kronos Dio luminometer (Atto). The signal was integrated for 1 min, and readings were taken every 10 min. Hcy, cystathionine, cysteine, GSH, GSSG, SAM and SAH quantiﬁcation by LC/ MS/MS. The standard stocks of SAM (Cayman Chemical, Ann Arbor, MI, USA) and SAH (Sigma-Aldrich) were dissolved in water (1 mg/mL) and stored at –30 °C until use. ARTICLE For the construction of calibration curves, a series of dilutions of SAM and SAH mixtures were prepared at the concentrations of 0.001, 0.01, 0.05, 0.1, 0.5, 1, 10, 50, 100, and 200 μM. μ To quantify the concentrations of SAM and SAH using the multiple reaction monitoring (MRM), LC/MS/MS conditions were optimized. The LC/MS/MS quantiﬁcations were conducted using a LC-ESI-TQMS (LCMS8030plus, Shimadzu, Kyoto, Japan). The target product ions were determined as m/z 250.10 for SAM (CE −15 V), m/z 136.05 for SAH (CE −20 V) from the precursor ions of [M + H]+ (m/z 399 for SAM, m/z 385 for SAH). p ( ) pp antibiotic–antimycotic (Thermo Fisher Scientiﬁc), 40 μM of 5-ﬂuoro-2′- deoxyuridine (FUdR) to avoid new eclosions, 5 mg/mL cholesterol, 10 µg/mL tobramycin (Denver Farma S.A.), 1 mM D-luciferin (Gold Biotechnology, St Louis MO, USA), and 0.05% Triton X-100 to increase cuticle permeabilization. All chemical compounds were purchased from Sigma-Aldrich unless otherwise speciﬁed. Population nematode bioluminescence was recorded in 35- mm plate dishes (CELLSTAR, Greiner Bio-One, Kremsmünster, Austria) with 1 mL of the luminescence medium, by means of an AB-2550 Kronos Dio luminometer (Atto). The signal was integrated for 1 min, and readings were taken every 10 min. The LC conditions were designed as previously described6, with some modiﬁcations: The elution program was set as a linear gradient of 95–10% of MeCN in water containing 0.1% HCOOH (0–15 min) followed by isocratic 10% MeCN in water containing 0.1% HCOOH (7 min), at a ﬂow rate of 0.2 mL/min. ZIC-HILIC (2.1 × 150 mm, Merck Millipore, Burlington, MA, USA) was selected as an analytical column at a temperature of 40 °C. For single-nematode measurements, Psur-5::luc::gfp nematodes at the L4 stage were selected as before, washed, and transferred directly to the liquid luminescence medium. A small, square piece of a transparent 96-well plate containing three × three wells was inserted inside a 35-mm dish plate to reduce the total volume and limit the nematode movement to the center of the plate. One nematode was placed in the center well with 200 μL of the luminescence medium. Plates were then transferred to the AB-2550 Kronos Dio luminometer. Single-nematode recordings were taken every 37 min with an integration time of 4 min. y p For the quantiﬁcation, a dilution series of SAM and SAH mixtures (3 μL injection) were subjected to MRM quantiﬁcation as described above. COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio Methods M l l 6a and b). For the experiments shown in Supplementary Fig. 5c–f, transfected conﬂuent cells were trypsinized and plated and very low density and allowed to grow a few days until ~30% conﬂuence. Cells Assay for effect of the inhibition of the methyl cycle on vertebrate circadian clock. Human U-2 OS cells stably transfected with a Bmal1-luciferase reporter vector25 and mouse PER2::LUC MEFs24 cell lines were cultivated as previously described6. Brieﬂy, cells were seeded into 35 mm dishes (Corning, New York, USA) and allowed to grow to conﬂuence in DMEM/F12 medium (Nacalai, Kyoto, Japan) containing penicillin/streptomycine/amphotericin (Nacalai). Cells were then shocked by dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) 200 nM for 2 h, followed by a medium change including 1 mM luciferine (Nacalai). 35 mm dishes were then transferred to an 8-dishes luminometer-incubator (Kronos Dio, Atto, Tokyo, Japan). Photons were counted in bins of 2 min at a frequency of 10 min. 10 COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio ARTICLE Cell resuspen- sions were submitted to 10 freeze-thaw cycles (liquid nitrogen/4 °C) to break the cell wall, with full-speed vortexing for 5 s between cycles. After the last thaw, samples were vortexed at full speed for 10 seconds then centrifuged at 12,000 g at 4 °C for 10 min. Two 0.3 ml aliquots of supernatant were transferred to VIVASPIN 500 3KDa cut-off ﬁlters (Sartorius) and centrifuged as recommended by the manufacturer. Eluates were kept at -80°C until quantiﬁcation by LC/MS/MS. Total cell counts (3.01–4.34 × 107) or optical density (0.514–0.646) were used as nor- malizers for Chlamydomonas or Ostreococcus, respectively. y C. elegans strain N2 (Bristol strain, wild-type was provided by the Caenorhabditis Genetics Center, University of Minnesota (cbs.umn.edu/cgc/ home). Stocks were maintained on plates with nematode growth medium (NGM) seeded with HB101 Escherichia coli strain, under 12-h/12-h LD/CW cycle (400/0 lx and CW (18.5/20 °C, Δ = 1.5 ± 0.125 °C) environmental cycles. Transgenic animals were generated by microinjection of a Psur-5::luc::gfp construct at 50 or 100 ng/μL with the pRF4 marker (100 ng/μL)26,65. For bioluminescence recordings, transgenic Psur-5::luc::gfp nematode populations were synchronized to the same developmental stage by the chlorine method66. The harvested eggs were cultured overnight in a 50-mL Erlenmeyer ﬂask with 3.5 mL of M9 buffer, 1× antibiotic-antimycotic (Thermo Fisher Scientiﬁc), and 10 µg/mL of tobramycin (Tobrabiotic, Denver Farma S.A., Buenos Aires, Argentina) at 110 rpm with a Vicking M23 shaker, in LD/CW (400/0 lx; 18.5/20 ° C, Δ = 1.5 °C ± 0.125 °C) conditions. The following day, L1 larvae were transferred to NGM plates at ZT1 and were grown for 48 h to the L4 stage under the same LD/CW cycle. Starting at ZT1 (1 h post lights on), the most ﬂuorescent nematodes were selected manually under a SMZ100 stereomicroscope equipped with an epi-ﬂuorescence attachment (Nikon, Minato, Tokyo) with a cool Multi- TK-LED light source (Tolket S.R.L., Buenos Aires, Argentina) to avoid warming the plate. Picking was performed in a room kept at a constant temperature (18 ° C). L4 larvae were selected after 48 h of light-dark/temperature entrainment and placed in a luminescence medium containing Leibovitz’s L-15 medium without phenol red (Thermo Fisher Scientiﬁc) supplemented with 1× g ( , yp p y Caenorhabditis Genetics Center, University of Minnesota (cbs.umn.edu/cgc/ home). References Chlamydomonas reinhardtii strain CBR carrying a codon-adapted luciferase reporter driven by the tufA promoter in the chloroplast genome68,69 was used. Culture preparation, bioluminescence monitoring, and data analysis were carried out as described previously69. Brieﬂy, 5-day-old algal cultures on HS agar medium were cut out along with the agar by using a glass tube, and transferred to separate wells of a 96-well microtiter plates. Luciferin (ﬁnal conc. 200 μM) and various concentrations of DZnep (Sigma-Aldrich) were added to the wells. Algae were synchronized by a single cycle of 12-h darkness/12-h light (30 μmol/m2/s) at 17 °C before bioluminescence monitoring using a custom-made luminometer- incubator70 in DD at 17 °C. Period was estimated by BioDare261. 7. Parveen, N. & Cornell, K. A. Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism. Mol. Microbiol. 79, 7–20 (2011). 8. Lee, J. E. et al. Mutational analysis of a nucleosidase involved in quorum- sensing autoinducer-2 biosynthesis. Biochemistry 44, 11049–11057 (2005). 9. Guranowski, A., Montgomery, J. A., Cantoni, G. L. & Chiang, P. K. Adenosine analogues as substrates and inhibitors of S-adenosylhomocysteine hydrolase. Biochemistry 20, 110–115 (1981). y 10. Chiang, P. K., Richards, H. H. & Cantoni, G. L. S-Adenosyl-L-homocysteine hydrolase: analogues of S-adenosyl-L-homocysteine as potential inhibitors. Mol. Pharmacol. 13, 939–947 (1977). Assay for effect of the inhibition of the methyl cycle on prokaryotic circadian clock. Vibrio harveyi luciferase encoded by luxA and luxB (luxAB) genes was used as a luminescence reporter in cyanobacterium Synechococcus elongatus PCC 7942. The cyanobacterial clock-regulated reporter strain kaiBCp::luxAB71 was selected for the test, expressing luxAB under the control of the promoter of the central clock genes kaiBC. Synechococcus strains were grown in modiﬁed BG11 media72 sup- plemented with 40 µg/mL of spectinomycin. The cultures grown on BG11 agar plates for 3 days at 30 °C under continuous cool-white illumination (LL) (50 µE/m2 s) were toothpicked onto fresh BG11 agar plates containing 0.015 g/L of L- hi i d diff i f d l i A h d hl id Assay for effect of the inhibition of the methyl cycle on prokaryotic circadian clock. Vibrio harveyi luciferase encoded by luxA and luxB (luxAB) genes was used as a luminescence reporter in cyanobacterium Synechococcus elongatus PCC 7942. The cyanobacterial clock-regulated reporter strain kaiBCp::luxAB71 was selected for the test, expressing luxAB under the control of the promoter of the central clock genes kaiBC. Data availability Assay for effect of the inhibition of the methyl cycle on plant and algae Assay for effect of the inhibition of the methyl cycle on plant and algae circadian clock. Ostreococcus tauri cells transgenically expressing a translational fusion of CCA1 to luciferase from the CCA1 promoter (CCA1-LUC)30 were grown, imaged, and analyzed as described previously49. Brieﬂy, cells were cultured under 12/12 h blue (Ocean Blue, Lee lighting ﬁlter 724) light/dark cycles (17.5 μE/ m2/s) in Keller medium in artiﬁcial sea water. Cells were then transferred to 96-well microplates (Lumitrac, Greiner Bio-one), at a density of ∼15 × 106 cells/ml and entrained for 7–10 days before recording. One day before recording, 150 μl Keller medium in artiﬁcial sea water was replaced with 150 μl of Keller medium in arti- ﬁcial sea water containing 333 μM luciferin (Km+). Bioluminescence was mea- sured on a TopCount (Packard) under constant darkness or constant red + blue LED light (5–12 μE/m2). The source data underlying plots shown in main ﬁgures are provided in Supplementary Data 1. All other data shown in this work are available from J.M. Fustin upon reasonable request. References Synechococcus strains were grown in modiﬁed BG11 media72 sup- plemented with 40 µg/mL of spectinomycin. The cultures grown on BG11 agar plates for 3 days at 30 °C under continuous cool-white illumination (LL) (50 µE/m2 s) were toothpicked onto fresh BG11 agar plates containing 0.015 g/L of L- methionine and different concentrations of 3-deazaneplanocin A hydrochloride (SML0305, Sigma-Aldrich) or sinefungin (Abcam, Cambridge, United Kingdom). After a single 12 h dark pulse for synchronization, in vivo luminescence rhythms were captured with a charge-coupled device (CCD) camera set up within a system to accommodate the agar plates containing the colonies71. Period was estimated by BioDare261. 11. Tseng, C. K. et al. Synthesis of 3-deazaneplanocin A, a powerful inhibitor of S- adenosylhomocysteine hydrolase with potent and selective in vitro and in vivo antiviral activities. J. Med. Chem. 32, 1442–1446 (1989). 12. Baric, I. et al. S-adenosylhomocysteine hydrolase deﬁciency in a human: a genetic disorder of methionine metabolism. Proc. Natl. Acad. Sci. USA 101, 4234–4239 (2004). 13. Yang, X. et al. Catalytic strategy of S-adenosyl-L-homocysteine hydrolase: transition-state stabilization and the avoidance of abortive reactions. Biochemistry 42, 1900–1909 (2003). y 14. Kusakabe, Y. et al. Structural insights into the reaction mechanism of S- adenosyl-L-homocysteine hydrolase. Sci. Rep. 5, 16641 (2015). 15. Brzezinski, K., Dauter, Z. & Jaskolski, M. High-resolution structures of complexes of plant S-adenosyl-L-homocysteine hydrolase (Lupinus luteus). Acta Crystallogr. D. Biol. Crystallogr. 68, 218–231 (2012). 16. Gomi, T. et al. Site-directed mutagenesis of rat liver S- adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding. J. Biol. Chem. 265, 16102–16107 (1990). Measurement of Hes7 oscillations in the mouse presomitic mesoderm. Pre- somitic mesoderm (PSM) tissues from three littermate pHes7-UbLuc Tg embryos were embedded in 0.35% LMP-agarose/culture medium (10% FBS-DMEM/F12), in a silicon mold mounted onto a φ35mm-glass bottom dish, and luciferin-containing medium with DZnep (Sigma-Aldrich) or vehicle (MilliQ water) were added. 17. Ault-Riche, D. B., Yuan, C. S. & Borchardt, R. T. A single mutation at lysine 426 of human placental S-adenosylhomocysteine hydrolase inactivates the enzyme. J. Biol. Chem. 269, 31472–31478 (1994). 18. Takata, Y. et al. Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670–22676 (2002). p g Time-lapse imaging of PSM was performed with an inverted microscope (IX81, Olympus, Tokyo, Japan) equipped with an Olympus x10 UPlanApo objective (N. A.: 0.8) and a VersArray cooled-CCD camera. Received: 30 October 2019; Accepted: 3 April 2020; Received: 30 October 2019; Accepted: 3 April 2020; g μ For protoplast isolation and luminescent imaging, plants expressing luciferase from the CCA1 promoter in the Col-0 background (kindly provided by Karen Halliday, University of Edinburgh) were grown in sterile soil under long-day conditions (16 h light/8 h dark) at 22 °C under 60–70 µmol/m2/s white LED tube lights (Impact T8). Protoplasts were isolated from 3-week old leaves by mechanically stripping the lower epidermis from the underlying mesophyll cells using magic tape, then releasing the mesophyll cells by incubating the remaining leaf tissue in a solution containing cellulase and pectinase R10 67. Protoplasts in a solution of 1 mM D-luciferin (Biosynth AG), 5% fetal bovine serum (Sigma- Aldrich), 50 µg/mL ampicillin, 140 mM NaCl, 115 mM CaCl2, 4.6 mM KCl, 1.86 mM MES pH 5.7 and 4.6 mM glucose were added to white, ﬂat-bottomed 96-well plates (Lumitrac, Greiner Bio-one) at a concentration of 2 × 105 cells/mL. DZnep (S7120, Selleck Chemicals LLC, Houston, TX, USA) was added to the protoplasts to achieve concentrations of 0, 0.125, 0.25, 0.5 or 1 µM from a 1 mM stock solution (prepared in distilled H2O) to a total volume of 200 µL per well. Plates were sealed with a clear adhesive lid (TopSeal-A, Perkin Elmer). Protoplast luminescence was read by a LB942 Tristar2 plate reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) every 50 min for 3 s per well, and kept under continuous red (630 nm) and blue (470 nm) LED light (5 µmol/m2/s each) at 19 °C. For protoplast isolation and luminescent imaging, plants expressing luciferase from the CCA1 promoter in the Col-0 background (kindly provided by Karen Halliday, University of Edinburgh) were grown in sterile soil under long-day conditions (16 h light/8 h dark) at 22 °C under 60–70 µmol/m2/s white LED tube lights (Impact T8). Protoplasts were isolated from 3-week old leaves by mechanically stripping the lower epidermis from the underlying mesophyll cells using magic tape, then releasing the mesophyll cells by incubating the remaining leaf tissue in a solution containing cellulase and pectinase R10 67. Protoplasts in a solution of 1 mM D-luciferin (Biosynth AG), 5% fetal bovine serum (Sigma- References 1. Cantoni, G. L. 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S i k M H S lf i id b li h f d i d References 16-bit images were acquired every 5 min with Image-Pro Plus (Media Cybernetics, Inc., Rockville, MD, USA), with 2×2 and 4×4 binning and exposures of 100 ms and 4 m 25 s for DIC and 19. Yamada, T. et al. Catalytic mechanism of S-adenosylhomocysteine hydrolase: roles of His 54, Asp130, Glu155, Lys185, and Aspl89. Int. J. Biochem. Cell Biol. 37, 2417–2435 (2005). chemiluminescent images, respectively. Raw imaging data were processed and quantiﬁed by ImageJ73. Period was estimated by BioDare261. chemiluminescent images, respectively. Raw imaging data were processed and quantiﬁed by ImageJ73. Period was estimated by BioDare261. 20. Sganga, M. W., Aksamit, R. R., Cantoni, G. L. & Bauer, C. E. Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus. Proc. Natl. Acad. Sci. USA 89, 6328–6332 (1992). Statistics and reproducibility. 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Further information on research design is available in the Nature Research Reporting Summary linked to this article. 12 COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio Author contributions Conceptualization, J.-M.F. and H.O.; Methodology, J.-M.F., M.V., C.R., S.J.C., T.K.T., Y.X., M.L.J., M.L.L., K.Y.-K., D.W., R.Ka., T.M., R.S., D.A.G., C.H.J., G.v.O., H.K.; investigation, J.-M.F., S.Y., C.R., M.Y., M.V., E.G., K.F., K.K., S.J.C., T.K.T., Y.X., M.L.J., R.Ko., M.L.L., K.Y.-K., T.M., D.A.G., G.v.O.; homology modeling, C.R.; original draft, J.-M.F.; reviewing & editing, J.-M.F., C.R., T.K.T, Y.X., K.Y.-K., D.W., R.Ka., T.M., R.S., D.A.G., C.H.J., H.K., G.v.O.; Funding Acquisition, J.-M.F., D.A.G., C.H.J., H.K., G.v.O and H.O.; Supervision, J.-M.F. Resources: S.T., L.H., H.K. Conceptualization, J.-M.F. and H.O.; Methodology, J.-M.F., M.V., C.R., S.J.C., T.K.T., Y.X., M.L.J., M.L.L., K.Y.-K., D.W., R.Ka., T.M., R.S., D.A.G., C.H.J., G.v.O., H.K.; investigation, J.-M.F., S.Y., C.R., M.Y., M.V., E.G., K.F., K.K., S.J.C., T.K.T., Y.X., M.L.J., R.Ko., M.L.L., K.Y.-K., T.M., D.A.G., G.v.O.; homology modeling, C.R.; original draft, J.-M.F.; reviewing & editing, J.-M.F., C.R., T.K.T, Y.X., K.Y.-K., D.W., R.Ka., T.M., R.S., D.A.G., C.H.J., H.K., G.v.O.; Funding Acquisition, J.-M.F., D.A.G., C.H.J., H.K., G.v.O and H.O.; Supervision, J.-M.F. Resources: S.T., L.H., H.K. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-020-0942-0 John O’Neill and Tokitaka Oyama for useful discussion and for kindly accepting to perform experiments. The authors thank G. Wolber, Freie Universität Berlin, Germany, for providing a LigandScout 4.2 license. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. p g The authors declare no competing interests. The authors declare no competing interests. Acknowledgements This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan: Grant-in-aid for Scientiﬁc Research on Innovative Areas (26116713, J.-M. F.; 17H06401, H.K.), for Young Scientists (26870283, J.-M. F.), for Scientiﬁc Research A (15H01843, H.O.; 18H04015, H.O.), a grant for Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (CREST/ JPMJCR14W3, H.O.). J.-M.F. was also supported by grants from the Kato Memorial Bioscience Foundation, the Senri Life Science Foundation (S-26003), the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Kyoto Uni- versity internal grant ISHIZUE, and H.O. was also supported by the Kobayashi Inter- national Scholarship Foundation. C.H.J. is supported by grants from the USA NIH/ NIGMS: GM067152 and GM107434. G.vO. is supported by a Royal Society University Research Fellowship (UF160685) and research grant (RGF\EA\180192). D.A.G. is sup- ported by grants from the National Research Agency (ANPCyT, PICT-2015-0572) and the National University of Quilmes. E.G. was supported by the Wellcome Trust- University of Edinburgh Institutional Strategic Support Fund. M.V. and R.S. are funded by a grant from the Deutsche Forschungsgemeinschaft (STA421/7-1). J.-M. F. is a UKRI Future Leaders Fellow (MR/S031812/1). We thank Adrienne K. Mehalow, Jay C. Dunlap, 46. Stipanuk, M. H. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annu. Rev. Nutr. 24, 539–577 (2004). 47. Nitanda, Y. et al. 3’-UTR-dependent regulation of mRNA turnover is critical for differential distribution patterns of cyclic gene mRNAs. FEBS J. 281, 146–156 (2014). 48. O’Neill, J. S. & Reddy, A. B. Circadian clocks in human red blood cells. Nature 469, 498–503 (2011). 49. Edgar, R. S. et al. Peroxiredoxins are conserved markers of circadian rhythms. Nature 485, 459–464 (2012). 50. Feeney, K. A. et al. Daily magnesium ﬂuxes regulate cellular timekeeping and energy balance. Nature 532, 375–379 (2016). 51. Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and ligand preparation: parameters, protocols, and inﬂuence on virtual screening enrichments. J. Comput. Aided Mol. Des. 27, 221–234 (2013). 52. Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018). 13 COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio ARTICLE Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s42003- 020-0942-0. Supplementary information is available for this paper at https://doi.org/10.1038/s42003- 020-0942-0. © The Author(s) 2020 Correspondence and requests for materials should be addressed to J.-M.F. or H.O. 14 COMMUNICATIONS BIOLOGY \| (2020) 3:211 \| https://doi.org/10.1038/s42003-020-0942-0 \| www.nature.com/commsbio
https://openalex.org/W4391968196	https://nmce.kntu.ac.ir/article_167675_89d2bb7371ea764c628febd51a9f23c3.pdf	English	null	Axial bearing capacity of helical piles in moist and saturated conditions using frustum confining vessel (FCV)	Annual report of the Board of Directors General Assembly of Shareholders	2,023	cc-by	9,111	Journal Homepage: https://nmce.kntu.ac.ir/ Journal Homepage: https://nmce.kntu.ac.ir/ RESEARCH PAPER Due to the increasing demand in construction and use of different types of piles on the one hand and the high cost of conducting large-scale tests on different types of piles, on the other hand, new methods have been proposed to study the behavior of different types of piles. Physical modeling provides the researcher the capability of studying model piles in the scaled environment at low costs. Among the different methods of physical modeling, the use of frustum confining vessels (FCV) has gained attraction in recent years. FCV is a cone-shaped vessel that can produce a stress distribution similar to the idealized linear stress distribution in depth. Helical piles are common types of deep foundations which were first used about 200 years ago. Helical Apiles are driven to the soil by applying a torque to the end of piles in the presence of vertical loads. Their quick and noise-free installation method, the minimal disturbance during the installation, and environmental compatibility make them popular for working in urban areas. In this research, using the finite element method, the optimal dimensions of FCV apparatus were selected, and the FCV apparatus with optimal dimensions were constructed. A total of 18 compression tests were performed on Anzali sand in different relative densities and moisture contents, using single-helix and three-helices piles. Results indicate that increasing the number of helices and relative density of soil increases the pile and sand contact and causes higher bearing capacity for helical piles. Soil saturation, on the other hand, significantly reduces the ultimate strength. Article history: Received: June 2022 Revised: November 2022 Accepted: December 2022 Keywords: Helical Pile, Axial Bearing Capacity, Physical Modeling, FCV Apparatus, Anzali Sand Keywords: Helical Pile, Axial Bearing Capacity, Physical Modeling, FCV Apparatus, Anzali Sand A helical pile is a circular pile with a blade. It is an old type of pile that has recently become attractive due to its quick and silent installation and minimal disturbances during installation [9, 10]. Helical piles installation is carried out by applying torque at the end of the pile in the presence of vertical pressure [9]. The first usage of helical piles was as anchors for electrical towers, subjected to uplift force [10]. * PhD Candidate, Department of Civil Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran. Corresponding author: Assistant Professor, Department of Civil Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran. * Professor, Department of Civil and Environment Engineering, Amirkabir University, Tehran, Iran. Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 Axial bearing capacity of helical piles in moist and saturated conditions using frustum confining vessel (FCV) Ali Jassim , Navid Ganjian , Abolfazl Eslami ** Keywords: Helical Pile, Axial Bearing Capacity, Physical Modeling, FCV Apparatus, Anzali Sand 1. Introduction Over the past few decades, the increasing demand from oil and gas industries for the construction of facilities nearshore and offshore yielded to an increasing attraction to the deep foundations, i.e., piles. The foundations of many structures, whether on the land such as high-rise buildings and chimneys or marine structures such as wind turbines and towers are subjected to tension and compression loads[1, 2]; therefore, the study of the bearing capacity of such foundations attracted attention in the recent decades. Many research has been conducted on the numerical and experimental ultimate bearing capacity of piles[1-14]. Physical modeling has been recognized as a beneficial tool for studying pile behavior. This is an important and sophisticated subject in the geotechnical field, that allows one to carry out many tests that are not expensive compared to field programs and offers the possibility of studying theoretical and practical viewpoints. Each apparatus used in physical modeling has its own advantages and disadvantages. Amongst the different physical modeling methods, Frustum Confining Vessel has gained increasing attraction in recent years due to its simplicity and the realistic stress distribution generation in depth. FCV apparatus is a truncated cone-shaped https://doi.org/10.61186/NMCE.2022.438 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. helical pile with two helices shows a performance equal to a steel pile with a shaft diameter equivalent to the helix diameter of the screw pile. In other words, using helical piles can yield approximately a 50% decrease in cost of materials. vessel that was first introduced by Horvath and Stole at Mc.Master University in 1996 [15]. FCV can convert the applied uniform base pressure to horizontal and vertical components. By converting the applied pressure to two perpendicular components, FCV can produce a near- linear stress gradient for vertical and horizontal stress [15]. FCV benefits from the advantages of both Centrifuge and Calibration chambers and is capable of mimicking the stress distribution condition of in-situ soil [16]. Zarrabi and Eslami [22] scrutinized the construction effects on the performance of piles, using physical modeling in FCV-AUT. Askari Fateh, Eslami, and Fahimifar [23] studied the failure mechanism of three helical piles made up of hollow galvanized iron pipes with one, two, and three helices. The experimental results were verified using two static analysis methods, CFEM (2006) and Vessic (1997). They concluded that the CFEM formulation has shown a more realistic prediction of the ultimate bearing capacity. 1. Introduction They also affirmed the conclusion made by the other researchers that depending on the helix spacing ratio, individual or cylindrical failure mechanisms may occur (Perko [24]). For helical piles in sand, the transition from cylindrical failure to individual bearing behavior occurs at a helix spacing ratio (S/D) of about three (Lutenegger [25]). As mentioned, the first FCV apparatus was introduced by Horvath and Stole at Mc.Master University in 1996. The next FCV devices were constructed at South Florida (1999), Amirkabir University of Technology (2012)[13], and Isfahan University of Technology (2019) [17]. The most recently constructed FCV apparatus was designed and fabricated by the authors at Baran Khak va Pey Company, Tehran, Iran. It has the largest top diameter amongst the existing FCV apparatus worldwide, providing the authors an invaluable tool for investigating the lateral and axial behavior of piles and the capability of testing piles with larger diameters (i.e., helical piles with larger helix diameters). The larger top diameter also minimizes errors related to scale effects and boundary effects. Additionally, the newly constructed FCV device is capable of testing model piles in saturated and near- saturated soil conditions (Similar to nearshore and offshore soil conditions). The readers are encouraged to refer to another paper by the authors[18] for more information regarding the newly constructed FCV apparatus, i.e., its dimensions, instrumentations, and saturating process. FCV apparatus provides a scaled environment for testing both conventional and newly developed piles. Shojaei et al. [26] performed a feasibility study on the use of a new type of self-expanded piles aiming to reduce the harsh effects of the pile installation with the aid of FCV-AUT. Also, Mortazavibak et al. [27] studied the bearing capacity of tapered helical piles as a brand-new type of pile using FCV-IUT. In their research, they measured the axial bearing capacity, stiffness, friction, and material efficiency of the proposed model pile. The geometric scale ratio (λL) in the FCV apparatus can be calculated using the following equation [28]: Many research has been carried out using FCV to study the different aspects of piles behavior. Zare and Eslami [13] studied the key features of FCV-AUT in terms of stress distribution in depth and performed a couple of axial loading tests on three driven steel piles. Results indicated a close agreement between the numerically predicted bearing capacity, using the ESA method, and the experimentally measured bearing capacity. A. Jassim et al. Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 Canadian foundation engineering manual (CFEM) provided the formulation for estimating the ultimate capacities of helical piles [29]. The CFEM formulation Can be summarized as follows: In the present research, the axial bearing capacity of the single-helix and multi-helices piles were evaluated through a comprehensive experimental program. Helical piles, used in this study, consist of steel pipes with one or three welded circular plates to the shaft of the pile with a conical shoe. The model piles were tested in the newly constructed FCV device at Baran Khak va Pey Co, Tehran, Iran, and the capability of the numerical methods to predict the ultimate bearing capacity of piles is evaluated by comparing the experimental and numerical results. To study the effects of the degree of saturation on the ultimate bearing capacity of helical piles, the results of the model piles tested in dry sand are compared with those of model piles tested in saturated sand. Additionally, the effects of installation depth, i.e., scale factor, were evaluated by increasing the uplift pressure in saturated sand conditions. (2) 𝑅= 𝑄𝑡+ 𝑄𝑓 (2) 𝑅= 𝑄𝑡+ 𝑄𝑓 Where Qt is the total multi-helix pile capacity, and Qf is the shaft skin resistance. 𝑄𝑡= ∑𝑄ℎ (3) Where; 𝑄ℎ= 𝐴ℎ(𝑆𝑢𝑁𝑐+ 𝛾𝐷ℎ𝑁𝑞+ 0.5𝛾𝐵𝑁𝛾) (4) Wh (3) 𝑄𝑡= ∑𝑄ℎ (3) Where; 𝑄𝑡= ∑𝑄ℎ Where; Where; 𝑄ℎ= 𝐴ℎ(𝑆𝑢𝑁𝑐+ 𝛾𝐷ℎ𝑁𝑞+ 0.5𝛾𝐵𝑁𝛾) (4) (4) 𝑄ℎ= 𝐴ℎ(𝑆𝑢𝑁𝑐+ 𝛾𝐷ℎ𝑁𝑞+ 0.5𝛾𝐵𝑁𝛾) 𝑄ℎ= 𝐴ℎ(𝑆𝑢𝑁𝑐+ 𝛾𝐷ℎ𝑁𝑞+ 0.5𝛾𝐵𝑁𝛾) (4) Where: Where: Ah: Projected helix area Ah: Projected helix area Su: Undrained shear strength : Unit weights Dh: Depth to helical plate B: Diameter of helical plate Nc, Nq, N: Bearing capacity factor for the local shear condition, presented in Table 10.1 of CFEM. 𝑓𝑠= 𝜎𝑣𝐾tan 𝛿 𝑓𝑠= 𝜎𝑣𝐾tan 𝛿 In which 𝜎𝑣 is the effective vertical stress at mid-depth of the soil, K = 2(1-sin ()). 𝛿, and  are the interface friction and frictional resistance angle, respectively. The axial bearing capacity of the helical pile is the summation of end bearing capacity and the skin friction of the shaft. The method of calculation of the end bearing capacity largely depends on the helix spacing. The individual bearing failure mechanism typically occurs where the helix spacing is greater than 3Dh, while the cylindrical shear failure mechanism occurs where the helix spacing is less than 1.5Dh[25]. Fig. 1 depicts the two possible failure mechanisms for helical piles. The analytical method is used to calculate the bearing capacities of piles used in this study. The computed values are then compared with those of the experimental ones. 1. Introduction 𝜆𝐿= 𝐿𝑚 𝜎𝑉𝛾𝑆 ⁄ (1) (1) Where Lm is the length of the model pile, σv is the vertical stress at the bottom of the pile (Installation depth), and γs is the unit weight of the soil sample. The corresponding scale factors for testing model piles in FCV apparatus were provided by Sedran et al. [28], and the scaling factors are provided in Table 1. Karimi and Eslami [19], Khazaei and Eslami [20], and Karimi et al. [21] investigated the effects of soil improvement techniques, such as soil densification and post-grouting, to compensate for the disturbances that occurred during pile installation. Different types of piles, such as open-ended piles, close-ended piles, H steel piles, and helical piles, have been scrutinized. They concluded that increasing the soil-pile interaction by post-grouting of the base and sides of the pile, as well as soil compaction yields, to an increase in soil-bearing capacity. Khazaei and Eslami [20] investigated the geotechnical behavior of different types of piles, including helical piles, with the aid of FCV-AUT. They concluded that a Table 1: Scaling factor for model pile testing in FCV Parameter Scaling Factor Length and Displacement 𝜆𝐿 Strain 𝜆𝐿 Stress 𝜆𝐿 2 Force 𝜆𝐿 2 Spring Stiffness 𝜆𝐿 Elastic Modulus 1 Table 1: Scaling factor for model pile testing in FCV 2 Where: D: Diameter of pile shaft D: Diameter of pile shaft There are well-known methods for calculating the axial bearing capacity of the helical pile: (1) Individual Bearing Method (IBM), (2) Cylindrical Shear Method (CSM), and (3) Torque Correlated Method. The first two methods are physics-based, while the last one is an empirical method that correlates the installation torque of the helical pile to the ultimate bearing capacity. L: Pile length over which the terms 𝜋𝐷 and 𝑓𝑠 are considered constant fs: sum of friction and adhesion between soil and pile (6) 𝑓𝑠= 𝜎𝑣𝐾tan 𝛿 (6) 2. Analytical Bearing Capacity of Helical Piles Nc, Nq, N: Bearing capacity factor for the local shear condition, presented in Table 10.1 of CFEM. Qf is calculated as follows: The axial bearing capacity of helical piles depends on the failure mechanism of the pile. The failure mechanism of a helical pile is governed by its inter-helix spacing ratio, which is defined as the ratio of the helix spacing to the helix plate diameter (S/Dh). Q 𝑄𝑓= ∑𝜋𝐷∆𝑙𝑓𝑠 (5) (5) Where: Where: 3. Numerical Modeling For this purpose, normal direction contact is defined as “Hard Contact”, while tangential direction contact is defined as a frictional behavior. Different skin friction angles are represented, as demonstrated in Table 3, and the developed models are evaluated in terms of vertical stress gradient in depth at the centerline of the device. (b) Fig. 3: (a) Max. Principle Stress and (b) Isosurface Stress plot for new FCV apparatus As seen in Figure 2, as the skin friction angle increases, the vertical stress distribution shows a more linear realistic trend in the depth of the FCV apparatus. Finally, based on the results of numerical simulations, the following dimensions were chosen, and the device was fabricated. More details of the modeling can be found in another paper by the authors, and the readers are encouraged to refer to it [18]. Based on the measured properties of Anzali sand, which is used in this study, the internal friction angle varies from 31 to 38 degrees. Therefore, the external surface of the soil and the internal surface of the FCV body can be considered as Fully Contacted. Wall Inclination Angle Bottom Diameter Top Diameter Height (°) (mm) (mm) (mm) 31 1700 500 1000 Table 3: Skin friction angles Equivalent Internal Friction Angle of Soil φ(°) Friction Coeff. 𝐭𝐚𝐧( 𝜹) Skin Friction Angle δ(°) 29.7 0.36 20 32.5 0.44 24 36 0.53 28 40 0.61 32 - - Fully Contact Fig. 2: Vertical stress gradient in depth Table 3: Skin friction angles 3. Numerical Modeling Prior to the fabrication of a new Frustum Confining Vessel capable of simulating the saturated sand conditions, a numerical optimization on the dimensions of the device was carried out. In this regard, reviewing the present FCV apparatuses all around the world, nine sets of dimensions are proposed (Table 2) and examined through finite element modeling in 2D-Axisymmetric modeling space via Abaqus CAE ®. Fig. 1: Pile resisting force in (a) Cylindrical failure mechanism and (b) Individual failure mechanism Fig. 1: Pile resisting force in (a) Cylindrical failure mechanism and (b) Individual failure mechanism 3 3 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. Table 2: Proposed dimensions Bottom Diameter mm Top Diameter mm Wall Angle Model ° 1050 400 72 Model 1 1150 500 72 Model 2 1250 600 72 Model 3 1420 400 63 Model 4 1520 500 63 Model 5 1620 600 63 Model 6 1600 400 59 Model 7 1700 500 59 Model 8 1800 600 59 Model 9 Table 2: Proposed dimensions In the model, the soil was defined using 4-node, reduced- integration, first-order, axisymmetric solid elements with hourglass control. The model was meshed using quadrilateral elements. Views of the directions of the maximum principle stresses, as well as the stress isosurfaces, are shown in Figure 3. (a) (b) Fig. 3: (a) Max. Principle Stress and (b) Isosurface Stress plot for new FCV apparatus (a) (b) Fi 3 ( ) M P i i l St d (b) I f St l t In order to consider the effects of Soil-Wall interaction on the stress distribution in depth of the device, the contact between the soil and the steel wall is defined using the Penalty method in two tangential and normal directions. In order to consider the effects of Soil-Wall interaction on the stress distribution in depth of the device, the contact between the soil and the steel wall is defined using the Penalty method in two tangential and normal directions. For this purpose, normal direction contact is defined as “Hard Contact”, while tangential direction contact is defined as a frictional behavior. Different skin friction angles are represented, as demonstrated in Table 3, and the developed models are evaluated in terms of vertical stress gradient in depth at the centerline of the device. 4.3. Installation Process of Piles the precise vertical displacement of the pile head during the experiment, one linear potentiometer with a stroke of 300 mm with a precision of measuring 0.1 mm is mounted to the pile head and the loading frame. To capture the applied load one S-Type load cell with a capacity of 10 tonf was placed between the pile head and the hydraulic actuator’s head. In the following, the real and schematic views of the experimental work setup are depicted. Helical pile installation is carried out by applying torque at the end of the pile in the presence of vertical pressure. Figure 5 shows a schematic view of the pile installation procedure. A rotary motor drive mounted on the head piles was used for the installation of helical piles into the sand. The servo-electric rotary drive was fixed on the top of the pile-driving platform, and the pushing force is supplied by the self-weight of the rotary drive, pile, and the upper part of the driving platform. The role of the pile-driving platform is to ensure that the pile is driven to the exact vertical position. The rotary speed of the pile during installation was kept constant at 25 rpm, and the linear speed of pile penetration in the soil sample was about 3-4 mm/sec, which is equal to about 7.2 to 9.6 mm/rev penetration speed. The FCV-BKP apparatus is the first and only FCV apparatus all around the world capable of testing model piles in saturated sand conditions, to the best of the author’s knowledge. More details of the construction process of the FCV-BKP apparatus are presented in another research paper by the authors, and readers are encouraged to refer to it for further information [18]. Fig. 5: Schematic view of FCV apparatus during pile installation 4.2. Model Piles Helical piles, used in this study, consist of steel pipe with one or three welded circular plates to the shaft of the pile with a conical shoe. In multi-helix piles, helices are spaced apart at a constant ratio, S/Dh=1.5. The model pile dimensions were obtained by scaling down the dimensions of a typical helical pile. The prototype pile has a shaft diameter and helix diameter of 406 mm and 1170 mm, respectively, which are common values in practice. For each test, the dimensions of the model pile were computed based on the scaling law. All the model piles had a length of 750 mm. Figure 4 depicts a layout of the piles used in this study, and Table 4 provides the geometric characteristics of the model piles. Fig. 4: (a) Layout of helical piles used in this study, (b) Schematic of helical pile Fig. 5: Schematic view of FCV apparatus during pile installation 4.1. Experimental Work Setup The axial bearing capacity of the helical pile is evaluated in the FCV apparatus. The FCV apparatus, used in this study, FCV-BKP, was constructed at Baran Khak va Pey Co, Tehran, Iran, by the authors. As mentioned in section 3, FCV-BKP has a height of 1000 mm and the top and bottom diameter of 500 mm and 1700 mm, respectively. To grant ease of access during sample preparation, the body of FCV-BKP was divided into two parts by the middle height of the device. To ensure the uniformity of applying the base pressure, a 1700 mm diameter cylinder with a height of 210 mm, equipped with a membrane at its bottom, was added to the FCV-BKP. The bottom pressure is applied by means of applying pressure to the water at the bottom of the apparatus using an air compressor capable of applying up to 600 kPa. The FCV- BKP was equipped with a loading frame used as a support for both the rotary motor drive for pile installation and the hydraulic actuator for applying the axial loads to the model piles during the test. To measure Fig. 2: Vertical stress gradient in depth 4 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. 4.5. Sample Preparation For this purpose, two valves were installed at the bottom of the FCV-BKP to limit the rate of intake water discharge to the soil sample, as shown in Fig. 7-c. The rate of water flow was kept constant during the process as low as reasonably practicable, about 0.2 lit/min and less. This is to ensure that the water entering the soil samples would have the least effect on the relative density of soils (Minimum disturbance). The saturation process was stopped when the volume of the water entering the soil sample was equal to or greater than 0.95xVvoid, and the water table level in the FCV apparatus stayed constant at the top of the specimen. The saturation process time varies from 2 to 3 days, based on the relative density of the soil samples. Fig. 6: Grain size distribution for Anzali sand Fig. 6: Grain size distribution for Anzali sand Fig. 6: Grain size distribution for Anzali sand Fig. 6: Grain size distribution for Anzali sand Table 4: Helical pile dimensions Table 4: Helical pile dimensions Table 4: Helical pile dimensions Pile Type Scale Factor 𝝀𝑳 Shaft Helix S H Diameter d (mm) Thickness e (mm) Diameter Dh (mm) Thickness e* (mm) No. Helix Spacing Length (mm) Prototype 1 1 406 50 1170 50 1 N.A 9750 Prototype 2 1 406 50 1170 50 3 1.5Dh 9750 BKP-N01 13 32 4 90 4 1 - 750 BKP-N02 13 32 4 90 4 3 1.5Dh 750 BKP-N03 22 32 4 54 4 1 - 750 BKP-N04 22 32 4 54 4 3 1.5Dh 750 Results of local relative densities proved the proper sample preparation method. The achieved relative densities have shown a maximum variation of less than 5% (Dr ± 2.5%) between the top and bottom layers. reasonably practicable, about 0.2 lit/min and less. This is to ensure that the water entering the soil samples would have the least effect on the relative density of soils (Minimum disturbance). The saturation process was stopped when the volume of the water entering the soil sample was equal to or greater than 0.95xVvoid, and the water table level in the FCV apparatus stayed constant at the top of the specimen. The saturation process time varies from 2 to 3 days, based on the relative density of the soil samples. FCV-BKP provides the capability of testing model piles in saturated conditions. 4.4. Soil Properties Interface skin friction between the soil particles and the internal wall of the device plays a key role in generating horizontal and vertical stress distribution in the FCV apparatus. Therefore, the FCV apparatus shall be best suited for simulating the soil-pile interactions in sandy soils, in which the soil strength relies on frictions of the particles rather than the cohesion between them. In this study, the dry sand of Anzali, a coastal city in the north of Iran, was used for sample preparation. According to the Unified Soil Classification System, ASTM D2487 [30], Anzali sand was classified as poorly graded sand (SP). Fig. 4: (a) Layout of helical piles used in this study, (b) Schematic of helical pile 5 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 4.5. Sample Preparation The specific gravity (Gs) of the solids was measured as 2.69. The mean grain size of the sand (D50), the coefficient of uniformity (CU), and the coefficient of curvature (CC) were found to be 0.215 mm, 1.62, and 0.97, respectively, based on ASTM D6913 [31]. The sand was poured into the apparatus to achieve three relative densities, loose state (Dr ≅ 25%), medium dense state (Dr ≅50%), and dense state (Dr ≅70%). The cohesion and the friction angle of the soil were evaluated through the triaxial consolidated drained test at three different states (Loose, Medium, and Dense) based on the instructions of ASTM D7181[32]. Fig. 6 shows the grain size distribution of Anzali sand. Obtained properties of the used sand are summarized in Table 5. The internal face of the FCV apparatus was marked at intervals of 50 mm to simplify accurate and homogeneous sample preparation. To overcome the internal friction force of the sand particles during sample preparation, water was added to the sand particles to achieve a moisture content of 4%. For sample preparation, the required weight of sand for each layer was calculated with regard to the under-compaction method; then the sand was poured into the FCV body and compacted to reach the desired surface level. The attained relative densities of sand samples were evaluated using the cylindrical mold with known volume, placed at the centerline of the device at different depths. p Fig. 6: Grain size distribution for Anzali sand Table 4: Helical pile dimensions Pile Type Scale Factor 𝝀𝑳 Shaft Helix S H Diameter d (mm) Thickness e (mm) Diameter Dh (mm) Thickness e* (mm) No. Helix Spacing Length (mm) Prototype 1 1 406 50 1170 50 1 N.A 9750 Prototype 2 1 406 50 1170 50 3 1.5Dh 9750 BKP-N01 13 32 4 90 4 1 - 750 BKP-N02 13 32 4 90 4 3 1.5Dh 750 BKP-N03 22 32 4 54 4 1 - 750 BKP-N04 22 32 4 54 4 3 1.5Dh 750 Results of local relative densities proved the proper sample preparation method. The achieved relative densities have shown a maximum variation of less than 5% (Dr ± 2.5%) between the top and bottom layers. FCV-BKP provides the capability of testing model piles in saturated conditions. Soil samples within the FCV body are saturated using the flooding method. 4.6. Testing Procedure 4.6. Testing Procedure Following the sample preparation, the base pressure was applied. The base pressure must be applied gradually in steps to make sure that the relative density of soil samples, especially in loose samples, is not affected. It should be noted that a sudden increase in the amount of applied base pressure may cause liquefaction in saturated sand samples. Therefore, to make sure that the generated excess pore water pressure is completely dissipated during applying the base pressure, the applied load at each step was kept constant for at least 20 minutes in saturated condition tests. After applying the base pressure, the pile was driven into the soil sample using a rotary motor drive. Then the axial load was applied to the pile end in accordance with the Quick Testing Procedure of ASTM (2013) standard D1143/D1134M 07 [33]. In this method, having in hand an estimate of the ultimate load capacity of the pile, the load is applied in an increment of 5% of the predicted capacity. During each load interval, the load is kept constant for a time interval of not less than 10 minutes for wet conditions and 20 minutes for saturated conditions. During the test, the applied load and the displacement of the head pile are measured and saved at a constant interval time of 1 sec using the Load Cell and Potentiometer, respectively. The test is interrupted when the displacement of the pile head reaches 50% of the helix diameter. At the end, the obtained data are filtered out using zero-phase digital filtering to make them smooth. (a) (b) (c) Fig. 7: (a) Schematic and dimensions of FCV, (b) FCV apparatus during the test, (c) View of the FCV apparatus valves (a) (b) (b) (c) Fig. 7: (a) Schematic and dimensions of FCV, (b) FCV apparatus during the test, (c) View of the FCV apparatu Fig. 7: (a) Schematic and dimensions of FCV, (b) FCV apparatus during the test, (c) View of the FCV apparatus 4.5. Sample Preparation Soil samples within the FCV body are saturated using the flooding method. For this purpose, two valves were installed at the bottom of the FCV-BKP to limit the rate of intake water discharge to the soil sample, as shown in Fig. 7-c. The rate of water flow was kept constant during the process as low as 6 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. Table 5: Anzali Soil Properties Properties Value Maximum unit weight, max (kN/m3) 15.46 Minimum unit weight, min (kN/m3) 13.36 Specific gravity, Gs 2.69 Mean grain size, D50 (mm) 0.215 Coefficient of uniformity, Cu 1.62 Coefficient of curvature, Cc 0.97 Loose Sand Properties Relative density, Dr (%) 25% Unit weight,  (kN/m3) 14.37 Saturated unit weight, sat (kN/m3) 18.43 Internal friction angle, 𝜑 31.0 Medium sand properties Relative density, Dr (%) 50% Unit weight,  (kN/m3) 14.90 Saturated unit weight, sat (kN/m3) 18.75 Internal friction angle, 𝜑 36.0 Dense sand properties Relative density, Dr (%) 70% Unit weight,  (kN/m3) 15.35 Saturated unit weight, sat (kN/m3) 19.02 Internal friction angle, 𝜑 38.0 Table 5: Anzali Soil Properties 4.7. Testing Program A parametric study with a total number of 18 tests was conducted on helical piles with different helix numbers to study the effects of the number of helices on the axial compressive bearing capacity of piles at different relative densities. The effects of saturation conditions and the effects of the installation depth of piles on the load- displacement behavior are studied as well. Table 6 shows the test descriptions for the current study. 4.7. Testing Program were categorized into three main classifications; (1) 6 tests in moist sand (W = 4%) with different relative densities, simulating installation depth of 10 to 11 m for pile, (2) 6 tests in saturated soils with different relative To investigate the axial compressive bearing capacity of helical piles, 18 experimental tests were performed. Tests 7 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. different relative densities (Dr, %) and for different helices. The axial bearing capacity of the pile at a certain condition, i.e., specific depth of installation and specific soil condition, is defined as the resisting force corresponding to the displacement of 15% of the helix diameter of the pile. densities, simulating installation depth of 10 to 11 m for pile, and (3) 6 tests in saturated soils, with different relative densities, simulating installation depth of 16.5 to 18.5 m for pile. In this study, the maximum applied base pressure for moist conditions was chosen to be 200 kPa, which was equal to simulating the installation depth of about 13 m to 14 m at the base of the device. This is the most common real-world depth for installing piles for geotechnical applications. To replicate the same installation depth of 13 m to 14 m at the device base for saturated sand conditions, based on the scale laws, the base pressure was selected to be 121 kPa. Table 7 summarizes the axial bearing capacity for tested piles, as well as the numerically predicted bearing capacity using CFEM formulation. The ratio of the predicted to measured load capacities for each test is provided in Table 7 as well. It can be seen that the numerical method provided a close correlation between the measured and numerical predicted results. The ratio of numerically predicted to experimentally measured ultimate bearing capacity is presented in Table 7. The average ratio of analytical to experimental bearing capacity for medium and dense samples is 1.18 with a standard deviation of 0.17, while, for loose sand, the average ratio is 0.61 with a standard deviation of 0.1. In the following sections, the effects of different parameters affecting the bearing compressive capacity of the helical pile, including the relative density of sand, number of helices, saturation condition, and installation depth, are evaluated. Tests were continued to a vertical displacement of half of the helix diameter of piles. 5. Tests Results and Discussion For different test conditions, the behavior of helical piles can be described using their load-displacement curves. Eighteen tests were performed in FCV-BKP apparatus at Table 6: Test schedule Test No. Soil Condition Helix No. Helix Diameter (mm) S/Dh Relative Density (Dr, %) Uplift Pressure (kPa) Equivalent simulation depth (m) State 1 1 moist Condition (Dry Sand with moist, W = 4%) 1 90 N/A Loose 20-25% 200 13~14 2 Medium 45-50% 3 Dense 65-70% 4 3 90 1.5 Loose 20-25% 5 Medium 45-50% 6 Dense 65-70% State 2 7 Saturated Sand 1 90 N/A Loose 20-25% 121 13~14 8 Medium 45-50% 9 Dense 65-70% 10 3 90 1.5 Loose 20-25% 11 Medium 45-50% 12 Dense 65-70% State 3 13 Saturated Sand 1 54 N/A Loose 20-25% 200 21.6~23.3 14 Medium 45-50% 15 Dense 65-70% 16 3 54 1.5 Loose 20-25% 17 Medium 45-50% 18 Dense 65-70% 5.1 Effects of Relative Density of Soil Effects of soil relative density (% Dr) on the bearing apacity of each model pile are shown in Figure 8. As can be seen, for both single-helix and multi-helices piles, th bearing capacity increases more from loose to medium state rather than from medium to dense state Additionally, it can be seen that soil densification is a be seen, for both single-helix and multi-helices piles, the bearing capacity increases more from loose to medium state rather than from medium to dense state. Additionally, it can be seen that soil densification is an be seen, for both single-helix and multi-helices piles, the bearing capacity increases more from loose to medium state rather than from medium to dense state. Additionally, it can be seen that soil densification is an 5.2 Effects of Helix Number To study the effects of helix numbers on the bearing capacity of piles, the load-displacement curves for all three states were plotted and presented in Figures 9 to 11. As illustrated, in State 1, for all relative densities, the load-displacement curve of the one-helix and three- helices piles have shown a similar pattern, especially for Loose and Medium sand. Therefore, the resisting force corresponding to the pile displacement of 15% of the helix diameter is approximately the same. This might be due to the fact that the failure behavior of multi-helices piles with S/Dh = 1.5 is mainly dominated by the cylindrical shear mechanism (CSM). The main discrepancy observed in the behavior of single-helix and three-helices piles is the initial slope of the curves. The initial linear slope for the multi-helices pile is steeper than the slope of the curves for the single-helix pile. The same trend was observed for model piles tested in saturated conditions. 5.1 Effects of Relative Density of Soil Effects of soil relative density (% Dr) on the bearing capacity of each model pile are shown in Figure 8. As can 8 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. appropriate method for increasing the soil-bearing capacity. besides the lubricant effects of the inter-particle water, with the decrease in the effective stress in depth in the presence of the water, a significant reduction in the bearing capacity of the soil was expected. The ultimate bearing capacity of the piles in saturated sand decreases by 60% on average compared to moist conditions. Additionally, comparing the initial slope of the load- displacement curves in moist and saturated conditions revealed that the soils beneath the helix in moist conditions are mobilized at low displacements such that at a displacement of 5% of the helix diameter, the resisting force is about 70% of the ultimate bearing capacity (corresponding force to displacement of 15% of helix diameter), while, in saturated sand condition, the force corresponded to displacement of 5% of helix diameter is about 30% of the ultimate bearing capacity. 5.4 Effects of Installation Depth of Pile Effects of the buried depth of piles on their axial bearing capacities, also known as scale effects, were studied by performing six tests in saturated conditions with an increased overburden pressure. In this respect, the uplift pressure at the bottom of the device was chosen to be 200 kPa for saturated tests (State 3), which generates a stress distribution equivalent to stress conditions at a depth of about 23 m on site. It is noteworthy to mention that to make the results of the tests at various installation depths comparable, the obtained values must be converted to their prototype scale. Given that, for each test the forces are multiplied by Land the displacements are multiplied byL (See Table 1). Figure 14 and Figure 15 compare the load-displacement curves for Single-Helix and Multi-Helix piles, respectively, at the prototype scale. The effects of installation depth on the performance of the helical piles can be inferred from the figures. As shown, increasing the installation depth from 10 m to 17.5 m yielded an average increase of about 95% 5.3 Effects of Water Content In the current study, to evaluate the effects of water content on the load-displacement behavior of piles, six tests in saturated conditions were carried out (State 2). In this regard, to make the results comparable, the geometric scale for saturated conditions tests was kept constant. Fig. 12 and Fig. 13 depict the comparison of load- displacement curves of State 1 and State 2 for single- helix and multi-helices piles, respectively. As can be inferred from the figures, the water content in saturated sand (State 2) plays a key role in decreasing the friction between the sand particles, yielding a significant decrease in ultimate bearing capacity and the initial slopes of the load-displacement curves. It is important to mention that Table 7: Ultimate bearing capacity of pile No. Soil Condition Helix No. Helix Dia. (mm) Relative Density (Dr, %) Geometric Scale Axial Bearing Capacity (kN) Ratio Analytic./ Exp. Prototype Bearing Capacity (kN) Analytical Experimental 1 Moist Condition (Dry Sand with moist, W = 4%) 1 90 Loose 13 16.17 20.8 0.78 3 515.2 2 Medium 32.51 34.5 0.94 5 830.5 3 Dense 43.82 37.9 1.16 6 405.1 4 3 90 Loose 16.17 23.5 0.69 3 971.5 5 Medium 32.51 36.1 0.90 6 100.9 6 Dense 43.85 50.0 0.88 8 450.0 7 Saturated Sand 1 90 Loose 13 3.55 7.5 0.47 1 267.5 8 Medium 18.58 14.2 1.31 2 399.8 9 Dense 25.24 18.1 1.39 3 058.9 10 3 90 Loose 3.58 6.3 0.57 1 064.7 11 Medium 18.62 14.9 1.25 2 518.1 Table 7: Ultimate bearing capacity of pile Table 7: Ultimate bearing capacity of pile 9 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. 12 Dense 25.32 20.2 1.25 3 413.8 13 Saturated Sand 1 54 Loose 23 2.26 4.00 0.57 2 116.0 14 Medium 11.28 8.50 1.33 4 496.5 15 Dense 15.51 12.05 1.29 6 374.5 16 3 54 Loose 2.42 4.30 0.56 2 274.7 17 Medium 11.45 9.60 1.19 5 078.4 18 Dense 15.67 12.15 1.29 6 427.4 (a) State 1, Left: Single-Helix, Right: Three-Helices Saturated Sand 23 15 Dense 15.51 12.05 1.29 6 374.5 16 3 54 Loose 2.42 4.30 0.56 2 274.7 17 Medium 11.45 9.60 1.19 5 078.4 18 Dense 15.67 12.15 1.29 6 427.4 (a) State 1, Left: Single-Helix, Right: Three-Helices (b) State 2, Left: Single-Helix, Right: Three-Helices (c) State 3, Left: Single-Helix, Right: Three-Helices Fig. 5.3 Effects of Water Content 9: Load-Displacement curve of the single-helix and multi-helix pile in moist conditions at diff ( ) ( ) -Displacement curve of the single-helix and multi-helix pile in moist conditions at different relative density (c) Dense (a) Loose (b) Medium ) Dense (a) Loose ( ) ( ) ( ) Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=121kPa) at a different relative ( ) ( ) ( ) Fig. 10: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=121kPa) at a different ( ) ( ) Fig. 10: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=12 of the single-helix and multi-helix pile in saturated sand (P=121kPa) at a different relative density (c) Dense (a) Loose (b) Medium (b) Medium (b) Medium Dense (a) Loose (b) Medium (c) Dense Fig. 11: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=200kPa) at a different relative density (a) Loose (b) Medium (c Fig. 11: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=200kPa) at a d ( ) ( ) ( ) Fig. 11: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=200kPa) at a differ ) ( ) ( ) Displacement curve of the single-helix and multi-helix pile in saturated sand (P=200kPa) at a different relative density (a) Loose (b) Medium (c) Dense Fig. 12: Load-Displacement curve of single-helix pile in moist conditions and saturated conditions at a different relative density (a) Loose (b) Medium (c) Dense Fig. 12: Load-Displacement curve of single-helix pile in moist conditions and saturated conditions at a different relative density (a) Loose (b) Medium (c) Dense Fig. 13: Load-Displacement curve of the multi-helix pile in moist conditions and saturated conditions at a different relative density (a) Loose (c) Dense (b) Medium (a) Loose (b) Medium (c) Dense Fig. 12: Load-Displacement curve of single-helix pile in moist conditions and saturated conditions at a different relative density (a) Loose (b) Medium (c) Dense Fig. 13: Load-Displacement curve of the multi-helix pile in moist conditions and saturated conditions at a different relative density (c) Dense (a) Loose (b) Medium ( ) ( ) e of single-helix pile in moist conditions and saturated conditions at a different relative density Fig. 5.3 Effects of Water Content 8: Effects of relative densities on load-displacement curves li i h h li (a) State 1, Left: Single-Helix, Right: Three-Helices (b) State 2, Left: Single-Helix, Right: Three-Helices Helix Right: Three Helices (b) St t 2 L ft Si (b) State 2, Left: Single-Helix, Right: Three-Helices (c) State 3, Left: Single-Helix, Right: Three-Helices Fig. 8: Effects of relative densities on load-displacement curves (c) State 3, Left: Single-Helix, Right: Three-Helices Fig. 8: Effects of relative densities on load-displacement curves (c) State 3, Left: Single-Helix, Right: Three-Helices Fig. 8: Effects of relative densities on load-displacement curves Fig. 8: Effects of relative densities on load-displacement curves 10 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. (a) Loose (b) Medium (c) Dense Fig. 9: Load-Displacement curve of the single-helix and multi-helix pile in moist conditions at different relative density (a) Loose (b) Medium (c) Dense Fig. 10: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=121kPa) at a different relative density (a) Loose (b) Medium (c) Dense Fig. 11: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=200kPa) at a different relative density (a) Loose (b) Medium (c) Dense Fig. 9: Load-Displacement curve of the single-helix and multi-helix pile in moist conditions at different relative density (a) Loose (b) Medium (c) Dense Fig. 10: Load-Displacement curve of the single-helix and multi-helix pile in saturated sand (P=121kPa) at a different relative density (a) Loose (b) Medium (c) Dense Fig. 9: Load-Displacement curve of the single-helix and multi-helix pile in moist conditions at different relative density (a) Loose (b) Medium (c) Dense Fig. 9: Load-Displacement curve of the single-helix and multi-helix pile in moist conditions at different relative density (a) Loose (b) Medium (c) Dense b) Medium (a) Loose Dense (a) oose (b) ed u (c) e se 9: Load-Displacement curve of the single-helix and multi-helix pile in moist conditions at different relative density ( ) ( ) of the single-helix and multi-helix pile in moist conditions at different relative density ( ) ( ) Fig. 6. Conclusions with a spacing ratio of 1.5 would be cylindrical shear failures. An experimental testing program was designed to study the bearing capacity of helical piles. A total of 18 tests were carried out using FCV-BKP, which is, to date, the only FCV apparatus capable of simulating the saturated conditions to the best of the author’s knowledge. The study focused on determining the effects of the water content of sands on the load-displacement behavior of helical piles, as well as the effects of the installation depth on the bearing capacity of helical piles. The obtained test results of axial compressive bearing capacity of a single-helix and multi-helices piles in the sand under different relative density conditions are compared. The findings of the experimental study can be summarized as follows: (4) Increasing the number of helices at low spacing ratio, S/Dh < 3, shows no significant effects on the ultimate bearing capacity of helical piles. The reason for this position is that at a low spacing ratio, the soil beneath each helix cannot be mobilized to resist the applied axial loads individually. (5) At a constant geometric scale factor, i.e., constant installation depth, increasing the water content has shown a significant effect on the ultimate bearing capacity. On average, the bearing capacity was reduced by 60%. The observation can be justified given the lubricant effects of the excess inter-particle water, as well as the reduced effective stress due to the presence of pore water pressure. (1) At a constant helix number, as the relative density increases, the bearing capacity increases, and soil densification can be considered as a potential method for strengthening soil. (1) At a constant helix number, as the relative density increases, the bearing capacity increases, and soil densification can be considered as a potential method for strengthening soil. (6) In saturated soil conditions, as the installation depth increased, both in single-helix and multi-helix piles, the ultimate bearing capacity increased. (7) The average ratio of analytical to experimental bearing capacity for medium and dense relative densities was 1.18 with a standard deviation of 0.17, while for loose sand conditions the average ratio was 0.61 with a standard deviation of 0.1. Totally these values confirm the accuracy of analytical methods. It should be noted that the soil sample preparation for loose sand is more susceptible to disturbance. Therefore, the ratios were calculated separately for loose sand conditions. 5.3 Effects of Water Content 12: Load-Displacement curve of single-helix pile in moist conditions and saturated conditions at a different relative density 12: Load-Displacement curve of single-helix pile in moist conditions and saturated conditions at a different relative de Fig. 12: Load-Displacement curve of single-helix pile in moist conditions and saturated conditions at a (a) Loose (c) Dense (b) Medium Fig. 13: Load-Displacement curve of the multi-helix pile in moist conditions and saturated conditions at a different relative density 11 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. (a) Loose (b) Medium (c) Dense Fig. 14: Load-Displacement curve of single-helix pile with different installation depths at a different relative density (d) Loose (e) Medium (f) Dense Fig. 15: Load-Displacement curve of the multi-helix pile with different installation depths at a different relative density (a) Loose (b) Medium (c) Dense Fig. 14: Load-Displacement curve of single-helix pile with different installation depths at a different relative density (a) Loose (b) Medium (c) Dense Fig. 14: Load-Displacement curve of single-helix pile with different installation depths at a different relative density (c) Dense (a) Loose (b) Medium (a) Loose (b) Medium (c) Dense Fig. 14: Load-Displacement curve of single-helix pile with different installation depths at a different relative density (d) Loose (e) Medium (f) Dense Fig. 15: Load-Displacement curve of the multi-helix pile with different installation depths at a different relative density Medium Loose Dense (a) Loose (b) Medium (c) Dense Fig. 14: Load-Displacement curve of single-helix pile with different installation depths at a different relative density a) Loose (b) Medium (c) Dense . 14: Load-Displacement curve of single-helix pile with different installation depths at a different relative density (a) Loose (b) Medium Fig. 14: Load-Displacement curve of single-helix pile with different installation d ( ) th different installation depths at a different relative density (d) Loose (f) Dense (e) Medium Medium Medium (f) (e) Dense Fig. 15: Load-Displacement curve of the multi-helix pile with different installation depths at a different relative densit References [16] A. Eslami, S. Moshfeghi, H. MolaAbasi, and M. M. Eslami, "Geotechnical engineering," in Piezocone and Cone Penetration Test (CPTu and CPT) Applications in Foundation Engineering, ed, 2020, pp. 1-23. [1] M. Sakr, A. Nazir, W. Azzam, and A. Sallam, "Model study of single pile with wings under uplift loads," Applied Ocean Research, vol. 100, 2020. [2] S. N. Rao, Y. V. S. N. Prasad, and M. D. Shetty, "The Behaviour of Model Screw Piles in Cohesive Soils," Soils and Foundations, vol. 31, pp. 35-50, 1991. [17] H. Mortazavibak, A. Halabian, H. Hashemalhosseini, M. Roshanzamir, A. Jafari, and B. Shabadagh, "Design Optimisation of the Size and Geometry of Frustum Confining Vessel," in 13TH INTERNATIONAL CONFERENCE ON THE MECHANICAL BEHAVIOUR OF MATERIALS, 2019, p. 298. [3] L. P. Andina Sprince, "Helical pile behavior and load transfer mechanism," presented at The 10th International Conference, Vilnius, Lithuania, 2010. [18] A. Jassim;, N. Ganjian;, and A. 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Harnish, "Helical pile installation torque and capacity correlations," Masters of Science Electroninc Thesis and Dissertation Repository. 2855, Civil Engineering, University of Western Ontario, 2015. [21] A. Karimi, A. Eslami, M. Zarrabi, and J. Khazaei, "Study of pile behavior by improvement of confining soils using frustum confining vessel," Scientia Iranica, vol. 24, pp. 1874-1882, 2017. [7] J. Khazaei and A. Eslami, "Behavior of Helical Piles – as a Geoenvironmental Choice – – by Frustum Confining Vessel," Advances in Science and Technology Research Journal, vol. 10, pp. 8-22, 2016. [22] M. Zarrabi and A. 6. Conclusions (2) In wet sand conditions, the soil beneath the helix is mobilized at low displacements such that at a displacement of 5% of the helix diameter, the resisting force is about 70% of the ultimate bearing capacity (corresponding force to a displacement of 15% of helix diameter). On the other hand, in saturated sand conditions, the force corresponded to a displacement of 5% of helix diameter is about 30% of the ultimate bearing capacity. (8) By increasing the installation depth in saturated sand conditions, the bearing capacity increases exponentially. (3) The obtained experimental results confirm the predicted failure mechanism for multi-helix piles with the spacing ratio of 1.5. Based on the literature, helical piles (3) The obtained experimental results confirm the predicted failure mechanism for multi-helix piles with the spacing ratio of 1.5. Based on the literature, helical piles 12 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. By a 75% increase in the installation depth, the bearing capacity increased by 95%. evaluation of pulling resistance," Soils and Foundations, vol. 58, pp. 355-369, 2018. [13] M. Zare and A. Eslami, "Study of deep foundation performances by frustum confining vessel (FCV)," International Journal of Civil Engineering, vol. 12, pp. 271- 280, 2014. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. [14] M. Zarrabi and A. Eslami, "Behavior of Piles under Different Installation Effects by Physical Modeling," International Journal of Geomechanics, vol. 16, 2016. [15] D. S. Robert G. Horvath, "Frustum confining vessel for testing model piles," Canadian Geotechnical Journal, vol. 33, p. 6, 1996. References Eslami, "Behavior of piles under different installation effects by physical modeling," International Journal of Geomechanics, vol. 16, p. 04016014, 2016. [8] D. Kim, K. Baek, and K. Park, "Analysis of the Bearing Capacity of Helical Pile with Hexagonal Joints," Materials (Basel), vol. 11, Oct 2 2018. [9] N. P. Kurian and S. J. Shah, "Studies on the behaviour of screw piles by the finite element method," Canadian Geotechnical Journal, vol. 46, pp. 627-638, 2009. [23] A. M. A. Fateh, A. Eslami, and A. Fahimifar, "Study of soil disturbance effect on bearing capacity of helical pile by experimental modelling in FCV," International Journal of Geotechnical Engineering, vol. 11, pp. 289-301, 2017. [10] R. S. Merrifield, "Ultimate Uplift Capacity of Multiplate Helical Type Anchors in Clay," Journal of Geotechnical and Geoenvironmental Engineering, vol. 137, pp. 704-716, 2011. [24] H. A. Perko, Helical Piles: A practical guide to design and installation: John Wiley & Sons, 2009. [11] A. Mohammadi, T. Ebadi, and M. R. Boroomand, "Physical Modelling of Axial Compressive Bearing Capacity of Instrumented Piles in Oil-Contaminated Sandy Soil," Iranian Journal of Science and Technology, Transactions of Civil Engineering, vol. 44, pp. 695-714, 2019. [25] A. J. Lutenegger, "Behavior of multi-helix screw anchors in sand," in Proceeding of the 14th Pan-American Conference on Soil Mechanics and Geotechnical Engineering, Toronto, 2011. [26] E. Shojaei, A. Eslami, and N. Ganjian, "Self- expanded piles: A new approach to unconventional piles [12] H. Nagai, T. Tsuchiya, and M. Shimada, "Influence of installation method on performance of screwed pile and 13 Numerical Methods in Civil Engineering, 8-2 (2023) 56-69 A. Jassim et al. development," Marine Georesources & Geotechnology, vol. 39, 2021. [27] H. Mortazavibak, A. Halabian, H. Hashemalhosseini, and M. Rowshanzamir, "An investigation on the axial response and material efficiency of tapered helical piles," Journal of Rock Mechanics and Geotechnical Engineering, vol. 13, 2021. [28] G. Sedran, D. F. Stolle, and R. G. Horvath, "An investigation of scaling and dimensional analysis of axially loaded piles," Canadian geotechnical journal, vol. 38, pp. 530-541, 2001. [29] C. G. S. F. 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https://openalex.org/W4220865569	https://www.intechopen.com/citation-pdf-url/80704	English	null	The New Institutional Approach as a Lens on Local Network Leadership	IntechOpen eBooks	2,022	cc-by	8,173	Anna Uster Anna Uster Abstract This chapter derives from an overview of key research findings and core concepts on network leadership, focusing on leading purpose-oriented networks. These are increasingly viewed as prominent modes of local service delivery as local government transitions to “local governance” and where local government mostly fol- lows a lead organization format. The literature encompassing local leadership empha- sizes the context of structures and processes for any leader’s action. This chapter treats the importance of the institutional factors in the era of local network governance, using the New Institutional approach, focusing especially on discursive institutional- ism, together with and network governance theory. As public managers are increas- ingly relying on inter-organizational networks providing public services, the manner they lead them is of great importance. The following chapter presents vital factors that may assist their effective leadership in an era of local network governance. Keywords: local networks, network leadership, new institutionalism, purpose-oriented networks, local government 1. Introduction The past three decades have witnessed changes in the structure, function, and leadership modes of public organizations, both at the local and national levels. These changes are captured in the literature by the Post New Public Governance (NPG) approach [1], a holistic view of government in which citizens and non- governmental actors become partners in the public management process [2–4]. “Focus on governance involves the use of institutions and structures of authority and collaboration to allocate resources and to coordinate and control joint action across the network as a whole” [5]. The literature describes these changes using various terms, such as collaborative governance, collaborative leadership and management, new public governance, co-governance, and meta-governance [1, 6–13]. Ansell and Gash [9] define governance as both the structure of “laws and rules that pertain to the provision of public goods” and as the process of “collec- tive decision making that includes both public and private actors” (p. 545). The move toward a collaborative mode of governance derives from the phenomenon known as the hollow state, “…a metaphor for the increasing use of third parties, often non-profits, to deliver social services and generally act in the name of the state” ([14], p. 360). 1 Leadership in a Changing World - A Multidimensional Perspective At the local level, this condition occurs due to the lack of local government (LG) capacity to provide multiple services to meet the needs and demands of the citizenry. Consequently, local government is growing increasingly conscious of the potential of working as a collaborative network to provide these services in the municipal arena [15, 16]. Collaboration enables external organizations such as non-profits, non-local public, private organizations, and citizens to share knowl- edge and experience to initiate novel solutions to different social, educational, and environmental wicked problems [17, 18]. p Local authorities now employ network modes of governance to include people and organizations which play a greater role in the provision of local services, gener- ally organized as a purpose-oriented network, defined as “a network comprised of three or more autonomous actors who participate in a joint effort based on a common purpose” ([19], p. 210). Berthod and Segato [20] and Lemaire et al. [21] proposed the term “purpose-oriented” networks to extend the well-known term of art “goal-directed networks”. 1. Introduction According to this line of research goal-directed refers to network members who have identified and agreed on a set of goals that guide the work of the network, which is not necessarily reality-based [22]. “Purpose- oriented networks’ (PONs) highlight the collective purpose that is “translated into actionable goals whose achievement can be monitored” (Carboni et al., p. 15), thus encompassing the complex reciprocity between the network members as well as the environment in which they operate. Such networks are understood to solve the wicked problems which characterize service delivery in local authorities. p y The idea underpinning PON’s is that by combining actors’ differing capabilities, skills, and resources the network’s outcomes will be improved [23]. In the local arena, these networks are generally headed by one lead organization, e.g., the local authority, which selects the other network partners, while coordinating decisions and activities [5]. This organization usually possesses sufficient resources and legitimacy to lead together with the capacity to take on most of the responsibilities of running and coordinating the network’s activities. Given local policy mak- ers’ increasing reliance on networks to achieve the provision of public goods and services, leadership constitutes a paramount challenge facing contemporary local governance. Thus, changes in leadership style and form are required [24, 25], and these, in turn, affect the establishment and coordination of these networks [26]. 2. Major challenges to local leadership in era of purpose-oriented networks Synchronizing collaboration activity and enhancing informational flow presents a primary challenge, due to the multiplicity of opinions and interests of the various network actors [27, 28]. Moreover, even with a strong lead-organization, network organizations are dynamic, not static [29, 30], often including and excluding certain actors or adapting to changing needs by altering the form of network governance [5]. Networks create numerous managerial dilemmas by diminishing the lead actor’s degree of control over adherence to public policy. Monitoring and coordinating pub- lic policy implementation, for example, while at the same time permitting autonomy to network actors concerning the delivery of the public service requires constant attention [1, 10, 31–33]. There is always a risk of poor coordination and of defection by one or more partners [34]. Furthermore, in certain cases, an action can increase conflict and create tension in a network. These tensions might lead to misunder- standing and a reluctance to engage with the lead organization in the future [35]. Different cultural characteristics may cause friction, diminishing a commitment from different management levels: the internal world of the organization employing 2 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 the participants and the external world of the network in which their organization is involved [36]. Cultural tensions may include different approaches to decision- making, levels of professionalism, and methods of providing service. When individual parties within the network expect different outcomes from the collabora- tion because of different norms or have different ways of communicating cultural friction is almost inevitable. In sum, these tensions create coordination fatigue, with the result that the coordination of network activities requires considerable time and effort. In addition, unequal distribution of power between network members can create cases in which powerful stakeholders influence network decision-making to favor certain interests, resulting in harm to the public interest and potential corrup- tion [37]. Naturally, these types of tensions may impair the local network’s ability to produce high-quality local services and perform effectively. p g q y p y Therefore, leading autonomous organizations, not directly subject to local authorities, raise questions about accountability, cost-effectiveness, and the ability to shape, implement, and monitor local policies reducing the quality of local services [38]. 2. Major challenges to local leadership in era of purpose-oriented networks To overcome these tensions and affect the overall local network, network leaders should possess the capacity to make decisions and mobilize the resources required to implement their policies [39]. As a result, considerable effort has been taken in the public administration literature on this issue, focusing on diverse coordinative, facilitative, and mobilizing leadership skills and behaviors required for effective local network leadership [28, 40–48]. The main thrust of this research holds that lead organization managerial and leadership behaviors exerted by the local authority with the aim of enhancing network collaboration may help minimize the mentioned these above-mentioned risks, thus adapting to complex and dynamic environments [49]. However, while most literature draws attention to leadership skills, less atten- tion has been paid to the characteristics of the specific local context as a crucial factor. Local government literature thus calls for a new local leadership style concentrating more on agenda-setting and network brokering in creating a vision, but less focused on policy implementation [50, 51]. This entrepreneurial model concentrates on context to mobilize and attract resources, generating new policies which establish collaborative networks with other governmental or non-govern- mental actors [25, 52–54]. Consideration of context is important because local leaders do not act in a homogeneous local environment and because various features distinguish local authorities from each other. Local government does not operate in a vacuum; it is embedded in the external political the environment within the local context [55–61]. This situation is quite evident in the local contexts where, in addition to structural characteristics, there is a cultural difference in the local authority. Together these factors shape the leadership environment influencing the leaders and their ability to govern [25]. 3. The new-institutional approach to local leadership: discursive and sociological and environmental factors “Political institutions do not determine the behavior of political actors, but provide the framework of understandings within which actors identify, compare and select courses of action.” Lowndes and Leach ([59], p. 560). “Political institutions do not determine the behavior of political actors, but provide the framework of understandings within which actors identify, compare and select courses of action.” Lowndes and Leach ([59], p. 560). The new-institutional perspective’s premise is that to understand the causes and consequences of different forms of leadership we should consider whether 3 Leadership in a Changing World - A Multidimensional Perspective the institution has constraining or enabling effects on the leadership behavior (in terms of formal and informal rules), and judge their level of effectiveness as well as the extent of their activity [50, 62]. Accordingly, different leaders respond differently to the same situation, depending on their environment. This environment includes structural factors, such as legislative rules and regulations, intra- and inter-organizational interactions. Thus, leaders behave contingent upon the locality’s context including local authority size, socio-economic status, central-local relationship as well as leaders’ structural position in various net- works [53, 55]. Further, cultural identity and norms are important factors influencing a leader’s ability to govern. This captures the new-sociological institutionalism which has become particularly important in research on norms and legitimacy, focusing on an understanding of the importance of how and why norms, formal rules and culture in institutions [63–65] to shape their leaders’ actions of the leaders. Parallel to this stream of new institutionalism, a discursive perspective on lead- ership arises. Discursive institutionalism emphasizes the role of ideas and discourse to dynamic reality. Discourse is considered the interactive process of conveying ideas. Therefore, discourse does not only consist of ideas or what is said but includes the context in which we map why and by whom a particular message is delivered. Applying the two forms of discourse (coordinative and communicative discourse) to leadership research enables us to argue that the first form, coordinative discourse, refers to the communication among the network actors themselves, focusing on central actors’ coordinative ability to lead the networks. The latter term refers to communicative discourse, that is, the communication and messages delivered from network actors and their leaders to the external stakeholders. 4. Discursive factors in network leadership research “This shift in governance structure often necessitates that public managers not only lead the agency in which they are employed, but also work within, and often lead, a network. These two different contexts in which public managers operate require different managerial and leadership approaches” [66]. Scholars propose that leadership in the network governance era be character- ized by certain specific skills and behaviors. Network management and network leadership study mostly emphasize the importance of facilitation behaviors, which increase cooperation and coordination between network members and thus change both the network’s rules and structure [9, 67–70]. Most studies focus on leadership skills as facilitating [48], framing and synthesizing [45], and bridging [43], and these capture the idea of coordinative discourse within the network. For example, Agranoff and McGuire [45] grouped network leadership behaviors into four categories based on their operational differences: activation, framing, mobi- lization, and synthesizing. Framing involves behaviors designed to establish work rules. Examples include ensuring individual roles are understood by all network members, asking network members to follow standard rules and regulations, and sharing the leadership role. Synthesizing regards behaviors promoting productive interactions among network participants by looking out for the personal welfare of network members, fostering trust, brainstorming, encouraging network members to use their own judgment in solving problems, and setting expectations for net- work members. Williams [71] noted several necessary leadership behaviors which promote communication inside the network: understanding of and empathy with the partners, trust-building, developing sustainable interpersonal relationships, 4 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 and communication aimed at establishing shared meanings and resolving conflicts. Bass [72] defined such behaviors as structuring work relationships utilizing encour- agement and rewards on one hand and sanctions on the other. The literature on collaborative leadership distinguishes between three facilitat- ing roles of collaborative leaders: that of convener (or steward), mediator, and catalyst [73]. Conveners facilitate and safeguard collaboration while maintaining project integrity. Mediators facilitate collaboration by managing conflict and arbitrating exchange between stakeholders. Catalyzers facilitate, help identify and realize value-creating opportunities. According to Ansell and Gash [73], “facilita- tive leadership will typically require leaders to play all three of these roles” (18–19). 4. Discursive factors in network leadership research Piatak, Romzek, LeRoux, and Johnston [74] examined the management of goal conflicts in public service delivery networks and found that the lead organization should play the dual role of both network manager and member. Their data sug- gested that network managers should exert formal, vertical authority, combining it with informal, horizontal interactions which build goal consensus, thus relieving goal conflict. They also underscore the important use of rewards and sanctions when leading successful networks [75]. g Provan and Kenis [5] proposed three modes of network governance which capture discursive coordinative communication in institutions: the network leader, or Participant-Governed Networks (participatory and internal coordination); Lead Organization-Governed Networks, featuring centralized and internal coordination, and; Network Administrative Organizations, emphasizing centralized and external coordination. In Participant-Governed Networks, for example, control and reflexive coordination of activities occur through direct collaboration in participatory deci- sion-making. By contrast, coordination in Lead Organization-Governed Networks one central leader coordinates, often drawing its power from resource dependencies or other types of obligation. The final mode, Network Administrative Organizations (NAO) is coordinated via a separate, neutral administrative body, acting as a central broker for the activities of the entire network and bridging between diverse actors Berthod et al. [76]. Coordinative and communicative discourse is well-manifested in the literature on internal and external legitimacy. Leading the networks requires maintaining both two types of legitimacies [44]. The former, the internal legitimacy of the network, is concentrated on the communication inside the network, developing trust-based ties between members, resolving conflicts to everyone’s satisfaction, and building communication mechanisms emphasizing the leader’s coordinative and facilitative ability. By contrast, building external legitimacy consists of seeking new members, promoting the network and its activities to outsiders, and mobilizing outside resources to achieve network goals matching the activating, mobilizing, and abili- ties of the leadership [43, 45]. These capture in communicative form the “discourse” in discursive institutionalism. Reviewing the literature we may conclude that a network leader should invest the effort to develop both forms of discourse: promoting the coordinative abilities inside the network and communicative abilities from the network outward toward external actors and the environment. 5. Environmental factors in network leadership research: the local authority context Contexts are the circumstances that form the setting for an event, statement, or idea, and in terms of which it can be fully understood ([77], p. 75). 5 5 Leadership in a Changing World - A Multidimensional Perspective As network collaborations are embedded in a specific context their functioning is logically dependent on that context [11, 75]. More specifically, an effective local network in one context may not be successful in another, even when they have a similar purpose [78]. Virtanen [79] distinguished between two types of scientific knowledge in the context of public administration: conceptual and factual. The former relates to frameworks and theories through which certain phenomena in public administra- tion are explained. The latter maintains that public administration is part of social reality and refers to such factors as actor and place, sector, culture, and institution. Public administration scholars study various contexts according to the research topic. For example, context is prominent where institutional embeddedness, environment, background, and settings are concerned [80–82], or administra- tive tradition and government capacity [83], and the network characteristics of decision-making [84]. In team leadership studies, context plays an important role in the relationship between shared leadership and performance [85–87]. p p p Public administration scholars note that structures and processes define the context in which leaders act [88]. According to Provan and Milward’s [89] net- works framework any change in the network’s environment originating outside, such as financial stability, challenges the network’s overall effectiveness [89–91]. Researchers have established that an integrated structure through network central- ization and direct mechanisms of external control has a positive effect on network effectiveness. However, these relationships are moderated by contextual conditions, such as stability and the availability of abundant resources. y y According to the contingency approach, the tasks and goals of collaborations affect a leader’s ability to collaborate successfully and promote collaborative innova- tion. For example, in their research on workforce development, Ansell and Gash [9] identified four contextual conditions influencing the efficacy of a collaboration leader: (1) access to resources; (2) the strength of the relationships with current and potential partners; (3) regional, state and local governance and service delivery infrastructures, and; (4) historical perceptions of workforce development shared by industry and economic development stakeholders. They found that local autonomy and conditions for economic competitiveness were the most important contextual characteristics for leading successful collaborations. 5. Environmental factors in network leadership research: the local authority context g Contextual factors may include environmental complexities under government regulation, legal constraints, or a combination of organizational culture, norms, and management practices [92]. Local government studies bestow great importance to community characteristics in context [93–96]. In fact, research has shown that a community’s socio-economic status affects the local leader’s capacity to govern [97], and studies have supported the argument that local efficiency is positively related to the level of education in the community; more educated residents tend to select more capable leaders and have a better understanding of the issues on which they vote [47, 93, 94], tend to be actively involved in local affairs [98] and press the lead- ers for more accountability and have better evaluative tools to cause the standard of service to conform to their expectations [99]. In his comparative project on local political leadership in Europe, Steyvers [55] focused on mayoral business orientation as an aspect of external networking to show that institutional form affects leadership behaviors while being highly con- tingent upon leadership context. The indicators of leadership context include the municipalities’ size and institutional position of municipalities in the intergovern- mental arena. Further, the cultural context of the community where the network operates can be crucial for leading effective networks [100–102]. More broadly, Klijn et al. [84] found that effective meso- (changes in the relationship between network organizations) and 6 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 micro-level characteristics of network management (the level of decision-making and implementation) highly depend on the cultural context. Uster Beeri and Vashdi [103] continue this line of thought, focusing on the importance of such contextual issues as socio-economic status and ethnicity in a local authority to the relationship between network leadership and effectiveness. They found that the manner in which the local authority leads the local network derives from network structure, which in turn impinges on effectiveness. A sample of 586 participants from 68 networks indicated that this association is contingent on the politico-cultural characteristics of the local authority and its socio-economic status in which the network exists. In general, local government research points to the differences in local authori- ties based on their size, economic stability, and type of population [25, 59, 94, 104]. 5. Environmental factors in network leadership research: the local authority context Local authorities differ enormously in their structural, political, cultural, and socio- economic indicators, not just cross-nationally, but also within a specific country, and these factors influence local leaders’ ability to govern [61, 95, 105, 106]. In sum, both the local political and cultural systems and the intergovernmental context within which the local networks are situated are crucial explanatory factors for local leaders` behaviors. 6. Structural factors: a leader’s position in the network Mouritzen and Svara [53] classified legislative-executive relations in terms of a combination of factors encompassing the acquisition and maintenance of the lead- ership position, the degree of control leaders has over appointment responsibilities, and the integration of leadership functions in the institutional position. g p p Some studies propose that leaders in a central position (when network is integrated through a central organization, and all members connect to a principal actor) better coordinate with other organizations to achieve network goals [107], using this central position to prevent free-riders while monitoring and controlling other network members [89, 107]. Further, they have an advantage over decen- tralized systems (with their multiplicity of players and linkages) in their ability to facilitate both integration and coordination [89, 108]. This is crucial to local purpose-oriented networks, which require better coordinative skills to achieve the network-level goal. Additionally, leaders holding central positions may more effi- ciently promote systems and integrate services [89] through an organized exchange of information and the coordination of collective action [109, 110]. The “brokerage” is still another structural position that could be a crucial factor for a network leader. While the leader may play a role of a broker, connect- ing disparate groups, he or she can more easily identify opportunities for creating new knowledge or products [111, 112], thus facilitating communication among diverse actors [113]. The position of broker plays a substantial and influential role in leadership ability, enabling governance by controlling access to resources and information across differing set of actors [114–118] while reducing the costs of interlocal cooperation [119]. According to Paquin and Howard-Grenville [120], brokers influence the network by “developing common goals, spurring actor inter- est and engagement, and/or defining norms of action” (p. 1625). More recent conceptualizations of brokerage regard this role not as a mediator between two actors, but as a function that improves the quality of the relationship between these actors [121]. For example, leaders holding a brokerage role tend to resolve conflicts between organizations better, increase the network’s social capital, and find resources to support collaboration [122]. Thus, the brokerage position enables leaders to act as a catalyst enhancing cooperation that builds and sustains connections in the network. 7 Leadership in a Changing World - A Multidimensional Perspective Therefore, an organization’s position in a network is essential to affecting its ability to lead effectively, enhancing its cooperative and coordinative skills. 7. Conclusions This chapter aimed at providing an overview of current thinking on network leadership and related factors affecting local governance. The study focused on the leadership of local purpose-oriented service delivery networks, as these are preva- lent in the local arena. To reiterate, the leadership of inter-organizational networks is concerns more than organizational leadership [123]. Such leadership requires organizational ability to achieve goals through collaboration with other organiza- tions, and as a result, challenges emerge and evolve, necessitating the autonomous organization to be reconstituted in network form. The question then arises: How can local leaders administer networks so as to provide improved local services? The literature as reviewed above proposes many factors which affect the ability to lead those networks. The focus here has drawn on the New Institutional lens which encompasses the whole cycle of factors unique to local government and considered relevant to local leaders’ ability to administer the networks. By combining the literature on network leadership in public administration with local governance the chapter offered insights on the structural, discursive, and environmental factors affecting a local lead organization’s ability to run the networks. g g y These factors include the local lead organization’s coordinative skills with the network itself. Such communicative abilities act as a bridge between the network and external stakeholders, thus enhancing its external legitimacy. Further, certain structural factors regarding a lead organization’s position in a network were shown to be essential in leading the network. Finally, examining network leadership in the local arena, attention was directed to local environmental factors. These envi- ronmental factors refer to locality characteristics such as the intergovernmental context, political, cultural features of the specific municipality in which the net- work runs, alongside its size and socio-economic status of its community, and are crucial for the local lead organization. Future research might examine the combina- tion of the factors discussed here in the context of conditions under which local lead organizations could bring about better functioning of local networks. 8 Author details Anna Uster Department of Political Science, The Max Stern Academic College of Emek Yezreel, Jezreel Valley, Israel Address all correspondence to: annau@yvc.ac.il © 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Address all correspondence to: annau@yvc.ac.il © 2022 The Author(s). 7. Conclusions Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 8 8 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 [5] Provan KG, Kenis P. Modes of network governance: Structure, management, and effectiveness. Journal of Public Administration Research and Theory. 2008;18(2):229-252 The New Institutional Approach as a Lens on Local Network Leadership DOI: http://dx.doi.org/10.5772/intechopen.101988 References [1] Osborne SP. The New Public Governance?: Emerging Perspectives on the Theory and Practice of Public Governance. London: Routledge; 2010 [1] Osborne SP. 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https://openalex.org/W2100520627	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0008712&type=printable	English	null	Role of Progesterone Receptor Polymorphisms in the Recurrent Spontaneous Abortions: Indian Case	PloS one	2,010	cc-by	5,521	Abstract Background: We attempt to ascertain if the 3 linked single nucleotide polymorphisms (SNPs) of the Progesterone Receptor (PR) gene (exon 1: G 1031 C; S344T, exon 4: G 1978 T; L660V and exon 5: C 2310 T; H770H) and the PROGINS insertion in the intron G, between exons 7 and 8, are associated with Recurrent Spontaneous Abortion (RSA) in the Indian population. Methodology/Principal Findings: A total of 143 women with RSA and 150 controls were sequenced for all the 8 exons looking for the above 3 linked SNPs of the PR gene earlier implicated in the RSA, as well as for any new SNPs that may be possibly found in the Indian population. PROGINS insertion was screened by electrophoresis. We did not find any new mutations, not observed earlier, in our population. Further, we did not find significant role of the 2 allele (representing the mutant allele at the three SNP loci) or the T2 allele (PROGINS insertion) in the manifestation of RSA. We also did not find an LD pattern between each of the 3 SNPs and the PROGINS insertion. Conclusions/Significance: The results suggest that the PR gene mutations may not play any exclusive role in the manifestation of RSA, and instead, given significantly higher frequency of the 2 allele among the normal women, we surmise if it does not really confer a protective role among the Indian populations, albeit further studies are required in the heterogeneous populations of this region before making any conclusive statement. raja T, Andal S, Tarakeswari S, Sirisha PVS, et al. (2010) Role of Progesterone Receptor Polymorphisms in the Recurrent Spontaneous LoS ONE 5(1): e8712. doi:10.1371/journal.pone.0008712 Received December 10, 2009; Accepted December 22, 2009; Published January 14, 2010 Copyright: 2010 Aruna et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was funded by Indian Statistical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bmr@isical.ac.in * E-mail: bmr@isical.ac.in Progesterone receptor (PR) mediates the physiologic effects of progesterone. Abstract The PR gene uses separate promoters and translational start sites to produce 2 isoforms, PRA and PRB, the only difference between the two being an additional 165 amino acids present in the N terminus of PRB. The human progesterone receptor (hPR) gene was localized to 11q22–q23 [8] and spans over 90 kb containing eight exons [9]. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da [10]. Meka Aruna1, Theeya Nagaraja1, Sadaranga Andal2, Surapaneni Tarakeswari3, Pisapati V. S. Sirisha1, Alla G. Reddy4, Kumarasamy Thangaraj4, Lalji Singh4, B. Mohan Reddy1* Meka Aruna1, Theeya Nagaraja1, Sadaranga Andal2, Surapaneni Tarakeswari3, Pisapati V. S. Sirisha1, Alla G. Reddy4, Kumarasamy Thangaraj4, Lalji Singh4, B. Mohan Reddy1* 1 Molecular Anthropology Group, Biological Anthropology Unit, Indian Statistical Institute, Hyderabad, India, 2 Lakshmi Fertility Clinic and Research Centre, Pogathota, Nellore, Andhra Pradesh, India, 3 Fernandez Hospital, Hyderabad, India, 4 Centre for Cellular and Molecular Biology, Hyderabad, India Role of Progesterone Receptor Polymorphisms in the Recurrent Spontaneous Abortions: Indian Case PLoS ONE \| www.plosone.org Introduction Recurrent Spontaneous Abortions (RSA) is defined as repeated occurrence of 3 or more miscarriages before 24th week of gestation [1]. The modern definition, however, is the spontaneous loss of 2 or more consecutive pregnancies before 20 weeks of gestation [2,3]. Implantation of the embryo is a critical event in pregnancy. In humans, peri-implantation pregnancy loss contributes to more than 20% of unexplained infertility. Deficient hormone levels result in aberrant growth and support of the uterine lining making it un-ideal for implantation. Three linked single nucleotide polymorphisms (SNPs) (exon 1: G 1031 C; S344T, exon 4: G 1978 T; L660V and exon 5: C 2310 T; H770H) in the PR gene were found to be associated with RSA [11]. The SNP in the exon 1 is reported to be apparently linked to the SNPs in exons 4 and 5 [11], which are in turn in linkage disequilibrium (LD) with PROGINS, a 306 base pairs (bp) insertion of PV/HS-1 Alu subfamily in intron G, between exons 7 and 8 in the codifying region of the hormone binding domain [11,12,13]. However, contrary to the expectation, Kurz et al. [14] suggest that PROGINS is not associated with idiopathic RSA. Thus, only two studies dealt directly with the association of PR mutations with RSA, one dealing with PROGINS insertion and other with the 3 SNPs. Polymorphic variants of hPR gene have been implicated in implantation failure [15,16]. There were also Progesterone, a 21 carbon steroid hormone, mainly produced in the ovaries, placenta, brain and the adrenal glands, is required for the maintenance of pregnancy and treatment with progesterone supplementation was observed to prevent abortions. It is mainly involved in the female menstrual cycle, pregnancy and embryo- genesis in most mammalian species. It stimulates and regulates various functions - i) helps in preparing the body for conception and pregnancy (implantation of the embryo, promoting uterine growth and suppressing myometrical contractility) [4–6] ii) acts as an anti-inflammatory agent and regulates the immune response [7] and iii) regulates estrogen levels and thus prevents endometrial cancer. PLoS ONE \| www.plosone.org 1 January 2010 \| Volume 5 \| Issue 1 \| e8712 PLoS ONE \| www.plosone.org Progesterone Receptor in RSA studies which did not show association of PR mutations in implantation failure [17] and preterm birth [18]. Results We did not find any new SNPs in hPR gene, other than the 3 found earlier. The three SNPs found in the PR gene – exon 1: G 1031 C, S344T; exon 4: G 1978 T, L660V; exon 5: C 2310 T, H770H – co-inherit. The genotype frequencies of the homozy- gotes for wild type allele (1), heterozygotes (1/2) and homozygotes for the rarer mutant allele (2) in the RSA women were observed to be 127 (88.8%), 15 (10.5%) and 1(0.7%), respectively, as compared to 122 (81.3%), 25 (16.7%), and 3(2%) in controls (Figure 1). The genotype frequencies were not significantly different between RSA and control women (x2 = 3.44, df = 2, p = 0.18). ( p ) The allele frequencies (Table 1) were significantly different between the cases and the controls in the pooled samples (x2 = 3.75, df = 1, p = 0.05) as well as between the RSA women with #3 abortions and controls (x2 = 4.88, df = 1, p = 0.03). However, this significance is due to an increased frequency of the 2 allele among the controls, not among the RSA women. Similar trend was seen when allele frequencies were calculated for the patients from LFC and FMH and for primary and secondary aborters separately. Although significant x2 values were observed to suggest association in the LFC data (x2 = 4.24, df = 1, p = 0.04), as well as for the primary aborters (x2 = 3.85, df = 1, p = 0.05) the perceived risk allele (2), contrary to the expectation, is observed in higher frequency among the controls, suggesting probably a protective role of this allele. Further analysis was carried out to check, if this trend can be statistically validated. Logistic regression was performed considering a possible protective role for the 2 allele. Although the odds ratio (OR) for the pooled RSA sample and the controls was only marginally significant (p = 0.056), OR for the RSA women with 2–3 abortions and the controls was significant (p = 0.03), suggesting a protective role for the allele (Table 2). Figure 1. Genotype distribution of the PR mutations; (1) wild type (G1031, G1978, and C2310) and (2) mutant allele. doi:10.1371/journal.pone.0008712.g001 GINS insertion (T2/T2) among the RSA women as compared to the frequencies of 143 (95.3%), 6 (4%) and 1 (0.7%) among the controls (Figure 2). Introduction To the best of our knowledge, most of the earlier studies on the role of the PR mutations in adverse reproductive outcomes were in isolation not in conjunction with the PROGINS insertion. Further, LD between the PROGINS and the SNPs has been assumed rather than empirically tested. Thus, the earlier studies concerning the role of hPR gene in RSA have been largely inconclusive. Figure 1. Genotype distribution of the PR mutations; (1) wild type (G1031, G1978, and C2310) and (2) mutant allele. doi:10.1371/journal.pone.0008712.g001 Given that most studies hitherto undertaken were on the Caucasian populations, it is not known if ethnicity has any role in the observed pattern of association of PR polymorphisms with adverse pregnancy outcomes. There were no studies on the role of these polymorphisms vis-a`-vis RSA in the Asian populations, especially from India, which is known for its unique population structure characterized by strictly endogamous castes and tribes. In view of this, we attempt to ascertain if the 3 linked SNPs of the hPR gene and the PROGINS insertion are associated with RSA in the relatively homogenous Indian sample. We also attempt to explore if we can find any novel SNPs in hPR gene that can be implicated in RSA. PLoS ONE \| www.plosone.org Results The genotype frequencies were not found to be significantly different between RSA and control women (x2 = 1.05, df = 2, p = 0.59). Allele frequencies for the T1 and T2 alleles were observed to be 0.982 and 0.018 and 0.973 and 0.027, respectively for the RSA and control women. The difference in the allele frequencies between RSA and control women was not significant in either the pooled sample or when RSA women with different number of abortions are separately considered. Statistical power (1-b) of these results was computed using GPower 3.1. Given the large sample (2N), the power obtained was significant at 80% for pooled RSA women (286) and controls (300) and 87% for RSA women with 2–3 abortions (250) and controls (300), conferring fair degree of reliability to the findings of this study. The analyses of PROGINS Alu insertion revealed genotypic frequencies of 133 (97.1%) homozygous wild type (T1/T1), 3 (2.2%) heterozygous (T1/T2) and 1 (0.7%) homozygous PRO- PLoS ONE \| www.plosone.org January 2010 \| Volume 5 \| Issue 1 \| e8712 January 2010 \| Volume 5 \| Issue 1 \| e8712 2 Progesterone Receptor in RSA Table 1. Allele frequencies of the 1 and the 2 alleles. Allele Freq PR mutation RSA pooled (n = 286) RSA (n = 250) (2–3 abortions) RSA (n = 36) ($4 abortions) RSA (n = 166) (LFC) RSA (n = 120) (FMH) RSA (n = 260) (1u aborters) RSA (n = 26) (2u aborters) Controls (300) 1 0.941 0.948 0.889 0.952 0.925 0.942 0.923 0.897 2 0.059 0.052 0.111 0.048 0.075 0.058 0.077 0.103 Allele Frequencies of the 1 and the 2 alleles in the RSA women (pooled, 2–3 abortions and 4 or more abortions) and the controls, RSA women from LFC and FMH and in the primary (1u) and secondary (2u) aborters. RSA women and controls (x2 = 3.75, DF = 1, p = 0.05). RSA women (2–3 abortions) and controls (x2 = 4.88, DF = 1, p = 0.03). RSA women ($4 abortions) and controls (x2 = 0.02, DF = 1, p = 0.88). RSA women (LFC) and controls (x2 = 4.24, DF = 1, p = 0.04). RSA women (FMH) and controls (x2 = 0.80, DF = 1, p = 0.37). RSA women (LFC) and (FMH) (x2 = 0.90, DF = 1, p = 0.34). Results RSA women (1u) and controls (x2 = 3.85, DF = 1, p = 0.05). RSA women (2u) and controls (x2 = 0.18, DF = 1, p = 0.67). RSA women (1u) and (2u) (x2 = 0.16, DF = 1, p = 0.69). doi:10.1371/journal.pone.0008712.t001 We did not find any significant increase in the frequency of either the 2 allele or the T2 allele among RSA women in our study of the Indian population and contrary to the expectations, we find a higher frequency of the 2 allele in the controls. Therefore, our results based on a relatively larger and more homogeneous Indian sample suggest that PR gene mutations may not play a significant role in the manifestation of RSA and, instead, prompt one to surmise if the 2 allele does not really confer a protective role among the Indian populations. Our results are not in agreement with previous reports of associations between progesterone receptor mutations and adverse reproductive out- comes including unexplained infertility [15], implantation failure after IVF/ET [16] and unexplained RSA [11]. Association between PR polymorphisms and adverse pregnancy outcomes was both reported [11,15,16] and refuted [14,17,18]. The discrepancy in the results could be due to multifactorial etiology as there may be other genes acting in conjunction with the PR, each with relatively small effect. To be able to detect the relatively small contribution of PR polymorphisms one would require very large sample size which has not been the case in most of the studies. Further, the effect of progesterone receptor is minimal after 6 weeks of gestation when compared with during the process of implantation [24]. Most RSA cases that one enrolls are usually much later than 6 weeks of gestation and therefore, PR mutations may not be the right candidates. Heterogeneity in study approaches may also contribute to this inconsistency. The mutant alleles considered together at the three SNP sites (2 allele) and the PROGINS insertion (T2 allele) are reported to be in LD [11,12,13]. LD analysis was carried out using Haploview version 4.1 [19] to check if the SNPs in the coding region and the insertion polymorphism in the intron G were in LD in our sample. Results Given the distance between the SNPs in exons 5 and 1 (76kb), our haploview analysis revealed an LD block (Figure 3) consisting of only the two SNPs in exons 4 and 5 (separated by 11 kb), even though the D9 value and the correlation coefficient for all pairs of comparisons between the three SNPs (i.e., between exons 5 and 4, exons 5 and 1, exons 4 and 1) is 1. The LD analysis suggests that the exons 1, 4 and 5 do not show complete LD with the PROGINS. Discussion Progesterone receptor mediated effects play a critical role in female reproduction [5–7,20–22]. We investigated mutations of the progesterone receptor gene and observed an increased frequency of the 2 allele in the controls (10.3%) when compared to the RSA women (5.9%). The frequency of 2.7% of the T2 allele in the controls in our study is relatively close to the frequency of 5.5% among the normal women from Hyderabad [23]. In our study, 3 women in the control group were homozygous for the 2 allele, yet they had no abortions suggesting that even the homozygous mutations are not sufficient to cause RSA. Haploview analysis suggests that even though the three SNPs show a D9 of 1, the LD block consists of the SNPs in the exons 4 and 5 only. Based on our genotyping results, we find that the insertion (T2 allele) is seen only among the individuals with at least one copy of the 2 allele suggesting a possible association between the 2 allele and the T2 allele, which is in concordance with the results of Haploview analysis revealing a partial LD. pp y y To the best of our knowledge, ours is by far the largest sample as far as the studies of PR mutations in RSA are concerned. For example, the studies concerning unexplained infertility [15] and RSA [11] that yielded positive association with PR mutations were based on only 26 and 42 cases. Even the studies that yielded negative results with reference to RSA [14], recurrent implanta- tion failure [17] and preterm birth [18] were with 125, 66 and 78 cases, respectively, whereas we used 143 cases and 150 controls. Given the large sample size, the statistical power of our study should be relatively higher. The 87% of power obtained with reference to odds ratio probably bears testimony to this, conferring fair degree of reliability to the findings of our study. We strongly believe that the ethnic differences in the nature of genetic predisposition to different complex genetic disorders could also lead to inconsistency in the pattern of association in our population, as has also been reported earlier with reference to other complex disorders in the Indian populations [25]. Thus the incongruent nature of our findings in the Indian population concerning the role of PR gene mutations in RSA Vis-a-Vis the non-Indian populations adds to the ever increasing body of Table 2. Discussion Odds ratio for the 2 allele in RSA pooled samples and in the 2–3 abortions. B6S.E. p-value OR 95.0% C.I. for OR Lower Upper Pooled RSA cases Vs controls 0.60160.314 0.056 1.824 0.986 3.374 RSA (2–3 abortions) Vs controls 0.74260.342 0.030 2.101 1.074 4.109 doi:10.1371/journal.pone.0008712.t002 Table 2. Odds ratio for the 2 allele in RSA pooled samples and in the 2–3 abortions. B6S.E. p-value OR 95.0% C.I. for OR Lower Upper Pooled RSA cases Vs controls 0.60160.314 0.056 1.824 0.986 3.374 RSA (2–3 abortions) Vs controls 0.74260.342 0.030 2.101 1.074 4.109 doi:10.1371/journal.pone.0008712.t002 Table 2. Odds ratio for the 2 allele in RSA pooled samples and in the 2–3 abortions. January 2010 \| Volume 5 \| Issue 1 \| e8712 3 Progesterone Receptor in RSA evidence on the novel patterns of genetic predisposition of Indian Dydrogesterone or Progesterone helps in prevention of RSA [26– 30]), PR expression levels (reduced levels have been reported in RSA women [31–33]), its transcription, its relation with estrogen receptor (ER) and its role in immune modulation along with th gene mutations in populations of diverse ethnic and geographi backgrounds before completely ruling out the role of PR in th manifestation of RSA. On the other hand, our results also suggest a probable protective role of the 2 allele. Since this study is th first of its kind in the populations of this region, one should awai results from more populations of diverse ethnic and geographi backgrounds before making any conclusive statement on the rol of PR in RSA, protective or otherwise. Materials and Methods Samples For the present study samples were collected from a relativel Figure 3. LD plot showing the confidence bounds color schem where dark grey suggests strong evidence of LD and light grey suggests an uninformative status of LD. The analysis reveals tha exons 4 and 5 inherit as an LD Block. doi:10.1371/journal.pone.0008712.g003 Figure 2. Genotype distribution of PROGINS insertion; (T1) wild type (no insertion) and (T2) insertion allele. doi:10.1371/journal.pone.0008712.g002 Progesterone Receptor in RSA Figure 3. LD plot showing the confidence bounds color scheme where dark grey suggests strong evidence of LD and light grey suggests an uninformative status of LD. The analysis reveals that exons 4 and 5 inherit as an LD Block. doi:10.1371/journal.pone.0008712.g003 Figure 2. Genotype distribution of PROGINS insertion; (T1) wild type (no insertion) and (T2) insertion allele. doi:10.1371/journal.pone.0008712.g002 Figure 3. Discussion LD plot showing the confidence bounds color scheme where dark grey suggests strong evidence of LD and light grey suggests an uninformative status of LD. The analysis reveals that exons 4 and 5 inherit as an LD Block. doi:10.1371/journal.pone.0008712.g003 Dydrogesterone or Progesterone helps in prevention of RSA [26– 30]), PR expression levels (reduced levels have been reported in RSA women [31–33]), its transcription, its relation with estrogen receptor (ER) and its role in immune modulation along with the gene mutations in populations of diverse ethnic and geographic backgrounds before completely ruling out the role of PR in the manifestation of RSA. On the other hand, our results also suggests a probable protective role of the 2 allele. Since this study is the first of its kind in the populations of this region, one should await results from more populations of diverse ethnic and geographic backgrounds before making any conclusive statement on the role of PR in RSA, protective or otherwise. Figure 2. Genotype distribution of PROGINS insertion; (T1) wild type (no insertion) and (T2) insertion allele. doi:10.1371/journal.pone.0008712.g002 PLoS ONE \| www.plosone.org Polymerase Chain Reaction and DNA sequencing DNA was isolated from the above samples following Sambrook et al., [35] protocol. PCR amplification of all the 8 exons of PR gene was carried out as per Schweikert et al. [11] and the PROGINS Alu insertion analysis was carried as per Gomes et al., [36]. Reactions were carried out in an ABI Gene Amp PCR system 9700. Cycle Sequencing of PCR products of the 8 exons was carried out with either the forward or the reverse primers using the Big-Dye Terminator ready reaction kit (Perkin Elmer) depending on the position of the mutation and analyzed on an ABI 3730 automated DNA Analyzer (Applied Biosystems) (Figure 4). The PROGINS amplification products were loaded in a 2% agarose gel, stained with ethidium bromide (1mg/ml), and electrophoresis was carried out at 100 volts for 30 minutes, in 0.56 TAE buffer (Figure 5). Peripheral blood samples (3–5 ml) were collected in EDTA coated vacutainers from 143 women with RSA of unknown etiology. Of these 83 were from LFC while remaining 60 were from FMH. All RSA women, with the mean age of 26 yrs (range 18–37 years of age) and with number of miscarriages ranging from 2 to 9, had regular menstrual cycles and were healthy. Karyotypes were normal. The women underwent careful clinical examina- tions, as well as analysis of tissue antibodies and auto-antibodies as prescribed by their doctors. Women normal for the above tests as well as for the blood glucose and thyroid stimulating hormone concentrations were enrolled for the study. A possible infectious etiology was also ruled out by assessing the reports of the microbiological cultures of the samples obtained from the cervix and uterine cavity. A total of 130 women had no previous children (primary aborters) while 13 had one or two children before the consecutive miscarriages (secondary aborters). Our control group consisted of 150 healthy women with no history of abortions and at least one live born child. Women in the control group were aged Samples For the present study, samples were collected from a relatively homogenous Telugu population from Andhra Pradesh, India. Women with RSA were recruited from two different hospitals - Lakshmi Fertility Clinic (LFC) in the suburban Nellore town and Fernandez Maternity Hospital (FMH) in metropolitan Hyderabad. These two hospitals not only represent two different socio economic strata of the patients, but are also separated geograph- ically by about 500kms. Control samples were collected from Hyderabad, Nellore and nearby villages so that they broadly represent matched ethnic and socio-economic backgrounds with evidence on the novel patterns of genetic predisposition of Indian populations to different complex diseases, underlining the importance of unique Indian population genetic structure. Despite the crucial role that progesterone plays in the maintenance of the pregnancy the genetic analysis of PR gene did not provide formidable evidence towards its association with RSA, either in the western or Indian populations so as to attach any prognostic value for RSA. However, since progesterone is essential for the development of a receptive endometrium, it is necessary to also consider: progesterone levels (as treatment with PLoS ONE \| www.plosone.org January 2010 \| Volume 5 \| Issue 1 \| e8712 January 2010 \| Volume 5 \| Issue 1 \| e8712 4 Progesterone Receptor in RSA Figure 4. Electropherograms of the two SNPs of hPR gene in exon 1 (G1031C) and exon 4 (G1978T). doi:10.1371/journal.pone.0008712.g004 Figure 4. Electropherograms of the two SNPs of hPR gene in exon 1 (G1031C) and exon 4 (G1978T). doi:10.1371/journal.pone.0008712.g004 between 18–45 years. Blood samples from the cases as well as the controls were collected with written informed consent and we had obtained approval for this study from the Indian Statistical Institute Review Committee for Protection of Research Risks to Humans. that of the cases. This framework facilitates to test if the results are consistent between the socioeconomically contrasting but genet- ically somewhat homogenous samples; screening a number of autosomal STR markers from 27 Telugu populations (castes and tribes) from different socioeconomic strata, Reddy et al [34] observed fair degree of homogeneity across the socioeconomic strata of the populations which form a compact cluster when compared to the populations of the other regions and linguistic groups. that of the cases. Samples This framework facilitates to test if the results are consistent between the socioeconomically contrasting but genet- ically somewhat homogenous samples; screening a number of autosomal STR markers from 27 Telugu populations (castes and tribes) from different socioeconomic strata, Reddy et al [34] observed fair degree of homogeneity across the socioeconomic strata of the populations which form a compact cluster when compared to the populations of the other regions and linguistic groups. Statistical analysis Allele frequencies were calculated by gene counting method for the case and control samples, women with #3 and $4 abortions, RSA women from LFC and FMH and primary and secondary aborters Figure 5. Gel picture showing bands for homozygotes (T1) allele, heterozygotes (T1/T2), and homozygote (T2) allele run on 2% agarose. Lane 3, 4, 5, and 6 are heterozygotes, and lane 13 is homozygous for the T2 allele and the remaining are homozygous for the T1 allele. doi:10.1371/journal.pone.0008712.g005 References 19. Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics;PubMed ID: 15297300]. 1. Reiss HE (1998) Reproductive Medicine: From A to Z. Oxford: Oxford University Press. y 2. Lanasa MC, Hogge WA (2000) X chromosome defects as an etiology of recurrent spontaneous abortion. Semin Reprod Med 18: 97–103. 2. Lanasa MC, Hogge WA (2000) X chromosome defects as a 20. Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240: 889–895. 3. Beever CL, Stephenson MD, Penaherrera MS, Jiang RH, Kalousek, et al. (2003) Skewed X-chromosome inactivation is associated with trisomy in women ascertained on the basis of recurrent spontaneous abortion or chromosomally abnormal pregnancies. Am J Hum Genet 72: 399–407. 21. Carson-Jurica MA, Schrader WT, O’Malley BW (1990) Steroid receptor family: structure and functions. Endocr Rev 11(2): 201–220. abnormal pregnancies. Am J Hum Genet 72: 399–407. 22. Bouchard P (1999) Progesterone and the progesterone receptor. J Reprod Med 44: 153–157. 4. Clarke CL, Sutherland RL (1990) Progestin regulation of cellular proliferation. Endocr Rev 11(2): 266–301. Endocr Rev 11(2): 266–301. 23. Govindan S, Ahmed SN, Vedicherla B, Kodati V, Jahen P, et al. (2007) Association of Progesterone gene polymorphism (PROGINS) with endometri- osis, uterine fibroids and breast cancer. Cancer Biomark 3: 73–78. 5. Pepe GJ, Albrecht ED (1995) Actions of placental and fetal adrenal steroid hormones in primate pregnancy. Endocr Rev 16(5): 608–648. 6. Graham JD, Clarke CL (1997) Physiological action of progesterone in target tissues. Endocr Rev 18(4): 502–519. 24. Fernandez-Valdivia R, Mukherjee A, Mulac-Jericevic B, Connely OM, De Mayo FJ, et al. (2005) Revealing progesterone’s role in uterine and mammary gland biology: insights from the mouse. Semin Reprod Med 23: 22–37. 7. Szekeres-Bartho J, Barakonyi A, Miko E, Polgar B, Palkovics T (2001) The role of gamma/delta T cells in the feto-maternal relationship. Semin Immunol 13: 229–233. 25. Dhandapany PS, Sadayappan S, Xue Y, Powell GT, Rani DS, et al. (2009) A common Cardiac Myosin Binding Protein C variant associated with cardiomy- opathies in South Asia. Nat Genet 41: 187–191. 8. Rousseau-Merck MF, Misrahi M, Loosfelt H, Milgrom E, Berger R (1987) Localization of the human progesterone receptor gene to chromosome 11q22– q23. Hum Genet 77: 280–282. 26. Chakravarty BN, Shirazee HH, Dam P, Goswami SK, Chatterjee R, et al. References (2005) Oral dydrogesterone versus intravaginal micronised progesterone as luteal phase support in assisted reproductive technology (ART) cycles: Results of a randomised study. J Ster Biochem & Mol Biol 97: 416–420. q 9. Misrahi M, Venencie PY, Saugier-Veber P, Sar S, Dessen P, et al. (1993) Structure of the human progesterone receptor gene. Biochim Biophys Acta 1216(2): 289–292. 27. El-Zibdeh MY (2005) Dydrogesterone in the reduction of recurrent spontaneous abortion. J Ster Biochem & Mol Biol 97: 431–434. ( ) 10. Misrahi M, Atger M, Lucd’Auriol, Loosfelt H, Meriel C, et al. (1987) Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA. Biochem Bioph Res Co 143(2): 740–748. 28. Gruber CJ, Huber JC (2005) The role of dydrogesterone in recurrent (habitual) abortion. J Ster Biochem & Mol Biol 97: 426–430. abortion. J Ster Biochem & Mol Biol 97: 426–430. 11. Schweikert A, Rau T, Berkholz A, Allera A, Daufeldt S, et al. (2004) Association of progesterone receptor polymorphism with recurrent abortions. Eur J Obstet Gynecol Reprod Biol 113: 67–72. 29. Omar MH, Mashita MK, Lim PS, Jamil MA (2005) Dydrogesterone in threatened abortion: Pregnancy outcome. J Ster Biochem & Mol Biol 97: 421–425. 12. Kieback DG, Tong X-W, Weigel NL, Agoulnick IU (1998) A genetic mutation in the progesterone receptor (PROGINS) leads to an increased risk of non- familial breast and ovarian cancer causing inadequate control of estrogen driven proliferation. J Soc Gynecol Invest 5: 40. 30. Szekeres-Bartho J, Balasch J (2008) Progestagen therapy for recurrent miscarriage. Hum Reprod Update 14(1): 27–35. 31. Lira S, Blanquet J, Tserotas K, Calzada L (2000) Endometrial progesterone and estradiol receptors in patients with recurrent early pregnancy loss of unknown etiology - preliminary report. Med Sci Monit 6(4): 759–762. p J y 13. Tong D, Fabjani G, Heinze G, Obermair A, Leodolter S, et al. (2001) Analysis of the human progesterone receptor gene polymorphism progins in Austrian ovarian carcinoma patients. Int J Cancer 95(6): 394–397. 32. Hickman TN, Shih LM, Zacur HA, Kurman RJ, Diener-west M, et al. (2002) Decreased progesterone receptor expression in the intermediate trophoblastic cells of spontaneous abortions. Fertil and Steril 77(5): 1001–1005. p J ( ) 14. Kurz C, Tempfer CB, Boecskoer S, Unfried G, Nagele F, et al. (2001) The PROGINS Progesterone Receptor Gene Polymorphism and Idiopathic Recurrent Miscarriage. J Soc Gynecol Invest 8: 295–298. 33. Figure 5. Gel picture showing bands for homozygotes (T1) allele, heterozygotes (T1/T2), and homozygote (T2) allele run on 2% agarose. Lane 3, 4, 5, and 6 are heterozygotes, and lane 13 is homozygous for the T2 allele and the remaining are homozygous for the T1 allele. doi:10.1371/journal.pone.0008712.g005 January 2010 \| Volume 5 \| Issue 1 \| e8712 January 2010 \| Volume 5 \| Issue 1 \| e8712 PLoS ONE \| www.plosone.org 5 Progesterone Receptor in RSA separately. x2 analysis and logistic regression were carried out using SPSS. GPower was employed to detect the power of the study. Linkage disequilibrium estimates, for the three SNP loci and the PROGINS insertion, were calculated using Haploview 4.1 software. Author Contributions Conceived and designed the experiments: MA BMR. Performed the experiments: MA TN PVSS. Analyzed the data: MA BMR. Contributed Conceived and designed the experiments: MA BMR. Performed the experiments: MA TN PVSS. Analyzed the data: MA BMR. Contributed Conceived and designed the experiments: MA BMR. Performed the experiments: MA TN PVSS. Analyzed the data: MA BMR. Contributed reagents/materials/analysis tools: SA ST AGR KT LS BMR. Wrote the paper: MA BMR. Participated in the discussion and approved the final manuscript: MA, TN, SA, ST, PVSS, AGR, KT, LS, BMR. Conceived and designed the experiments: MA BMR. Performed the experiments: MA TN PVSS. Analyzed the data: MA BMR. Contributed reagents/materials/analysis tools: SA ST AGR KT LS BMR. Wrote the paper: MA BMR. Participated in the discussion and approved the final manuscript: MA, TN, SA, ST, PVSS, AGR, KT, LS, BMR. reagents/materials/analysis tools: SA ST AGR KT LS BMR. Wrote the paper: MA BMR. Participated in the discussion and approved the final manuscript: MA, TN, SA, ST, PVSS, AGR, KT, LS, BMR. References Babalioglu R, Varol FG, Ilhan R, Yalcin O, Cizmecioglu F (1996) Progesterone profiles in luteal-phase defects associated with recurrent spontaneous abortions. J Assist Reprod Genet 13: 306–309. g J y 15. Pisarska MD, Carson SA, Casson PR, Tong X, Buster JE, et al. (2003) A mutated progesterone receptor allele is more prevalent in unexplained infertility. Fertil Steril 80: 651–653. 34. Reddy BM, Naidu VM, Madhavi VK, Thangaraj K, Kumar V, et al. (2005) Microsatellite diversity in Andhra Pradesh, India: Genetic stratification versus social stratification. Hum Biol 77: 803–823. 16. Cramer DW, Hornstein MD, McShane P, Powers RD, Lescault PJ, et al. (2003) Human progesterone receptor polymorphisms and implantation failure during in vitro fertilization. Am J Obstet Gynecol 189: 1085–1092. 35. Sambrook J, Fitsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Press. 17. Coulam CB, Jeyendran RS, Roussev R (2008) Association of progesterone receptor polymorphisms with recurrent implantation failure after in vitro fertilization and embryo transfer. J Assist Reprod Genet 25: 119–122. 36. Gomes MTV, Castro RA, Villanova FE, da Silva IDCG, Baracat EC, et al. (2007) The progesterone receptor gene polymorphism, PROGINS, may be a factor related to the development of uterine fibroids. Fertil Steril 87(5): 1116–1121. y J p 18. Luo G, Morgan T, Bahtiyar MO, Snegovskikh VV, Schatz F, et al. (2008) Single Nucleotide Polymorphisms in the Human Progesterone Receptor Gene and Spontaneous Preterm Birth. Reprod Sci 15(2): 147–155. 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https://openalex.org/W2910582897	https://zenodo.org/records/2549000/files/PK_article_30755.pdf	English	null	A remarkable new species of Pamianthe (Amaryllidaceae) from the Department of Cauca, Colombia	PhytoKeys	2,019	cc-by	3,861	Alan W. Meerow1, Philip A. Silverstone-Sopkin2, Alejandro Zuluaga-Tróchez2, Jhon A. Sánchez-Taborda3 1 USDA-ARS-SHRS, National Germplasm Repository, 13601 Old Cutler Road, Miami, Florida 33158, USA 2 Departamento de Biología, Universidad del Valle, Calle 13 # 100-00, Cali, Valle, Colombia 3 Fundación Ecohábitats, Calle 78N # 19-157, Popayán, Cauca, Colombia 1 USDA-ARS-SHRS, National Germplasm Repository, 13601 Old Cutler Road, Miami, Florida 33158, USA 2 Departamento de Biología, Universidad del Valle, Calle 13 # 100-00, Cali, Valle, Colombia 3 Fundación Ecohábitats, Calle 78N # 19-157, Popayán, Cauca, Colombia Corresponding author: Alan W. Meerow (alan.meerow@ars.usda.gov) Academic editor: L. Peruzzi \| Received 22 October 2018 \| Accepted 4 December 2018 \| Published 17 Janu Citation: Meerow AW, Silverstone-Sopkin PA, Zuluaga-Tróchez A, Sánchez-Taborda JA (2019) A remarkable new species of Pamianthe (Amaryllidaceae) from the Department of Cauca, Colombia. PhytoKeys 115: 73–82. https://doi. org/10.3897/phytokeys.115.30755 Citation: Meerow AW, Silverstone-Sopkin PA, Zuluaga-Tróchez A, Sánchez-Taborda JA (2019) A remarkable new species of Pamianthe (Amaryllidaceae) from the Department of Cauca, Colombia. PhytoKeys 115: 73–82. https://doi. org/10.3897/phytokeys.115.30755 Abstract A new saxicolous species of Amaryllidaceae tentatively assigned to the tribe Clinantheae, Pamianthe ecollis Silverst., Meerow & Sánchez-Taborda, is described from the western slope of the Cordillera Occidental in the department of Cauca, Colombia. The new species differs from the two hitherto known species of Pamianthe in its yellow flowers and in its nearly obsolete perianth tube. The near loss of the perianth tube may be correlated with a change in pollinator. The new species lacks a bulb; it produces a large number of winged seeds that are wind-dispersed. A key to the species of Pamianthe is provided. This is the first record of the genus Pamianthe for Colombia. The phylogenetic position of the genus Pamianthe is discussed. Keywords Amaryllidaceae, biodiversity, Cauca, Clinantheae, Colombia, Pamianthe, Andes PhytoKeys 115: 73–82 (2019) doi: 10.3897/phytokeys.115.30755 http://phytokeys.pensoft.net PhytoKeys 115: 73–82 (2019) doi: 10.3897/phytokeys.115.30755 http://phytokeys.pensoft.net PhytoKeys 115: 73–82 (2019) doi: 10.3897/phytokeys.115.30755 http://phytokeys.pensoft.net ble new species of Pa RESEARCH ARTICLE Copyright Alan W. Meerow et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Resumen Se describe una nueva especie de Amaryllidaceae tentativamente perteneciente a la tribu Clinantheae, Pamianthe ecollis Silverst., Meerow & Sánchez-Taborda, procedente de la vertiente occidental de la cor dillera Occidental en el departamento del Cauca, Colombia. La nueva especie difiere de las dos especies conocidas de Pamianthe por su perianto amarillo que tiene un tubo casi ausente. La reducción del tubo del Copyright Alan W. Meerow et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) 74 perianto probablemente tiene correlación con un cambio en el polinizador. La nueva especie carece de un bulbo, y produce numerosas semillas aladas que se dispersan por el viento. Se provee una clave a las espe cies de Pamianthe. Este es el primer registro del género Pamianthe para Colombia. Se discute la posición filogenética del género Pamianthe. Introduction Amaryllidaceae J. St.-Hil. is a cosmopolitan family represented in Colombia by nine native genera and 26 native species, including a monotypic endemic genus, Plagiolirion Baker (Meerow and Silverstone-Sopkin 1995). Some of the Colombian species have restricted ranges and are in danger of extinction or may already be extinct (Silverstone- Sopkin 2011). Recent field work in the Cordillera Occidental of the Andes, in the de partment of Cauca, has resulted in the discovery of a new species of Amaryllidaceae that also seems to be narrowly distributed. Vegetative and floral morphology and nrDNA ITS sequences indicate that this species represents a novelty in the genus Pamianthe Stapf. Stapf (1933a, 1933b) published the genus Pamianthe in honor of Major Albert Pam, who cultivated bulbs in England that he received from Peru in 1928. There are five published species names that have been assigned to this genus: P. andreana (Baker) Stapf, P. cardenasii Traub, P. parviflora Meerow, P. peruviana Stapf, and P. quitoensis (Herb.) Stapf. Pamianthe quitoensis was transferred to the genus Leptochiton Sealy, as L. quitoensis (Herb.) Sealy, and P. andreana is considered a synonym of this species. Pamianthe cardenasii has been placed in the synonymy of P. peruviana (Meerow 1984). Thus, the genus Pamianthe, as previously recognized, includes only two species, P. parviflora, known only from Ecuador (Meerow 1984), and P. peruviana (the type spe cies), known from Perú and Bolivia. The new species described in this paper is the third species of the genus and the first record from Colombia. It is also the first species of the tribe Clinantheae, to which Pamianthe has been assigned (Meerow et al. 2000; Leiva and Meerow 2016), discovered north of Ecuador. Results Taxonomic treatment Pamianthe ecollis Silverst., Meerow & Sánchez-Taborda, sp. nov. urn:lsid:ipni.org:names:77193890-1 Figs 1, 2 Diagnosis. This species differs from both Pamianthe parviflora Meerow and P. peru viana Stapf in having a yellow perianth and staminal cup (versus white) and in nearly lacking a perianth tube. Additionally, it differs from P. parviflora in having shorter pedicels, a longer ovary, and more numerous ovules, and from P. peruviana in having much longer pedicels, more flowers per umbel, much shorter tepals, a shorter staminal cup that is not exserted from the perianth, and a smaller fruit. p p Type. COLOMBIA. Cauca: Municipio Argelia, road between Nuevo Horizonte and La Montaña, north of the Serranía El Pinche, Cordillera Occidental, western slope (Fig. 3), 2839 m, 4 Feb 2018, J. A. Sánchez-Taborda 2870 (holotype: CUVC 67719!, 67720!, mounted on two sheets; isotype: CAUP). GPS coordinates are withheld to discourage poaching; they are available to bonafide researchers upon request. Type. COLOMBIA. Cauca: Municipio Argelia, road between Nuevo Horizonte and La Montaña, north of the Serranía El Pinche, Cordillera Occidental, western slope (Fig. 3), 2839 m, 4 Feb 2018, J. A. Sánchez-Taborda 2870 (holotype: CUVC 67719!, 67720!, mounted on two sheets; isotype: CAUP). GPS coordinates are withheld to discourage poaching; they are available to bonafide researchers upon request. g p gi p q Description. Terrestrial saxicolous herbs (Fig. 1A); bulb absent, roots emerge from base of pseudostem, and are thick, possibly with a velamen layer (Fig. 1B). Leaves (Fig. 1C) sessile, attached alternately to an elongate pseudostem; lamina lo rate, 82.7–104.5 × 5.5–6.3 cm, margin entire, glabrous, narrowing distally (but not acuminate), apex acute, with a conspicuous midrib. Scape cylindrical, 45–46 cm long; intact bracts not seen (bracts withered and damaged in dried specimens); in florescence pseudoumbellate, flowers oriented at right angles from apex of pedicels. Flowers (Fig. 1D–F) 9–10, of which 3–4 are at anthesis simultaneously; pedicels in flowers at anthesis 7–9 cm long; perianth tube nearly obsolete (ca. 1.8 mm long); limb crateriform, ca. 3.3 cm in diam; tepals 6, yellow, glabrous; outer tepals with green tips and very narrow green abaxial mid-longitudinal stripe, valvate, ellipti cal, ca. 3.2 × 1.4–1.5 cm, apiculate, apex thickened, ca. 2.3 mm long, with salient adaxial apiculum (Fig. 2B) ca. 1.3 × 1.4 mm, which is densely glandular-papillate (Fig. 2C); inner tepals imbricate at base, ovate, broader than outer tepals, ca. Methods Photographs of the flower in alcohol and seeds of Pamianthe ecollis were taken with a Nikon model DS-Ri1U3 digital camera, using a Nikon model SMZ-1500 stereo dis secting microscope at the Laboratorio de Imágenes del Postgrado en Ciencias-Biología de la Universidad del Valle; floral and seed measurements were made with NIS Ele ments Br, version 4.20 software. DNA extraction, amplification and sequencing protocols were as described in Mee row et al. (2000, 2006). The ITS sequence of P. ecollis was aligned with a previous ITS alignment of the tribe Clinantheae (Meerow et al. 2000; Meerow 2010) using the program MAFFT (Katoh and Standley 2013). A branch and bound parsimony analysis was run us ing PAUP v. 4.10 (Swofford 2002), followed by generation of Jackknife support percent ages. The ITS sequence of P. ecollis is deposited in GenBank (Genbank Acc. MH979036). 75 A remarkable new species of Pamianthe... Results 2.8 × 2.1 cm, apex rounded, thickened and papillate on adaxial surface, but not apiculate and lacking adaxial protuberance. Stamens 6, basally connate into immaculate yel low staminal cup attached to the adaxial base of inner tepals (Fig. 2A), ca. 5 mm long (measured from base to tip of tooth), not exserted, with 2 deltoid to rounded teeth between each 2 free filaments; free filaments yellow, ca. 5 mm long, attached to border of staminal cup, included, strongly incurved; anthers grouped in center of flower (but not connivent), brown with yellow borders, ca. 7.1 mm long, linear, dorsifixed, versatile, longitudinally dehiscent; pollen yellow. Style (in the only flower preserved in ethanol) apparently immature (flower protandrous), curved, ca. 10 mm long, included (hidden below the grouped anthers), stigma 3-lobed, lobes papillate; 76 Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) Figure 1. Pamianthe ecollis. A Pamianthe ecollis growing in its native habitat, on a steep, rocky bank B Base of plant C Habit D Inflorescence E Flower, lateral view F Flower, front view A photo by Fredy Gómez-Ortiz B photo by Laura Clavijo C–F type collection, photographs taken in the field by Jhon A. Sánchez-Taborda. Figure 1. Pamianthe ecollis. A Pamianthe ecollis growing in its native habitat, on a steep, rocky bank B Base of plant C Habit D Inflorescence E Flower, lateral view F Flower, front view A photo by Fredy Gómez-Ortiz B photo by Laura Clavijo C–F type collection photographs taken in the field by Jhon A Sánchez Taborda Figure 1. Pamianthe ecollis. A Pamianthe ecollis growing in its native habitat, on a steep, rocky bank B Base of plant C Habit D Inflorescence E Flower, lateral view F Flower, front view A photo by Fredy Gómez-Ortiz B photo by Laura Clavijo C–F type collection, photographs taken in the field by Jhon A. Sánchez-Taborda. B ovary green, 3-angled, oblong, ca. 40 × 9 mm, 3-loculed, placentation axile, ovules oblong, ca. 1.6 × 0.5 mm, ca. 100 per locule (Fig. 2D), biseriate, ovules of each row alternating with those of the other row. Fruit (Fig. 2E): unopened fruit not available for measurement; dehiscent fruit 3-valved, valves broad-elliptic to obovate, base ob tuse, apex short-beaked, dry, smooth, glabrous, ca. 38 × 29 mm. Seeds (Fig. Results 2E, F) as many as 233 in one capsule, alate, glabrous, seed body dark brown, wing light brown, flat, thin, membranous, shape of entire seed (including wing) narrowly to broadly falcate, (12–) 15–18 × 5–9 mm. A remarkable new species of Pamianthe... 77 p Figure 2. Pamianthe ecollis. A Androecium, with staminal cup B Tip of outer tepal, showing apex and adaxial protuberance C Adaxial protuberance, showing glandular papillae D Opened ovary with ovules (ovules in two locules are visible) E Infructescence of living plant F Seeds, showing variation in shape A–D, F photographs by Juan Felipe Ortega-Giraldo, Laboratorio de Imágenes del Postgrado en Ciencias- Biología, Universidad del Valle, Cali, Colombia E photo by Laura Clavijo. Figure 2. Pamianthe ecollis. A Androecium, with staminal cup B Tip of outer tepal, showing apex and adaxial protuberance C Adaxial protuberance, showing glandular papillae D Opened ovary with ovules (ovules in two locules are visible) E Infructescence of living plant F Seeds, showing variation in shape A–D, F photographs by Juan Felipe Ortega-Giraldo, Laboratorio de Imágenes del Postgrado en Ciencias- Biología, Universidad del Valle, Cali, Colombia E photo by Laura Clavijo. Figure 2. Pamianthe ecollis. A Androecium, with staminal cup B Tip of outer tepal, showing apex and adaxial protuberance C Adaxial protuberance, showing glandular papillae D Opened ovary with ovules (ovules in two locules are visible) E Infructescence of living plant F Seeds, showing variation in shape A–D, F photographs by Juan Felipe Ortega-Giraldo, Laboratorio de Imágenes del Postgrado en Ciencias- Biología, Universidad del Valle, Cali, Colombia E photo by Laura Clavijo. Distribution and ecology. Pamianthe ecollis is known only from the type locality (Fig. 3). The general habitat is cloud forest. The forest at this site includes the follow ing genera: trees: Clusia L., Hedyosmum Sw., Ocotea Aubl.; shrubs: Miconia Ruiz & Pav., Palicourea Aubl.; herbs: Anthurium Schott, Besleria L., Kohleria Regel, Peperomia 78 Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) Figure 3. Map of Colombia showing the distribution of Pamianthe ecollis (black circle). Figure 3. Map of Colombia showing the distribution of Pamianthe ecollis (black circle). Ruiz & Pav., and Sphaeradenia Harling. Epiphytes were predominantly bromeliads and orchids. The new species is common at this site (Fredy Gómez-Ortiz pers. com.). However, this species does not grow within closed forest. The seeds of P. Discussion A strict consensus tree cladogram (Fig. 4) based on ITS sequences of the tribe Cli nantheae places the new species of Pamianthe as sister to P. peruviana with 92% jack knife support, in a subclade that is sister to a second subclade comprising Clinanthus Herb. and Paramongaia Velarde. However, with ITS there is no support for Pamianthe as part of Clinantheae (jackknife support = 42%; Fig. 4). Preliminary super matrix trees from sequence capture with anchored bait enrichment (Meerow, unpublished data) suggest that Pamianthe is in fact sister to the tribes Clinantheae, Eucharideae, and Hymenocallideae, rather than the first branch in Clinantheae. yi Pamianthe ecollis resembles the two other species of Pamianthe in its staminal cup morphology, with the free portion of the staminal filaments attached to the rim of the cup (not below the rim), two lobes or teeth between each two staminal filaments, and the staminal filaments strongly curved inward, as well as numerous, biseriate, winged, wind-dispersed seeds. Leaf width and the conspicuous midvein are similar to that of P. peruviana. It differs from both of the two hitherto known species in having a yel low perianth and staminal cup (versus white in the other two species) and in its nearly obsolete perianth tube. Moreover, P. parviflora has a shorter ovary (10 mm versus 40 mm in P. ecollis) and fewer ovules per locule (about 20 versus about 100 in P. ecollis). Pamianthe peruviana additionally differs in having fewer flowers (2–4, usually 2, versus 9–10 in P. ecollis), shorter pedicels (1.5–3 cm long versus 7–9 cm long in P. ecollis), free tepals much longer (outer tepals 10–12 cm long, inner tepals 9–11 cm long, versus 3.2 and 2.8 cm long in P. ecollis), staminal cup 8 cm long and long-exserted (versus ca. 0.5 cm long and included in P. ecollis), and larger fruit (8 cm long, 5 cm wide, versus 3.8 cm long, 2.9 cm wide in P. ecollis). The elongate (12–25 cm long) perianth tube in P. peruviana, which contains three nectar-bearing internal channels (Traub 1972), may be correlated with pollination by sphingid moths. The nearly obsolete perianth tube of P. ecollis may be associated with a change in pollinators; in a tubeless perianth, nectar would be available to short- tongued insects, such as bees. No flower visitors have been observed.h The glandular papillae (Fig. A remarkable new species of Pamianthe... 79 Results ecollis, which are adapted for anemochory, and a photograph of the population at the type locality (Fig. 1A), indicate that this species inhabits open areas on steep banks near creeks. Plants from the type collection were growing near a waterfall. Plants from a later col lection, from which herbarium specimens were not prepared, were growing on an apparently disturbed, open slope on rocky substrate. The roots of the plants are super ficial, immersed in a thick layer of moss, and grasp the surface of the rock. Thus, this species is a lithophyte. Ruiz & Pav., and Sphaeradenia Harling. Epiphytes were predominantly bromeliads and orchids. The new species is common at this site (Fredy Gómez-Ortiz pers. com.). However, this species does not grow within closed forest. The seeds of P. ecollis, which are adapted for anemochory, and a photograph of the population at the type locality (Fig. 1A), indicate that this species inhabits open areas on steep banks near creeks. Plants from the type collection were growing near a waterfall. Plants from a later col lection, from which herbarium specimens were not prepared, were growing on an apparently disturbed, open slope on rocky substrate. The roots of the plants are super ficial, immersed in a thick layer of moss, and grasp the surface of the rock. Thus, this species is a lithophyte. p p y Phenology. Plants were collected in flower in February and in fruit in August.hi gl g Etymology. The specific epithet is from Latin, e (without), collum (neck), adjecti val form collis, referring to the almost absent perianth tube of this species. Preliminary conservation status. Since nothing is known of the distribution of this species apart from the type locality, it is best to place it in the category Data Defi cient (IUCN 2012, 2017). Preliminary conservation status. Since nothing is known of the distribution of this species apart from the type locality, it is best to place it in the category Data Defi cient (IUCN 2012, 2017). A remarkable new species of Pamianthe... Key to the species of the genus Pamianthe Key to the species of the genus Pamianthe 1 Perianth and staminal cup yellow, perianth tube nearly obsolete.................... ............................Pamianthe ecollis Silverst., Meerow & Sánchez-Taborda – Perianth and staminal cup white, perianth with a well-developed tube.........2 2 Pedicels 5–6 cm long; perianth tube less than 2 cm long; outer tepals less than 3 cm long; staminal cup less than 2 cm long..... Pamianthe parviflora Meerow – Pedicels 1.5–3 cm long; perianth tube more than 11 cm long; outer tepals more than 8 cm long; staminal cup more than 7 cm long.............................. ......................................................................... Pamianthe peruviana Stapf Discussion 2C) on the adaxial protuberance of the outer tepals apparently have a secretory function. They probably play a role in pollinator attrac tion; they may produce a substance that is gathered by insect visitors, or they may function as osmophores. Possible osmophores have been reported in the Chilean al lioid amaryllid Gilliesia Lindl. (Rudall et al. 2002). The flat, alate seeds are most likely wind-dispersed, suggesting that these plants inhabit open areas within the cloud forest vegetation; seeds of Amaryllidaceae of closed lowland tropical forest, such as Eucharis Planch. & Lind., are relatively few per locule, subglobose, and wingless, and probably are bird-dispersed, and in one case possibly water-dispersed (Silverstone-Sopkin 2011). The Clinantheae, which is sister to the tribe Hymenocallideae (Meerow et al. 2000), was not previously known to extend to Colombia. We hypothesize that the three rare species of Pamianthe may represent the remnants of a once more broadly dis tributed epiphytic and lithophytic lineage in the tribe that were isolated as the Andes Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) 80 Figure 4. Strict branch and bound parsimony consensus tree of the Clinantheae, based on ITS sequences with jackknife support values. Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) 80 Figure 4. Strict branch and bound parsimony consensus tree of the Clinantheae, based on ITS sequences, with jackknife support values. rose to their present position, and moist forests contracted on the western slopes. We are confident that rigorous analysis of our next generation sequence data will success fully resolve the current ambiguous phylogenetic position of the genus. rose to their present position, and moist forests contracted on the western slopes. We are confident that rigorous analysis of our next generation sequence data will success fully resolve the current ambiguous phylogenetic position of the genus. Acknowledgments Field work by Jhon A. Sánchez-Taborda was financed by the Fondo de Alianzas para los Ecosistemas Críticos (CEPF), Conservación Internacional, Fundación Ecohábi tats, and the Corporación Autónoma Regional del Cauca (CRC). We thank the A remarkable new species of Pamianthe... 81 Laboratorio de Imágenes del Postgrado en Ciencias-Biología de la Universidad del Valle (Cali, Colombia) for permission to photograph the flower and seeds of the new species; photographs in this lab were taken by Juan Felipe Ortega-Giraldo. Martín Llano-Almario arranged Figures 1 and 2. Fredy Gómez-Ortiz collected a plant in fruit at the type locality. Laura Clavijo photographed the fruiting plant. Thanks to Liliana Paz y Luis Alfonso Ortega from the Fundación Ecohabitats for their work in designing and managing the project that allowed the collection of this new species, and to Asociación Agroambiental Santa Clara-El Pinche for their support during the field work. References IUCN (2012) IUCN Red List categories and criteria: Version 3.1, ed. 2. IUCN Species Sur vival Commission, Gland, Switzerland and Cambridge, UK. IUCN (2017) Guidelines for using the IUCN red list categories and criteria. Version 13. Pre pared by the Standards and Petitions Subcommittee. https://www.iucnredlist.org/resourc es/redlistguidelines [Accessed 3 December 2018] Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Im provements in performance and usability. Molecular Biology and Evolution 30(4): 772– 780. https://doi.org/10.1093/molbev/mst010 Leiva S, Meerow AW (2016) A new species of Clinanthus from northern Peru (Asparagales, Amaryllidaceae, Amarylloideae, Clinantheae). PhytoKeys 63: 99–106. https://doi. org/10.3897/phytokeys.63.8895 Meerow AW (1984) Two new species of pancratioid Amaryllidaceae from Peru and Ecuador. Brittonia 36(1): 18–25. https://doi.org/10.2307/2806286 Meerow AW (2010) Convergence or reticulation? Mosaic evolution in the canalized American Amaryllidaceae. In: Seberg O, Petersen G, Barfod AS, Davis JI (Eds) Diversity, Phylogeny and Evolution in the Monocotyledons, Aarhus University Press, Aarhus, 145–168. Meerow AW, Silverstone-Sopkin PA (1995) The rediscovery of Plagiolirion horsmannii Baker (Amaryllidaceae). Brittonia 4(4): 426–431. https://doi.org/10.2307/2807573 Meerow AW, Guy CL, Li Q-B, Yang S-L (2000) Phylogeny of the American Amaryllidace ae based on nrDNA ITS sequences. Systematic Botany 25(4): 708–726. https://doi. org/10.2307/2666729 Meerow AW, Francisco-Ortega J, Kuhn DN, Schnell RJ (2006) Phylogenetic relationships and biogeography within the Eurasian clade of Amaryllidaceae based on plastid ndhF and nrD NA ITS sequences: Lineage sorting in a reticulate area? Systematic Botany 31(1): 42–60. https://doi.org/10.1600/036364406775971787 Rudall PJ, Bateman RM, Fay MF, Eastman A (2002) Floral anatomy and systematics of Alliace ae with particular reference to Gilliesia, a presumed insect mimic with strongly zygomor phic flowers. American Journal of Botany 89(12): 1867–1883. https://doi.org/10.3732/ ajb.89.12.1867 Alan W. Meerow et al. / PhytoKeys 115: 73–82 (2019) 82 Silverstone-Sopkin PA (2011) Los muertos vivientes, la historia natural de cuatro lirios amazónicos del suroccidente de Colombia (Eucharis y Plagiolirion, Amaryllidaceae). Pro grama Editorial, Universidad del Valle, Cali, Colombia, 98 pp. Stapf O (1933a) Pamianthe peruviana. The Gardeners´ Chronicle and Agricultural Gazette ser. 3(93): 106. tapf O (1933b) Pamianthe peruviana. Curtis´s Botanical Magazine 156, tab. 9315: 1–4.f Stapf O (1933b) Pamianthe peruviana. Curtis´s Botanical Magazine 156, tab. 9315: 1–4. Swofford DL (2002) PAUP* Phylogenetic analysis using parsimony (and other methods), v. Stapf O (1933b) Pamianthe peruviana. Curtis s Botanical Magazine 156, tab. 9315: 1 4. Swofford DL (2002) PAUP Phylogenetic analysis using parsimony (and other methods), v. Swofford DL (2002) PAUP Phylogenetic analysis using parsimony (and other methods), v. References 4 0 b 10 Si A i S d l d MA d DL (2002) PAUP Phylogenetic analysis using parsimony (*and other methods), v. 4.0 beta 10. Sinauer Associates, Sunderland, MA. Traub HP (1972) Pamianthe cardenasii. Plant Life 28: 46.
https://openalex.org/W2300034374	https://jyx.jyu.fi/bitstream/123456789/72205/1/93471artikkelin%20teksti15404911020200508.pdf	Finnish	null	Nomadeja ja aktiivisia kansalaisia	Aikuiskasvatus	2,003	public-domain	654	Year: 2003 Rights url: http://rightsstatements.org/page/InC/1.0/?language=en This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Heikkinen, Anja Title: Nomadeja ja aktiiveja kansalaisia. Pääkirjoitus, Aikuiskasvatus 2/2003 Please cite the original version: Heikkinen, A. (2003). Nomadeja ja aktiiveja kansalaisia. Pääkirjoitus, Aikuiskasvatus 2/2003. Aikuiskasvatus, (2). https://doi.org/10.33336/aik.93471 Heikkinen, A. (2003). Nomadeja ja aktiiveja kansalaisia. Pääkirjoitus, Aikuiskasvatus 2/2003. Aikuiskasvatus, (2). https://doi.org/10.33336/aik.93471 NOMADEJA JA AKTIIVISIA KANSALAISIA AIKUISKASVATUS 2/2003 PÄÄKIRJOITUS K ansallisvaltioiden ja kansalaisuuden itsestään selvät merkitykset näyttävät olevan muuttumassa tavalla, joka muistuttaa niiden aktiivisen tekemisen aikakausia. Aikuiskasvatuspolitiikka, -käytäntö ja -tutkimus ovat mukana muutoksessa: onko niissä kehitteillä kansalaisuuden uusi muunnos, joka yhdistää globaaleille markkinoille kelpaavan kuluttaja- tuottaja-työllistyjän ylikansallisesti toimivaan kulttuuriseen nomadiin? K Kansallisvaltio, jota halutaan sotilaallisesti, taloudellisesti ja poliittisesti puo- lustaa ja jonka uskotaan puolustavan asukkaitaan, tarvitsee kansan ja kansa- laiset. Ne näyttävät puolestaan tarvitsevan edustuksen, näkyväksi tekemisen ja henkilöitymisen. Valtion, kansan ja kansalaisuuden suhteet ovat saaneet maailmassa monenlaisia muotoja. Ilkka Liikanen on tutkimuksissaan osoitta- nut fennomaanien pitkän ja esimerkillisen projektin itsensä, ajattelunsa ja toi- mintatapansa tekemiseksi suomalaisen kansan edustukseksi. Kansanliikkeiden ja järjestöjen tuli osaltaan tukea valtion ja kansalaisen suhteen välittömyyttä. Yhdysvaltoihin sopivaa kansalaisuutta taas muotoiltiin Putnamin mukaan yh- teisöllisyyttä rakentavissa liikkeissä ja itsekasvatuksellisissa ohjelmissa, joi- den murentuminen uhkaa kansallisvaltiollista yhteisyyttä. Kansallisvaltioiden rakentamisen vaihtoehdoille näyttää silti olleen yhteistä niihin kelpaavien kan- salaisten systemaattisen ja massaluonteisen kasvattamisen institutionalisoi- minen. Se käynnistyi yhtäältä lasten ja nuorten koulumaisesta kasvatuksesta, toisaalta alueellisesti tai sosiaalisesti rajattujen yhteisöjen elämäntapojen ja asenteiden muovaamisesta aatteellisen, poliittisen ja ammatillisen valistuksen kautta. Anja Heikkinen Akateeminen intelligentsia on perinteisesti pyrkinyt tai joutunut muotoile- maan sosiaalisen osallistumisen ja asemoitumisen käsitteellisiä ja ideologisia perusteita. Kansallisvaltioyhteiskuntien ja kansallisten talouksien rakentami- seen liittyi myös kasvatuksen ja sivistyksen tieteellisten käsitteiden ja mallien kehittäminen. Kun kansa ja sivistyneistö ovat tuottaneet toisiaan, on tulkinta sivistyksestä ollut keskeinen. Juha Siltala on kuvannut suomalaisen sivisty- neistön sisäistä, psykohistoriallista itsensä muotoilun projektia, jossa se on etsinyt kansaa itsestään ja itseään kansasta. Sivistyneistön tulkintamahdolli- suudet ovat riippuneet sen suhteesta kansallisiin poliittisiin ja taloudellisiin eliitteihin tai sellaisiksi pyrkiviin ryhmiin. 98 P erinteinen suomalainen aikuiskasvatus on määritellyt itsensä sivistyk- sen edustukseksi, sivistyneistön ja kansan itseymmärryksen mahdollis- tavaksi toimintakentäksi. Kuitenkin sen sivistyskäsitystä ovat hallinneet länsimaisen opillisen eliitin henkiset arvot ja usko tieteeseen ja tiedolliseen oppimiseen. Vaihtoehtoisiin tulkintoihin pyrkivät ohjelmat näyttävät sopeutu- P AIKUISKASVATUS 2/2003 PÄÄKIRJOITUS neen dominoivaan sivistysdiskurssiin synnyttäen puhetta esimerkiksi teknil- lisestä ja ammatillisesta sivistyksestä tai vaikkapa sydämen sivistyksestä. Aikuiskasvatuksen paraatipuolella sivistyneistön, taloudellisen ja poliitti- sen eliitin ylläpitämä itseymmärrys on edelleen voimissaan. Kansalliseen ai- kuisten koulutustason nostamisohjelmaan pyrkivät koulutusinstituutiot eivät epäröi sovittaa sosiaali- ja työvoimapoliittista alihankintatehtäväänsä vapaan sivistystyön kunniakkaisiin kansakunnan rakentamisen ja sivistymisen perin- teisiin. Erityisesti Irakin sota on tuonut näkyviin talouden ja politiikan ylikan- sallistumiseen sisältyvän sotilaallisen ulottuvuuden. Kansalaisen uuden muun- noksen on haluttava puolustaa jotain kansallisvaltioon samaistumatonta enti- teettiä ja uskottava sen puolustavan itseään. NOMADEJA JA AKTIIVISIA KANSALAISIA Sivistysdiskurssin muuntumi- nen, jopa korvautuminen, yhtäältä markkinataloudellisella oppimis- ja työllis- tymisdiskurssilla, toisaalta itserefleksiivisellä yksilödiskurssilla ilmentää myös sivistyneistön muuntumista. Millaista tiedollis-ideologista edustusta uudelle kansalaisuudelle se rakentaa uusissa sivistyksen tulkinnoissa ja millaisiin ta- loudellis-poliittisiin liittoutumiin sen on projektissaan nojauduttava? Zygmunt Bauman jatkaa teoksessaan Notkea moderni länsimaisten sosiologien ja post- modernien ajattelijoiden vuosia kestänyttä kertomusta kehkeytyvästä uuspai- mentolaisuudesta kansalaisuuden ylittävänä maailmassa olemisen tapana. Mil- laista aikuiskasvatuksen itseymmärrystä oikeuttaa paikkaan ja aikaan sitoutu- mattoman, toiseen ja vieraaseen alati uteliaan avoimesti ja vastavuoroisesti suhtautuvan intellektuellin esikuva? T ässä Aikuiskasvatus-lehden teemanumerossa herätellään keskustelua kansan ja kansalaisuuden vanhoista ja uusista muunnoksista. Samalla kokeillaan sisällön uutta jäsennystä, joka toivottavasti viestii lukijoille ja kirjoittajille toimituskunnan pyrkimyksiä lehden kehittämiseksi keskustele- vampaan suuntaan. Kaikkien numeroiden rungoksi odotamme aikuiskasvatus- tutkimusta ilmentäviä tieteellisiä artikkeleita, aikuiskasvatuksen käytännöistä nousevia ja ajankohtaisia poliittisia kysymyksiä käsitteleviä kirjoituksia sekä teoriaa, käytäntöjä ja politiikkaa kommentoivaa keskustelua ja puheenvuoroja. Myös kirja-arviointien toivomme edustavan monipuolisesti teoriaa, politiik- kaa ja käytäntöjä. T 99
https://openalex.org/W2514469050	https://www.scielo.br/j/ean/a/r6XgDz4MBBZtTGjYDrK64bP/?lang=pt&format=pdf	Portuguese	null	The Impact of homophobia on adolescent health	Escola Anna Nery	2,015	cc-by	6,080	Esc Anna Nery 2015;19(4):664-670 Esc Anna Nery 2015;19(4):664-670 Resumo Objetivo: Objetivou-se conhecer os tipos de violência sofrida por adolescentes homossexuais e compreender a influência da homofobia na saúde dessa população. Métodos: Foram realizadas entrevistas, em profundidade, com nove adolescentes residentes em um município do interior de São Paulo que se autodeclararam homossexuais. Resultados: Os tipos de violência sofridos e referidos pelos adolescentes foram: física, verbal, psicológica e sexual. A homofobia provoca percepções negativas sobre si mesmo e a não adoção de hábitos de vida saudáveis relacionados aos cuidados com alimentação, prática de atividades físicas, padrão de sono e ideações suicidas. Foram relatadas situações de homofobia nos serviços de saúde. Conclusão: Adolescentes homossexuais são vulneráveis a diferentes tipos de violência. A dificuldade de acesso aos serviços de saúde é um fator concorrente ao aumento da vulnerabilidade. A contribuição deste estudo reside na problematização de tópicos que podem auxiliar na construção do cuidado integral dos adolescentes homossexuais. 1. Universidade de São Paulo. Ribeirão Preto, SP, Brasil. Palavras-chave: Saúde do Adolescente; Violência; Homossexualidade. O impacto da homofobia na saúde do adolescente The Impact of homophobia on adolescent health El impacto de la homofobia en la salud del adolescente Taison Regis Penariol Natarelli1 Iara Falleiros Braga1 Wanderlei Abadio de Oliveira1 Marta Angélica Iossi Silva1 Abstract Objective: This study had the purpose of recognizing the types of violence suffered by homosexual adolescents and understanding the effects of homophobia on the health of this group. Methods: In-depth interviews were conducted with nine adolescents that live in a country town on the state of São Paulo who declared themselves homosexuals. Results: The respondents reported cases of physical, verbal, psychological and sexual violence. Homophobia causes negative perceptions about themselves and the non-adoption of healthy lifestyle related to their nutrition, physical activity practice, sleep patterns and suicidal ideas. Homophobic situations were also described at health units. Conclusion: Homosexual adolescents are more vulnerable to different types of violence and the difficulty of access to health services increases the vulnerability. The relevance of this study is on the problematization of topics that can promote the construction of comprehensive care of this group. Keywords: Adolescent Health; Violence; Homosexuality. 1. Universidade de São Paulo. Ribeirão Preto, SP, Brasil. Autor correspondente: Taison Regis Penariol Natarelli. E-mail: taison.natarelli@hotmail.com Recebido em 04/04/2015. Aprovado em 14/12/2015. DOI: 10.5935/1414-8145.20150089 EEAN .e PESQUISA \| RESEARCH INTRODUÇÃO ser objeto de discriminação ou violência, dentro ou fora da família. Realidade divergente para determinados grupos de adolescentes brasileiros, principalmente os homossexuais, expostos a violação de direitos humanos e a diversos tipos de violência8. Segundo o Ministério da Saúde9, em 2012 foram registrados 4.851 casos de homofobia, sendo que a maioria (61,16%) das vítimas tinha idade entre 15 e 29 anos. Esses dados evidenciam a relevância e magnitude da problemática da homofobia e, apontam, também, os adolescentes como integrantes de um grupo vulnerável. Em uma análise global, pode-se inferir que os direitos à liberdade e à segurança, parecem negados a população LGBT, de forma geral e ao adolescente. Adolescência é um período do desenvolvimento humano, compreendido cronologicamente entre 10 e 19 anos de idade1, sendo histórico, cultural e socialmente definida e marcada pelos aspectos das transformações físicas e comportamentais. Essas transformações são fundamentais para que o ser humano atinja a maturidade e se insira na sociedade como adulto, mas, sobretudo, a adolescência é uma etapa da vida que agrega sujeitos detentores de direitos que merecem ser vistos como atores ativos na sociedade, capazes de ter e incorporar valores e atitudes cidadãs que os permitam conviver de forma autônoma2. Nesse período, também há uma maior exposição a diferentes situações de conflito, violência e exclusão3, considerando-se a ampliação do convívio e contato social. Assim, a adolescência se refere ao encontro de situações sociais, históricas e culturais com a transformação dos sujeitos, tornando-os ao mesmo tempo singulares e coletivos, buscando sua identidade, inclusive sexual, e lugar no mundo. Nessa busca, a sexualidade revela- se como um elemento constitutivo do desenvolvimento e do processo de adolescer. Tomando-se por referência o conceito ampliado de saúde, onde este, para além dos aspectos biológicos, compreende que os sujeitos apresentam sobre sua saúde e qualidade de vida, às características de sua personalidade e a indissociação entre o físico, mental e social, vemos que a saúde é resultante de diversas condições e determinantes10. Além disso, a homofobia pode interferir na socialização, nos hábitos e comportamentos cotidianos, na alimentação, lazer, acesso a serviços de saúde, dentre outros, culminando em algumas situações, em prejuízos para o bem-estar dos adolescentes.i A homofobia, nesse contexto, surge como um conceito polissêmico e um fenômeno plural e faz referência a um conjunto de emoções e comportamentos negativos de uma pessoa ou grupo em relação aos homossexuais. INTRODUÇÃO Ela é, também, um dispositivo de controle que reforça a ideia de naturalização da normalidade relacionada à orientação heterossexual e que se manifesta nas relações sociais por meio de agressões físicas, verbais, psicológicas e sexuais4. Associada aos sintomas psicopatológicos e sentimentos negativos que provoca (medo, incomodo, ódio, repúdio), mas também em relação ao preconceito, a discriminação e a violência contra lésbicas, gays, bissexuais, travestis, transexuais e transgêneros, a homofobia, também, se associam às relações de poder e de gênero que se fazem presentes na sociedade4,5. A literatura científica indica que a homofobia é um dos determinantes para a saúde dos adolescentes. Um estudo com 300 adolescentes não heterossexuais, desenvolvido no Canadá com o objetivo de conhecer diferentes formas de bullying homofóbico e modelar relações entre o fenômeno e seu processo de internalização e questões de autoestima, verificou que a homofobia causa efeitos negativos sobre o bem-estar, a qualidade de vida e a saúde dos adolescentes11. Evidencia-se ainda, a associação entre a orientação sexual e ideações e tentativas de suicídio na adolescência, pois os homossexuais têm mais chances de pensarem e tentarem suicídio, comparativamente em relação aos heterossexuais5. A violência, enquanto um complexo processo relacionado à dinâmica social, afeta a integridade física, moral, mental ou espiritual das pessoas6. Ela é multicausal, na medida em que se relaciona à evolução da civilização e aos instintos de sobre vivência, bem como pode assumir um caráter eminentemente social, resultante das diferenças e desigualdades existentes entre as pessoas. Assim, a importância deste estudo se dá no aprofundamento das questões que envolvem as consequências da homofobia na saúde dos adolescentes. Além disso, problematiza-se a atuação do enfermeiro no cuidado direto ou indireto ao usuário adolescente homossexual, consoante aos princípios e diretrizes do Sistema Único de Saúde (SUS). Esses profissionais, também, devem conhecer e considerar as especificidades da demanda dos adolescentes homossexuais. Atuação que exige compreensões sobre os processos que levam a sua exclusão perante a sociedade, os tipos de violência às quais são submetidos e seus riscos. Adensando, dessa forma, a produção científica brasileira nessa direção, visto que existem lacunas nos estudos nacionais acerca do tema, especialmente, na área de conhecimento da enfermagem.i Para a área da saúde interessa a abordagem das vio lências enquanto um processo social. Resumen Objetivo: Conocer los tipos de violencia sufrida por adolescentes homosexuales y comprender la influencia de la homofobia en la salud de ese grupo. Métodos: Fueron realizadas entrevistas en profundidad con nueve jóvenes residentes en un municipio del interior de São Paulo que se declararon homosexuales. Resultados: Los entrevistados relataron casos de violencia física, verbal, psicológica y sexual. La homofobia causa percepciones negativas acerca de si mismo y la no adopción de hábitos de vida saludables relacionados a los cuidados con la alimentación, práctica de actividades físicas, patrones de sueño e ideas suicidas. También fueron relatadas situaciones de homofobia en los servicios de salud. Conclusión: Adolescentes homosexuales son más vulnerables a diferentes tipos de violencia. La dificultad de acceso a los servicios de salud aumenta la vulnerabilidad. La contribución de este estudio está en la problematización de tópicos que puedan auxiliar la construcción del cuidado integral de estos individuos. Palabras clave: Salud del Adolescente; Violencia; Homosexualidad. Autor correspondente: Taison Regis Penariol Natarelli. E-mail: taison.natarelli@hotmail.com Recebido em 04/04/2015. Aprovado em 14/12/2015. DOI: 10.5935/1414-8145.20150089 Escola Anna Nery 19(4) Out-Dez 2015 664 Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI Tipos da violência Os adolescentes referiram serem vítimas de diversos tipos de violência física, verbal, psicológica e sexual. Como principais cenários para ocorrência da homofobia os adolescentes referiram: a escola, a família, e a comunidade. O contato com o participante inicial foi possível, uma vez que os pesquisadores já apresentavam uma relação por meio de um trabalho sistemático de prevenção junto a essa população em uma praça no município estudado. Em um primeiro contato foi detalhado o objetivo da pesquisa, as condições de participação, a necessidade do Termo de Consentimento Livre e Esclarecido e mediante sua concordância, a realização da entrevista em local de sua preferência. A violência física não foi considerada tão frequente quanto a verbal e a psicológica, sendo mais constante contra os adolescentes homossexuais do sexo masculino. Entrementes, nesse sentido, além da agressão física, propriamente dita, foram identificados relatos pessoais e de terceiros, a respeito de ameaças de agressão dessa natureza e tentativas de homicídio. Os dados foram coletados por meio de entrevistas semiestruturadas. Foi utilizado um roteiro que incluía questões sobre a percepção do adolescente em relação à: violência contra adolescentes homossexuais no cotidiano; a violência contra o próprio adolescente entrevistado; as acusas dessa violência e a influência dessa violência para a saúde do adolescente. As entrevistas individuais foram gravadas, sendo, posteriormente, transcritas. Sempre têm, as pessoas falam, 'ah eu prefiro ficar aqui [praça/ponto de encontro LGBT] à noite porque em casa meu pai me bate, discute comigo, todo dia' (A1). As físicas também tão predominando bastante. Na TV, no jornal, é frequente, quase todo dia tem uma notícia, um relato. [...] Sentir medo mesmo, de 'vou pra escola posso apanhar, ou toda hora vou ser zoado' (A4). Foram realizadas nove entrevistas individuais em profundi dade, com duração de cerca de uma hora cada, no período entre novembro de 2013 e julho de 2014. A idade dos adolescentes variou entre 13 e 19 anos de idade, com uma média de 17,4 anos, sendo cinco do sexo masculino (55,6%) e quatro do sexo femi nino (44,4%). Todos os entrevistados eram estudantes, sendo um (11,1%) do Ensino Fundamental II, cinco (55,6%) do Ensino Médio, e três (33,3%) do Ensino Superior. Três adolescentes (33,3%) declararam trabalhar como ator/maquiador, represen tante de atendimento/tatuadora e aprendiz de confeiteiro. Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI Dessa forma, objetivou-se conhecer os tipos de violência sofrida por adolescentes homossexuais e a repercussão das práticas homofóbicas na saúde dessa população. O tratamento e a análise dos dados coletados foram desenvolvidos por meio do referencial da análise de conteúdo, em sua modalidade temática. A análise levou à ordenação e classificação dos dados por unidades de registros, referenciadas por temas, e refinados em expressões de síntese, as categorias empíricas que explicitaram a realidade, segundo a visão dos entrevistados. A saturação teórica das respostas foi utilizada como determinante do número suficiente de participantes. Dessa forma, foram identificados os seguintes temas: "Tipos de Violência" e "Influência na Saúde". MÉTODOS Considerando os objetivos deste estudo, o desenvolvimento da investigação se deu por meio de uma abordagem qualitativa. O campo de estudo foi um município do interior do estado de São Paulo. Os critérios de inclusão dos participantes foram: ser adolescentes entre 10 e 19 anos de idade, residentes no município paulista e que se autodeclaravam homossexuais, de ambos os sexos. O projeto da pesquisa foi analisado e aprovado pelo Comitê de Ética em Pesquisa com Seres Humanos (CEP) da EERP/USP, Protocolo CAAE n. 16304213.3.0000.5393, respeitando-se as questões éticas necessárias para a sua realização. A participação dos adolescentes foi voluntária e aos menores de 18 anos de idade, além do seu assentimento, também, foi solicitado consentimento de seus pais ou responsáveis. A seleção dos participantes da pesquisa se deu por meio da técnica de bola de neve (snowball)12. Essa técnica se inicia a partir de um ator, ou um grupo de atores que indicarão novos sujeitos para participar do estudo, estes indicarão outros e, assim, sucessivamente, possibilitando ao pesquisador a imersão em seu círculo social. A técnica de bola de neve pode ser, particularmente, útil para estudar populações de difícil acesso e em situações de vulnerabilidade ou na abordagem de temas e tópicos delicados, como é o caso da pesquisa em questão e do seu objeto de estudo12. INTRODUÇÃO Neste debate, não se reduz a abordagem da área à problematização de modelos de assistência às vítimas de diferentes violências, mas essa abordagem procura auxiliar no desenvolvimento de estratégias de prevenção do fenômeno, incluindo-o na agenda de deba tes e de reflexões sobre como intervir em sua complexidade. Tal perspectiva ultrapassa o modelo curativo e biomédico de saúde, realçando a concepção holística e ampliada de saúde e seus correlatos como a integralidade, a intersetorialidade e a promoção da saúde6. Pressupõe-se que, adolescentes homossexuais, configu ram-se como população vulnerável, tanto pela condição ado lescente, quanto pela violência e exclusão que estão expostos, por diferirem em termos de comportamento e orientação sexual do padrão hegemônico fixado social, cultural e historicamente. Noutra perspectiva, o Estatuto da Criança e do Adolescente7 (ECA) assegura que nenhuma criança ou adolescente deve Escola Anna Nery 19(4) Out-Dez 2015 Escola Anna Nery 19(4) Out-Dez 2015 665 Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI Influência na saúde A violência psicológica, ou simbólica, foi tratada tanto como a mais preponderante, quanto àquela que compõe o cotidiano do adolescente homossexual, que enfrenta no dia a dia, situações de preconceito, opressão, tratamento diferenciado, dentre outras formas de exclusão. Os adolescentes evidenciaram nas entrevistas a dimensão e a complexidade das situações de homofobia com as quais convivem por meio de diferentes formas de manifestação, que acarretam prejuízos à sua saúde. Isso pode ser mensurado em termos do comprometimento da saúde mental e nas dificuldades para adotar hábitos de vida saudáveis. Os adolescentes demonstram uma percepção negativa de si mesmos, que podem contribuir para que eles negligenciem práticas de autocuidado, não consigam manter hábitos saudáveis e podem até desenvolver ideação suicida. Esses aspectos são ilustrados nos trechos a seguir: Na rua todo mundo encara, tipo, as pessoas não são discretas, às vezes, os olhares são muito estranhos, tipo, é uma coisa assim, as pessoas cerram o olho e encaram você e você fica tipo, se sentindo desconfortável, às vezes, um pouco, sabe? Observado demais (A2). Às vezes, por exemplo, tem uma cadeira para sentar, uma poltrona, cabem duas pessoas, sentam um homem e uma mulher, o segurança passa perto e não fala nada, senta a gente, não dá dois minutos 'Ó, por favor, se retirem, que essa cadeira é pra uma pessoa só', então fica aquele clima chato sabe? (A5). O Stress é muito grande, com relação a isso [...]. Eu já vomitei, porque sabe, quando você fica tão.... Chorando e mal, e você fica mal mesmo, e aí dá aquela reviravolta no estomago e volta tudo, eu já vomitei uma vez, foi estranho e é o stress, é bastante (A2). Eu perdi bastante amigo, [...] eu tinha até um melhor amigo que andava comigo bastante, parou de falar comigo quando soube, foi triste (A6). Eu com 13 anos eu entrei em depressão, [...] comecei a me sentir mal, comecei a ficar doente, eu comecei a ficar com anemia, porque eu comecei a parar de comer. E foi logo em seguida, que eu tive um câncer, dai eu me curei, e começou a acontecer essas coisas, então, eu não podia ficar doente, então, piorou muito a minha saúde isso, porque numa época que era para eu estar o melhor com o meu corpo, cuidar do meu corpo, eu comecei a fazer o contrário, a prejudicar o meu corpo. Tipos da violência Para efeito de apresentação e para manter resguardada a identidade dos participantes, eles serão designados pela letra A seguida do número atribuído a cada adolescente (Adolescente 1 = A1, e, assim, sucessivamente). Com o homem, eu até acho que eles apanham bem mais que as mulheres, e não conseguem sair na rua, não conseguem sair de mão dada, dar um beijo, com certeza eles vão sofrer agressão mais física do que verbal [...] (A5). Colocaram ele [adolescente gay] lá, e dois seguranças e esse cara, que eles falaram depois que foi contratado pra exterminar as 'bichinhas' do shopping né, bateram nele, deram socos, [...] bateram, acho que deram soco no estô mago, chute, [...] e falaram pra ele nunca mais voltar (A6). A violência verbal foi um tipo de violência em que os adolescentes significaram como de muito sofrimento. Nesses Escola Anna Nery 19(4) Out-Dez 2015 666 Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI casos, essa violência foi caracterizada, principalmente, pelo uso de ofensas e termos pejorativos referentes à condição sexual do adolescente, mas também do uso de palavras para oprimir e pressionar o adolescente. O homem de certa forma, ele tem fetiche por duas mulheres juntas [...], então as mulheres são discriminadas, mas de uma forma mais abusiva, [...] porque eu não duvido que um homem chegue e agarre duas lésbicas para fazer loucura com elas [...]. Várias vezes a gente passou de mão dada na rua e vários policiais passaram olhando, um já mexeu com a gente, [...] com um olhar, sabe, diferente, um olhar mais sexual. [...]. Eu sinto como violência, as pessoas pensam que não é tão abusivo, não é tão invasivo, mas só de você olhar para uma pessoa com um sentido diferente, dá a entender que, é abusivo pra gente (A5). [Na escola] Tem sempre as brincadeiras, fica tipo, 'aí veadinho, veadinho', etc. (A1). Se eu já sofri homofobia também foi só verbal, é mais, é constante, assim de, na rua tipo, xingar tipo, 'sapatão', eu estou acostumada (A2). Uma vez a gente estava na rua e por algum motivo ela estava mal, e aí eu abracei ela [namorada], [...] e aí chegou um cara todo louco, sei lá, alucinado, falando assim 'é, vocês acham que vocês são homem e mulher? Tipos da violência Vocês não são!', e eu só tinha dado um abraço nela, e aí começou a ficar aquele clima tenso (A5). Ah, já teve muitos comentários de assédio, já teve propostas sexuais, muitas propostas sexuais, ofereciam muito dinheiro 'não, muito obrigado'[...]. Eu era menor na época, depois de maior também aconteceu (A7). Também chegam ameaças, direto, [...] às vezes, chega até a violência sexual, porque, às vezes, algumas pessoas acham que nós somos inferiores e, por isso acham que tem direitos sobre o que é nosso. [...]. Que a gente não vai poder fazer nada (A9). Mais verbal, mexe um pouco com o psicológico da pessoa, tanto é que, tipo, é, não é fácil para um homossexual ser esculachado na frente de outras pessoas por causa de sua opção, isso acaba mexendo um pouco com a sua autoes tima, ele acaba se desvalorizando um pouco mais (A8). DISCUSSÃO Os adolescentes, ainda, relataram a presença de ideais e comportamentos homofóbicos dentro dos serviços de saúde e entre seus profissionais, tratados como elementos capazes de dificultar o acesso à saúde e a um atendimento integral. [Em uma consulta médica] Eles acharam que eu era homem, antes, sabe, foi meio que, foi uma situação assim meio desagradável porque o médico chamou [...], então por eu não ter a aparência feminina ele pensou que eu não fosse eu, ou alguma coisa assim, ai foi uma situaçãozinha desconfortável (A2). Já aconteceu de enfermeira não querer tirar sangue, ou pedir para outra pessoa tirar sangue sabe, por eu acreditar que seja medo [...] A pessoa que eu conheço que sofreu isso, ela nunca mais tirou sangue, ela nunca mais foi no médico, todo mundo fala para ela ir no médico, mas ela morre de vergonha de ir no médico e acontecer de novo (A3). A violência verbal, aquela que utiliza de palavras como meio de agressão, humilhação, exclusão, no caso do adolescente homossexual, também se baseia na relação de poder e domínio do agressor sobre a vítima, podendo levá-lo a não aceitação de sua própria orientação sexual e a quadros e comportamentos que indicam algum sofrimento psíquico. Ela [ginecologista] não faz pergunta diretamente para você, você fica com receio de falar alguma coisa, e, às vezes, ela deixa de te dar um diagnóstico certo por falta de informação. [...] eu acho que devia partir da parte deles perguntar isso para a gente, porque nem sempre a gente se sente a vontade de falar 'ó, não, então, eu não tenho relações com homens, eu tenho com mulher', entendeu? É complicado... (A5). De acordo com uma pesquisa, realizada com os participantes da Parada do Orgulho, acerca da violência sofrida, dentre os 320 entrevistados, 40% declararam a ocorrência de situações de discriminação verbal na escola ou faculdade ao longo da vida. Pouco mais da metade do conjunto que foi entrevistado relatou ter vivido situações em que eles mesmos ou colegas muito próximos foram colocados no lugar de "bicha" ou de "sapatão" na infância ou no início da adolescência14. DISCUSSÃO inteiro, então eu, sabe, te deixa mal, se você deixar levar pelo preconceito, te deixa muito mal, afeta muito a saúde, afetou muito a minha saúde (A3). inteiro, então eu, sabe, te deixa mal, se você deixar levar pelo preconceito, te deixa muito mal, afeta muito a saúde, afetou muito a minha saúde (A3). Tentam se matar e tudo, tem bastante gente assim (A6). Este estudo objetivou conhecer os tipos de violência sofrida por adolescentes homossexuais e compreender a repercussão de práticas homofóbicas na saúde dessa população. Nesse sentido, a respeito dos tipos de violência sofrida pelos adolescentes, encontra-se a homofobia em suas mais variadas formas de manifestação e tipos de agressão, quais sejam: física, verbal, psicológica e sexual. As violências verbais e psicológicas foram as mais referidas. Essas violências acontecem nos contextos familiar, escolar e comunitário. A homofobia é recorrente e compõe o cotidiano dos adolescentes. Por seu turno, o impacto à saúde dessa população pode ser apreciado a partir de dois aspectos: 1) a percepção dos adolescentes homossexuais sobre as violências às quais são submetidos; e 2) as repercussões das práticas homofóbicas na saúde do adolescente. Tentam se matar e tudo, tem bastante gente assim (A6). Isso me prejudicou que, eu entrei em depressão por causa disso, e eu já tentei suicídio várias vezes por causa disso também, eu estou começando a superar, e aconteceu já faz vários anos o episódio mais grave. [...]. Eu conheço pessoas que começaram a virar anoréxicos e ter bulimia, entre outros transtornos, então realmente afeta diretamente a saúde das pessoas. [...]. Eu mesmo tava chegando num ponto que minhas costelas já estavam todas aparecendo (A9). Sobre os tipos de violência sofrida, numa dimensão explorativa, salienta-se que a do tipo física é definida pelo uso da força - para "disciplinar" a criança ou o adolescente, impetrada por alguém que está em relação de poder ao outro, podendo ou não deixar lesões externas ou internas ou ambas13. Pensando nos padrões da sociedade heteronormativa na qual vivemos, a agressão física contra os adolescentes homossexuais surge objetivando a punição por um comportamento "desajustado", buscando a mudança desse comportamento, no caso, da identidade sexual do próprio adolescente. Destacam-se ainda nas falas dos adolescentes a ocorrência e a prevalência de casos letais de violência física contra o adolescente homossexual, como por exemplo, as tentativas de homicídio, de afogamento e atropelamento, relatadas pelos adolescentes7,8. Influência na saúde Eu tinha vergonha de me exercitar, dai eu comecei a ter problema com gordura, e como é que chama? Diabetes [...]. Eu já não dormia porque eu sabia que eu ia sofrer preconceito no outro dia e ficava pensando nisso o tempo Eu tive uma rejeição, [...] meu pai biológico, ele não aceitou, [...] ele não conversou comigo durante um ano. [...]. Sofri essa rejeição, ele brigou comigo, não quis mais falar comigo (A7). A violência sexual, ainda que não presente na maioria dos relatos, foi destacada como um risco em potencial aos adolescentes homossexuais, principalmente aos do sexo feminino, atribuindo ainda uma prevalência maior aos casos de assédio sexual, ameaças e tentativas de abuso. Escola Anna Nery 19(4) Out-Dez 2015 667 Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI DISCUSSÃO Eu vejo que acontece muito isso, de as pessoas às vezes negarem atendimento a essas pessoas, e fazerem questão, mesmo estando escrito o nome social, que é o nome que a pessoa escolheu para si, fazer questão de usar o nome de registro da pessoa pra chamar ela, que na verdade é proibido de acordo com as leis e isso acontece muito mesmo, até nos serviços do SUS que está escrito só o nome social, não tem sexo nem nada no cartão, e isso acontece direto, então eu percebi que sim, tem mesmo preconceito (A9). A violência psicológica, por seu turno, foi apontada pelos adolescentes, não apenas como a mais prevalente, mas também como a que causa maior sofrimento, bem como maiores agravos, podendo levar a ideações e tentativas de suicídio. Esse dado está em consonância com outros achados da literatura5 que, quando compara estatisticamente os adolescentes heterossexuais com os não heterossexuais, verifica que esses últimos apresentam mais chances de pensar e tentar suicídio. A violência sexual foi citada pelos adolescentes, em forma de ameaças, tentativas de abuso e assédio, seja pelos seus pares ou por adultos. Ressalta-se que nesses casos, os adolescentes Escola Anna Nery 19(4) Out-Dez 2015 Escola Anna Nery 19(4) Out-Dez 2015 668 Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI relataram terem se sentidos violados no momento da agressão. Esse tipo de violência pode ser compreendido sob duas formas - abuso sexual e exploração sexual - como todo ato, de qualquer natureza, atentatório ao direito humano ao desenvolvimento sexual da criança e do adolescente, praticado por agente em situação de poder e de desenvolvimento sexual desigual em relação aos adolescentes vítimas15. Ou seja, o ódio e repúdio aos homossexuais, bem como a ideia de superioridade a eles, especialmente aos adolescentes, que são particularmente vulneráveis, contribuem para provocar esse tipo de agressão. obstáculo para que a população LBGT tenha acesso a serviços de saúde e receba um cuidado mediante suas necessidades de saúde específicas17. Nessa direção, uma revisão integrativa da literatura sobre o papel da enfermagem diante da homossexualidade masculina revelou um baixo número de publicações sobre o tema e que, enquanto área, a enfermagem trabalha com elementos da promoção a saúde, por meio das orientações e da escuta qualificada. Contudo, ainda foram identificadas lacunas relacionadas à necessidade de se oferecer um cuidado que acolha homossexuais, travestis e transexuais. DISCUSSÃO Explicitando o protagonismo da enfermagem, o estudo revelou que a atenção integral dessa população deve ser repensada dentro da lógica do compromisso social da profissão, seus aspectos éticos e o grande contingente de profissionais na atenção primária à saúde18.i Outro aspecto relevante observado na análise dos resultados refere-se à influência da homofobia para a saúde do adolescente, principalmente, no que se diz respeito a sua saúde mental, pois ela contribui para o surgimento de comportamentos depressivos, ansiedades e medos excessivos, ideações e tentativas de suicídio, quadros que indicam sofrimentos psíquicos, cuja origem está nos episódios de violência vivenciados. Isso revela um dos efeitos perversos da homofobia que é o processo de internalização da violência. Experiências dessa natureza afetam as ações e a maneira de pensar de quem a sofre, além de interferirem na adoção de hábitos de vida saudáveis e no autocuidado, como por exemplo, alimentação, atividade física e padrão de sono, inadequados, causando sinais e sintomas somáticos como dores na cabeça, no estômago e no corpo, vômito e desmaios, dentre outros relatados pelos adolescentes. Reitera-se que é atribuição dos profissionais pertencentes a instituições de saúde assegurar os direitos das crianças e adolescentes, independente de questões gênero e orientação sexual. Entretanto, percebe-se uma reprodução de práticas homofóbicas contra a população homossexual. Nesse sentido, as equipes de saúde devem estar preparadas para a atenção à saúde do adolescente, especialmente adolescentes homossexuais, no que se refere à saúde e à violência, para que se possam construir uma rede de prevenção, e apoio social a esta população, como salientado pelos adolescentes participantes deste estudo. A linha de cuidado para a atenção integral à saúde da criança, do adolescente e suas famílias em situação de violência, por intermédio do acolhimento, atendimento, notificação e seguimento na rede de cuidado e de proteção social, visa à continuidade do atendimento, à articulação dos diversos setores envolvidos, além do fortalecimento da responsabilização e envolvimento dos serviços e seus profissionais, nos casos de violência, como se configura a homofobia. Quando o adolescente começa a apresentar comportamen tos considerados inadequados pela sociedade heteronormativa, o indivíduo começa a ser exposto a discursos homofóbicos, dentre outras formas de violência, como a simbólica, cujo in tuito é "coagir" o sujeito a assumir seu papel de gênero (como um "homem" ou uma "mulher" deveriam agir)5. DISCUSSÃO O adolescente homossexual ao se perceber "diferente" de seus pares, passa a acumular pensamentos negativos a respeito de si mesmo, internalizando a homofobia, que pode levá-lo a adotar com portamentos de risco, que são comuns entre a maioria dos adolescentes, mas que representam um peso maior ao se tratar de homossexuais5. Pode-se dizer que a condição LGBT incorre em hábitos corporais ou mesmo práticas sexuais que podem guardar alguma relação com o grau de vulnerabilidade dessas pessoas. No entanto, o maior e mais profundo sofrimento é aquele decorrente da discriminação e preconceito16. Impacto da homofobia na saúde do adolescente Natarelli TRP, Braga IF, Oliveira WA, Silva MAI Natarelli TRP, Braga IF, Oliveira WA, Silva MAI Natarelli TRP, Braga IF, Oliveira WA, Silva MAI capacitados e orientados para lidar com a homofobia, adotando posturas marcadas pela prevenção, identificação, acolhimento, atendimento, notificação e encaminhamento dos casos de violência contra o adolescente homossexual, visando um atendimento integral e livre de preconceitos, como preconizado pelo SUS. Tal perspectiva amplia a abordagem dessa temática contemporânea, ultrapassando estudos centrados no uso de substâncias psicoativas ou de doenças sexualmente transmissíveis relacionados à população LGBT. 4. Miskolci R, Balieiro FF. O drama público de Raul Pompeia: sexualidade e política no Brasil finissecular. Rev. Bras. Ci. Soc. [online]. 2011 fev; [citado 2014 out 08]; 26(75): 73-88. Disponível em: http://www.scielo. br/scielo.php?script=sci_arttext&pid=S0102-69092011000100004&ln g=en&nrm=iso. 5. Teixeira-Filho FS, Rondini CA. Ideações e tentativas de suicídio em adolescentes com práticas sexuais hetero e homoeróticas. Saúde Soc. 2012 jul/set;21(3):651-667. 6. Minayo MCS. Violência e educação: impactos e tendências. Revista Pedagógica. 2013 jul/dez;15(31):249-264. 7. Lei n(o) 8.069, de 13 de julho de 1990. Dispõe sobre o Estatuto da Criança e do Adolescente e dá outras providências. Diário Oficial da República Federativa do Brasil. Brasília (DF), 17 de julho de 1990: Seção 1:1. No entanto, os resultados deste estudo devem ser interpretados considerando algumas limitações. O tipo de recrutamento utilizado favorece a abordagem para se conhecer questões relacionadas a grupos vulneráveis, podendo se construir uma abordagem mais profunda do contexto das relações sociais, especificamente, mas pode-se ter incluído adolescentes com maior exposição às situações de violência, por exemplo, o que pode não refletir a experiência da maioria dos adolescentes homossexuais. Não foi previsto e possível explorar aspectos de gênero na análise dos dados, ou seja, se a maneira como meninos e meninas vivenciam a homofobia difere ou possui semelhanças e como cada gênero interpreta essas situações. 8. Fundo das Nações Unidas para a Infância (UNICEF). O direito de ser adolescente: Oportunidade para reduzir vulnerabilidades e superar desigualdades. Brasília: Unicef; 2011. p. 182. 9. Ministério da Saúde (BR). SUS vai registrar casos de agressão por homofobia [online]. Brasília: Ministério da Saúde; 2013 [citado 2014 out 08]. Disponível em: http://potal.saude.gov.br/portal/saude/area. cfm? 10. Dalmolin BB, et al. Significados do conceito de saúde na perspectiva de docentes da área da saúde. Esc Anna Nery [online]. 2011; [citado 2014 out 08]; 15(2):389-394. Disponível em: http://www.scielo.br/ scielo.php?script=sci_arttext&pid=S1414-81452011000200023&ln g=en&nrm=iso. Impacto da homofobia na saúde do adolescente Recomenda-se que outras pesquisas, com diferentes delineamentos, explorarem esses e outros aspectos relacionados à saúde do adolescente homossexual. Por exemplo, para investigações sistemáticas sobre a temática da homofobia, especificamente, sugere-se a formação de grupos focais com meninos e meninas homossexuais e heterossexuais. Tais iniciativas podem suplantar a carência de estudos que sejam capazes de embasar a prática do profissional que presta assistência direta aos adolescentes e, também, a implementação de políticas públicas voltadas para a promoção da saúde e proteção dos homossexuais. 11. Blais M, Gervais J, Hebert M. Homofobia internalizada como um mediador parcial entre bullying homofóbico e auto-estima entre os jovens das minorias sexuais em Quebec (Canadá). Cienc saude colet. [online]. 2014 mar; [citado 2014 out 08]; 19(3): 727- 735. Disponível em: http://www.scielo.br/scielo.php?script=sci_ arttext&pid=S1413-81232014000300727&lng=en&nrm=iso. 12. Hanneman RA, Riddle M. Introduction to social network methods [online]. Riverside: University of California; 2009 [citado 2014 out 13]. Disponível em: http://faculty.ucr.edu/~hanneman/nettext/ Introduction_to_Social_Network_Methods.pdf 13. Algeri S, Souza LM. Violência contra crianças e adolescentes: um desafio no cotidiano da equipe de enfermagem. Rev. Latino-Am. Enferm. 2006 jan/fev;14(4):625-631. 14. Facchini R, França IL. Convenções de gênero, sexualidade e violência: pesquisa com participantes de eventos do Orgulho LGBT de São Paulo - 2009. Latitude. 2013 jan/jun;7(1):13-32. REFERÊNCIAS 15. Secretaria de Direitos Humanos, Conselho Nacional dos Direitos da Criança e do adolescente. Plano Nacional de Enfrentamento da Violência Sexual contra Crianças e Adolescentes. Brasilia: CONANDA; 2013. 1. United Nations Children's Fund - UNICEF. The state of the world's children 2011 - Adolescence: an age of opportunity. New York: United Nations Children's Fund; 2011. p. 34. 1. United Nations Children's Fund - UNICEF. The state of the world's children 2011 - Adolescence: an age of opportunity. New York: United Nations Children's Fund; 2011. p. 34. 2. Silva MAI. Adolescence: resignify it to understand it and act. Rev Enferm UFPE on line. [online]. 2012 mar; [citado 2014 out 08]; 6(3): 1-3. Disponível em: http://www.revista.ufpe.br/revistaenfermagem/index. php/revista/article/view/2646/pdf_1089. 2. Silva MAI. Adolescence: resignify it to understand it and act. Rev Enferm UFPE on line. [online]. 2012 mar; [citado 2014 out 08]; 6(3): 1-3. Disponível em: http://www.revista.ufpe.br/revistaenfermagem/index. php/revista/article/view/2646/pdf_1089. 16. Ministério da Saúde (BR). Política nacional de saúde integral de lésbicas, gays, bissexuais, travestis e transexuais. 1a ed. Brasília (DF): Ministério da Saúde; 2013. 17. Campos-Arias A, Herazo E, Cogollo Z. Homophobia among nursing students. Rev. Esc. Enferm. USP. 2010 mai/jun;44(3):826-830. 3. Borges ZN, Perurena FCV, Passamani GR, Bulsing M. Patriarcado, heteronormatividade e misoginia em debate: pontos e contrapontos para o combate à homofobia nas escolas. Latitude. 2013 jan/jun; 7(1): 61-76. 3. Borges ZN, Perurena FCV, Passamani GR, Bulsing M. Patriarcado, heteronormatividade e misoginia em debate: pontos e contrapontos para o combate à homofobia nas escolas. Latitude. 2013 jan/jun; 7(1): 61-76. 18. Matoso LML. O papel da enfermagem diante da homossexualidade masculina. Saúde (Santa Maria). 2014 jul/dez; 40(2): 27-34. CONCLUSÃO Os adolescentes homossexuais encontram-se em situação de vulnerabilidade e são expostos a diferentes tipos de violência. A saúde dessa população é afetada pela homofobia, que provoca quadros e comportamentos que caracterizam sofrimento mental e interfere na adoção de comportamentos e hábitos de vida saudáveis. A contribuição original deste estudo, nesse sentido, reside na problematização de práticas de cuidado e atenção integral à saúde, direcionadas aos adolescentes homossexuais, destacando o enfermeiro como profissional estratégico para a propagação desse tipo de práticas. A realidade relatada pelos adolescentes, com referência a homofobia presente nos serviços de saúde, também foi encontrada na literatura. Estudos sugerem que profissionais de saúde apresentam dificuldades para lidar com os adolescentes em geral, além de altos níveis de homofobia, posicionamento conservador e inflexível, atitudes negativas e discriminatórias contra homossexuais17. Atitudes como estas influenciam o comportamento do profissional que, muitas vezes, atende o paciente homossexual de maneira rápida e superficial, sem se aprofundar em questões relativas à saúde sexual e limitam a possibilidade de prestar um cuidado integral a esses pacientes. Sendo assim, a homofobia pode ser caracterizada como um Nessa direção, observa-se que essas práticas devem acompanhar as necessidades desse público por abordagens terapêuticas que valorizem a singularidade e a expressão da sexualidade, ao mesmo tempo, que orientam e avaliam a necessidade de apoio ou serviços formais de saúde mental. Para tanto, os profissionais de saúde que lidam com adolescentes, especialmente os enfermeiros da atenção primária, devem ser Escola Anna Nery 19(4) Out-Dez 2015 Escola Anna Nery 19(4) Out-Dez 2015 669 670 Escola Anna Nery 19(4) Out-Dez 2015 670
https://openalex.org/W2139271485	https://europepmc.org/articles/pmc4344550?pdf=render	English	null	Antimalarial pharmacology and therapeutics of atovaquone	The journal of antimicrobial chemotherapy/Journal of antimicrobial chemotherapy	2,013	cc-by	7,367	# The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. *Corresponding author. Tel: +441517053151; Fax: +441517053371; E-mail: biagini@liverpool.ac.uk †These authors contributed equally to the study. Atovaquone is used as a ﬁxed-dose combination with proguanil (Malarone) for treating children and adults with uncomplicated malaria or as chemoprophylaxis for preventing malaria in travellers. Indeed, in the USA, between 2009 and 2011, Malarone prescriptions accounted for 70% of all antimalarial pre-travel prescriptions. In 2013 the patent for Malarone will expire, potentially resulting in a wave of low-cost generics. Furthermore, the malaria scientiﬁc community has a number of antimalarial quinolones with a related pharmacophore to atovaquone at various stages of pre-clinical development. With this in mind, it is timely here to review the current knowledge of atovaquone, with the purpose of aiding the decision making of clinicians and drug devel- opers involved in the future use of atovaquone generics or atovaquone derivatives. Keywords: malaria, drug development, mechanism of action, resistance, drug interactions Laboratories. More meaningful studies could be carried out at this time due to the development of test systems using the human parasite Plasmodium falciparum in vitro or in Aotus monkeys. The aim of this study was to design a quinone with good antimalarial activity against P. falciparum combined with good metabolic stability in humans. Several 2-cyclohexyl- 3-hydroxy-1,4-naphthoquinone analogues (2 and 3) were synthesized with the metabolically labile 4′ position of the cyclo- hexyl ring substituted with a range of groups.15,16 Several of these quinones demonstrated a potency of ≏1 nM towards P. falciparum in vitro, but only atovaquone (4) was inert to human liver microsomes.17,18 The trans isomer of atovaquone is substantially more potent than the corresponding cis isomer. The chemical synthesis of atovaquone was originally disclosed in 1991 in US patent no. 4981874. This route gave a poor yield of 4% atovaquone calculated from only the last two steps (Figure 2a).19 20 J Antimicrob Chemother 2013; 68: 977–985 doi:10.1093/jac/dks504 Advance Access publication 4 January 2013 Introduction vivax 1980s - Designed to investigate the metabolic stability of quinones in humans by Wellcome Research Laboratories Atovaquone - Only quinone found to be inert to human microsomes 1. R = -(CH2)8C(C5H11)2 OH Cl C(CH3)3 Atovaquone O O OH Cl O O R OH 4. R = 3. R = 2. R = ATOVAQUONE 366.837 5.8 (m), 4.74 (p) 54.37 99.9% 2.2–3.2 Half-life (days) PPB (1-90 mg/mL) Solubility (water) (g/L) PSA Log P MW Insoluble (m), 7.96e–04 (p) Figure 1. Historical development of atovaquone and its pharmacokinetic properties. MW, molecular weight; m, measured; p, predicted; PSA, polar surface area; PPB, plasma protein binding. O O R OH Atovaquone - Only quinone found to be inert to human microsomes Cl 4. R = Atovaquone O O OH Cl Cl Figure 1. Historical development of atovaquone and its pharmacokinetic properties. MW, molecular weight; m, measured; p, predicted; PSA, polar surface area; PPB, plasma protein binding. synthesis of atovaquone, as it is higher yielding and does not involve the use of heavy metals.21 infection, a key role of the parasite mitochondrion is to provide orotate for pyrimidine biosynthesis through the activity of dihy- droorotate dehydrogenase (DHODH). Consistent with this, inhibition of the bc1 complex by atovaquone affects the concentrations of metabolites in the pyrimidine biosynthetic pathway.34,35 Indeed, transgenic P. falciparum parasites expressing ubiquinone- independent yeast DHODH have been shown to display an atovaquone-resistant phenotype.36 In addition, a recent study sug- gests that a further cellular consequence of mitochondrial inhib- ition by atovaquone is the inhibition of purine biosynthesis.37 Blood-stage parasite death as a result of atovaquone is relatively slow compared with other antimalarials such as artemisinin and chloroquine.25,38 This feature appears to be consistent with other mitochondrial-acting antimalarials and is possibly due to the drug acting only on late trophozoites and not on the earlier ‘ring’ stages.25 Atovaquone is, however, active against liver stages, resulting in its utility as a prophylactic drug; however, it is not believed to be active against ‘dormant’ hypnozoites.8,39 A common problem with all the routes so far is that large amounts of the potentially useful, yet signiﬁcantly less potent cis isomer of atovaquone are disregarded, as only the trans isomer is required. There are two literature procedures that address this problem. Introduction Reacting the cis isomer of atovaquone, atovaquone intermediates or isomeric mixtures thereof with a strong acid results in a clean epimerization to the corresponding trans isomer and thus high yields of trans atovaquone.22 Heating the cis isomer at reﬂux in organic solvent also causes this transformation.23 With the patent relating to Malarone due to expire in 2013, the synthesis of atovaquone will be exploited to its full potential as generic versions of the drug are likely to become common- place. This will in turn have a marked effect on the cost, as cur- rently the high cost of atovaquone is frequently prohibitive in its use by the endemic population within countries affected by malaria. Increased availability and use of the drug will also have an effect on the clinical efﬁcacy of atovaquone, and factors such as access, sustainability and resistance need to be considered.24 Fur- thermore, the malaria scientiﬁc community has a number of anti- malarial quinolones with a pharmacophore related to atovaquone at various stages of pre-clinical development.25–30 Introduction Atovaquone is the end product of half a century of research by many groups who researched the antiparasitic properties of nu- merous structurally related compounds.1–6 Currently, atovaquone is used as a ﬁxed-dose combination with proguanil (Malarone) for the treatment of children and adults with uncomplicated malaria or as a chemoprophylactic agent for preventing malaria in travel- lers.7,8 In the USA, between 2009 and 2011, Malarone accounted for 70% of all antimalarial pre-travel prescriptions.9 p p p The development of atovaquone as an antimalarial drug began more than 50 years ago when the outbreak of World War II caused substantial shortages in the supply of quinine.10 Intense efforts in the USA led to thousands of structurally diverse compounds being investigated, several of which were hydroxynaphthoquinones. Modest antimalarial activity when administered to ducks infected with Plasmodium lophurae resulted in a robust optimization programme generating more than 300 quinones, some of which demonstrated greater activity than quinine in the duck assay. However, when administered to malaria patients these compounds were devoid of any activity due to poor absorption and rapid metabolism.11,12 Attempts to solve these problems and produce an orally active quinine were unsuccessful both then and when the problem was revisited in the 1960s.13 Research in the 1960s did, however, lead to the de- velopment of lapinone (1), which was given intravenously and had activity against Plasmodium vivax (Figure 1).14 Williams and Clark20 then published a variant of this method- ology (Figure 2b) in which oxalate (11) was used to produce racemic compound (9) with a 43% yield and the ester by-product (12) with a 38% yield. Conversion into atovaquone was then achieved as described in Figure 2(a). The disadvantages of this process are the column chromatography required to separate (9) from (12) and the same poor yield problem still prevails in the ﬁnal two steps. Both processes described so far also involve the use of silver nitrate, a heavy metal that can be difﬁcult to remove and whose use is tightly regulated. The recently patented (WO 2010/001379) synthesis seen in Figure 2(c) offers an improved The use of quinones as antimalarial agents was then reinves- tigated in the 1980s by a group at the Wellcome Research 977 Review 1960s - Lapinone - showed clinical activity against P. Mechanism of parasite resistance to atovaquone/ malarone Although the crystal structure of the P. falciparum cytochrome bc1 complex is not available, details of atovaquone binding to cytochrome b have been elucidated based on studies performed on model organisms and molecular modelling. These studies, which include electron paramagnetic resonance spectroscopy of the Rieske [2Fe2S] cluster, site-directed mutagenesis of model organism cytochrome b and gene sequencing of atovaquone-resistant Plasmodium species, demonstrate that atovaquone is most likely a competitive inhibitor of the parasite’s cytochrome b quinol oxidation (Qo) site (Figure 3).28,40 Mode of action Atovaquone is a competitive inhibitor of ubiquinol, speciﬁcally inhibiting the mitochondrial electron transport chain at the bc1 complex.31 Inhibition of bc1 activity results in a loss of mitochon- drial function.32,33 During the intra-erythrocytic stage of 978 JAC Review Malarone drug failure has been associated with a missense point mutation at position 268 in cytochrome b, exchanging tyrosine for serine (Y268S) or, less frequently, asparagine (Y268N).41 – 45 Position 268 in cytochrome b is highly conserved across all phyla and is located within the ‘ef’ helix component f th Q it hi h i t ti l i l d i bi i l binding. The resultant atovaquone-resistant growth IC50 (half- maximal inhibitory concentration) phenotype of these mutants is some 1000-fold higher than susceptible strains; however, this is accompanied by an ≏40% reduction in the Vmax of the bc1 complex, suggestive of a signiﬁcant ﬁtness t t th it 46 + + O O Cl O O Cl Cl O O Cl Cl O O OH Cl 5 6 7 8 9 10 O Cl + O O C l O O Cl Cl OH O O O O Cl O O Cl + 11 8 9 12 Cl + O O O O OH Cl Base N S OH Cl O O N S DCC O O S Cl N O O OH Cl 13 14 15 16 17 88% 80% 80% 1. AcCl, AlCl3, CS2 2. Br2, NaOH, Na2S2O5 HO2C Cl AgNO3, CH3CN AgNO3, CH3CN (NH4)2S2O8, H2O Atovaquone (b) (c) (a) crystallisation CH3CN KOH/MeOH (NH4)2S2O8 Adogen 464 HO2C 48:38 ratio cis:trans Atovaquone Isomer separation 48:38 ratio cis:trans Figure 2. Synthetic routes used to synthesize atovaquone. (a) The original synthesis of atovaquone. (b) Williams and Clarke atovaquone synthesis. (c) Improved atovaquone synthesis. Review JAC + + O O Cl O O Cl C O O Cl Cl O O OH Cl 5 6 7 8 9 10 1. AcCl, AlCl3, CS2 2. Br2, NaOH, Na2S2O5 HO2C Cl AgNO3, CH3CN (NH4)2S2O8, H2O Atovaquone (a) crystallisation CH3CN KOH/MeOH + + O O Cl 5 6 7 1. AcCl, AlCl3, CS2 2. Mode of action Br2, NaOH, Na2S2O5 HO2C Cl A (a) (a) O Cl O Cl l O O OH Cl 8 9 (NH4)2S2O8, H2O Atovaquone KOH/MeOH 7 l O O OH Cl 8 9 Atovaquone KOH/MeOH O O Cl Cl 10 crystallisation CH3CN Cl O O Atovaquone Malarone drug failure has been associated with a missense point mutation at position 268 in cytochrome b, exchanging t i f i (Y268S) l f tl i binding. The resultant atovaquone-resistant growth IC50 (half- maximal inhibitory concentration) phenotype of these t t i 1000 f ld hi h th tibl t i O O 10 O Cl + O O C l O O Cl Cl OH O O O O Cl O O Cl + 11 8 9 12 Cl + O O O O OH Cl Base N S OH Cl O O N S DCC O O S Cl N O O OH Cl 13 14 15 16 17 88% 80% 80% AgNO3, CH3CN Atovaquone (b) (c) (NH4)2S2O8 Adogen 464 HO2C 48:38 ratio cis:trans Atovaquone Isomer separation 48:38 ratio cis:trans Figure 2. Synthetic routes used to synthesize atovaquone. (a) The original synthesis of atovaquone. (b) Williams and Clarke atovaquone synthesis. (c) Improved atovaquone synthesis. O Cl + O O C l O O Cl Cl OH O O O O Cl O O Cl + 11 8 9 12 AgNO3, CH3CN (b) (NH4)2S2O8 Adogen 464 (b) Cl O O Cl O O Cl 12 O O Cl Cl O O Cl O O Cl + 9 12 AgNO3, CH3CN (NH4)2S2O8 Adogen 464 O Cl + OH O O 11 (c) 11 Cl + O O O O OH Cl Base N S OH Cl O O N S DCC O O S Cl N O O OH Cl 13 14 15 16 17 88% 80% 80% (c) HO2C 48:38 ratio cis:trans Atovaquone Isomer separation 48:38 ratio cis:trans Figure 2. Synthetic routes used to synthesize atovaquone. (a) The original synthesis of atovaquone. (b) Williams and Clarke atovaquone synthesis. (c) Improved atovaquone synthesis. Cl + O O O O OH Cl Base N S OH Cl O O N S DCC O O S Cl N O O OH Cl 13 14 15 16 17 88% 80% 80% (c) HO2C 48:38 ratio cis:trans Atovaquone Isomer separation 48:38 ratio cis:trans Figure 2. Synthetic routes used to synthesize atovaquone. (a) The original synthesis of atovaquone. Mode of action The positions of haem bl (cyt b) and the ISP [2Fe2S] cluster are also shown. (b) ISP:[2Fe2S] ISP:H181 b:E272 bl Atv (a) Figure 3. (a) Cartoon representation of the yeast cytochrome bc1 complex (3CX5.PDB), with atovaquone modelled at the Qo site (boxed area).83 The bc1 complex is a structural and functional homodimer with a molecular mass of ≏480 kDa, consisting of 10 discrete subunits per monomer in yeast and P. falciparum.28 The electron-transferring catalytic unit of one monomer is highlighted; cytochrome b is represented in orange, cytochrome c1 in blue and the Rieske iron-sulphur protein (ISP) in green. Haem groups (cyt b and cyt c1) are shown in red. The remaining subunits of the complex are rendered in grey. (b) Molecular model of atovaquone (Atv) bound to the Qo site of the bc1 complex. Subunits are coloured as in panel (a). Atovaquone was modelled into the Qo site of cytochrome b as described by Fisher et al.46 Hydrogen-bonding interactions between the naphthoquinone head group of atovaquone and side chains of Glu-272 (cyt b) and His-181 (ISP) are indicated by yellow lines. The positions of haem bl (cyt b) and the ISP [2Fe2S] cluster are also shown. Figure 3. (a) Cartoon representation of the yeast cytochrome bc1 complex (3CX5.PDB), with atovaquone modelled at the Qo site (boxed area).83 The bc1 complex is a structural and functional homodimer with a molecular mass of ≏480 kDa, consisting of 10 discrete subunits per monomer in yeast and P. falciparum.28 The electron-transferring catalytic unit of one monomer is highlighted; cytochrome b is represented in orange, cytochrome c1 in blue and the Rieske iron-sulphur protein (ISP) in green. Haem groups (cyt b and cyt c1) are shown in red. The remaining subunits of the complex are rendered in grey. (b) Molecular model of atovaquone (Atv) bound to the Qo site of the bc1 complex. Subunits are coloured as in panel (a). Atovaquone was modelled into the Qo site of cytochrome b as described by Fisher et al.46 Hydrogen-bonding interactions between the naphthoquinone head group of atovaquone and side chains of Glu-272 (cyt b) and His-181 (ISP) are indicated by yellow lines. The positions of haem bl (cyt b) and the ISP [2Fe2S] cluster are also shown. the biological target, it is likely that new bc1-target antimalarials will require marriage with a partner drug, unless the candidate drugs possess biologically distinct polypharmacology. Mode of action It is well documented that atovaquone monotherapy gives rise to de novo resistance very rapidly.47,48 However, the under- lying reason for this phenomenon has not been determined and, as discussed in the next section, may be partially explained by pharmacodynamic/pharmacokinetic considerations (related to the physicochemical properties of atovaquone combined with a slow rate of sterilization) as well as hitherto untested con- siderations related to the molecular target such as, e.g. the effect of an increased mutation rate of mitochondrially encoded genes such as cytochrome b compared with nuclear encoded genes.49 Mode of action (b) Williams and Clarke atovaquone synthesis. (c) Improved atovaquone synthesis. (c) O O Cl Cl O O S Cl N 16 80% 48:38 ratio cis:trans Cl + O O N S OH Cl O O N S DCC O O S Cl N 13 14 15 88% 80% (c) HO2C 15 13 O O OH Cl Atovaquone eparation Base O O OH Cl 17 80% Isomer s 48:38 ratio cis:trans 48:38 ratio cis:trans Isomer separation Base O Atovaquone Atovaquone 48:38 ratio cis:trans Figure 2. Synthetic routes used to synthesize atovaquone. (a) The original synthesis of atovaquone. (b) Williams and Clarke atovaquone synthesis. (c) Improved atovaquone synthesis. Figure 2. Synthetic routes used to synthesize atovaquone. (a) The original synthesis of atovaquone. (b) Willi (c) Improved atovaquone synthesis. binding. The resultant atovaquone-resistant growth IC50 (half- maximal inhibitory concentration) phenotype of these mutants is some 1000-fold higher than susceptible strains; however, this is accompanied by an ≏40% reduction in the Vmax of the bc1 complex, suggestive of a signiﬁcant ﬁtness cost to the parasite.46 Malarone drug failure has been associated with a missense point mutation at position 268 in cytochrome b, exchanging tyrosine for serine (Y268S) or, less frequently, asparagine (Y268N).41 – 45 Position 268 in cytochrome b is highly conserved across all phyla and is located within the ‘ef’ helix component of the Qo site, which is putatively involved in ubiquinol 979 Review (a) (b) ISP:[2Fe2S] ISP:H181 b:E272 bl Atv Figure 3. (a) Cartoon representation of the yeast cytochrome bc1 complex (3CX5.PDB), with atovaquone modelled at the Qo site (boxed area).83 The bc1 complex is a structural and functional homodimer with a molecular mass of ≏480 kDa, consisting of 10 discrete subunits per monomer in yeast and P. falciparum.28 The electron-transferring catalytic unit of one monomer is highlighted; cytochrome b is represented in orange, cytochrome c1 in blue and the Rieske iron-sulphur protein (ISP) in green. Haem groups (cyt b and cyt c1) are shown in red. The remaining subunits of the complex are rendered in grey. (b) Molecular model of atovaquone (Atv) bound to the Qo site of the bc1 complex. Subunits are coloured as in panel (a). Atovaquone was modelled into the Qo site of cytochrome b as described by Fisher et al.46 Hydrogen-bonding interactions between the naphthoquinone head group of atovaquone and side chains of Glu-272 (cyt b) and His-181 (ISP) are indicated by yellow lines. Pharmacokinetics The pharmacokinetic parameters of atovaquone in the currently utilized formulation (Malarone, 250 mg atovaquone+100 mg proguanil) have been determined (Figure 4).52 The median ato- vaquone plasma AUC (h/mM), t1/2 (h), Cmax (mM) and Tmax (h) were 295, 87.2, 3.74 and 3.25, respectively, following a single dose and 254, 55.9, 13.8 and 4.00, respectively, upon reaching steady state. The similar AUC values observed between single- dose and steady-state dosing suggest no unexpected accumula- tion of atovaquone following repeated administration, although this may be due to saturation of plasma atovaquone concentra- tions, and an increase in atovaquone concentrations in tissues cannot be ruled out. Furthermore, it has been reported that an in vitro atovaquone- resistant parasite line has been generated in the laboratory possessing wild-type cytochrome b.50 The mechanism under- pinning the parasite’s atovaquone-resistant phenotype in this strain remains to be elucidated. The speed of development of resistance to a new antimalarial is an important consideration. According to the Medicines for Malaria Venture (MMV) target product proﬁles (TPPs), pre-clinical development of new bc1-acting antimalarials must show activity against a panel of multidrug-resistant antimalarial parasites that include atovaquone-resistant isolates. There are also in vitro speed of development of resistance assays that are available that can be used to guide go/no-go development decisions.51 Whether the observed rapid onset of de novo resistance seen in atovaquone is based on the physicochemical property of the molecule or whether it is based on inherent issues relating to Atovaquone IC50 against susceptible malaria in vitro is very low, ranging from 1 to ≏3.5 nM.31,53,54 This has resulted in the belief that atovaquone plasma concentrations (around 1–10 mM; see Figure 4) are sufﬁcient to produce total suppres- sion of malaria. However, atovaquone shows extremely high levels of plasma protein binding (.99.5%) and therefore the con- centration of unbound atovaquone is likely to be signiﬁcantly lower.55 Extrapolations of pharmacokinetic/pharmacodynamic 980 JAC Review Figure 4. Atovaquone plasma concentration–time proﬁle after a single dose of Malarone in 13 healthy individuals. Reproduced with permission from the study by Thapar et al.52 Figure 4. Atovaquone plasma concentration–time proﬁle after a single dose of Malarone in 13 healthy individuals. Reproduced with permission from the study by Thapar et al.52 dynamics using in vitro data should therefore be treated with caution. Absorption Absorption of atovaquone shows dose limitations, with maximum absorption observed using 750 mg tablets.59 Poor drug solubility was suggested as the cause of this limit to ab- sorption, and this led to the development of an atovaquone liquid suspension formulation that showed improved Pneumo- cystis pneumonia treatment success compared with the tablet formulation.60 Pharmacokinetics six clinical trials, the interpatient variability of atovaquone bio- availability is substantial and has been determined to be 107%, which is likely due to the drug’s low solubility and the effects of food.61–63 At present, there are no established minimum effective plasma concentrations of atovaquone for malaria prophylaxis. However, a clear correlation between atovaquone steady-state plasma concentration and treatment success has been estab- lished in Pneumocystis pneumonia in patients with AIDS.56 Ato- vaquone plasma concentrations of 10 to ,15 mg/mL and 15 to ,20 mg/mL resulted in 79% and 95% treatment success, re- spectively. Furthermore, there have been case reports of atova- quone treatment failure in antimalarial therapy that were not explained by drug resistance mutations, and patients with a body weight .100 kg have a marked increased chance of treat- ment failure compared with ,100 kg patients, both of which suggest drug concentration may be a factor in determining treatment failure.42,57,58 The prediction of atovaquone therapy failure and resistance selection using drug concentration para- meters has the potential to improve current patient therapy and an investigation determining a pharmacokinetic/pharmaco- dynamic relationship is warranted. The oral absorption of atovaquone increased when taken with a high-fat meal (two slices of toast with 56 g of butter, with 3.9-fold exposure compared with fasting), whereas a minimal- fat meal (two slices of toast) had minimal impact on absorp- tion.63 Consequently it is recommended that atovaquone be taken with a high-fat meal. However, a recent in vitro study showed that the atovaquone IC50 increased 20-fold when serum used in the assay was taken from a subject recently given a high-fat meal compared with serum from a fasting subject (0.5–12 ng/mL, P,0.01).64 A correlation between high serum triglyceride concentrations and high atovaquone IC50 was observed, suggesting reduced free (unbound) atovaquone concentrations due to increased drug–fat binding. The clinical relevance of this ﬁnding is unknown, but the impact to atova- quone pharmacokinetics is likely to be transient and is unlikely to outweigh the beneﬁt of increased atovaquone absorption. Dissolution of atovaquone tablets increases in the presence of milk, and therefore the presence of milk in meals may increase atovaquone bioavailability in patients.62 This may provide an al- ternative strategy to high-fat meals when aiming to maximize the bioavailability of atovaquone, although this has not been shown clinically. Distribution Atovaquone is highly bound to plasma protein (.99.5%) and shows a high afﬁnity for human serum albumin, although the low drug clearance rate suggests that atovaquone may also The bioavailability of 750 mg atovaquone when taken with food was 23% in HIV-infected patients.61 Combining data from 981 Review accumulate in tissues, where it is protected from biliary clear- ance.55 In a study of atovaquone population pharmacokinetics, the volume of distribution of atovaquone was 7.98 L/kg, al- though individual values were markedly linked to body weight; the volume of distribution shows a linear increase with increased patient body weight.61 that atovaquone did not alter the pharmacokinetics of the anti- epileptic drug phenytoin, another highly protein-bound drug, which is susceptible to displacement interactions.69 The evidence that atovaquone can compete with other drugs for plasma protein binding is lacking, although further investigations are required to fully understand this potential factor in atovaquone pharmacokinetics. Atovaquone exposure is markedly decreased when taken con- comitantly with the antibiotic drug rifampicin and therefore co-administration of atovaquone and rifampicin is not recom- mended.70 The mechanism behind this interaction is not fully understood, although the ability of rifampicin to induce activity in metabolism enzymes and drug transporters is assumed to be responsible. However, no metabolite of atovaquone has been identiﬁed in humans, and the impact of individual enzymes and transporters on atovaquone disposition is unclear. Metabolism Under normal conditions, there is no evidence that atovaquone is signiﬁcantly metabolized in humans, or that metabolism is required for drug elimination. It may be possible that certain enzymes could be induced and therefore lead to increased ato- vaquone biotransformation, but this has not been demonstrated. Elimination y p q p There is evidence that atovaquone can inhibit cytochrome P450 enzymes, although data have been generated in vitro and the relevance to clinical drug interactions is unknown. Atova- quone inhibited the metabolism of 50 mM of 7-benzyloxy-4- (triﬂuoromethyl)-coumarin (BFC) by recombinant CYP3A4, with an IC50 of 4.7 mM.52 Similarly, sulfamethoxazole metabolism by recombinant CYP2C9 was inhibited by atovaquone, with an inhib- ition constant (Ki) of 15 mM.71 However, when atovaquone was pre-incubated with human serum and centrifuge ﬁltered to remove protein before use, no CYP2C9 inhibitory activity was observed. A recent case study described an HIV-infected female with a marked increase in plasma concentrations of the antiretroviral drugs etravirine (+55%) and unboosted saquinavir (+274%) following atovaquone/proguanil prophylaxis.72 In the same study, raltegravir plasma concentrations were unchanged following atovaquone/proguanil prophylaxis. The evidence that atovaquone/proguanil prophylaxis increases exposure of etravirine and saquinavir (both cytochrome P450 substrates) but not ralte- gravir (no afﬁnity for cytochrome P450 enzymes) suggests atova- quone, proguanil or both drugs may be inhibiting cytochrome P450 activity.73–75 Atovaquone pharmacokinetics are characterized by an extremely long elimination half-life of ≏50–84 h.59,63,65 Elimination is pri- marily via the liver, with almost undetectable amounts (,0.6%) of drug being eliminated via the kidneys.66 More than 90% of the drug excreted in bile was in the parent form. Elimin- ation of atovaquone is complicated by the possibility of entero- hepatic recirculation of the drug, which may help explain atovaquone pharmacokinetic proﬁles where a reduction and then an increase in drug concentration is seen with time. In a study of atovaquone population pharmacokinetics, the oral clearance of atovaquone was increased in patients with higher body weights, with 60% increased clearance seen in an 80 kg patient compared with a 40 kg patient.61 In the same study, the average oral clearance of atovaquone was higher in Oriental (8.49 L/h) and Malay (9.13 L/h) subjects compared with White (1–7.6 L/h) subjects.61 Conclusions Despite the extensive use of atovaquone/proguanil, there remains a considerable knowledge gap concerning its pharma- cology. The rollout of generics following the expiration of this patent will undoubtedly see an increase in atovaquone/proguanil usage that will be closely followed by an increase in treatment failures. Clearly, if the community is to manage this issue and develop improved derivatives, more effort needs to be directed towards understanding the pharmacokinetic/pharmacodynamic mechanisms underpinning atovaquone/proguanil treatment failure. 14 Fawaz G, Haddad FS. The effect of lapinone (M-2350) on P. vivax infection in man. Am J Trop Med 1951; 31: 569–71. 15 Hudson AT, Randall AW, Fry M et al. Novel anti-malarial hydroxynaphthoquinones with potent broad-spectrum anti-protozoal activity. Parasitology 1985; 90: 45–55. 16 Hudson AT, Pether MJ, Randall AW et al. In vitro activity of 2-cycloalkyl-3-hydroxy-1,4-naphthoquinones against Theileria, Eimeria and Plasmodia species. Eur J Med Chem 1986; 21: 271–5. 17 Hudson AT, Dickins M, Ginger CD et al. 566C80: a potent broad spectrum anti-infective agent with activity against malaria and opportunistic infections in AIDS patients. Drugs Exp Clin Res 1991; 17: 427–35. Safety and toxicology Atovaquone has been found to be generally well tolerated and causes few side effects. Adverse events are generally mild and include rash, fever, vomiting, diarrhoea, abdominal pain and headache. Indeed, overdoses as large as 31500 mg have been reported to cause little or no symptomatology.81 8 Lalloo DG, Hill DR. Preventing malaria in travellers. BMJ 2008; 336: 1362–6. 9 LaRocque RC, Rao SR, Lee J et al. Global TravEpiNet: a national consortium of clinics providing care to international travelers—analysis of demographic characteristics, travel destinations, and pretravel healthcare of high-risk US international travelers, 2009–2011. Clin Infect Dis 2012; 54: 455–62. A signiﬁcant concern for the development of novel antimalar- ials targeting the parasite bc1 is host mitochondrial toxicity. In animal models this manifests itself as acute toxicity (presumed to be cardiotoxicity). Current development projects use in vitro counter-screens such as human bc1 screening or human cell lines grown on galactose, making these cells more reliant on mitochondrial metabolism by circumventing the Crabtree effect.82 However, these projects are hampered by the absence of industry standards relating to pre-clinical or clinical mitochon- drial toxicity. 10 Fieser LF, Richardson AP. Naphthoquinone antimalarials. II. Correlation of structure and activity against P. lophurae in ducks. J Am Chem Soc 1948; 70: 3156–65. 11 Fieser LF, Heymann H, Seligman AM. Naphthoquinone antimalarials. XX. Metabolic degradation. J Pharmacol Exp Ther 1948; 94: 112–24. 12 Fieser LF, Chang FC, Dauben WG et al. Naphthoquinone antimalarials. XVIII. Metabolic oxidation products. J Pharmacol Exp Ther 1948; 94: 85–96. 13 Fieser LF, Schirmer JP, Archer S et al. Naphthoquinone antimalarials. XXIX. 2-Hydroxy-3-(omega-cyclohexylalkyl)-1,4-naphthoquinones. J Med Chem 1967; 10: 513–7. JAC JAC JAC Review (Malarone) for chemoprophylaxis against malaria. J Antimicrob Chemother 2007; 60: 929–36. (Malarone) for chemoprophylaxis against malaria. J Antimicrob Chemother 2007; 60: 929–36. Therefore, clearance of UGT2B7 substrates, such as the anti-HIV drug efavirenz, may also be inﬂuenced by atovaquone, and further investigations are warranted in this area.78 4 Looareesuwan S, Chulay JD, Canﬁeld CJ et al. Malaronew (atovaquone and proguanil hydrochloride): a review of its clinical development for treatment of malaria. Am J Trop Med Hyg 1999; 60: 533–41. Atovaquone did not alter the exposure of the anti-HIV prote- ase inhibitor drug indinavir in healthy volunteers.79 Indinavir is a substrate of the drug efﬂux transporter ABCB1, and the absence of any effect of atovaquone on indinavir pharmacokinetics sug- gests that atovaquone is not altering the activity of ABCB1, al- though this has not been conﬁrmed.80 5 Spencer CM, Goa KL. Atovaquone—a review of its pharmacological properties and therapeutic efﬁcacy in opportunistic infections. Drugs 1995; 50: 176–96. 6 Haile LG, Flaherty JF. Atovaquone—a review. Ann Pharmacother 1993; 27: 1488–94. 7 Osei-Akoto A, Orton LC, Owusu-Ofori S. Atovaquone-proguanil for treating uncomplicated malaria. Cochrane Database Syst Rev 2005; Issue 4: CD004529. Transparency declarations None to declare. 21 Patent WO/2010/001379. A process for preparing atovaquone and associate intermediates. http://patentscope.wipo.int/search/en/ WO2010001379 (8 November 2012, date last accessed). Acknowledgements 18 Patent WO9320044. 1,4 Naphthoquinone derivatives with anti- protozoal and anti-parasitic activity. http://www.freepatentsonline.com/ EP0634996.html (8 November 2012, date last accessed). We acknowledge grant support from the Leverhulme Trust, Wellcome Trust, EU FP7, Medical Research Council (MRC) and Medicines for Malaria Venture (MMV). 19 Patent 4981874. Medicaments. http://www.google.com/patents/ US4981874 (8 November 2012, date last accessed). 20 Williams DR, Clark MP. Synthesis of atovaquone. Tetrahedron Lett 1998; 39: 7629–32. Drug interactions Atovaquone is highly bound to plasma protein (.99.5%) and shows a high afﬁnity for human serum albumin.55 Furthermore, the half-life of atovaquone is long, ranging from ≏50 to 84 h, and the major limiting factor to atovaquone clearance is prob- ably its plasma protein binding.59,63,65 This suggests that any drug that reduces atovaquone plasma protein binding may po- tentially alter atovaquone tissue distribution and/or clearance. However, the authors can ﬁnd no published articles investigating the drug-mediated displacement of atovaquone from plasma protein and the clinical impact of these interactions, and this area requires further research. The interaction observed between atovaquone and antiretrovirals, where efavirenz, lopina- vir and ritonavir (all highly protein-bound drugs) reduced atova- quone plasma concentrations in HIV-infected patients, may involve atovaquone plasma protein displacement, although this was not demonstrated.67 This emphasizes the importance of establishing the interactions between antimalarials, including atovaquone, and antiretrovirals. Co-administration of atovaquone and the nucleoside reverse transcriptase inhibitor zidovudine increased the exposure (33% increase in AUC0–8, P,0.05) and decreased the oral clear- ance (25% reduction, P,0.05) of zidovudine in HIV-infected patients.76 Furthermore, patients taking atovaquone showed a trend towards lower zidovudine/glucuronide plasma concentra- tions (6% reduction in AUC0-8, P,0.1) and a signiﬁcant decrease in the ratio between zidovudine/glucuronide and plasma concen- trations (30% reduction, P,0.05). Atovaquone exposure was un- changed when co-administered with zidovudine. The atovaquone-mediated 33% increase in zidovudine expos- ure is itself unlikely to cause increased haematological toxicity, although caution is advised in patients taking additional drugs with similar toxicity proﬁles to zidovudine.76 Also, increased zidov- udine plasma concentrations and reduced zidovudine glucuroni- dation may potentially lead to increased formation of the cytochrome P450-mediated zidovudine metabolite 3′-amino-3′- deoxythymidine, which shows a 7-fold higher toxicity in bone marrow cells compared with the parent drug.77 The potential for atovaquone to displace other protein-bound drugs has been investigated. A case study was recently published that describes a potential interaction between the anticoagulant drug warfarin and atovaquone, where the author suggests that atovaquone caused an increase in free warfarin concentrations to super-therapeutic levels.68 A separate investigation found The increased exposure and decreased clearance of zidovudine suggests that atovaquone is inhibiting the glucuronidation of zidov- udine. The primary enzyme involved in zidovudine glucuronidation is uridine 5′-diphospho-glucuronosyltransferase (UGT) 2B7.78 982 References 28 Barton V, Fisher N, Biagini GA et al. Inhibiting Plasmodium cytochrome bc1: a complex issue. Curr Opin Chem Biol 2010; 14: 440–6. 47 Chiodini PL, Conlon CP, Hutchinson DB et al. Evaluation of atovaquone in the treatment of patients with uncomplicated Plasmodium falciparum malaria. J Antimicrob Chemother 1995; 36: 1073–8. 29 Winter RW, Kelly JX, Smilkstein MJ et al. Optimization of endochin-like quinolones for antimalarial activity. 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https://openalex.org/W2887128163	https://europepmc.org/articles/pmc6158747?pdf=render	English	null	Tunable order–disorder continuum in protein–DNA interactions	Nucleic acids research	2,018	cc-by	9,992	ABSTRACT bind proteins. This arises from two specific factors: the large negative charge density on DNA and the necessity to scan millions of very similar sequences to find their target site and promote regulation (1–3). The negative charge density from the phosphates on DNA requires the DBDs to pos- sess equal but positively charged residues in close vicinity in their 3D structure to promote efficient binding (‘electro- static complementarity’ (4)). This co-evolved protein struc- tural feature in turn leads to large frustration (5), a phe- nomenon that is also observed in the active sites of many proteins and enzymes (5–7). The energetic or electrostatic frustration helps the DBDs to sample structurally differ- ent conformations in equilibrium as the unfavorable inter- actions just about keeps the protein folded. Some confor- mations possess the right orientation of charged residues for specific and tight binding, while the other conformations promote non-specific and weak binding. This inherent plas- ticity enables DBDs to quickly explore numerous and ener- getically degenerate sequences with ease (8–11).ii DNA-binding protein domains (DBDs) sample di- verse conformations in equilibrium facilitating the search and recognition of speciﬁc sites on DNA over millions of energetically degenerate competing sites. We hypothesize that DBDs have co-evolved to sense and exploit the strong electric potential from the array of negatively charged phosphate groups on DNA. We test our hypothesis by employing the intrinsically disordered DBD of cytidine repressor (CytR) as a model system. CytR displays a graded increase in structure, stability and folding rate on increasing the osmolarity of the solution that mim- ics the non-speciﬁc screening by DNA phosphates. Electrostatic calculations and an Ising-like statisti- cal mechanical model predict that CytR exhibits fea- tures of an electric potential sensor modulating its dimensions and landscape in a unique distance- dependent manner, while DNA plays the role of a non-speciﬁc macromolecular chaperone. Accord- ingly, CytR binds its natural half-site faster than the diffusion-controlled limit and even random DNA con- forming to an electrostatic-steering binding mecha- nism. Our work unravels for the ﬁrst time the syn- ergistic features of a natural electrostatic potential sensor, a novel binding mechanism driven by elec- trostatic frustration and disorder, and the role of DNA in promoting distance-dependent protein struc- tural transitions critical for switching between spe- ciﬁc and non-speciﬁc DNA-binding modes. C⃝The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT The extent of specific and non-specific interactions between DBDs and DNA is in turn determined by the protein primary sequence, the folded structure and even by the folding mechanism. This can lead to sev- eral interesting folding–binding–translocation–regulation mechanisms––folding-rate acceleration (12), folding-upon- binding/binding-upon-folding (13,14) or combination of the two (15), synergistic folding (14,16,17), ‘fly-casting’ (18,19), ‘monkey-bar’ binding (20), conformational switch- ing (21–26), homo- versus heterodimerization (27) and pro- tein co-localization (28–30)––all of which determine the ex- tent of time the protein spends bound to DNA, its 1D dif- fusion coefficient relative to the time spent freely diffusing in solution (14,31–34) and hence fine-tuned expression of genes. In many cases, the differences between specific and non-specific binding poses are subtle (8,11,22,35) and can result in distinct cooperative effects (21,36). Sneha Munshi1,†, Soundhararajan Gopi1,†, Gitanjali Asampille2, Sandhyaa Subramanian1, Luis A. Campos3, Hanudatta S. Atreya2 and Athi N. Naganathan1,* 1Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India, 2NMR Research Centre, Indian Institute of Science, Bangalore 560012, India and 3National Biotechnology Center, Consejo Superior de Investigaciones Cient´ıﬁcas, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain Received June 20, 2018; Revised July 24, 2018; Editorial Decision July 28, 2018; Accepted July 31, 2018 Published online 11 August 2018 Published online 11 August 2018 8700–8709 Nucleic Acids Research, 2018, Vol. 46, No. 17 doi: 10.1093/nar/gky732 *To whom correspondence should be addressed. Tel: +91 44 2257 4140; Fax: +91 44 2257 4102; Email: athi@iitm.ac.in †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. Kinetics Ionic strength and urea-dependent kinetic experiments were performed at 298 K, pH 7.0 in a Chirascan SF.3 Stopped Flow instrument (Applied Photophysics Ltd.; dead-time ∼2 ms) coupled to a thermostated water bath. The sole tyro- sine in CytR (Y53) was excited at 280 nm, kinetic traces col- lected, averaged (from at least six repeats with one-minute equilibration between individual repeats) and fit to single- exponential functions. The starting buffer for ionic strength dependent folding and unfolding experiments was at 11 and 2500 mM ionic strength, respectively. The starting protein concentrations were ∼200 M with the final concentrations (after mixing) of ∼18 M. The kinetics of association was monitored by recording anisotropy traces of Alexa-532 labeled udp half-site with ex- citation and emission wavelengths of 530 and 570 nm, re- spectively. Both DNA and CytR were dissolved in 50 mM sodium phosphate buffer, 30 mM sodium chloride and 1 mM ethylenediaminetetraacetic acid, pH 6.0. Binding was initiated by 1:1 mixing of Alexa-532 labeled DNA with ex- cess CytR DBD mimicking pseudo-first order conditions. For each protein concentration, six traces were recorded at an interval of one minute and averaged. In the case of fully folded proteins or ligands that carry excess positive charges, the large negative electrostatic po- tential of DNA is expected to merely ‘pull’ them toward the center of attraction thus promoting binding. However, what happens when the protein is less structured or disordered, as is the case for CytR? If they are frustrated electrostati- cally due to the presence of excess positive charges, the intu- itive expectation is that the non-specific electrostatic poten- tial should promote folding of such disordered proteins in a distance-dependent manner due to progressively stronger charge screening as the protein approaches DNA. We test this hypothesis in the current work through salt-screening experiments on CytR and a statistical mechanical model. We identify a unique mechanism determining the heteroge- neous binding of CytR to DNA, a feature that could also be prevalent in other disordered and even folded proteins. Protein expression, purification and spectroscopy The protocol for overexpression of CytR, purification and spectroscopic measurements is outlined in detail in (36). The 2D [15N, 1H]-HSQC spectra were recorded at 298 K on a Bruker Avance III 800 MHz nuclear magnetic reso- nance (NMR) spectrometer equipped with a cryogenically cooled triple resonance probe. The spectra were acquired with 4 transients and 256 complex points, with an average measurement time of 20 minutes at protein concentrations of 200–400 M. wi = N j=1 exp −G j,DNA ρ j/RT , where ρj is the folded status of the residue j in microstate i, N is the number of residues, R is 8.314 J mol−1 K−1 and T is the temperature. The free energy contribution due to the in- teraction of residue j with DNA (ΔGj,DNA) includes van der Waals interactions as identified using a G¯o-like approach employing a 5 ˚A heavy atom distance cutoff and electro- static interactions between the residue j of CytR and ev- ery phosphate group on DNA (non-specific charge–charge interactions). The bound conformation of CytR was mod- eled in PyMOL (54) with the LacR structure (1CJG) as the INTRODUCTION DNA-binding protein domains (DBDs) are structurally and conformationally distinct from their counterparts that Nucleic Acids Research, 2018, Vol. 46, No. 17 8701 One underlying theme to all of the mechanisms reported above is the conformational malleability of DBDs. The co- evolved but frustrated landscape of DBDs can thus mani- fest as complex binding thermodynamics (37–40), anoma- lous heat capacity profiles (41), downhill-like folding mech- anistic behaviors (14) and large dynamics even in the DNA- bound form (42,43). An extreme case is that of a DBD fold- ing upon binding to DNA (or vice versa), while it remains disordered in the absence of DNA. Such a phenomenon is frequently observed in protein–protein interactions where one partner remains folded while the other protein domain folds upon binding (44,45). Cytidine repressor (CytR) DBD (referred to as CytR) is an intrinsically disordered protein (IDP) that binds its target udp half-site with a weak affinity (46), and promiscuously to multiple other sites (47), despite assuming a folded-like structure on binding. In fact, a de- tailed analysis revealed that CytR is highly frustrated elec- trostatically, samples multiple conformations in equilibrium driven by non-specific and a continuous collapse transition, but this conformational heterogeneity is also translated into binding heterogeneity (36), reminiscent of the ‘fuzzy’ com- plexes in protein–protein interactions (48). Differential scanning calorimetry (DSC) and variable-barrier (VB) model The scanning calorimetry experiments were recorded in a MicroCal VP-Capillary DSC with an automated sample in- jector as described before (36). The variable-barrier (VB) model analysis was performed on the absolute heat capacity thermograms of CytR at the three explored ionic strength conditions by fixing the Freire folded baseline (49). The final parameters at [43, 600 and 1300] mM ionic strength condi- tions are: α = [1554.9, 585.1 and 121.1] kJ mol−1; T0 = [291.7, 313.6 and 337.3] K; β = [−173.7, −12.14 and 0.12] kJ mol−1; f = [0.535, 0.631 and 0.903]. MATERIALS AND METHODS The Wako–Saitˆo–Mu˜noz–Eaton (WSME) model (50,51) in its latest version includes contributions from intramolecu- lar van der Waals interactions, electrostatics and solvation apart from conformational entropy (52,53) (see Support- ing Methods). In the current work, the basic WSME model terms of CytR DBD (PDB ID: 2L8N) are supplemented with an extra weighting term on residue j. Protein expression, purification and spectroscopy Charge screening promotes structure in CytR To explore the predictions experimentally, we systematically increase the ionic strength of the buffer by adding salt and probe for the effect on secondary and tertiary structure of the protein. The far-UV CD monitored secondary structure increases continuously on adding salt ranging from −6000 deg. cm2 dmol−1 at 298 K and 11 mM ionic strength buffer to −15 000 deg. cm2 dmol−1 at 2.5 M ionic strength condi- tions (Figure 2A). In fact, the signal at 298 K approaches that of the folded PurR or LacR (which are fully folded in the absence of DNA) at the highest salt concentrations in- dicating a fully folded domain. The apparent melting tem- perature also shows an increasing trend ranging from ∼303 K at 11 mM, ∼340 K at 1.7 M and finally to 345 K at 2.5 M ionic strength conditions (Figure 2A). A shorter CytR construct, which does not include the long unstructured N- and C-terminal residues, exhibits a similar increase in struc- ture and stability highlighting that the stability modulation is an intrinsic feature of the sequence region that folds in the presence of DNA (Supplementary Figure S2). Analytical ultracentrifugation (AUC) The calculation of overall partition function, free en- ergy profiles and residue probabilities are described in detail elsewhere (50,52). The final model parameters are––mean interaction energy per native contact (ξ) = −217.1 J mol−1, entropic cost for fixing a residue in native conformation (Sconf) = −33.31 J mol−1 K−1 per residue and the temper- ature independent heat capacity per native contact (Ccont p ) template and energy minimized in GROMACS (55). A se- ries of poses spatially displaced from DNA were generated in PyMOL and used for model predictions on the role of DNA. The calculation of overall partition function, free en- ergy profiles and residue probabilities are described in detail elsewhere (50,52). The final model parameters are––mean interaction energy per native contact (ξ) = −217.1 J mol−1, entropic cost for fixing a residue in native conformation (Sconf) = −33.31 J mol−1 K−1 per residue and the temper- ature independent heat capacity per native contact (Ccont p ) DNA (K18, K20, R43, K46) are as frustrated, if not more, as those residues that come together to form favorable inter- actions with the DNA backbone (K13, R28 and K35; Fig- ure 1A and B). TK electrostatic interaction energy calcula- tions (57,58) reveal that CytR exhibits unfavorable interac- tions between helices 1 and 3, i.e. long-range interactions that hold the protein together. p g This can be observed in the plots of the electrostatic in- teraction energy as a function of ionic strength: increas- ing ionic strength screens local interactions making them more unfavorable while promoting long-range (non-local) charge–charge interactions (Figure 1C). Beyond 500 mM ionic strength, both the interactions contribute equally to the overall stability. Electrostatic potential calculations in fact indicate that as the ionic strength is increased, the un- favorable interactions are progressively screened thus re- ducing electrostatic frustration (Supplementary Figure S1). These observations hint that an intrinsic conflict between local and non-local electrostatic interactions could be one of the fundamental factors contributing to the low stability and structure of CytR. = −2.33 J mol−1 K−1. An uniform dielectric constant (ε = 29) was used to scale both intramolecular (εprot; between the charged residues on the protein (52,56)) and intermolecular (εprot,DNA; between protein and DNA) electrostatic interac- tions. The latter was also varied from the 29 to 74.3 with little overall changes in the model predictions. Electrostatic calculations The net electrostatic interaction energy (pH 7.0, 310 K, 100 mM ionic strength) between folded CytR and DNA was cal- culated at different distances employing a simplified Debye– H¨uckel formalism (52). Tanford–Kirkwood (TK) electro- static calculations were carried out as before (57,58) to ex- tract the pair-wise charge–charge interaction energies of CytR at varying ionic strength conditions. The electrostatic potential around CytR and DNA was calculated with the Adaptive Poisson−Boltzmann Solver (59,60). The charges were assigned using the PDB2PQR module employing AM- BER charge set, while the residue protonation states at pH 7 were assigned using the PROPKA routine. The non-linear PB equations were numerically solved at 310 K, on a 193 × 193 × 193 ˚A grid with 100 grid points per ˚A2 for sur- face construction. The dielectric constant was set to default (78.5 for solvent and 2.0 for protein interior) and the ion radii were set to 2 ˚A. The net electrostatic potential of the molecules was calculated at 2 ˚A from the molecular surface. Analytical ultracentrifugation (AUC) Sedimentation velocity experiments were carried out in Op- tima XL-I (ultraviolet (UV)–VIS absorbance at 280 nm and interference detection) at 48 000 rpm and at 298 K at a CytR concentration of ∼50 M. The sedimentation velocity pro- files were collected at different time intervals and the sedi- mentation coefficients calculated using SEDFIT with cor- rected buffer densities and viscosities. 8702 Nucleic Acids Research, 2018, Vol. 46, No. 17 Figure 1. Electrostatic frustration in CytR (A). The large positive surface potential on the DNA-binding face. (B) Left: Same orientation as in panel (A) but with the residues labeled. Right: Identity of the positively charged residues that do not bind DNA but are still frustrated. (C) Charge–charge interaction energy of folded CytR as a function of ionic strength and as calculated from the TK algorithm. Non-local interactions are identified with a sequence separation >4. Figure 1. Electrostatic frustration in CytR (A). The large positive surface potential on the DNA-binding face. (B) Left: Same orientation as in panel (A) but with the residues labeled. Right: Identity of the positively charged residues that do not bind DNA but are still frustrated. (C) Charge–charge interaction energy of folded CytR as a function of ionic strength and as calculated from the TK algorithm. Non-local interactions are identified with a sequence separation >4. template and energy minimized in GROMACS (55). A se- ries of poses spatially displaced from DNA were generated in PyMOL and used for model predictions on the role of DNA. The calculation of overall partition function, free en- ergy profiles and residue probabilities are described in detail elsewhere (50,52). The final model parameters are––mean interaction energy per native contact (ξ) = −217.1 J mol−1, entropic cost for fixing a residue in native conformation (Sconf) = −33.31 J mol−1 K−1 per residue and the temper- ature independent heat capacity per native contact (Ccont p ) = −2.33 J mol−1 K−1. An uniform dielectric constant (ε = 29) was used to scale both intramolecular (εprot; between the charged residues on the protein (52,56)) and intermolecular (εprot,DNA; between protein and DNA) electrostatic interac- tions. The latter was also varied from the 29 to 74.3 with little overall changes in the model predictions. template and energy minimized in GROMACS (55). A se- ries of poses spatially displaced from DNA were generated in PyMOL and used for model predictions on the role of DNA. Electrostatic frustration in CytR Electrostatic frustration in CytR Unfavorable electrostatic interaction is a characteristic fea- ture of DBDs that bind DNA. In this regard, the folded CytR (i.e. the structure in the presence of DNA) exhibits a unique feature wherein specific residues that are far from CytR possesses a single tyrosine (Y53) thus allowing us to probe for the changes in the tertiary environment on Nucleic Acids Research, 2018, Vol. 46, No. 17 8703 Figure 2. Charge screening promotes a continuum of structural states in CytR (A and B). Far-UV and near-UV CD unfolding curves at varying ionic strength conditions. MRE represents mean residue ellipticity in units of deg. cm2 dmol−1. (C and D) Global singular value decomposition (SVD) of temperature–wavelength fluorescence data of CytR unfolding at different ionic strength conditions. The first and second basis spectra (which shows a red shift) are displayed in black and gray, respectively. The amplitudes of the second basic spectra as a function of temperature are shown in panel (D) highlighting the temperatures at which they change sign depending on the stability conditions. (E) Overlay of 15N,1H-HSQC spectra at 43 mM (green), 430 mM (blue) and 1000 mM (red) ionic strength conditions. (F and G) Corrected sedimentation velocity distributions and the apparent Stokes radii (RS) of CytR at the two extreme ionic strength conditions. The dashed horizontal line in panel (G) signals the dimensions of a perfect sphere. Figure 2. Charge screening promotes a continuum of structural states in CytR (A and B). Far-UV and near-UV CD unfolding curves at varying ionic strength conditions. MRE represents mean residue ellipticity in units of deg. cm2 dmol−1. (C and D) Global singular value decomposition (SVD) of temperature–wavelength fluorescence data of CytR unfolding at different ionic strength conditions. The first and second basis spectra (which shows a red shift) are displayed in black and gray, respectively. The amplitudes of the second basic spectra as a function of temperature are shown in panel (D) highlighting the temperatures at which they change sign depending on the stability conditions. (E) Overlay of 15N,1H-HSQC spectra at 43 mM (green), 430 mM (blue) and 1000 mM (red) ionic strength conditions. (F and G) Corrected sedimentation velocity distributions and the apparent Stokes radii (RS) of CytR at the two extreme ionic strength conditions. The dashed horizontal line in panel (G) signals the dimensions of a perfect sphere. ure 2E and F). Electrostatic frustration in CytR The dimensions of CytR at high salt are as compact as that expected for a perfect sphere, i.e. 12.8 ˚A, suggestive of a well-folded protein domain. ionic strength modulation. The near-UV CD spectral mag- nitude of CytR increases with salt and shifts to the right mirroring far-UV CD observations providing a clear indi- cation that the overall tertiary structure increases concomi- tantly with secondary structure (Figure 2B). The fluores- cence emission of tyrosine exhibits a small red-shift on in- creasing temperatures providing an alternate probe to mon- itor structural changes. This component of the spectra can be extracted by performing singular value decomposition of the global temperature–wavelength–ionic strength data (Figure 2C) (36). Such an analysis reveals that the red-shift dominates the observed spectrum at ∼283 K in the pres- ence of urea wherein the protein is fully unfolded, while it increases to ∼301 K at 43 mM ionic strength and eventu- ally to just ∼319 K at 1.7 M ionic strength (Figure 2D). The large difference in the apparent inflection points at 1.7 M (∼22 K comparing against far- or near-UV CD under identical conditions) indicates that though the protein gains structure with increasing ionic strength, the changes are de- coupled thermodynamically. ionic strength modulation. The near-UV CD spectral mag- nitude of CytR increases with salt and shifts to the right mirroring far-UV CD observations providing a clear indi- cation that the overall tertiary structure increases concomi- tantly with secondary structure (Figure 2B). The fluores- cence emission of tyrosine exhibits a small red-shift on in- creasing temperatures providing an alternate probe to mon- itor structural changes. This component of the spectra can be extracted by performing singular value decomposition of the global temperature–wavelength–ionic strength data (Figure 2C) (36). Such an analysis reveals that the red-shift dominates the observed spectrum at ∼283 K in the pres- ence of urea wherein the protein is fully unfolded, while it increases to ∼301 K at 43 mM ionic strength and eventu- ally to just ∼319 K at 1.7 M ionic strength (Figure 2D). The large difference in the apparent inflection points at 1.7 M (∼22 K comparing against far- or near-UV CD under identical conditions) indicates that though the protein gains structure with increasing ionic strength, the changes are de- coupled thermodynamically. Evidence for continuous structural acquisition The slow relaxation rates even at low ionic strength con- ditions potentially arise from a highly frustrated (or rough) landscape. The rates increase would therefore be a mani- festation of reduced electrostatic frustration (i.e. smoother landscape) at higher IS conditions. strength conditions can be modulated to tune the relative stability of states, the corresponding relaxation rates should be chevron-like if the transition is over a large free-energy barrier. Ionic strength-modulated kinetics of CytR surpris- ingly reveals a continuous increase in relaxation rates with increasing structure and stability: the rates range from ∼100 s−1 at 43 mM to nearly 500 s−1 at 1.7 M with no evidence for a chevron-like behavior (Figure 3B and C). Moreover, a roll-over in rates is not evident near the apparent chemical denaturation midpoint on perturbation with urea at 1.3 and 1.72 M ionic strength conditions, providing additional evi- dence for a non-two-state transition (Supplementary Figure S4). The slow relaxation rates even at low ionic strength con- ditions potentially arise from a highly frustrated (or rough) landscape. The rates increase would therefore be a mani- festation of reduced electrostatic frustration (i.e. smoother landscape) at higher IS conditions. measured absolute heat capacity at the lowest temperature and the expectation for a folded domain (Freire baseline) at even 1.3 M ionic strength conditions, highlighting the pres- ence of significant residual enthalpic fluctuations.i gi pl To quantify the differences, we fit the heat capacity pro- files to the VB model of Mu˜noz and Sanchez-Ruiz (49) that provides estimates of thermodynamic barrier heights and conformational widths of the native ensembles. At the ap- parent midpoint temperatures (T0), the probability densi- ties of CytR are unimodal at 43 and 600 mM ionic strength (i.e. one-state-like folding with thermodynamic barrier ≤0), while exhibiting signs of a small thermodynamic barrier (∼0.1 kJ mol−1) at 1300 mM ionic strength conditions (Fig- ure 3E). The small barrier appears to coarsely separate the folded-like and unfolded-like conformations (a broad free energy well) and is consistent with the progressively better two-state model fits to the heat capacity profile (but with crossing baselines; Supplementary Figure S5) and the ap- pearance of the sharper excess heat capacity with increasing salt. Evidence for continuous structural acquisition The gain in CytR structure is also found to be indepen- dent of the nature of salt added thus ruling out specific binding (Figure 3A). The range of salt concentrations re- quired to promote structural transitions in CytR are 2–3 orders of magnitude more than that reported in Ribonu- clease P protein that folds in the presence of salt. Ribonu- clease P protein folds in a cooperative manner on increas- ing the phosphate or sulphate concentration in buffer from 0 M to a mere 20 mM suggestive of specific binding (61). The larger range of salt concentrations required to induce structural changes in CytR is evidence that the stabiliza- tion mechanism is most likely driven by non-specific electro- static screening of unfavorable charge–charge interactions within the protein (Figure 1), similar to observations in acid-denatured BBL (62,63). The question then is: does salt stabilize folded or binding-competent conformations over disordered states in a two-state-like manner or does it con- tribute to a continuous stabilization of folded-like confor- mations, akin to a continuous or second-order transition (64)? p y y At low ionic strength, the 1H-15N HSQC peaks of CytR are cluttered indicating heterogeneity in chemical environ- ment (green in Figure 2E) that decreases with increasing ionic strength (blue and red in Figure 2E and Supplemen- tary Figure S3). At 1 M ionic strength, the peaks are as dis- persed as in the NMR spectrum obtained in the presence of DNA (46) indicating that the structural changes encompass the entire protein. The gain in structure and stability is in- tuitively expected to go hand-in-hand with a compaction of the native ensemble at 298 K. AUC experiments accordingly result in relative CytR dimensions (RS) of 18.4 and 12.5 ˚A at low and high ionic strength conditions, respectively (Fig- ( ) Chevron-like kinetic behaviors are typical of two-state systems; in other words, upon denaturant addition the ob- served rate constants decrease, reach a minimum and in- crease again (65). This arises from the fact that at the mid- point of unfolding transition the rate determining barrier height is maximal in a two-state-like system, while decreas- ing on either side of it. In the case of CytR, since the ionic 8704 Nucleic Acids Research, 2018, Vol. 46, No. 17 Figure 3. Evidence for a continuous structural acquisition (A). Evidence for continuous structural acquisition Non-specific charge screening promotes structural gain in CytR as monitored by far-UV CD at 222 nm with various ions. (B) Observed relaxation rates from stopped-flow experiments in the folding (red), unfolding (blue) direction and from extrapolation of rates estimated from urea-induced changes in equilibrium at 43, 1300 and 1720 mM ionic strength conditions (green). (C) The amplitudes following the color-code in panel (B). (D) Absolute heat capacity profiles of CytR at the specified ionic strength conditions. FB and MP stand for the Freire and Makhatadze–Privalov unfolded baselines, respectively. (E and F) Probability densities at T0 and 298 K from a VB model analysis of the heat capacity profiles following the color code in panel (D). Inset to panel (F): asymmetry factor, a measure of structural compactness or the native ensemble width, at different ionic strength conditions. Figure 3. Evidence for a continuous structural acquisition (A). Non-specific charge screening promotes structural gain in CytR as monitored by far-UV CD at 222 nm with various ions. (B) Observed relaxation rates from stopped-flow experiments in the folding (red), unfolding (blue) direction and from extrapolation of rates estimated from urea-induced changes in equilibrium at 43, 1300 and 1720 mM ionic strength conditions (green). (C) The amplitudes following the color-code in panel (B). (D) Absolute heat capacity profiles of CytR at the specified ionic strength conditions. FB and MP stand for the Freire and Makhatadze–Privalov unfolded baselines, respectively. (E and F) Probability densities at T0 and 298 K from a VB model analysis of the heat capacity profiles following the color code in panel (D). Inset to panel (F): asymmetry factor, a measure of structural compactness or the native ensemble width, at different ionic strength conditions. strength conditions can be modulated to tune the relative stability of states, the corresponding relaxation rates should be chevron-like if the transition is over a large free-energy barrier. Ionic strength-modulated kinetics of CytR surpris- ingly reveals a continuous increase in relaxation rates with increasing structure and stability: the rates range from ∼100 s−1 at 43 mM to nearly 500 s−1 at 1.7 M with no evidence for a chevron-like behavior (Figure 3B and C). Moreover, a roll-over in rates is not evident near the apparent chemical denaturation midpoint on perturbation with urea at 1.3 and 1.72 M ionic strength conditions, providing additional evi- dence for a non-two-state transition (Supplementary Figure S4). Evidence for continuous structural acquisition The probability densities at 298 K are expectedly uni- modal at the three ionic strength conditions, with extracted sharpness of the native probability distribution (quantified by the asymmetry factor f) increasing from 0.53 at 43 mM, 0.63 at 600 mM to 0.90 at 1300 mM indicative of a more compact ensemble at high ionic strength conditions (inset to Figure 3F). In other words, salt is predicted to progres- sively or continuously fold CytR with distinct ensembles at different stabilization conditions akin to a one-state system p ) g The observations above hint that the protein folds in a continuous manner with increasing ionic strength condi- tions. An avenue to test this expectation is to perform scan- ning calorimetry experiments at varying ionic strength con- ditions or extent of charge screening and model the underly- ing the distribution of states through statistical approaches (49,66). The absolute heat capacity profiles show dramatic differences in the presence/absence of pre-transition base- lines and excess heat capacity and are incompatible with the expectation from a two-state-model (Figure 3D and Supple- mentary Figure S5). There is a large difference between the Nucleic Acids Research, 2018, Vol. 46, No. 17 8705 Figure 4. The electrostatic potential of DNA and its chaperone-like role. In all calculations, a distance of zero represents the DNA-bound CytR confor- mation. (A) The electrostatic potential of B-DNA (in kBT/e units) can be felt up till 20–25 ˚A from the molecular surface acting as a guiding funnel for charged molecules. (B) The electrostatic interaction energy (in kJ mol−1) between folded CytR and DNA as a function of distance between the two. (C) A schematic of the approach employed to calculate distance-dependent structural stability features of CytR from DNA (dark gray surface) through the WSME model. The most-distal pose (red circle) is assumed to have a melting temperature of 305 K as experimentally identified in the absence of DNA (Figure 2). The WSME model predicts the thermodynamic features as the protein is continuously moved toward DNA (green and blue circles). (D) Pre- dicted mean residue folding probabilities of CytR, a measure of global structure, with (filled circles) and without (open circles) intermolecular electrostatic terms as a function of protein–DNA distance. CytR folds only when it is very close to the DNA (∼5 ˚A or less) in the absence of intermolecular electrostatic terms (open circles), guided purely by intermolecular van der Waals interactions. Evidence for continuous structural acquisition (E) One-dimensional free energy profiles of CytR as a function of number of structured residues at varying distances from DNA. (F) Predicted changes in melting temperature of CytR as function of distance from DNA. (G) The distribution of folded CytR populations at different distances for numerous relative orientations from >345 000 1D free energy profiles. Note that multiple conformational states of CytR are possible even at a CytR–DNA distance of 5 ˚A. Figure 4. The electrostatic potential of DNA and its chaperone-like role. In all calculations, a distance of zero represents the DNA-bound CytR confor- mation. (A) The electrostatic potential of B-DNA (in kBT/e units) can be felt up till 20–25 ˚A from the molecular surface acting as a guiding funnel for charged molecules. (B) The electrostatic interaction energy (in kJ mol−1) between folded CytR and DNA as a function of distance between the two. (C) A schematic of the approach employed to calculate distance-dependent structural stability features of CytR from DNA (dark gray surface) through the WSME model. The most-distal pose (red circle) is assumed to have a melting temperature of 305 K as experimentally identified in the absence of DNA (Figure 2). The WSME model predicts the thermodynamic features as the protein is continuously moved toward DNA (green and blue circles). (D) Pre- dicted mean residue folding probabilities of CytR, a measure of global structure, with (filled circles) and without (open circles) intermolecular electrostatic terms as a function of protein–DNA distance. CytR folds only when it is very close to the DNA (∼5 ˚A or less) in the absence of intermolecular electrostatic terms (open circles), guided purely by intermolecular van der Waals interactions. (E) One-dimensional free energy profiles of CytR as a function of number of structured residues at varying distances from DNA. (F) Predicted changes in melting temperature of CytR as function of distance from DNA. (G) The distribution of folded CytR populations at different distances for numerous relative orientations from >345 000 1D free energy profiles. Note that multiple conformational states of CytR are possible even at a CytR–DNA distance of 5 ˚A. (67) explaining why the folding kinetics does not exhibit a chevron-like behavior. Evidence for continuous structural acquisition The maximum of the distribution along the order parameter carries additional information on the nature of the ensemble populated; the mode moves from a positive value at 43 mM to successively lower en- thalpy values at 600 and 1300 mM (Figure 3F). The confor- mational behavior of CytR therefore smoothly shifts from disordered and high enthalpy ensemble to molten-globule- like to a low enthalpy compact ensemble at 298 K but still exhibiting downhill features by mere modulation of salt concentration in the buffer. (67) explaining why the folding kinetics does not exhibit a chevron-like behavior. The maximum of the distribution along the order parameter carries additional information on the nature of the ensemble populated; the mode moves from a positive value at 43 mM to successively lower en- thalpy values at 600 and 1300 mM (Figure 3F). The confor- mational behavior of CytR therefore smoothly shifts from disordered and high enthalpy ensemble to molten-globule- like to a low enthalpy compact ensemble at 298 K but still exhibiting downhill features by mere modulation of salt concentration in the buffer. or charge screening, it is tempting to speculate that the dis- ordered CytR folds as it approaches DNA in a distance- dependent manner, funneled by the long-range negative electrostatic potential of DNA. Direct evidence for this unique mechanism is challeng- ing to explore experimentally as it requires single-molecule methods with precise Angstrom-level control of intermolec- ular distances while at the same time probing for the degree of foldedness of CytR (three-color single-molecule FRET potentially). However, the non-specific nature of the screen- ing effect indicates that it should be possible to model this behavior. We take recourse to the WSME model (50,51), an ensemble-based statistical mechanical model, wherein the phase space of a protein residue is simply represented as native (binary 1) or non-native (binary 0), allowing for an instantaneous ensemble of 2N microstates for a N-residue protein. We extend the classical WSME model (with in- tramolecular interactions) to include protein–DNA interac- tions by re-weighting the statistical weights of those residues that interact with DNA through both van der Waals in- teractions, and specific (52) and non-specific charge–charge interactions between the protein positive charges and the backbone phosphates of DNA (as obtained from the mod- eled bound structure, see ‘Materials and Methods’ section). The electrostatic potential of DNA and its chaperone-like role True to this, CytR binds random DNA with a similar affinity to the udp half-site (K1/2 ∼10 M; red in Figure 5B) and even the PurR complement with two overlapping titration profiles (K1/2,1 ∼0.4 M and K1/2,2 > 20 M; green in Figure 5B) suggestive of two different binding modes (Figure 5B and Supplementary Figure S7). Taken together, our results con- firm that the large electrostatic potential of DNA drives the binding of CytR in a non-specific manner. In the calculation above, we have assumed that the rela- tive orientation of CytR does not change as it diffuses to- ward DNA. This will not hold true as the protein is free to sample conformations in a plane orthogonal to DNA approach axis, particularly when it is far from DNA. To simulate this expectation, we consider the possible orien- tations of CytR (with respect to DNA) at spacings of 5◦ in all the three dimensions resulting in >82 000 potential binding poses far from DNA and >20 000 poses close to DNA. For each pose and at specific distances (5, 10, 13, 15 and 20 ˚A), 1D free energy profiles are generated for a to- tal of 345 022 1D free energy profiles (see ‘Materials and Methods’ section). The predicted distributions of confor- mational states at specific distances (Figure 4G) follow the overall trend shown in Figure 4E. It is interesting to note that a large conformational distribution is likely even at 5 ˚A from DNA, thus hinting at the molecular origins of the heterogeneous binding reported in experiments (36). i It is well established that DBDs need to switch conforma- tions in going from non-specific to specific binding modes. What has not been clear is the source of activation energy for such a process that determines the relative populations and interconversion rates to enable efficient balance be- tween 1D sliding and 3D hopping modes. Our experiments and calculations on CytR hint at an answer to this question: the activation energy to drive transitions could be effectively derived from the electrostatic potential of DNA in addition to the random solvent kicks. When CytR is far from DNA, the large electrostatic frustration promotes only unfolded- like conformations (Figure 5C). The electrostatic potential of DNA and its chaperone-like role The observed continuum of conformational behavior with increased charge screening raises the question of why CytR displays this distinct feature. Classic non-linear Poisson– Boltzmann calculations reveal that the electrostatic poten- tial of even 10–24 base-pair DNA fragments extends to nearly 20–25 ˚A at 100 mM bulk ionic strength conditions (Figure 4A) (68,69). The interaction energy between the folded conformation of CytR and DNA is also progres- sively favorable with decreasing distances between the two (Figure 4B). Given the continuous folding of CytR with salt 8706 Nucleic Acids Research, 2018, Vol. 46, No. 17 The model is parameterized by merely reproducing the apo- CytR melting temperature of ∼305 K, i.e. when CytR is po- sitioned at large distances from DNA (>30 ˚A, unbound), thus resulting in a disordered conformational behavior (red circle in Figure 4C). The role of non-specific charge–charge interactions at varying distances is directly calculated by merely moving the protein toward DNA along a specific axis (empty circles Figure 4C). In all cases discussed below, a distance of zero represents the bound conformation (∼6 ˚A from the DNA surface). different from ‘fly-casting’ (wherein a disordered charged segment latches on to the DNA from a distance driving folding (18,19)) and is more akin to the ‘conformational se- lection’ mechanism of binding (70,71) or the ‘electrostatic steering’ observed in simulations of Ets-DNA binding (72) and experimentally in the classic Barnase–Barstar interac- tions (73). The only difference is that in the ‘continuous con- formational selection’ we infer here, the protein can elec- trostatically pre-organize itself when it is relatively far from DNA without the need for a disordered protein segment to bind DNA. This pre-organization helps the protein to in- creasingly sample folded-like and potentially binding com- petent poses as it approaches DNA enabling rapid binding. CytR is disordered in the absence of DNA with very little secondary structure (Figure 2). As the protein ap- proaches DNA driven by the strong electrostatic potential, the unfavorable charge–charge interactions are increasingly screened thus promoting a folding transition with the folded probability (PF) going from 0.3 at >20 ˚A to ∼0.5 at ∼13–15 ˚A and finally to 1 when close to DNA even before complete binding (<8–9 ˚A) (Figure 4D). The electrostatic potential of DNA and its chaperone-like role This is also manifested in the CytR free energy profile that goes from near downhill- like in the unfolded side when far from DNA (>20–25 ˚A) to molten-globule-like with a flat free energy profile (∼13–15 ˚A) and eventually to downhill-like toward the folded side in the vicinity of DNA (<8–9 ˚A) (Figure 4E), exactly as ob- served in experiments with increased salt screening (Figure 3). The corresponding melting temperatures increase con- tinuously from 305 K when far from DNA to near 350 K when fully bound with a Tm of ∼320 K at intermediate distances, very similar to the higher melting temperatures observed in salt-screening experiments (Figure 4F). On re- moving the specific and non-specific charge–charge interac- tions between the protein and DNA, CytR exhibits a tran- sition toward the folded state only at very short distances (<5 ˚A) indicating pure packing effects between protein and DNA driving folding (black in Figure 4D and F). The over- all model predictions are insensitive to the magnitude of the dielectric constant of the intervening medium (Supplemen- tary Figure S6), highlighting the robustness of the folding– binding mechanism. p p pp g p g In such a mechanism, the binding event should be ex- tremely rapid funneled by large electrostatic forces. In fact, the association between CytR and its native udp comple- ment is faster than the dead-time of the stopped flow in- strument (∼2 ms) under pseudo-first order conditions even at 278 K wherein the association rate is slow due to in- creased solvent viscosity (Figure 5A and Supplementary Figure S7). This sets a lower bound on kon to be ∼1 × 109 M−1 s−1 at 278 K, thus being faster than the diffusion con- trolled limit (104–106 M−1 s−1) and characteristic of electro- static steering (73). It is important to note that anisotropy experiments on cMyb (IDP)–KIX (ordered protein) com- plex formation reveal distinct binding kinetic phases despite exhibiting equilibrium dissociation constants of 1–10 M (74), very similar to CytR–DNA complex at 293 K (blue in Figure 5B, K1/2 ∼10 M from inflection point analysis) (36); the comparison highlights that CytR also dissociates rapidly from DNA contributing to the low binding affin- ity (36). The second expectation from the proposed mech- anism is that such non-specific electrostatic forces should also enable CytR to bind random DNA sequences. The electrostatic potential of DNA and its chaperone-like role As it is driven closer to DNA, the free energy landscape of CytR exhibits multi- ple minima as a result of conflict between destabilizing na- tive interactions and stabilizing long-range protein–DNA interactions; many more structured conformations are thus possible contributing to an efficient sampling of the confor- mational space (in case of CytR, non-specifically collapsed states would also contribute (36)). Finally, as the protein is Rapid and non-specific DNA binding What advantage does this mechanism provide to binding? It is important to note that the proposed mechanism is slightly Nucleic Acids Research, 2018, Vol. 46, No. 17 8707 Figure 5. Rapid non-specific binding of CytR to DNA driven by ‘continuous conformational selection’ and electrostatic steering. (A) Stopped-flow kinetic anisotropy traces of excess CytR binding to Alexa-532 labeled udp half-site (300 nM) mimicking pseudo-first order conditions at 278 K. Note that ‘DNA’ stands for the anisotropy of labeled DNA in the absence of protein (blue), while the other colors represent the kinetic traces at the indicated final protein concentrations. (B) Binding isotherms of CytR to different DNA sequences (circles) at 293 K. The data have been shifted vertically for ease of viewing. (C) A schematic of the proposed continuous conformational–selection mechanism. CytR is disordered when it is far from DNA due to unfavorable intramolecular interactions. CytR folds continuously as it approaches DNA, i.e. it gains structure and reduces its dimensions, driven by the favorable electrostatic potential of DNA that screens out unfavorable intraprotein charge–charge interactions. At closer distances, the intrinsic conflict between intra- and intermolecular interactions contribute to an increased sampling of structured states thus allowing the protein to rapidly explore both specific- and non-specific binding poses. Figure 5. Rapid non-specific binding of CytR to DNA driven by ‘continuous conformational selection’ and electrostatic steering. (A) Stopped-flow kinetic anisotropy traces of excess CytR binding to Alexa-532 labeled udp half-site (300 nM) mimicking pseudo-first order conditions at 278 K. Note that ‘DNA’ stands for the anisotropy of labeled DNA in the absence of protein (blue), while the other colors represent the kinetic traces at the indicated final protein concentrations. (B) Binding isotherms of CytR to different DNA sequences (circles) at 293 K. The data have been shifted vertically for ease of viewing. (C) A h i f h d i f i l l i h i C i di d d h i i f f A d f bl i l l Figure 5. Rapid non-specific binding of CytR to DNA driven by ‘continuous conformational selection’ and electrostatic steering. (A) Stopped-flow kinetic anisotropy traces of excess CytR binding to Alexa-532 labeled udp half-site (300 nM) mimicking pseudo-first order conditions at 278 K. Rapid and non-specific DNA binding Note that ‘DNA’ stands for the anisotropy of labeled DNA in the absence of protein (blue), while the other colors represent the kinetic traces at the indicated final protein concentrations. (B) Binding isotherms of CytR to different DNA sequences (circles) at 293 K. The data have been shifted vertically for ease of viewing. (C) A schematic of the proposed continuous conformational–selection mechanism. CytR is disordered when it is far from DNA due to unfavorable intramolecular interactions. CytR folds continuously as it approaches DNA, i.e. it gains structure and reduces its dimensions, driven by the favorable electrostatic potential of DNA that screens out unfavorable intraprotein charge–charge interactions. At closer distances, the intrinsic conflict between intra- and intermolecular interactions contribute to an increased sampling of structured states thus allowing the protein to rapidly explore both specific- and non-specific binding poses. very near at the DNA surface, the electrostatic frustration is reduced promoting folded-like conformations albeit with occasional transitions to partially structured states. to three macroscopic states––partially structured, unfolded- like and non-specifically collapsed––thus dramatically in- creasing the conformational space that is sampled. The ‘continuous conformational selection’ mechanism need not be restricted to protein–DNA interactions, but even to protein–protein or protein–membrane interactions driven by non-specific electrostatic complementarity. In fact, a specific class of proteins termed DNA-mimic pro- teins exhibits a similar and extensive charge complemen- tary surface as DNA (77). Even ordered protein–DNA and disordered protein–protein interactions (driven by exten- sive charge complementarity) exhibit distance-dependent trends in terms of their interaction energy (35,78). Our in- terpretations further highlight the role of DNA and particu- larly its electrostatic potential in determining the conforma- tional behavior of proteins from a distance. The conforma- tional landscape of CytR is likely to have evolved to specif- ically fold only when it senses a favorable electrostatic field with the DNA playing the role of a passive macromolec- ular chaperone. In other words, a ‘folding funnel’ (79) ap- pears only in the vicinity of DNA while being non-existent at other conditions. Such tunable ‘conditional order’ likely enables tight regulation and compartmentalizing function- ality to specific regions within the cell. REFERENCES 1. Berg,O.G., Winter,R.B. and Von Hippel,P.H. (1981) Diffusion-driven mechanisms of protein translocation on nucleic-Acids.1. models and theory. Biochemistry, 20, 6929–6948.i 25. Zhou,H.X. (2011) Rapid search for specific sites on DNA through conformational switch of nonspecifically bound proteins. Proc. Natl. Acad. Sci. U.S.A., 108, 8651–8656. 2. Halford,S.E. and Marko,J.F. (2004) How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res., 32, 3040–3052. 26. Tempestini,A., Monico,C., Gardini,L., Vanzi,F., Pavone,F.S. and Capitanio,M. (2018) Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching. Nucleic Acids Res., 46, 5001–5011. 3. 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Funding for open ac- cess charge: Wellcome Trust/DBT India Alliance. Conflict of interest statement. None declared. 22. Roy,R., Kozlov,A.G., Lohman,T.M. and Ha,T. (2007) Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein. J. Mol. Biol., 369, 1244–1257.i 23. Murugan,R. (2010) Theory of site-specific DNA-Protein interactions in the presence of conformational fluctuations of DNA binding domains. Biophys. J., 99, 353–359. p y 24. Marcovitz,A. and Levy,Y. (2011) Frustration in protein-DNA binding influences conformational switching and target search kinetics. Proc. Natl. Acad. Sci. U.S.A., 108, 17957–17962.i CONCLUSIONS We effectively find that intramolecular charge screening promotes folded-like conformations in CytR at the expense of unfolded conformations in a purely non-specific man- ner. The conformational behavior of CytR thus smoothly transitions from being disordered to molten-globule-like to downhill (free energy profile toward the folded well; Fig- ures 2 and 3). This arises from an intrinsic conflict in the charge patterning of CytR with local and non-local effects displaying opposite trends on charge screening (Figure 1). Given that even folded DBDs exhibit a similar and ex- treme salt-sensitivity (40,75,76), our observations here sug- gest that such tunable conformational landscape could be a generic feature of DBDs. Statistical mechanical model- ing of the interaction behavior highlights the dramatic role of the long-range electrostatic potential of DNA in influ- encing the conformational landscape and thus the folding of CytR in a uniquely distance-dependent manner (Fig- ure 4). It is important to note that the native conforma- tional landscape of CytR (in the absence of DNA and at 310 K) is close to the collapse transition midpoint (36). 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https://openalex.org/W4385308388	https://rumahjurnal.or.id/index.php/BANSI/article/download/461/215	Indonesian	null	The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru)	Jurnal Bisnis Manajemen Akuntansi	2,023	cc-by-sa	7,920	Abstract The purpose of this study is to determine the effect of independence, due professional care and accountability on audit quality. The research was conducted at the Public Accounting Firm (KAP) in the city of Pekanbaru which is registered with the Indonesian Association of Public Accountants (IAPI). The sample used in this study is 40 auditors obtained by convenience sampling. The data analysis used is multiple linear regression with the help of the Statistical Package for Social Science (SPSS) version 21 program. The results of this study indicate that the variables of independence, due professional care, and accountability have significant effect on audit quality. Keywords: Independence, Due Professional Care, Accountability, Audit Quality Abstrak Tujuan dari penelitian ini adalah untuk mengetahui pengaruh independensi, due professional care dan akuntabilitas terhadap kualitas audit. Penelitian di lakukan pada Kantor Akuntan Publik (KAP) di kota Pekanbaru yang terdaftar pada Ikanatan Akuntan Publik Indonesia (IAPI). Sampel yang digunakan dalam penelitian ini sejumlah 40 auditor yang diperoleh berdasarkan convenience sampling. Analisis data yang digunakan adalah regresi linier berganda dengan menggunakan bantuan program Statistical Package for Social Science (SPSS) versi 21. Hasil penelitian ini menunjukkan bahwa variabel independensi, due professional care dan akuntabilitas berpengaruh signifikan terhadap kualitas audit The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) Anton1, Magdalena Panjaitan2 1,2Institut Bisnis dan Teknologi Pelita Indonesia Email : 1anton.st.maharajo@lecturer.pelitaindonesia.ac.id, 2magdalenapanjaitan99@gmail.com Anton1, Magdalena Panjaitan2 1,2Institut Bisnis dan Teknologi Pelita Indonesia Email : 1anton.st.maharajo@lecturer.pelitaindonesia.ac.id, 2magdalenapanjaitan99@gmail.com Jurnal BANSI (Bisnis, Manajemen dan Akuntasi) Vol. 3 No. 1 Tahun. 2023 Jurnal BANSI (Bisnis, Manajemen dan Akuntasi) Vol. 3 No. 1 Tahun. 2023 The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) Anton1, Magdalena Panjaitan2 1,2Institut Bisnis dan Teknologi Pelita Indonesia Email : 1anton.st.maharajo@lecturer.pelitaindonesia.ac.id, 2magdalenapanjaitan99@gmail.com 1. Pendahuluan Seiring dengan perkembangan media informasi yang semakin transparan mengharuskan setiap perusahaan untuk lebih kompoten dalam menyajikan laporan keuangan. Laporan yang disajikan harus sesuai dengan prinsip-prinsip akuntansi yang berlaku sehingga dapat di terima dengan baik oleh pihak-pihak yang berkepentingan dalam pengambil keputusan. Begitu juga dengan pihak eksternal perusahaan yang akan memberikan penilaian terkait laporan keuangan perusahaan tersebut. Laporan keuangan merupakan rincian yang menjelaskan keadaan dimana keberhasilan perusahaan dalam hal keuangan perusahaan melalui laporan keuangan internal para pihak dalam periode tertentu sehingga dengan situasi dan kondisi yang ada dapat diambil keputusan [1]. Menurut FASB (Financial Accounting Standards Boards) laporan keuangan perusahaan harus memiliki dua karakteristik penting yaitu relevan (relevance) dan dapat diandalkan (reliable) [2]. Akan tetapi dua karakteristik ini sangat sulit untuk diukur, sehingga dibutuhkan peranan auditor sebagai penghubung antara investor (pengguna laporan keuangan) dan perusahaan (penyedia laporan keuangan). Oleh karena itu perlu dilakukan pemeriksaan (audit) atas laporan keuangan dalam menentukan opini atas laporan keuangan. Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 47 Menurut [3] audit merupakan suatu proses sistematik untuk memperoleh dan mengevaluasi bukti secara objektif mengenai pernyataan-pernyataan tentang kegiatan dan kejadian ekonomi, dengan tujuan untuk menetapkan tingkat kesesuaian antara pernyataan-pernyataan tersebut dengan kriteria yang telah ditetapkan, serta penyampaian hasil-hasilnya kepada pemakai yang berkepentingan. Proses audit terdiri dari penyelidikan mencari catatan akuntansi dan bukti lain yang mendukung laporan keuangan tersebut. Pengumpulan bukti dapat dilakukan dengan memahami sistem pengendalian internal perusahaan, mengamati dokumen, mengamati aset, bertanya didalam maupun diluar perusahaan. Auditor harus menghimpun bukti sebanyak- banyaknya agar dapat menentukan apakah laporan keuangan telah cukup melengkapi gambaran situasi keuangan dan kegiatan perusahaan selama periode audit. Audit atas laporan keuangan haruslah di lakukan oleh pihak yang independen, profesional dan bertanggung jawab yaitu Akuntan Publik (AP). Profesi akuntan publik merupakan salah satu profesi yang diberikan kepercayaan oleh pihak manajemen dan pihak ketiga untuk membuktikan laporan keuangan yang disajikan manajemen terbebas dari salah saji material. Kepercayaan ini harus dijaga dengan menunjukan kinerja yang profesional. Untuk menjaga profesionalisme sebagai akuntan publik, maka seorang akuntan publik harus mengacu pada standar auditing yang telah ditetapkan oleh IAPI, yaitu standar umum, standar pekerjaan lapangan dan standar pelaporan. Standar umum merupakan cerminan kualitas pribadi yang harus dimiliki oleh seorang auditor yang mengharuskan auditor untuk memiliki keahlian dan pelatihan teknis yang cukup guna menunjang dalam melaksanakan prosedur audit. 1. Pendahuluan Sementara, untuk KAP dikenakan peringatan tertulis dengan disertai kewajiban untuk melakukan perbaikan terhadap sistem pengendalian mutu KAP dan dilakukan tinjauan oleh BDO Internasional Limited. Hardiyanto menyatakan, KAP belum mengimplementasikan kebijakan unsur pelaksanaan keterikatan dalam sistem pengendalian mutu. KAP seharusnya memiliki sistem pengendalian mutu, yakni bertanggung jawab memastikan kualitas dari audit tersebut [4]. Selanjutnya masalah audit juga terjadi pada tahun yang sama yang melibatkan akuntan publik dan kantor akuntan publik yaitu; PT. Sunprima Nusantara Pembiayaan (SNP Finance), sebuah perusahaan multifinance yang masih memiliki anak perusahaan Columbia di Indonesia membuat perekayasaan laporan keuangan yang dapat merugikan banyak bank. Dalam mengaudit laporan keuangan anak perusahaan Columbia Group, KAP Deloitte Indonesia mengaudit laporan keuangannya diindikasi dengan kelalaian. Kasus SNP Finance bisa menjadi pembelajaran bagi akuntan publik dan kantor akuntan publik agar tidak lagi melakukan pelanggaran dalam mengaudit laporan keuangan. Ketua Dewan Komisioner OJK Wimboh Santoso mengatakan akan terus melakukan monitoring kepada lembaga auditor keuangan publik dan industri keuangan agar tidak menyalahi prosedur. “Ini agar penegak hukum supaya ada efek jera. Kalau dicabut ya sudahlah, kami akan lanjutkan sesuai dengan ketentuan. Ini supaya yang lain memetik pelajaran dari yang terjadi oleh akuntan publik dan kantor akuntan publik”. OJK telah mengenakan sanksi administratif berupa pembatalan pendaftaran kepada Kantor Akuntan Publik Satrio Bing Eny & Rekan (KAP SBE) beserta dua akuntan publik, yakni Akuntan Publik (AP) Marlinna dan Akuntan Publik (AP) Merliyana Syamsul. y Kasus terakhir yang terjadi pada PT. Asabri yang dimana adanya dugaan yang merugikan negara hingga di atas Rp 10 Triliun. Badan Pemeriksa Keuangan (BPK) masih mengaudit kerugian negara atas kasus dugaan korupsi pada PT. Asabri, BPK menaksir kerugian negara dalam kasus tersebut mencapai Rp 16 triliun. Mengatasi kasus ini, Kementrian BUMN turut menggandeng Badan Pemeriksaa Keuangan (BPK). BPK sedang mengumpulkan data dan informasi, diperkirakan potensi kerugian Rp 10 sampai Rp 16 Triliun. Setelah semua data terverifikasi secara keseluruhan, BPK berencana menyerahkan data persoalan PT. Asabri itu kepada Komisi Pemberantasan Korupsi (KPK) untuk kemudian ditindaklanjuti. Fenomena yang sering terjadi tentang banyaknya kasus perusahaan yang jatuh karena tingkat kegagalan laporan keuangan yang dikaitkan dengan kegagalan auditor. Peranan kualitas audit sangat penting karena dengan hasil audit yang berkualitas dapat meningkatkan kredibilitas laporan keuangan yang dihasilkan sehingga memperkecil risiko dari informasi yang tidak kredibel pada laporan keuangan. Dampak dari suatu kegagalan audit adalah rusaknya kredibilitas dan kepercayaan kepada kantor akuntan publik, akuntan publik yang bersangkutan, dan profesi audit pada umumnya. 1. Pendahuluan Sedangkan standar pekerjaan lapangan dan standar pelaporan mengatur auditor dalam hal pengumpulan data dan kegiatan lain yang dilaksanakan selama melakukan audit [4]. p g p g y g Standar Audit (SA) ini mengatur tentang tanggung jawab keseluruhan auditor independen ketika melaksanakan audit atas laporan keuangan berdasarkan Standar Audit (SA). Secara spesifik, Standar Audit (SA) ini menetapkan tujuan keseluruhan auditor independen, serta menjelaskan sifat dan ruang lingkup suatu audit yang didesain untuk memungkinkan auditor independen mencapai tujuan tersebut [5]. Auditor harus memiliki sikap mental independen dan selalu berpikir kritis terhadap bukti audit sehingga informasi yang digunakan dalam pengambilan keputusan akurat dan hasil audit dapat berkualitas. Kemampuan auditor untuk menghasilkan kualitas audit yang tinggi akan meningkatkan reputasinya sehingga auditor diharapkan mampu menghasilkan laporan keuangan yang andal dan dapat dipercaya untuk digunakan sebagai dasar dalam pengambilan keputusan. Kualitas audit adalah karakteristik audit yang dapat memenuhi standar auditing dan juga standar pengendalian mutu yang telah menggambarkan praktik audit serta menjadi ukuran dari kualitas dalam pelaksanaan tugas untuk memenuhi tanggung jawab profesinya [6]. Auditor dituntut untuk selalu memberikan kualitas audit yang tinggi dalam setiap penugasannya, sehingga mengharuskan auditor memastikan tidak ada kekeliruan material ketika melakukan proses audit sebelum memberikan opininya. Namun pada kenyataannya, beberapa tahun belakangan ini banyak permasalahan akan rendahnya kualitas audit yang dihasilkan oleh akuntan publik sehingga menjadi sorotan dalam masyarakat. Salah satunya adalah kasus PT Garuda Indonesia (Persero) Tbk (GIAA) tahun 2018, Kementrian Keuangan (Kemenkeu) memberikan sanksi pada Kantor Akuntan Publik (KAP) Tanubrata, Sutanto, Fahmi, Bambang & Rekan & Akuntan Publik Kasner Sirumapea, yang merupakan auditor dari laporan keuangan tahun 2018 dari PT Garuda Indonesia (Persero) Tbk (GIAA). Kemenkeu tim Pusat Pembinaan Profesi Keuangan (PPPK) menetapkan sanksi berupa pembekuan izin selama 12 bulan pada Kasner Sirumapea yang berlaku sejak 27 Juli 2019. Kasner dinilai melakukan pelanggaran berat yang berpotensi berpengaruh signifikan terhadap opini Laporan Auditor Independen (LAI). Kasner melakukan pelanggaran tiga hal. Pertama, dia belum secara tepat menilai substansi transaksi untuk kegiatan perlakuan akuntansi terkait pengakuan piutang dan pendapatan lain-lain secara sekaligus di awal. Ini melanggar Standar Audit 315. Kemudian Kasner dikatakan belum sepenuhnya mendapatkan bukti audit yang cukup dan tepat untuk menilai ketepatan perlakuan akuntansi sesuai dengan substansi transaksi dari perjanjian yang melandasi transaksi tersebut. Ini melanggar Standar Audit 500. Ketiga, akuntan publik belum mempertimbangkan fakta-fakta setelah tanggal pelaporan keuangan, sebagai dasar pertimbangan ketepatan perlakuan. Ini Jurnal BANSI (Bisnis Manajemen dan Akutansi) 48 melanggar Standar Audit 560. 1. Pendahuluan Inti dari masalah yang belakangan sering terjadi kepada kantor akuntan publik dan auditornya yaitu kurangnya independensi, due professional care dan akuntabilitas akan mempengaruhi kinerja audit yang ditentukan oleh proses audit yang dijalankan oleh auditor. p g j y g p y g j Independensi yang merupakan kejujuran dalam diri auditor dalam mempertimbangkan fakta dan adanya pertimbangan yang objektif tidak memihak dalam diri auditor dalam merumuskan dan menyatakan pendapatnya. Independensi dapat diartikan sikap mental yang bebas dari pengaruh, tidak dikendalikan oleh pihak lain, tidak tergantung pada orang lain [7]. Penelitian yang telah dilakukan oleh [6], [2] dan [8] yang menyatakan bahwa independensi berpengaruh terhadap kualitas audit. Berbeda dengan hasil penelitian yang telah dilakukan oleh [9] menyatakan bahwa independensi tidak berpengaruh terhadap kualitas audit. Due professional care mengacu pada kemahiran profesional yang cermat dan seksama. Kemahiran profesional menuntut auditor untuk selalu berpikir kritis terhadap bukti audit yang ditemukannya. Menurut [10] due professional care dapat diartikan sebagai sikap yang cermat dan seksama dengan berpikir kritis serta melakukan evaluasi terhadap bukti audit, berhati-hati dalam tugas, tidak ceroboh dalam melakukan pemeriksaan dan memiliki keteguhan dalam melaksanakan tanggung jawab. Hasil dari penelitian yang telah dilakukan oleh [11], [12] dan [7] yang menyatakan bahwa due professional care berpengaruh terhadap kualitas audit. Berbeda dengan hasil penelitian yang telah dilakukan oleh [13] yang menyatakan bahwa due professional care tidak berpengaruh terhadap kualitas audit. Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 49 Akuntabilitas merupakan wujud kewajiban seseorang untuk mempertanggung jawabkan pengelolaan atas kewenangan yang dipercayakan kepadanya guna pencapaian tujuan yang ditetapkan. Menurut [2] menjelaskan akuntabilitas adalah bentuk dorongan psikis yang membuat seseorang bertanggung jawab atas semua tindakan dan keputusan yang diambilnya. Penelitian yang telah dilakukan oleh [2] menyatakan bahwa akuntabilitas berpengaruh terhadap kualitas audit. Sejalan dengan penelitian yang dilakukan oleh [14] menyatakan akuntabilitas memiliki pengaruh kepada kualitas audit, hasil itu berhasil memperlihatkan terdapatnya etika auditor yang dipegang teguh dari seseorang auditor pada saat bekerja akan meningkatkan akuntabilitas auditor untuk menuntaskan laporan auditnya, sehingga seorang auditor mampu memberikan hasil yang berkualitas. Berbeda dengan hasil penelitian yang telah dilakukan oleh [15] menyatakan bahwa akuntabilitas tidak mempunyai pengaruh kepada kualitas audit. Teori Keagenan (Agency Theory) Teori keagenan yang dikembangkan oleh Jensen dan Meckling [12] menjelaskan tentang adanya konflik kepentingan antara manajemen selaku agen dan pemilik selaku principal. Principal ingin mengetahui segala informasi yang sebenarnya termasuk aktivitas manajemen yang terkait dengan investasi atau dananya dalam perusahaan dengan meminta laporan pertanggungjawaban pada agen (manajemen). Namun manajemen cenderung melakukan kecurangan dalam pembuatan laporan pertanggungjawaban perusahaan agar kinerja perusahaan terlihat baik. Terlihat adanya kepentingan yang berbeda antara manajemen dan pemakai laporan keuangan, untuk mengatasi masalah ini diperlukan pengujian oleh pihak ketiga (auditor) untuk mengurangi kecurangan yang mungkin dilakukan. g Agency theory membantu auditor sebagai pihak ketiga untuk memahami adanya konflik kepentingan yang muncul antara agen dan principal. Principal selaku investor bekerja sama dengan agen atau manajemen perusahaan. Diharapkan dengan adanya auditor independen, tidak ada kecurangan dalam laporan keuangan yang dibuat oleh manajemen, sekaligus dapat mengevaluasi kinerja agen sehingga dihasilkan informasi yang relevan dan berguna dalam pengambilan keputusan investasi. Dalam hal ini sebagai pondasi dalam melakukan pekerjaan auditor harus mengetahui lebih dalam pengetian, standar audit, tujuan, opini dan kualitas audit yang akan dijelaskan lebih lanjut. Teori Behaviorisme Teori behaviorisme adalah sebuah teori yang dicetuskan oleh Gage dan Berliner tentang perubahan tingkah laku sebagai hasil dari pengalaman [16]. Teori ini sangat menekankan pada perilaku yang dapat diamati dan diukur. 1. Pendahuluan Berdasarkan latar belakang penelitian di atas, maka tujuan dari penelitian ini adalah 1) mengetahui dan mengevaluasi pengaruh independensi terhadap kualitas audit, 2) mengetahui dan mengevaluasi pengaruh due professional care terhadap kualitas audit, dan 3) mengetahui dan mengevaluasi pengaruh akuntabilitas terhadap kualitas audit Adapun manfaat dari penelitian ini bagi 1) institusi sebagai masukan untuk pimpinan KAP dalam menjaga dan meningkatkan kualitas auditnya, karena jika tidak memperhatikan kualitas audit yang diberikan atau bahkan melakukan kesalahan hingga dikenakan sanksi pembekuan maka reputasi KAP dan auditornya akan dipertaruhkan, 2) auditor diharapkan dapat digunakan sebagai motivasi bagi para akuntan untuk dapat lebih independen, professional dan akuntabilitas dalam melaksanakan tugas dan tanggung jawabnya, serta memberikan hasil audit yang dapat dipercaya bagi masyarakat luas, 3) masyarakat diharapkan dapat meningkatkan kepercayaan masyarakat terhadap laporan keuangan yang dihasilkan oleh auditor, khususnya mengenai independensi, due professional care dan akuntabilitas terhadap kualitas audit yang dihasilkan, dan 4) peneliti dapat menambah wawasan mengenai kualitas audit dan sebagai acuan ataupun bahan masukan untuk melakukan penelitian selanjutnya yang berhubungan dengan judul penelitian ini. Kualitas Audit [3] mengatakan bahwa‚ kualitas audit merupakan probabilitas auditor untuk menemukan kesalahan yang ada pada laporan keuangan klien dan melaporkannya dalam laporan auditan. IAI Due Professional Care Menurut [19] due professional care diartikan sebagai sikap yang cermat dan seksama dengan berpikir kritis serta melakukan evaluasi terhadap bukti audit, berhati-hati dalam tugas, tidak ceroboh dalam melakukan pemeriksaan dan memiliki keteguhan dalam melaksanakan tanggung jawab. Dalam pelaksanaan audit dan penyusunan laporannya, auditor wajib menggunakan kemahiran profesionalnya dengan cermat dan seksama (due professional care) Penggunaan kemahiran profesional dengan cermat dan seksama memungkinkan auditor untuk memperoleh keyakinan memadai bahwa laporan keuangan bebas dari salah saji material, baik yang disebabkan oleh kekeliruan maupun kecurangan. Ketelitian profesional sepantasnya menghendaki penerapan ketelitian dan kecakapan yang secara patut diduga akan dilakukan oleh pemeriksaan yang bijaksana dan kompeten dalam keadaan yang sama. Akuntabilitas Akuntabilitas berarti pertanggungjawaban atau keadaan untuk dipertanggungjawabkan atau keadaan untuk diminta pertanggunganjawab. Akuntabilitas sebagai bentuk dorongan psikologi yang membuat seseorang berusaha mempertanggungjawabkan semua tindakan dan keputusan yang diambil kepada lingkungannya dalam melaksanakan tanggungjawabnya sebagai profesional setiap auditor harus senantiasa menggunakan pertimbangan moral dan profesional dalam semua kegiatan yang dilakukannya [20]. Menurut [14] akuntabilitas adalah suatu kewajiban kepada seseorang agar memberikan pertanggungjawaban pengelolaannya untuk wewenang yang sudah dipertanggungkan terhadapnya. Jika seorang auditor memahami akan tanggung jawabnya maka dia bisa melaksanakan pekerjaannya dengan sebaik mungkin serta mengabdikan dirinya kepada suatu profesi merupakan sebuah tanggung jawab yang sudah dibentuk dengan sukarela didalam pribadi seseorang. 50 Jurnal BANSI (Bisnis Manajemen dan Akutansi) 50 (Ikatan Akuntan Indonesia) menyatakan bahwa, Audit yang dilaksanakan seorang auditor dapat dikatakan berkualitas jika memenuhi standar auditing yang berlaku umum dan standar pengendalian mutu. Standar auditing tersebut dijadikan pedoman oleh auditor dalam melaksanakan pekerjaannya. Selain auditor, KAP juga harus mematuhi standar auditing yang telah ditetapkan oleh IAPI. Oleh karena itu, KAP harus membuat kebijakan dan prosedur pengendalian mutu untuk memberikan keyakinan memadai tentang kesesuaian penugasan audit dengan standar auditing yang telah ditetapkan. Prosedur pengendalian mutu yang ditetapkan oleh KAP secara individual berpengaruh terhadap pelaksanan penugasan audit sedangkan secara keseluruhan akan berpengaruh pada pelaksanaan praktik KAP. Independensi Independensi dalam The CPA Handbook menurut E.B. Wilcox [17] adalah merupakan suatu standar auditing yang penting karena opini akuntan independen bertujuan untuk menambah kredibilitas laporan keuangan yang disajikan oleh manajemen. Menurut [18] independensi kejujuran dalam diri auditor dalam mempertimbangkan fakta dan adanya pertimbangan yang objektif tidak memihak dalam diri auditor dalam merumuskan dan menyatakan pendapatnya. Independensi dapat diartikan sikap mental yang bebas dari pengaruh, tidak dikendalikan oleh pihak lain, tidak tergantung pada orang lain . Dalam SA bagian 210 Standar Perikatan Audit [5] disebutkan bahwa‚ dalam semua hal yang berhubungan dengan perikatan, independensi dalam sikap mental harus dipertahankan oleh auditor. Auditor harus bersikap independen karena ia melaksanakan pekerjaannya untuk kepentingan umum. Oleh karena itu independensi auditor merupakan hal yang penting karena pendapat yang diberikan oleh auditor sangat mempengaruhi banyak pihak, misalnya pemerintah, investor, pengamat ekonomi, masyarakat, manajer dan serikat buruh. Kerangka Pemikiran Berdasarkan uraian pengaruh antar variabel independen terhadap variabel dependen, dapat digambarkan kerangka pemikiran sebagai berikut: Gambar 1. Kerangka Pemikiran Gambar 1. Kerangka Pemikiran Pengaruh Independensi terhadap kualitas audit Independensi sendiri adalah ketidakberpihakan auditor terhadap pimpinan maupun obyek yang di audit. Menurut Mulyadi, Kualitas audit yang baik dipengaruhi oleh sikap independen dari Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 5 51 51 seorang auditor tersebut [20]. Hasil penelitian yang dilakukan oleh [6], [2] dan [8] yang menyatakan bahwa independensi berpengaruh terhadap kualitas audit. Berbeda dengan hasil penelitian yang telah dilakukan oleh [9] menyatakan bahwa independensi tidak berpengaruh terhadap kualitas audit. Berdasarkan uraian diatas, maka dirumuskan hipotesis : H1 : Independensi berpengaruh terhadap kualitas audit. Tempat dan Waktu Penelitian Tempat dilakukannya penelitian ini adalah di Kantor Akuntan Publik (KAP) yang berada di kota Pekanbaru. Waktu penelitian mulai dari bulan November 2022 sampai dengan Februari 2023. Pengaruh Akuntabilitas terhadap kualitas audit Menurut [20] akuntabilitas merupakan suatu keadaan yang dirasakan oleh auditor bahwa pekerjaan yang dilakukan telah sesuai dengan prosedur dan standar akuntan publik sehingga dapat dipertanggungjawabkan mengenai simpulan yang dibuat untuk pihak-pihak yang berkepentingan. Hasil penelitian yang telah dilakukan oleh [2] menyatakan bahwa akuntabilitas berpengaruh terhadap kualitas audit. Sejalan dengan penelitian yang dilakukan oleh [14] menyatakan akuntabilitas memiliki pengaruh kepada kualitas audit. Berbeda dengan hasil penelitian yang telah dilakukan oleh [15] menyatakan bahwa akuntabilitas tidak mempunyai pengaruh kepada kualitas audit. Berdasarkan uraian diatas, maka dirumuskan hipotesis : H3 : Akuntabilitas berpengaruh terhadap kualitas audit. Pengaruh Due Professional Care terhadap kualitas audit Due professional care merupakan hal penting yang harus diterapkan setiap akuntan publik dalam melaksanakan pekerjaan profesionalnya agar dicapai kualitas audit yang memadai [21]. Hasil dari penelitian yang telah dilakukan oleh [11], [12] dan [7] yang menyatakan bahwa due professional care berpengaruh terhadap kualitas audit. Berbeda dengan hasil penelitian yang telah dilakukan oleh [13] yang menyatakan bahwa due professional care tidak berpengaruh terhadap kualitas audit. Berdasarkan uraian diatas, maka dirumuskan hipotesis : H2 : Due professional care berpengaruh terhadap kualitas audit. H2 : Due professional care berpengaruh terhadap kualitas audit. Uji Normalitas Uji normalitas menggunakan uji normalitas PP-Plot dan uji Kolmogrov-Smirnov. Jika hasil uji menunjukkan nilai Probabilitas > 0,05 maka distribusi dari model regresi adalah normal. Jika Probabilitas < 0,05 maka distribusi dari model regresi adalah tidaknormal. Jika data tidak berdistribusi normal, maka pengujian akan dilanjutkan dengan menggunakan aplikasi SmartPLS. Uji Multikolinearitas j Uji Multikolinearitas bertujuan untuk menguji apakah model regresi ditemukan adanya korelasi antar variabel bebas (Independen). Uji Asumsi Klasik Asumsi klasik merupakan persyaratan yang harus terpenuhi pada analisis regresi berganda yang harus memenuhi asumsi yang disyaratkan untuk memenuhi uji asumsi normalitas, uji multikolinieritas dan uji heteroskedastisitas. Teknik Analisis Data Metode analisis data yang digunakan pada penelitian ini yaitu analisis regresi berganda. Teknik analisis data yang digunakan untuk mengolah data adalah menggunakan SPSS (Statistic Package For Social Science) versi 21. Uji Reliabilitas Uji ini menggunakan reliabilitas konsistensi internal yaitu metode Cronbach Alpha. Apabila nilai cronbach alpha dari hasil pengujian > 0,6 maka dapat dikatakan bahwa variabel penelitian reliabel. Uji Validitas Tingkat validitas dapat diukur dengan membandingkan antara nilai r hitung dengan r tabel, apabila rhitung > rtabel maka pernyataan pada kuesioner dikatakan valid tetapi jika rhitung < rtabel maka pernyataan pada kuesioner dikatakan tidak valid. Uji Kualitas Data j Uji kualitas data adalah uji yang disyaratkan dalam penelitian yang menggunakan instrumen dalam bentuk kuesioner. Uji kualitas data pada data primer ini meliputi uji validitas dan uji reliabilitas. Analisis Statistik Deskriptif Statistik Deskriptif digunakan untuk memberikan gambaran data berupa distribusi frekuensi serta ukuran statistik lainnya pada responden. Penyajian data dapat berupa tabel, grafik dan diagram. 52 Jurnal BANSI (Bisnis Manajemen dan Akutansi) 52 Jurnal BANSI (Bisnis Manajemen dan Akutansi) convenience sampling, yaitu teknik pengambilan sampel dengan menyebar sejumlah kuesioner dengan menggunakan kuesioner yang kembali dan dapat diolah [23]. Operasional Variabel Operasional Variabel Penelitian menggunakan variabel independen (X) yang terdiri dari independensi, due professional care dan akuntabilitas sedangkan variabel dependen (Y) adalah kualitas audit. Instrumen Penelitian Instrumen yang digunakan dalam penelitian ini berbentuk kuesioner dengan menggunakan skala sikap (skala likert) dengan 5 pilihan (opsi) Populasi dan Sampel Menurut [22] Populasi merupakan wilayah generalisasi yang terdiri atas objek atau subjek yang mempunyai kualitas dan karakteristik tertentu yang ditetapkan oleh peneliti untuk dipelajari dan kemudian ditarik kesimpulannya. Dalam penelitian ini, populasi yang digunakan merupakan seluruh auditor yang bekerja pada 8 Kantor Akuntan Publik (KAP) di Pekanbaru. Menurut [22] sampel merupakan bagian dari jumlah dan karakteristik yang dimiliki oleh populasi tersebut. Metode penetapan sampel yang digunakan dalam penelitian ini adalah 52 Jurnal BANSI (Bisnis Manajemen dan Akutansi) convenience sampling, yaitu teknik pengambilan sampel dengan menyebar sejumlah kuesioner dengan menggunakan kuesioner yang kembali dan dapat diolah [23]. 52 Jurnal BANSI (Bisnis Manajemen dan Akutansi) Uji Kelayakan Model (Uji F) Dengan tingkat signifikan sebesar 5%. Jika hasilnya signifikan, maka H0 ditolak dan Ha diterima dan sebaliknya jika hasilnya tidak signifikan, maka H0 diterima dan Ha ditolak. Kriteria pengujian yang dipergunakan adalah jika F hitung < F tabel, maka H0 diterima dan Ha ditolak dan jika F hitung > F tabel, maka H0 ditolak dan Ha diterima Koefisien Deteriminasi (R2) Pengujian ini bertujuan untuk mengetahui besar presentase dari variabel independen secara bersama–sama terhadap variabel dependen dengan melihat besarnya nilai koefisien deteriminasi (R2). Nilai koefisien determinasi antara 0% sampai dengan 100%, dimana semakin mendekati 100% semakin baik determinasi dari persamaan regresi. Uji Hipotesis (Uji t) Pengujian t bertujuan untuk memastikan variabel independen yang terdapat dalam persamaan berpengaruh terhadap variabel dependen. Pengujian dilakukan dengan membandingkan antara t hitung dengan t tabel. Untuk menentukan t tabel ditentukan dari tingkat signifikansi, 5% dengan df = (n-k-1), dimana n adalah jumlah responden dan k merupakan jumlah variabel independen. Kriteria pengujian yang digunakan adalah jika t hitung (Tstatistics) < t tabel atau jika nilai Sig (=Pvalues) > 5% , maka H0 diterima dan Ha ditolak dan jika t hitung (Tstatistics) ≥ t tabel atau jika nilai Sig (Pvalues) < 5%, maka H0 ditolak dan Ha diterima Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) Analisis Regresi Berganda Analisis Regresi Berganda Analisis ini digunakan untuk menentukan signifikasi pengaruh independensi, due professional care dan akuntabilitas terhadap kualitas audit di kantor akuntan publik Pekanbaru. Model persamaan regresi yang digunakan dalam penelitian ini adalah : KA b I d b DP f b Ak Dimana: KA = Kualitas Audit a = Bilangan Konstanta b1, b2, b3 = Koefisien Regresi Ind = Independensi DProf = Due Professional Care Ak = Akuntabilitas e = Error Term Uji Heteroskedastisitas Pengujian ini dapat dilihat dari hasil uji glejser. Jika nilai signifikansi lebih besar dari 0,05 maka tidak terjadi heteroskedastisitas. Sebaliknya Jika nilai signifikansi lebih kecil dari 0,05, maka terjadi heteroskedastisitas. 53 53 asil Uji analisis deskripsi data dapat dilihat Tabel 6 berikut ini: j p Analisis deskripsi data yang disajikan dalam penelitian ini meliputi Minimum, aksimum, Mean (M), dan Standar Deviasi (Std. Deviation). 3. Hasil Dan Pembahasan Data – data diperoleh melalui penyebaran kuesioner dengan mendatangi langsung 7 Kantor Akuntan Publik. Sampel yang diperoleh dalam penelitian ini adalah sebanyak 40 orang auditor. Berikut tabel karakteristik responden : Tabel 1. Karakteristik Responden berdasarkan Jenis Kelamin No Jenis Kelamin Jumlah Persentase 1 Pria 24 60% 2 Wanita 16 40% Jumlah 40 100% No Tingkat Pendidikan Jumlah Persentase 1 Diploma 2 5% 2 S1 24 60% 3 S2 14 35% 4 S3 0 0% Tabel 1. Karakteristik Responden berdasarkan Jenis Kelamin Tabel 2. Karakteristik Responden berdasarkan Pendidikan Tabel 3. Karakteristik Responden Berdasarkan Jabatan Tabel 4. Karakteristik Responden Berdasarkan Masa Bekerja No Masa Bekerja Jumlah Persentase 1 <1 tahun 0 0% 2 1-5 tahun 18 45% 3 >5tahun 22 55% Total 40 100% Tabel 5. Karakteristik Responden berdasarkan keikutsertaan dalam Kursus/Diklat/ Bimtek di bidang pemeriksaan (auditing) No Kursus/Diklat/Bimtek di bidang auditing Jumlah Persentase 1 Tidak Pernah 2 5% 2 Pernah 14 35% 3 Jarang 6 15% 4 Sering 10 25% 5 Sangat Sering 8 20% Total 40 100% Uji Analisis deskriptif Jumlah 40 100% No Jabatan Jumlah Persentase 1 Auditor Senior 12 30% 2 Auditor Junior 24 60% 3 Manager 0 0% 4 Supervisor 0 0% 5 Partner 4 10% Total 40 100% Tabel 5. Karakteristik Responden berdasarkan keikutsertaan dalam Kursus/Diklat/ Bimtek di bidang pemeriksaan (auditing) No Kursus/Diklat/Bimtek di bidang auditing Jumlah Persentase 1 Tidak Pernah 2 5% 2 Pernah 14 35% 3 Jarang 6 15% 4 Sering 10 25% 5 Sangat Sering 8 20% Total 40 100% Uji Analisis deskriptif j p Analisis deskripsi data yang disajikan dalam penelitian ini meliputi Minimum Maksimum, Mean (M), dan Standar Deviasi (Std. Deviation). Hasil Uji analisis deskripsi data dapat dilihat Tabel 6 berikut ini: Hasil Uji analisis deskripsi data dapat dilihat Tabel 6 berikut ini: Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 55 Tabel 6. Uji Analisis Deskriptif N Minimum Maximum Mean Std. Deviation N Minimum Independensi 40 29,00 50,00 40,8500 4,42922 40 29,00 Due_Professional_Care 40 28,00 45,00 36,1500 4,35919 40 28,00 Akuntabilitas 40 13,00 25,00 20,8750 3,59621 40 13,00 Kualitas_Audit 40 36,00 59,00 50,8000 5,37420 40 36,00 Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 55 55 Tabel 6. 3. Hasil Dan Pembahasan Uji Analisis Deskriptif Dari Tabel 6, dapat dilihat dengan jumlah sampel sebanyak 40 responden, didapatkan nilai minimum, nilai maksimum, rata-rata dan standar deviasi dari keseluruhan variabel penelitian. Nilai minimum untuk Independensi, Due Professional Care, Akuntabilitas dan Kualitas Audit masing-masing sebesar 29,00; 28,00; 13,00; dan 36.00. Nilai maksimum masing-masing variabel adalah 50.00; 45,00; 25,00; dan 59,00. Variabel Independensi memiliki nilai rata-rata (mean) sebesar 40,8500 dengan nilai standar deviasi (Std. Deviation) sebesar 4,42922. Variabel Due Professional Care mempunyai nilai rata-rata sebesar 36,1500 dengan nilai dengan standar deviasi sebesar 4,35919. Variabel Akuntabilitas mempunyai nilai rata-rata sebesar 20.8750 dengan standar deviasi 3,59621. Variabel Kualitas Audit dengan nilai rata-rata 50,8000 dengan standar deviasi 5,3742. Hal ini dapat diartikan bahwa semakin besar nilai standar deviasi maka data sampel semakin menyebar (bervariasi) dari rata-ratanya. Dimana karakter sampel dalam penelitian ini mempunyai nilai cukup konsisten untuk dapat diterima sebagai karakter sampel yang sebenarnya. Sehingga hal ini dapat digunakan sebagai bahan atau kebijakan untuk mengambil suatu keputusan dalam penelitian ini. Uji Kualitas Kelayakan Data Uji Validitas Suatu instrumen yang digunakan dalam penelitian dapat dikatakan valid jika nilai r ≥ 0,30 (Sugiyono, 2010: 178). Sebaliknya, jika nilai r < 0,30 maka instrumen tersebut dinyatakan tidak valid. Berdasarkan uji validitas yang dilakukan dapat dilihat pada lampiran, dapat diketahua bahwa seluruh butir pernyataan yang digunakan untuk mengukur variabel dependen, yaitu Kualitas Audit, kemudian variabel independen, yaitu Independensi, Due Professional Care dan Akuntabilitas adalah valid. Tabel 7. Validitas Variabel Kualitas Audit No Indikator Butir Pernyataan Rhitung Rtabel Hasil Uji 1 Melaporkan semua kesalahan klien Y.1 0,590 0,3120 Valid Y.2 0,521 0,3120 Valid 2 Pemahaman terhadap sistem informasi akuntansi klien. Y.3 0,406 0,3120 Valid 3 Komitmen yang kuat dalam menyelesaikan audit Y.4 0,398 0,3120 Valid Y,5 0,540 0,3120 Valid 4 Berpedoman pada prinsip auditing dan prinsip akuntansi Y.6 0,493 0,3120 Valid Y.7 0,434 0,3120 Valid Y.8 0,447 0,3120 Valid 5 Tidak percaya begitu saja terhadap pernyataan klien Y.9 0,487 0,3120 Valid Y.10 0,434 0,3120 Valid 6 Sikap hati-hati dalam pengambilan keputusan Y.11 0,498 0,3120 Valid Y.12 0,465 0,3120 Valid Tabel 7. Validitas Variabel Kualitas Audit Tabel 7. Validitas Variabel Kualitas Audit 56 Jurnal BANSI (Bisnis Manajemen dan Akutansi) 56 Jurnal BANSI (Bisnis Manajemen dan Akutansi) Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 57 Tabel 8. Validitas Variabel Independensi Tabel 9. Validitas Variabel Due Professional Care No Indikator Butir Pernyataan Rhitung Rtabel Hasil Uji 1 Spektisme Profesional X2.1 0,623 0,3120 Valid X2.2 0,612 0,3120 Valid X2.3 0,428 0,3120 Valid X2.4 0,486 0,3120 Valid X2.5 0,407 0,3120 Valid 2 Keyakinan yang memadai X2.6 0,545 0,3120 Valid X2.7 0,399 0,3120 Valid X2.8 0,444 0,3120 Valid X2.9 0,514 0,3120 Valid Tabel 10. Uji Kualitas Kelayakan Data Uji Validitas Validitas Variabel Akuntabilitas No Indikator Butir Pernyataan Rhitung Rtabel Hasil Uji 1 Motivasi menyelesaikan X3.1 0,621 0,3120 Valid 2 Usaha atau daya pikir dalam menyelesaikan pekerjaan X3.2 0,590 0,3120 Valid 3 Keyakinan akan hasil kerjanya X3.3 0,658 0,3120 Valid X3.4 0,669 0,3120 Valid X3.5 0,608 0,3120 Valid No Indikator Butir Pernyataan Rhitung Rtabel Hasil Uji 1 Lama hubungan dengan klien (Audit Trenue) X1.1 0,375 0,3120 Valid X1.2 0,315 0,3120 Valid 2 Tekanan dari klien Auditor X1.3 0,374 0,3120 Valid X1.4 0,545 0,3120 Valid X1.5 0,471 0,3120 Valid 3 Telaah dari rekan auditor (Peer Review) X1.6 0,637 0,3120 Valid X1.7 0,559 0,3120 Valid 4 Jasa non audit X1.8 0,461 0,3120 Valid X1.9 0,611 0,3120 Valid X1.10 0,399 0,3120 Valid Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 57 57 57 Tabel 9. Validitas Variabel Due Professional Care No Indikator Butir Pernyataan Rhitung Rtabel Hasil Uji 1 Spektisme Profesional X2.1 0,623 0,3120 Valid X2.2 0,612 0,3120 Valid X2.3 0,428 0,3120 Valid X2.4 0,486 0,3120 Valid X2.5 0,407 0,3120 Valid 2 Keyakinan yang memadai X2.6 0,545 0,3120 Valid X2.7 0,399 0,3120 Valid X2.8 0,444 0,3120 Valid X2.9 0,514 0,3120 Valid Tabel 10. Validitas Variabel Akuntabilitas No Indikator Butir Pernyataan Rhitung Rtabel Hasil Uji 1 Motivasi menyelesaikan X3.1 0,621 0,3120 Valid 2 Usaha atau daya pikir dalam menyelesaikan pekerjaan X3.2 0,590 0,3120 Valid 3 Keyakinan akan hasil kerjanya X3.3 0,658 0,3120 Valid X3.4 0,669 0,3120 Valid X3.5 0,608 0,3120 Valid Uji Reliabilitas j Uji Reliabilitas adalah alat untuk mengukur suatu kuesioner yang merupakan indikator dari variabel atau konstruk. Butir kuesioner dikatakan reliabel (layak) jika cronbach’s alpha > 0,06 dan dikatakan tidak reliabel jika cronbach’s alpha < 0.06 (Ghozali, 2012 : 47). T b l 11 Uji R li bilit Tabel 11. Uji Reliabilitas Variabel Cronbach’s Alpha Batas Reliabel Keterangan Kualitas Audit (Y) 0,651 0.60 Reliabel Independensi (X1) 0,619 0.60 Reliabel Due Professional Care (X2) 0.603 0.60 Reliabel Akuntabilitas (X3) 0,615 0.60 Reliabel 58 Jurnal BANSI (Bisnis Manajemen dan Akutansi) Berdasarkan pengujian reliabilitas menunjukkan bahwa nilai cronbach alpha untuk variabel Kualitas Audit sebesar 0,651; Independensi sebesar 0,619, variabel Due Professional Care sebesar 0,603 dan variabel Akuntabilitas sebesar 0,615. Oleh karena itu dapat dikatakan bahwa seluruh variabel dalam penelitian ini adalah reliabel. Uji Asumsi Klasik Tabel 12. Uji Normalitas Data One-Sample Kolmogorov-Smirnov Test Unstandardized Residual N 40 Normal Parametersa,b Mean ,0000000 Std. Deviation 3,63913341 Most Extreme Differences Absolute ,088 Positive ,076 Negative -,088 Kolmogorov-Smirnov Z ,558 Asymp. Sig. (2-tailed) ,915 a. Test distribution is Normal b. Calculated from data. One-Sample Kolmogorov-Smirnov Test Berdasarkan hasil penelitian ini telah lulus dari uji asumsi klasik dimana hasil uji normalitas diketahui bahwa nilai Asymp. Sig (two tailed) 0,915 lebih besar dari 0,05 atau 5%, maka dapat dinyatakan bahwa data residual berdistribusi normal. Tabel 13. Uji Multikolinearitas Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. Collinearity Statistics B Std. Error Beta Tolerance VIF 1 (Constant) 19,924 6,034 3,302 ,002 Independensi ,757 ,206 ,599 3,680 ,001 ,443 2,258 Due_Professio nal_Care -,501 ,205 -,390 -2,440 ,020 ,460 2,175 Akuntabilitas ,863 ,179 ,555 4,826 ,000 ,889 1,125 Tabel 13. Uji Multikolinearitas Hasil uji menunjukkan bahwa seluruh variabel bebas yaitu Independensi, Due Professional Care dan Akuntabilitas nilai Tolerance yang lebih besar dari 10% (0,10). Sementara dari nilai Variance Inflation Factor (VIF) dari seluruh variabel bebas yaitu Independensi, Due Professional Care dan Akuntabilitas menunjukkan nilai VIF yang lebih kecil dari 10. Dengan demikian dapat disimpulkan bahwa tidak terjadi multikolinearitas antar variabel. Tabel 14. Uji Heteroskedastisitas Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) ,727 3,288 ,221 ,826 Independensi ,013 ,112 ,028 ,116 ,908 Due_Professional_Care ,095 ,112 ,203 ,848 ,402 Akuntabilitas -,082 ,097 -,145 -,841 ,406 59 59 Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) Dasar pengambilan keputusan pada uji heterokedastisitas adalah jika nilai signifikan (kolom Sig) lebih besar dari 0,05, maka kesimpulannya adalah tidak terjadi heterokedastisitas. Pada tabel di atas dapat dilihat bahwa seluruh nilai signifkan masing-masing variabel independen lebih besar dari 0,05. Dapat disimpulkan bahwa tidak terjadi heterokedastisitas. Hal ini juga dapat dilihat pada Gambar 2 sebagai berikut: Gambar 2. Scatterplot Gambar 2. Scatterplot Gambar 2. Scatterplot Uji Regresi Linear Berganda j g g Uji regresi digunakan untuk mengetahui arah hubungan antara variabel independen dengan variabel dependen apakah masing-masing variabel independen berhubungan positif atau negatif dan untuk memprediksi nilai dari variabel dependen jika nilai variabel independen mengalami kenaikan atau penurunan [22]. Hasil pengujian linear berganda dapat di lihat pada tabel output berikut ini : Tabel 15. Analisis Regresi Linear Berganda Tabel 15. Analisis Regresi Linear Berganda Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 19,924 6,034 3,302 ,002 Independensi ,757 ,206 ,599 3,680 ,001 Due_Professional_Care -,501 ,205 -,390 -2,440 ,020 Akuntabilitas ,863 ,179 ,555 4,826 ,000 Dari output pada tabel di atas, dapat diperoleh hasil analisis uji regresi sebagai berikut: KA = 19,924 + 0,757Ind -0,501DProf + 0,863Ak + e Tabel 15. Analisis Regresi Linear Berganda Dari output pada tabel di atas, dapat diperoleh hasil analisis uji regresi sebagai berikut: KA = 19,924 + 0,757Ind -0,501DProf + 0,863Ak + e Berdasarkan persamaan diatas, interprestasinya adalah 1) nilai konstanta sebesar 19,924 , berarti jika semua variabel bebas (X) memiliki nilai konstan, maka variabel terikat (Y) yaitu kualitas audit akan berubah nilainya sebesar 19,924, 2) nilai koefisien (β1Ind) dari Independensi (X1) adalah 0,757 berarti bahwa setiap peningkatan independensi satu satuan, maka variabel kualitas audit (Y) akan meningkat sebesar 0,757 dengan asumsi variabel bebas lain dari model regresi adalah tetap, 3) nilai koefisien (β2DProf) dari due professional care (X2) adalah -0,501 berarti bahwa setiap peningkatan due professional care satu satuan, maka variabel kualitas audit (Y) akan menurun sebesar 0,501 dengan asumsi variabel bebas lain dari model regresi adalah tetap, dan 4) nilai koefisien (β3Ak) dari akuntabilitas (X3) adalah 0,863 berarti bahwa setiap 60 Jurnal BANSI (Bisnis Manajemen dan Akutansi) peningkatan akuntabilitas satu satuan, maka variabel kualitas audit (Y) akan meningkat sebesar 0,863 dengan asumsi variabel bebas lain dari model regresi adalah tetap. peningkatan akuntabilitas satu satuan, maka variabel kualitas audit (Y) akan meningkat sebesar 0,863 dengan asumsi variabel bebas lain dari model regresi adalah tetap. Uji Model (F) Tabel 16. Uji Model (F) ANOVAa Model Sum of Squares df Mean Square F Sig. 1 Regression 704,487 3 234,829 16,368 ,000b Residual 516,488 36 14,347 Total 1220,975 39 a. Dependent Variable: Kualitas_Audit b. Predictors: (Constant), Akuntabilitas, Due_Professional_Care, Independensi b. Uji Regresi Linear Berganda Predictors: (Constant), Akuntabilitas, Due_Professional_Care, Independensi Dengan memperhatikan bahwa nilai Fhitung (16,368) > Ftabel (2,866), dan nilai signifikansi yang lebih kecil dari 0,05 (0,000a < 0,05), maka dapat disimpulkan bahwa independensi, due professional care dan akuntabilitas berpengaruh signifikan terhadap kualitas audit pada Kantor Akuntan Publik (KAP) di Kota Pekanbaru. Atau dapat disimpulkan bahwa model regresi dapat digunakan untuk memprediksi variabel dependen. Uji Koefisien Determinasi (R2) Tabel 17. Uji Koefisien Determinasi (R2) Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 ,760a ,577 ,542 3,78773 a. Predictors: (Constant), Akuntabilitas, Due_Professional_Care, Independensi b. Dependent Variable: Kualitas_Audit Uji Koefisien Determinasi (R2) Uji Koefisien Determinasi (R2) Berdasarkan hasil uji R2 diperoleh angka koefisien determinasi dengan Adjusted R Square sebesar 0,542 atau 54,2 %. Hal itu menunjukan bahwa kualitas audit dipengaruhi oleh variabel independensi, due professional care dan akuntabilitas sebesar 54,2 %, sedangkan sisanya 45,8 % merupakan kontribusi variabel lain yang tidak masuk dalam penelitian ini. Pengaruh Due Professional Care terhadap Kualitas Audit Berdasarkan pengujian yang telah dilakukan, diketahui bahwa Due Professional Care berpengaruh negative dan signifikan terhadap kualitas audit pada Kantor Akuntan Publik (KAP) di Pekanbaru. Hal ini disebabkan karena kurang cermat dan kurang pengetahuan dari auditor untuk mengevaluasi dan mengumpulkan bukti audit secara objektif. Diharapkan supaya setiap auditor mampu mengikuti kursus/diklat/bimtek dibidang pemeriksaan (auditing). Hasil ini sejalan dengan penelitian yang dilakukan oleh [13] menyatakan bahwa Due Professional Care berpengaruh negatif terhadap Kualitas Audit. Pengaruh Independensi terhadap Kualitas Audit Berdasarkan pengujian yang telah dilakukan, diketahui bahwa independensi berpengaruh positif dan signifikan terhadap kualitas audit pada Kantor Akuntan Publik (KAP) di Pekanbaru. Kualitas audit dapat dicapai oleh auditor jika auditor memiliki independensi yang baik, serta jaminan atas keandalan laporan yang diberikan oleh auditor tersebut dapat dipercaya oleh semua pihak yang berkepentingan. Auditor harus memiliki kemampuan untuk mengumpulkan setiap informasi yang dibutuhkan dalam pengambilan keputusan audit. Tidak dapat dipungkiri bahwa sikap independen merupakan hal yang melekat pada diri auditor, sehingga independen seperti telah menjadi syarat mutlak yang harus dimiliki seorang auditor. Dengan demikian, semakin tinggi independensi seorang auditor maka semakin tinggi pula kualitas audit yang dihasilkannya. Hasil penelitian ini menguatkan penelitian sebelumnya oleh [6] menyatakan bahwa Independensi berpengaruh positif dan signifikan terhadap Kualitas Audit. Hasil yang sama juga dinyatakan dari penelitian yang dilakukan oleh [8] dimana Independensi berpengaruh positif dan signifikan terhadap Kualitas Audit. Pengaruh Akuntabilitas terhadap Kualitas Audit Berdasarkan pengujian yang telah dilakukan, diketahui bahwa akuntabilitas berpengaruh positif dan signifikan terhadap kualitas audit pada Kantor Akuntan Publik (KAP) di Pekanbaru. Hal ini karena akuntabilitas sangat penting bagi auditor sebagai faktor yang mempengaruhi kualitas audit. Akuntabilitas menunjukkan bahwa auditor dapat menyelesaikan audit dengan benar dan tepat waktu, yakin bahwa pekerjaan ini diperiksa dengan teliti, ditinjau oleh supervisor/manajer/pimpinan, dan bertanggung jawab kepada pemberi kerja. Hasil ini sesuai dengan penelitian yang dilakukan oleh [2] menyatakan bahwa akuntabilitas berpengaruh positif terhadap Kualitas Audit. Hasil yang sama juga dinyatakan dari penelitian yang dilakukan oleh [14] dimana akuntabilitas berpengaruh positif terhadap Kualitas Audit. Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 61 61 Berdasarkan tabel di atas dapat diketahui bahwa hasil hipotesis variabel ketiga, yaitu akuntabilitas diperoleh t hitung = 4,826 > t tabel = 1,688 dengan signifikan 0,000 < 0,05. Dengan demikian maka hipotesis akuntabilitas dapat diterima, yaitu akuntabilitas berpengaruh positif dan signifikan terhadap kualitas audit. Uji Hipotesis (t) Uji Hipotesis (t) Tabel 18. Uji Hipotesis (t) Variabel T tabel T hitung Sig Keputusan Hasil Independensi 1,688 3,680 ,001 H1 diterima Berpengaruh positif dan signifikan Due_Professional_Care 1,688 -2,440 ,020 H2 diterima Berpengaruh negatif dan signifikan Akuntabilitas 1,688 4,826 ,000 H3 diterima Berpengaruh positif dan signifikan Uji Hipotesis (t) Tabel 18. Uji Hipotesis (t) Variabel T tabel T hitung Sig Keputusan Hasil Independensi 1,688 3,680 ,001 H1 diterima Berpengaruh positif dan signifikan Due_Professional_Care 1,688 -2,440 ,020 H2 diterima Berpengaruh negatif dan signifikan Akuntabilitas 1,688 4,826 ,000 H3 diterima Berpengaruh positif dan signifikan Berdasarkan tabel di atas dapat diketahui bahwa hasil hipotesis variabel pertama, yaitu independensi memperoleh t hitung = 3,680 > t tabel = 1,688 dengan signifikan 0,001 < 0,05. Dengan demikian maka hipotesis independensi dapat diterima, yaitu independensi berpengaruh positif dan signifikan terhadap kualitas audit. Berdasarkan tabel di atas dapat diketahui bahwa hasil hipotesis variabel kedua, yaitu due professional care diperoleh t hitung = -2,440 > t tabel = 1,688 dengan signifikan 0,020 < 0,05. Dengan demikian maka hipotesis due professional care dapat diterima, yaitu due professional care berpengaruh negatif dan signifikan terhadap kualitas audit. Berdasarkan tabel di atas dapat diketahui bahwa hasil hipotesis variabel pertama, yaitu independensi memperoleh t hitung = 3,680 > t tabel = 1,688 dengan signifikan 0,001 < 0,05. Dengan demikian maka hipotesis independensi dapat diterima, yaitu independensi berpengaruh positif dan signifikan terhadap kualitas audit. Berdasarkan tabel di atas dapat diketahui bahwa hasil hipotesis variabel kedua, yaitu due professional care diperoleh t hitung = -2,440 > t tabel = 1,688 dengan signifikan 0,020 < 0,05. Dengan demikian maka hipotesis due professional care dapat diterima, yaitu due professional care berpengaruh negatif dan signifikan terhadap kualitas audit. Berdasarkan tabel di atas dapat diketahui bahwa hasil hipotesis variabel kedua, yaitu due professional care diperoleh t hitung = -2,440 > t tabel = 1,688 dengan signifikan 0,020 < 0,05. Dengan demikian maka hipotesis due professional care dapat diterima, yaitu due professional care berpengaruh negatif dan signifikan terhadap kualitas audit. Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) Daftar Pustaka [1] S. Adzra, “Pengaruh Due Professional Care, Locus Of Control Dan Ethical Sensitivity Terhadap Kualitas Audit (Studi Empiris Pada Kantor Akuntan Publik Di Kota Palembang),” Universitas Sriwijaya, 2021. [1] S. Adzra, “Pengaruh Due Professional Care, Locus Of Control Dan Ethical Sensitivity Terhadap Kualitas Audit (Studi Empiris Pada Kantor Akuntan Publik Di Kota Palembang),” Universitas Sriwijaya, 2021. g) j y [2] A. D. Laksita and S. Sukirno, “Pengaruh Independensi, Akuntabilitas, Dan Objektivitas Terhadap Kualitas Audit,” Nominal Barom. Ris. Akunt. dan Manaj., vol. 8, no. 1, pp. 31– 46, 2019, doi: 10.21831/nominal.v8i1.24497. [2] A. D. Laksita and S. Sukirno, “Pengaruh Independensi, Akuntabilitas, Dan Objektivitas Terhadap Kualitas Audit,” Nominal Barom. Ris. Akunt. dan Manaj., vol. 8, no. 1, pp. 31– 46, 2019, doi: 10.21831/nominal.v8i1.24497. [3] Mulyadi, Auditing. Jakarta: Salemba Empat, 2014. [3] Mulyadi, Auditing. Jakarta: Salemba Empat, 2014. [4] L. Khairiyah, “Pengaruh Indepedensi, Kompetensi Dan Fee Audit Terhadap Kualitas Audit (Studi Kasus Pada Auditor Di Kantor Akuntan Publik Kota Medan),” Universitas Islam Negeri Sumatera Utara, 2020. [5] Ikatan Akuntan Publik Indonesia (IAPI), “Standar Audit 210 (Revisi 2021) Persetujuan Atas Ketentuan Perikatan Audit Berdasarkan Standar Audit,” Standar Prof. Akuntan Publik ( SA 210) 2021, vol. 200, no. Revisi, pp. 1–22, 2021. [6] N. O. Haryanto and C. Susilawati, “Pengaruh Kompetensi, Independensi, dan Profesionalisme Auditor Internal Terhadap Kualitas Audit,” J. Akunt. Bisnis, vol. 16, no. 1, pp. 42–55, 2018, doi: 10.24167/jab.v16i2.1694. pp j [7] A. R. Sa’adah and A. E. Challen, “Pengaruh Independensi Auditor, Due Professional Care, Fee Audit Dan Perikatan Audit Terhadap Kualitas Audit ,” J. Revenue, vol. 3, no. 1, pp. 1–9, 2022. [8] M. L. Nandawardani, “Pengaruh Independensi, Kompotensi, Pengalaman Kerja, Audit Tenure Dan Workload Terhadap Kualitas Audit (Studi Empiris Pada Auditor KAP DI Surakarta Dan Yogyakarta),” Universitas Muhammadiyah Surakarta, 2021. [9] S. Rebecca, “Pengaruh Kompetensi, Independensi, Dan Etika Profesi Auditor Terhadap Kualitas Audit (Studi Empiris Pada Kantor Akuntan Publik Di Wilayah Jakarta Pusat),” STIE INDONESIA, 2019. [10] S. Agoes, Auditing Petunjuk Praktis Pemeriksaan Akuntan, Buku 1 Edi. Jakarta: Salemba Empat, 2010. [11] N. P. Oktavinarni, “Pengaruh Kompetensi, Independensi, Dan Due Professional Care Auditor Terhadap Kualitas Audit Pada Kantor Akuntan Publik Di Wilayah Surabaya Timur,” Universitas Islam Negeri Sunan Ampel Surabaya, 2018. [12] N. K. Megayani, N. Nyoman, A. Suryandari, and A. A. P. G. B. A. Susandya, “Pengaruh Independensi, Due Professional Care dan Locus of Control Terhadap Kualitas Audit Dengan Pengalaman Auditor Sebagai Variabel Moderasi Pada KAP di Provinsi Bali,” J. 4. Kesimpulan Berdasarkan hasil analisis data dan pembahasan yang telah diuraikan di atas, maka kesimpulan dari penelitian ini bahwa independensi dan akuntabilitas berpengaruh positif dan signifikan terhadap kualitas audit. Sedangkan due professional care berpengaruh negatif dan signifikan terhadap kualitas audit. Berdasarkan hasil tersebut diharapkan auditor, senantiasa meningkatkan independensi yang dimiliki serta terus menjaga akuntabilitas dan tetap bekerja sesuai dengan Standar Profesional Akuntan Publik (SPAP). Penelitian ini memiliki keterbatasan diantaranya adalah 1) Data yang digunakan dalam penelitian ini hanya berupa kuesioner yang disebarkan kepada responden, 2) Ruang lingkup penelitian ini hanya dilakukan di Kantor Akuntan Publik (KAP) di Pekanbaru, dan 3) Masih terdapat variabel independen lain yang mempengaruhi variabel kualitas audit yang belum diteliti. Adapun saran-saran terkait dalam penelitian ini adalah 1) Peneliti selanjutnya diharapkan agar menambah jumlah sampel dan memperluas wilayah pengambilan sampel. Responden pada peneliti selanjutnya hendaknya diperluas, tidak hanya Kantor Akuntan Publik (KAP) di 62 Jurnal BANSI (Bisnis Manajemen dan Akutansi) 62 Pekanbaru melainkan di seluruh Provinsi Riau, 2) Bagi peneliti selanjutnya diharapkan agar melakukan pengecekan terlebih dahulu Kantor Akuntan Publik yang terdaftar di IAPI melalui telepon untuk memastikan dan mengantisipasi alamat yang salah dan pindah atau tidak aktif lagi, 3) Bagi auditor agar dapat menjaga independensi, due professional care dan akuntabilitas serta meningkatkan pengalaman dengan cara mengikuti pelatihan-pelatihan, seminar-seminar dan peningkatan pendidikan profesi agar meningkatkan kualitas audit, dan 4) Bagi Kantor Akuntan Publik (KAP) disarankan agar dapat menjaga independensi, due professional care dan akuntabilitas auditor karena ketiga faktor tersebut dapat meningkatkan kualitas audit yang tentunya akan memberikan dampak positif kepada Kantor Akuntan Publik (KAP) secara keseluruhan ke depannya Pekanbaru melainkan di seluruh Provinsi Riau, 2) Bagi peneliti selanjutnya diharapkan agar melakukan pengecekan terlebih dahulu Kantor Akuntan Publik yang terdaftar di IAPI melalui telepon untuk memastikan dan mengantisipasi alamat yang salah dan pindah atau tidak aktif lagi, 3) B i dit d t j i d d i d f i l d k t bilit t Pekanbaru melainkan di seluruh Provinsi Riau, 2) Bagi peneliti selanjutnya diharapkan agar melakukan pengecekan terlebih dahulu Kantor Akuntan Publik yang terdaftar di IAPI melalui telepon untuk memastikan dan mengantisipasi alamat yang salah dan pindah atau tidak aktif lagi, 3) Bagi auditor agar dapat menjaga independensi, due professional care dan akuntabilitas serta meningkatkan pengalaman dengan cara mengikuti pelatihan-pelatihan, seminar-seminar dan peningkatan pendidikan profesi agar meningkatkan kualitas audit, dan 4) Bagi Kantor Akuntan Publik (KAP) disarankan agar dapat menjaga independensi, due professional care dan akuntabilitas auditor karena ketiga faktor tersebut dapat meningkatkan kualitas audit yang tentunya akan memberikan dampak positif kepada Kantor Akuntan Publik (KAP) secara keseluruhan ke depannya 3) Bagi auditor agar dapat menjaga independensi, due professional care dan akuntabilitas serta meningkatkan pengalaman dengan cara mengikuti pelatihan-pelatihan, seminar-seminar dan peningkatan pendidikan profesi agar meningkatkan kualitas audit, dan 4) Bagi Kantor Akuntan Publik (KAP) disarankan agar dapat menjaga independensi, due professional care dan akuntabilitas auditor karena ketiga faktor tersebut dapat meningkatkan kualitas audit yang tentunya akan memberikan dampak positif kepada Kantor Akuntan Publik (KAP) secara keseluruhan ke depannya Daftar Pustaka Ris. Akunt. dan Keuang., vol. 8, no. 1, pp. 133–150, 2020. [13] W. A. Ningtyas, Aris, and M. Abdul, “Independensi, Kompetensi, Pengalaman Kerja, Dan Due Professional Care: Pengaruhnya Terhadap Kualitas Audit Yang Dimoderasi Dengan Etika Profesi (Studi Empiris Pada Kantor Akuntan Publik Se-Jawa Tengah Dan Diy),” Ris. Akunt. dan Keuang. Indones., vol. 1, no. 1, pp. 75–88, 2016, doi: 10.23917/reaksi.v1i1.1971. [14] A. N. S. A. Ningsih, D. J. Kirana, and W. A. Andriyanto, “Pengalaman Audit, Fee Audit, dan Akuntabilitas Terhadap Kualitas Audit,” Pros. BIEMA (bus. Manag. Econ. Account. Natl. Semin., vol. 1, pp. 1460–1476, 2020. pp [15] A. A. Ismiyati, “Pengaruh Kompetensi, Independensi, Dan Akuntabilitas Terhada Anton, The Effect Of Independence, Due Professional Care And Accountability On Audit Quality (Study On KAP Pekanbaru) 63 Kualitas Audit Dengan Etika Auditor Sebagai Variabel Moderasi (Studi Empiris Pada Kantor Akuntan Publik Di Provinsi Banten),” J. Ris. Akunt. Tirtayasa, vol. 4, no. 1, pp. 89–101, 2019, doi: 10.48181/jratirtayasa.v4i1.5504. [16] j y [16] R. M. Kosanke, “Faktor - Faktor Yang Mempengaruhi Kualitas Audit Pada Inspektorat Provinsi Sulawesi Utara,” pp. 119–140, 2019. [17] Suharti and T. Apriyanti, “Pengaruh Kompetensi Dan Independensi Terhadap Kualitas Audit Dengan Etika Auditor Sebagai Variabel Moderasi,” STIE Pelita Indonesia, 2019. [18] A. A. Arens, M. S. Beasley, and R. S. Eder, Auditing dan Jasa Assurance : Pendekatan Terintegrasi, 15th ed. Erlangga, 2015. [19] S. Agoes, Auditing-Petunjuk Praktis Pemeriksaan Oleh Akuntan Publik. Jakarta: Salemb Empat, 2013. [20] I. Faturochman, “Pengaruh Independensi, Kompetensi, dan Akuntabilitas terhadap Kualitas Audit Badan Pemeriksa Keuangan Pemerintah Kota Magelang,” Universitas Islam Indonesia Yogyakarta, 2019. [21] I. R. Bawono and E. M. Singgih, “Faktor-Faktor Dalam Diri Auditor Dan Kualitas Audit: Studi Pada Kap ‘Big Four’’ Di Indonesia,’” J. Akunt. dan Audit. Indones., vol. 14, no. 2, 2010. [22] Sugiyono, Metode Penelitian Kuantitatif, Kualitatif dan R&D. Bandung: PT Alfabeta, 2017. [23] I. Ghozali, Aplikasi Analisis Multivariete Dengan Program IBM SPSS, Edisi 8. Universitas Diponegoro, 2013. [24] I. Ghozali, Aplikasi Analisis Multivariate dengan Program SPSS. Cetakan Kelima. Semarang: Universitas Diponegoro, 2012.
https://openalex.org/W2157501255	https://pure.eur.nl/files/47610633/68.pdf	English	null	A Genome Wide Association Study Links Glutamate Receptor Pathway to Sporadic Creutzfeldt-Jakob Disease Risk	PloS one	2,015	cc-by	8,682	* c.vanduijn@erasmusmc.nl RESEARCH ARTICLE Academic Editor: Gianluigi Zanusso, University of Verona, ITALY Academic Editor: Gianluigi Zanusso, University of Verona, ITALY Copyright: © 2015 Sanchez-Juan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A Genome Wide Association Study Links Glutamate Receptor Pathway to Sporadic Creutzfeldt-Jakob Disease Risk Pascual Sanchez-Juan1, Matthew T. Bishop2, Gabor G. Kovacs3, Miguel Calero4,5, Yurii S. Aulchenko6,7,8, Anna Ladogana9, Alison Boyd10, Victoria Lewis10, Claudia Ponto11, Olga Calero4, Anna Poleggi9, Ángel Carracedo12,13, Sven J. van der Lee6, Thomas Ströbel3, Fernando Rivadeneira6,14, Albert Hofman6, Stéphane Haïk15, Onofre Combarros1, José Berciano1, Andre G. Uitterlinden6,14, Steven J. Collins10, Herbert Budka3, Jean-Philippe Brandel15, Jean Louis Laplanche16, Maurizio Pocchiari9, Inga Zerr11, Richard S. G. Knight2, Robert G. Will2, Cornelia M. van Duijn6* 1 Neurology Department, University Hospital “Marqués de Valdecilla”. Instituto de Investigación “Marqués de Valdecilla” IDIVAL and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED). Santander, Spain, 2 The National Creutzfeldt-Jakob disease Research and Surveillance Unit, University of Edinburgh, United Kingdom, 3 Institute of Neurology, Medical University Vienna, Vienna, Austria, 4 Chronic Disease Programme and CIBERNED. Carlos III Institute of Health. Madrid. Spain, 5 Alzheimer Disease Research Unit, CIEN Foundation, Carlos III Institute of Health, Alzheimer Center Reina Sofia Foundation, Madrid, Spain, 6 Department of Epidemiology, Erasmus Medical Centre, Rotterdam, the Netherlands, 7 Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia, 8 Novosibirsk State University, Novosibirsk, Russia, 9 Department of Cell Biology and Neurosciences Instituto Superiore di Sanità, Roma, Italy, 10 Department of Pathology, The University of Melbourne, Parkville, 3010, Australia, 11 Department of Neurology, Clinical Dementia Centre, University Medical Center and German Center for Neurodegenerative Diseases (DZNE)—site Göttingen, Göttingen, Germany, 12 Fundación Pública Galega de Medicina Xenómica, CIBERER, Grupo de Medicina Xenómica-Universidad de Santiago de Compostela, Santiago de Compostela, Spain, 13 Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, KSA, 14 Department of Internal Medicine, Erasmus Medical Center, Rotterdam, the Netherlands, 15 Sorbonne Universités, UPMC Univ Paris 06 UMR S 1127, and Inserm, U 1127, and CNRS UMR 7225, and ICM, F-75013, Paris, France; AP-HP, Hôpital de la Pitié Salpêtrière, Cellule Nationale de Référence des maladies de Creutzfeldt-Jakob, F-75013, Paris, France, 16 Service de biochimie et biologie moleculaire, Laboratoire associé au CNR "ATNC", Hôpital Lariboisiére, AP-HP, Paris, France OPEN ACCESS Citation: Sanchez-Juan P, Bishop MT, Kovacs GG, Calero M, Aulchenko YS, Ladogana A, et al. (2015) A Genome Wide Association Study Links Glutamate Receptor Pathway to Sporadic Creutzfeldt-Jakob Disease Risk. PLoS ONE 10(4): e0123654. doi:10.1371/journal.pone.0123654 Abstract We performed a genome-wide association (GWA) study in 434 sporadic Creutzfeldt-Jakob disease (sCJD) patients and 1939 controls from the United Kingdom, Germany and The Netherlands. The findings were replicated in an independent sample of 1109 sCJD and 2264 controls provided by a multinational consortium. From the initial GWA analysis we se- lected 23 SNPs for further genotyping in 1109 sCJD cases from seven different countries. Five SNPs were significantly associated with sCJD after correction for multiple testing. Subsequently these five SNPs were genotyped in 2264 controls. The pooled analysis, in- cluding 1543 sCJD cases and 4203 controls, yielded two genome wide significant results: rs6107516 (p-value=7.62x10-9) a variant tagging the prion protein gene (PRNP); and rs6951643 (p-value=1.66x10-8) tagging the Glutamate Receptor Metabotropic 8 gene (GRM8). Next we analysed the data stratifying by country of origin combining samples from Data Availability Statement: All relevant data are within the paper and its Supporting Information files. A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD the pooled analysis with genotypes from the 1000 Genomes Project and imputed genotypes from the Rotterdam Study (Total n=12967). The meta-analysis of the results showed that rs6107516 (p-value=3.00x10-8) and rs6951643 (p-value=3.91x10-5) remained as the two most significantly associated SNPs. Rs6951643 is located in an intronic region of GRM8, a gene that was additionally tagged by a cluster of 12 SNPs within our top100 ranked re- sults. GRM8 encodes for mGluR8, a protein which belongs to the metabotropic glutamate receptor family, recently shown to be involved in the transduction of cellular signals trig- gered by the prion protein. Pathway enrichment analyses performed with both Ingenuity Pathway Analysis and ALIGATOR postulates glutamate receptor signalling as one of the main pathways associated with sCJD. In summary, we have detected GRM8 as a novel, non-PRNP, genome-wide significant marker associated with heightened disease risk, pro- viding additional evidence supporting a role of glutamate receptors in sCJD pathogenesis. the pooled analysis with genotypes from the 1000 Genomes Project and imputed genotypes from the Rotterdam Study (Total n=12967). The meta-analysis of the results showed that rs6107516 (p-value=3.00x10-8) and rs6951643 (p-value=3.91x10-5) remained as the two most significantly associated SNPs. Rs6951643 is located in an intronic region of GRM8, a gene that was additionally tagged by a cluster of 12 SNPs within our top100 ranked re- sults. GRM8 encodes for mGluR8, a protein which belongs to the metabotropic glutamate receptor family, recently shown to be involved in the transduction of cellular signals trig- gered by the prion protein. Pathway enrichment analyses performed with both Ingenuity Pathway Analysis and ALIGATOR postulates glutamate receptor signalling as one of the main pathways associated with sCJD. In summary, we have detected GRM8 as a novel, non-PRNP, genome-wide significant marker associated with heightened disease risk, pro- viding additional evidence supporting a role of glutamate receptors in sCJD pathogenesis. 222887) Project number: FP7-KBBE-2007-2A). The study was performed within the recently established Clinical Dementia Center at the University Medical Center Göttingen and was partly supported by grants from the EU Joint Programme – Neurodegenerative Disease Research (JPND - DEMTEST "Biomarker based diagnosis of rapid progressive dementias- optimization of diagnostic protocols", 01ED1201A). This study was funded by the Robert Koch Institute through funds from the Federal Ministry of Health (grant no. 1369-341). Italy: The Italian Registry of CJD and related disorders is funded by the Ministry of Health, National Centre for Disease Prevention and Control, Central Actions.This work was partly supported by grant from the EU Joint Programme – Neurodegenerative Disease Research (JPND - DEMTEST “Biomarker based diagnosis of rapid progressive dementias-optimization of diagnostic protocols”, 01ED1201A). The Netherlands: the generation and management of genome-wide association study (GWAS) genotype data for the Rotterdam Study is supported by the Netherlands Organization of Scientific Research NWO Investments (nr. 175.010.2005.011, 911- 03-012). This study is funded by the Research Institute for Diseases in the Elderly (014-93-015; RIDE2), the Netherlands Genomics Initiative (NGI)/Netherlands Organisation for Scientific Research (NWO) project nr. 050-060-810. The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. YSA is supported by Russian Science Foundation (RSCF) grant 14-14-00313. Spain. PSJ was supported by a grant from FIS (PI12/02288) and JPND project DEMTEST (PI11/03028). AC is supported by PI13/01136 Acción Estratégica de Salud del Instituto de Salud Carlos III e INNOPHARMA. Australia: The Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR) is funded by the Commonwealth Department of Health. SJC is supported by a NHMRC Practitioner Fellowship (#APP1005816). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist Introduction Sporadic Creutzfeldt-Jakob disease (sCJD), although rare, with a yearly incidence of one to two cases per million, is the most common form of human prion disease. This group of disorders is characterized by spongiform changes in the brain, as well as accumulation of misfolded, often protease-resistant, conformers (PrPSc) of the normal prion protein (PrPC). The PrPC gene (PRNP) plays a central role in prion disease susceptibility. Expression of PrPC is indispensable for disease transmission [1] and the polymorphism coding for methionine (M) or valine (V) at codon 129 (PRNP M129V) has been linked to disease risk [2]. Homozygosity at PRNP M129V has been consistently associated to sCJD, being one of the strongest common genetic risk fac- tors reported for neurodegenerative diseases. The remarkable disease-determining effect of this PRNP polymorphism is observed in variant CJD, a subtype acquired from dietary exposure to bovine spongiform encephalopathy [3], where all definite and probable clinical cases studied to date have been PRNP129MM [4]. Similar to other diseases, several genetic association studies with candidate genes have been performed on sCJD susceptibility [5]. Only one previous genome wide association study (GWAS) of sCJD risk has been published to date, showing that the PRNP locus was strongly as- sociated with disease risk, specifically with rs1799990 (PRNP M129V) [6]. To further scrutinize genomic variations related to sCJD risk we have performed a three- stage GWAS encompassing a total of 1,543 sCJD cases and 4,203 controls, as well as a meta- analysis encompassing data from the 1000 Genomes Project and imputations from the Rotter- dam Study. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by: UK: the National CJD Research and Surveillance UK Unit is funded by the Department of Health and the Scottish Government Health Department. The National CJD Research and Surveillance Unit is funded by the Policy Research Programme in the Department of Health. Germany: This work was supported by a grant from the European Commission (Protecting the food chain from prions: shaping European priorities through basic and applied research (PRIORITY, N° PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 1 / 14 Results Demographic and clinical features of the sCJD case populations are shown in S1 Table. The Q-Q plots for autosomal and X chromosome SNPs are given in S1 Fig; during discovery (stage one) the genomic inflation factor λ was 1.053 for autosomal and 1.057 for X chromosome SNPs. Competing Interests: The authors have declared that no competing interests exist. S2 Table shows the top 100 SNPs associated with sCJD at discovery stage, sorted by allelic differences p-value. A total of 23 SNPs were taken forward to replication. In stage two, we suc- cessfully genotyped 22 of the 23 SNPs with the Sequenom iPLEx GOLD platform in an indepen- dent population of 1,109 samples of sCJD. Table 1 shows that five SNPs remained significant 2 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD Table 1. (Continued) ( ) Stage one: Discovery Stage two: Replication in independent sCJD Cases SNP CJD risk allele in Discovery population GENE tagged Controls Frequencies of sCJD risk allele (n = 1939) Cases Frequencies of sCJD risk allele (n = 434) P1df* Cases Frequencies of sCJD risk allele (n = 1109) P-value** rs9830696 T NA 0.120 0.173 1.02E- 05 0.104 0.0711 sCJD: Sporadic Creutzfeldt-Jakob Disease In bold SNPs signiﬁcant after Bonferroni correction (Replication P-value <0.0023) * P-value for allellic diferences adjusted by Country of origin by PCA *Chi Squared p-value from comparison between Allele frequencies of independent sCJD cases and stage one controls NA: SNP in intergenic region. doi:10.1371/journal.pone.0123654.t001 doi:10.1371/journal.pone.0123654.t001 after Bonferroni correction. In stage three we genotyped those five SNPs in a population of 2,264 independent controls with Sequenom iPLEx GOLD. S2 Fig depicts neatly separated geno- type clusters of the five SNPs studied in stage three. Only two SNPs were successfully replicated at stage three and reached genome wide significant p-values after meta-analysis of discovery and replication results: rs6107516 (p-value = 7.62x10-9) tagging PRNP and in linkage disequilib- rium (LD) with PRNP M129V (rs1799990), and rs6951643 (p-value = 1.66x10-8) an intronic SNP within GRM8 (Glutamate Receptor Metabotropic 8) in chromosome 7. Table 2 shows dif- ferences in allelic frequencies in the discovery, replication and pooled population sets including 1,543 sCJD cases and 4,203 controls. The A allele of rs6951643 was associated with a 1.27 fold increased risk of sCJD (95%CI = 1.17–1.38), and this effect was consistently observed across all tested populations (Table 3). after Bonferroni correction. In stage three we genotyped those five SNPs in a population of 2,264 independent controls with Sequenom iPLEx GOLD. S2 Fig depicts neatly separated geno- type clusters of the five SNPs studied in stage three. Only two SNPs were successfully replicated at stage three and reached genome wide significant p-values after meta-analysis of discovery and replication results: rs6107516 (p-value = 7.62x10-9) tagging PRNP and in linkage disequilib- rium (LD) with PRNP M129V (rs1799990), and rs6951643 (p-value = 1.66x10-8) an intronic SNP within GRM8 (Glutamate Receptor Metabotropic 8) in chromosome 7. Table 2 shows dif- ferences in allelic frequencies in the discovery, replication and pooled population sets including 1,543 sCJD cases and 4,203 controls. A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD Table 1. SNPs genotyped in stage two. Stage one: Discovery Stage two: Replication in independent sCJD Cases SNP CJD risk allele in Discovery population GENE tagged Controls Frequencies of sCJD risk allele (n = 1939) Cases Frequencies of sCJD risk allele (n = 434) P1df Cases Frequencies of sCJD risk allele (n = 1109) P-value** rs10061929 A FLJ43080 0.159 0.224 4.58E- 05 0.200 5.89E-05 rs10915708 T NA 0.140 0.207 1.35E- 06 0.158 0.0626 rs11075924 C NA 0.498 0.565 5.52E- 05 0.500 0.8808 rs11245373 T NA 0.037 0.069 2.48E- 05 0.049 0.0238 rs12102156 T NA 0.863 0.912 2.00E- 05 0.849 0.1457 rs12188818 C NA 0.796 0.859 6.44E- 05 0.809 0.2544 rs12419710 A NA 0.210 0.279 2.52E- 05 0.219 0.3746 rs17060736 G NA 0.112 0.160 7.13E- 05 0.121 0.3134 rs17115017 A GRIA1 0.050 0.082 2.23E- 04 0.072 5.00E-04 rs17833759 C GRIN2B 0.085 0.140 7.74E- 07 0.095 0.1955 rs196940 C ERN1 0.758 0.818 6.19E- 05 0.758 0.9631 rs2240344 G NA 0.432 0.600 4.47E- 19 0.443 0.4194 rs2627829 A INPP4B 0.939 0.970 1.83E- 04 0.931 0.2188 rs392184 T MACROD2 0.052 0.096 2.00E- 06 0.07 0.003 rs565559 T NA 0.553 0.618 2.02E- 04 0.579 0.057 rs6027482 C LOC100131710 0.187 0.247 6.69E- 05 0.181 0.5476 rs6107516 C PRNP 0.752 0.827 6.92E- 06 0.804 3.27E-06 rs6463269 G NA 0.084 0.134 3.53E- 06 GENOTYPE FAILED - rs6496239 T NA 0.805 0.862 4.96E- 05 0.821 0.1195 rs6820644 T NA 0.357 0.433 3.40E- 06 0.647 0.7566 rs6951643 A GRM8 0.507 0.592 8.09E- 06 0.573 6.79E-07 rs9521699 A COL4A2 0.137 0.194 3.84E- 05 0.173 2.00E-04 (Continued) Stage two: Replication in independent sCJD Cases PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 3 / 14 Fine mapping in search for functional variants linked to rs6951643 was attempted sequencing the 11 exons of GRM8 gene and the corre- sponding intronic flanking regions in 96 sCJD patients. We found 8 intronic SNPs (rs73231278, rs62468898, rs1008274, rs111546739, rs17685327, rs6951643, rs2074012, rs35648111) and one exonic non-coding SNP (rs34182595). Functional prediction analysis of these genetic variants by FuncPred or RESCUE-ESE showed no indication of regulatory impli- cations. In the Gtex eQTL database we found no eQTL associated to any of the GRM8 SNPs. There is no co-expression of GRM8 and PRNP in blood eQTLs in Genenetwork. However, based on PITA prediction we found that rs34182595, which is an insertion/deletion SNP in were 12 tagging GRM8 (S2 Table). Pathway analysis using IPA showed that glutamate receptor signalling was the most over-represented canonical pathway within our top results (p-value = 8.01x10-5) (Fig 1). Analysis with ALIGATOR software showed (S3 Table) that the GO category glutamate receptor activity was the 4th most represented amongst our top results. Three genes from the GO category "glutamate receptor pathway" (S2 Table) were within our top 100 SNPs (GRM8, GRIN2B and GRIA1). Fine mapping in search for functional variants linked to rs6951643 was attempted sequencing the 11 exons of GRM8 gene and the corre- sponding intronic flanking regions in 96 sCJD patients. We found 8 intronic SNPs (rs73231278, rs62468898, rs1008274, rs111546739, rs17685327, rs6951643, rs2074012, rs35648111) and one exonic non-coding SNP (rs34182595). Functional prediction analysis of these genetic variants by FuncPred or RESCUE-ESE showed no indication of regulatory impli- cations. In the Gtex eQTL database we found no eQTL associated to any of the GRM8 SNPs. There is no co-expression of GRM8 and PRNP in blood eQTLs in Genenetwork. However, based on PITA prediction we found that rs34182595, which is an insertion/deletion SNP in Table 4. Meta-analysis by country of origin. The A allele of rs6951643 was associated with a 1.27 fold increased risk of sCJD (95%CI = 1.17–1.38), and this effect was consistently observed across all tested populations (Table 3). p p Rs6107516 (p-value = 3.00x10-8) and rs6951643 (p-value = 3.91x10-5) remained as the two most statistically significant associations in a meta-analysis of the discovery data adjusted for PCAs and analysis of replication samples stratified by country. This meta-analysis included discovery and replication samples plus data from the 1000 Genomes Project and imputed geno- types from the Rotterdam Study (Total n = 12967), (Table 4). Within the top 100 SNPs, there Table 2. SNPs studied in stage three. Discovery Population Replication Population Pooled Samples ID CJD risk allele in Discovery population GENE tagged Case, Control Frequencies of CJD risk allele (n = 434, n = 1939) P-value P-value* OR (95%CI) Case, Control Frequencies of CJD risk allele (n = 1109, n = 2264) P-value OR (95%CI) Case.Control Frequencies of CJD risk allele (n = 1543, n = 4203) P-value OR (95%CI) rs10061929 A FLJ43080 0.224, 0.159 4.30E- 06 4.58E-05 1.53 (1.27–1.83) 0.200, 0.196 0.6882 1.03 (0.90–1.17) 0.207, 0.179 0.0007 1.20 (1.08–1.33) rs17115017 A GRIA1 0.082, 0.050 3.00E- 04 2.23E-04 1.69 (1.27–2.24) 0.072, 0.077 0.4434 1.08 (0.89–1.31) 0.075, 0.065 0.0597 1.17 (0.99–1.37) rs6107516 C PRNP 0.827, 0.752 2.07E- 06 6.92E-06 1.58 (1.31–1.91) 0.804, 0.766 5.00E- 04 1.25 (1.10–1.42) 0.811, 0.759 7.62E- 09 1.35 (1.22–1.50) rs6951643 A GRM8 0.592, 0.507 6.73E- 06 8.09E-06 1.41 (1.21–1.64) 0.573, 0.529 7.00E- 04 1.19 (1.08–1.32) 0.578, 0.519 1.66E- 08 1.27 (1.17–1.38) rs9521699 A COL4A2 0.194, 0.137 2.41E- 05 3.84E-05 1.51 (1.25–1.83) 0.173, 0.173 0.9723 1.00 (0.88–1.15) 0.179, 0.157 0.0045 1.17 (1.05–1.31) * Adjusted by Country of origin by PCA. doi:10.1371/journal.pone.0123654.t002 ID PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 4 / 14 MarkerName Effect estimates of discovery analysis corrected for population substructure by PCA Meta-analysis of effect estimates of replication analysis stratiﬁed by country of origin Meta-analysis of discovery and replication β SE P-value β SE P-value β Direction* β SE P-value rs6107516 0.4243 0.0944 6.92E-06 0.2327 0.0637 0.000262 ₊₊₊₊₊ 0.2927 0.0528 3.00E-08 rs17115017 -0.5363 0.1453 0.000223 -0.1420 0.1061 0.181 ₋₋₋₊₊ -0.2792 0.0857 0.001123 rs9521699 -0.4087 0.0993 3.84E-05 -0.0233 0.0723 0.7468 ₊₋₊₋₋ -0.157 0.0585 0.007242 rs6951643 -0.3472 0.0778 8.09E-06 -0.1033 0.0545 0.05821 ₋₊₊₊₊ -0.1837 0.0447 3.91E-05 rs10061929 -0.3737 0.0917 4.58E-05 -0.0289 0.0701 0.6801 ₋₋₋₊₊ -0.1562 0.0557 0.005048 * Concordance between discovery population βs and each of the ﬁve replication populations; PCA, Principal Component Analysis; SE, standard Error. doi:10 1371/journal pone 0123654 t004 Table 3. - rs6951643 pooled genotypes by country. Population GG n (%) AG n (%) AA n (%) A allele n (%) G allele n (%) CONTOLS UK 364 (24.6) 741 (50.0) 377 (25.4) 1495 (50.4) 1469 (49.6) NL 118 (22.4) 265 (50.3) 144 (27.3) 553 (52.5) 501 (47.5) Spain 488 (22.3) 1097 (50.0) 608 (27.7) 2313 (52.7) 2073 (47.3) CASES UK 28 (10.5) 155 (58.3) 83 (31.2) 321 (60.3) 211 (39.7) NL 23 (18.1) 62 (48.8) 42 (33.1) 146 (57.5) 108 (42.5) Germany 66 (16.7) 217 (54.8) 113 (28.5) 443 (55.9) 349 (44.1) Italy 43 (15.0) 140 (48.8) 104 (36.2) 348 (60.6) 226 (39.4) Australia 10 (20.8) 26 (54.2) 12 (25.0) 50 (52.1) 46 (47.9) France 23 (15.3) 77 (51.3) 50 (33.3) 177 (59.0) 123 (41.0) Spain 35 (17.2) 110 (54.2) 58 (28.6) 226 (55.7) 180 (44.3) Austria 11 (20.0) 27 (49.1) 17 (30.9) 61 (55.5) 49 (44.5) ALL CONTOLS 970 (23.1) 2103 (50.0) 1129 (26.9) 4361 (51.9) 4043 (48.1) ALL CASES 239 (15.6) 814 (53.1) 479 (31.3) 1772 (57.8) 1292 (42.2) HapMap Frequencies (CEU) 27 (23.9) 59 (52.2) 27 (23.9) 113 (0.50) 113 (0.50) doi:10.1371/journal.pone.0123654.t003 Table 3. - rs6951643 pooled genotypes by country. were 12 tagging GRM8 (S2 Table). Pathway analysis using IPA showed that glutamate receptor signalling was the most over-represented canonical pathway within our top results (p-value = 8.01x10-5) (Fig 1). Analysis with ALIGATOR software showed (S3 Table) that the GO category glutamate receptor activity was the 4th most represented amongst our top results. Three genes from the GO category "glutamate receptor pathway" (S2 Table) were within our top 100 SNPs (GRM8, GRIN2B and GRIA1). A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD were 12 tagging GRM8 (S2 Table). Pathway analysis using IPA showed that glutamate receptor signalling was the most over-represented canonical pathway within our top results (p-value = 8.01x10-5) (Fig 1). Analysis with ALIGATOR software showed (S3 Table) that the GO category glutamate receptor activity was the 4th most represented amongst our top results. Three genes from the GO category "glutamate receptor pathway" (S2 Table) were within our top 100 SNPs (GRM8, GRIN2B and GRIA1). Fine mapping in search for functional variants linked to rs6951643 was attempted sequencing the 11 exons of GRM8 gene and the corre- sponding intronic flanking regions in 96 sCJD patients. We found 8 intronic SNPs (rs73231278, rs62468898, rs1008274, rs111546739, rs17685327, rs6951643, rs2074012, rs35648111) and one exonic non-coding SNP (rs34182595). Functional prediction analysis of these genetic variants by FuncPred or RESCUE-ESE showed no indication of regulatory impli- cations. In the Gtex eQTL database we found no eQTL associated to any of the GRM8 SNPs. There is no co-expression of GRM8 and PRNP in blood eQTLs in Genenetwork. However, based on PITA prediction we found that rs34182595, which is an insertion/deletion SNP in Table 3. - rs6951643 pooled genotypes by country. Population GG n (%) AG n (%) AA n (%) A allele n (%) G allele n (%) CONTOLS UK 364 (24.6) 741 (50.0) 377 (25.4) 1495 (50.4) 1469 (49.6) NL 118 (22.4) 265 (50.3) 144 (27.3) 553 (52.5) 501 (47.5) Spain 488 (22.3) 1097 (50.0) 608 (27.7) 2313 (52.7) 2073 (47.3) CASES UK 28 (10.5) 155 (58.3) 83 (31.2) 321 (60.3) 211 (39.7) NL 23 (18.1) 62 (48.8) 42 (33.1) 146 (57.5) 108 (42.5) Germany 66 (16.7) 217 (54.8) 113 (28.5) 443 (55.9) 349 (44.1) Italy 43 (15.0) 140 (48.8) 104 (36.2) 348 (60.6) 226 (39.4) Australia 10 (20.8) 26 (54.2) 12 (25.0) 50 (52.1) 46 (47.9) France 23 (15.3) 77 (51.3) 50 (33.3) 177 (59.0) 123 (41.0) Spain 35 (17.2) 110 (54.2) 58 (28.6) 226 (55.7) 180 (44.3) Austria 11 (20.0) 27 (49.1) 17 (30.9) 61 (55.5) 49 (44.5) ALL CONTOLS 970 (23.1) 2103 (50.0) 1129 (26.9) 4361 (51.9) 4043 (48.1) ALL CASES 239 (15.6) 814 (53.1) 479 (31.3) 1772 (57.8) 1292 (42.2) HapMap Frequencies (CEU) 27 (23.9) 59 (52.2) 27 (23.9) 113 (0.50) 113 (0.50) doi:10.1371/journal.pone.0123654.t003 Table 4. Meta-analysis by country of origin. A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD Fig 1. Canonical pathway analysis performed by IPA software including those genes tagged by SNPs with p-value <0.001 in our GWAs analysis. The horizontal axis represents the pathways identified. The ratio (vertical axis, right) is calculated by the numbers of genes in a given pathway that meet cutoff criteria, divided by total numbers of genes that make up that pathway. The orange line stands for the threshold above which there are statistically significantly (by default P<0.05). The vertical axis (left) shows the −log of the p-value calculated based on Fisher’s exact test. Fig 1. Canonical pathway analysis performed by IPA software including those genes tagged by SNPs with p-value <0.001 in our GWAs analysis. The horizontal axis represents the pathways identified. The ratio (vertical axis, right) is calculated by the numbers of genes in a given pathway that meet cutoff criteria, divided by total numbers of genes that make up that pathway. The orange line stands for the threshold above which there are statistically significantly (by default P<0.05). The vertical axis (left) shows the −log of the p-value calculated based on Fisher’s exact test. Fig 1. Canonical pathway analysis performed by IPA software including those genes tagged by SNPs with p-value <0.001 in our GWAs analysis. The horizontal axis represents the pathways identified. The ratio (vertical axis, right) is calculated by the numbers of genes in a given pathway that meet cutoff criteria, divided by total numbers of genes that make up that pathway. The orange line stands for the threshold above which there are statistically significantly (by default P<0.05). The vertical axis (left) shows the −log of the p-value calculated based on Fisher’s exact test. doi:10.1371/journal.pone.0123654.g001 exon 11 in partial LD with rs6951643 (D' = 0.41; r2 = 0.16), is located in a target site for micro- RNAs (miR-103 and miR-107), a finding that has been previously reported [7]. We then performed an immunohistochemical study in brain samples from 48 sCJD pa- tients. Immunoreactivity for mGluR8 was observed in neurons and microglial cells (both in the white and grey matter) While in neurons we did not observe obvious differences we found a trend towards higher combined score of mGluR8 immunoreactivity in microglia related to the A allele at rs6951643 (Fig 2); however, these differences were not statistically significant (ordi- nal regression A-carriers versus A-non-carriers adjusting by c129 and levels of microglia in temporal cortex; p-value = 0.093). S3 Fig shows semi-quantitative assessments of mGluR8 expression in microglia (0 to 3) in the temporal region across the three rs6951643 genotypes. We found no association between rs6951643 genotypes and patient’s age of disease onset or disease duration. PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 MarkerName Effect estimates of discovery analysis corrected for population substructure by PCA Meta-analysis of effect estimates of replication analysis stratiﬁed by country of origin Meta-analysis of discovery and replication β SE P-value β SE P-value β Direction* β SE P-value rs6107516 0.4243 0.0944 6.92E-06 0.2327 0.0637 0.000262 ₊₊₊₊₊ 0.2927 0.0528 3.00E-08 rs17115017 -0.5363 0.1453 0.000223 -0.1420 0.1061 0.181 ₋₋₋₊₊ -0.2792 0.0857 0.001123 rs9521699 -0.4087 0.0993 3.84E-05 -0.0233 0.0723 0.7468 ₊₋₊₋₋ -0.157 0.0585 0.007242 rs6951643 -0.3472 0.0778 8.09E-06 -0.1033 0.0545 0.05821 ₋₊₊₊₊ -0.1837 0.0447 3.91E-05 rs10061929 -0.3737 0.0917 4.58E-05 -0.0289 0.0701 0.6801 ₋₋₋₊₊ -0.1562 0.0557 0.005048 * Concordance between discovery population βs and each of the ﬁve replication populations; PCA, Principal Component Analysis; SE, standard Error. doi:10.1371/journal.pone.0123654.t004 Table 4. Meta-analysis by country of origin. PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 5 / 14 Discussion In this study we report a non-PRNP genetic risk variant for sCJD, GRM8. Moreover, building on findings from previous studies [8], pathway analyses yielded glutamate receptor signalling as one of the main pathways linked to sCJD pathogenesis. A previous GWAS of prion diseases employed 1,259 sCJD samples, in addition to other dis- ease subtypes and 6,015 shared controls [6]. In the sCJD sub-group and in a meta-analysis in- cluding all prion cases only variants in PRNP were found to be significantly associated with disease risk. Several SNPs outside PRNP were identified, but in contrast to our multi-national disease cohort the association with sCJD was not homogeneous across the different geographi- cal groups. Our main novel finding (rs6951643) was not statistically significant in that analysis. One possible explanation for this discrepancy is the fact that both studies have limited power to detect small effects due to the overall restricted case numbers included for study and there- fore a SNP with a relatively modest OR of 1.27 might not be detected by all analyses. The expe- rience and insights drawn from GWAS performed in more prevalent disorders, such as Alzheimer’s disease, shows that increasing sample size and performing meta-analysis might be essential in order to discover and confirm, relatively small-effect genetic risk factors. However, it is worth re-iterating that in our study, the association with rs6951643 was consistent across PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 6 / 14 A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD Fig 2. Representative photomicrographs of a sCJD case with AA (a, c) and a sCJD case with GG (b, d) rs6951643 genotype showing differences in mGluR8 immunostaining of microglial cells in the temporal cortex (a, b) and temporal white matter (c, d). Bar represents 50 μm for all images. d i 10 1371/j l 0123654 002 Fig 2. Representative photomicrographs of a sCJD case with AA (a, c) and a sCJD case with GG (b, d) rs6951643 genotype showing differences in mGluR8 immunostaining of microglial cells in the temporal cortex (a, b) and temporal white matter (c, d). Bar represents 50 μm for all images. the geographically diverse sCJD population tested, showing an over-representation of the A al- lele in cases, and across the different genotyping methods used. doi:10.1371/journal.pone.0123654.g002 doi:10.1371/journal.pone.0123654.g002 the geographically diverse sCJD population tested, showing an over-representation of the A al- lele in cases, and across the different genotyping methods used. PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 Discussion A limitation of our primary analysis was the fact that we were not able to adjust by country of origin at the replication stage. In order to overcome this caveat we performed a meta- analysis including control data from the 1000 Genomes Project and imputed data from the Rot- terdam Study. In the meta-analysis of the stratified analysis by country of origin the association of both SNPs became less significant suggesting that some signal might be caused by popula- tion stratification. Despite the fact that both variants were nominally significant in the meta- analysis of the replication data, the PRNP variant was the only genome wide significantly asso- ciated to sCJD in the pooled meta-analysis. However, this meta-analysis is not perfect either, as ideally the cases and controls should originate from the same population and in our analysis we attempted to match on country of origin (see S4 Table) and this approach is statistically less powerful because of the uneven distribution of cases and controls, as might reflect the fact that the PRNP SNP p-value is also less significant in the meta-analysis than in the primary analysis. Still we acknowledge that the results are a call for caution and the association between GRM8 genetic variants and sCJD should be confirmed in larger analysis with extensive control for population stratification. 7 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD GRM8 encodes for mGluR8, a protein that belongs to the metabotropic glutamate receptor family, a family that has recently been linked to the transduction of physiological and cytotoxic signals mediated by PrPC. MGluR1 and mGluR5 have been shown to interact with PrPC, with such associations appearing important for promoting neurite outgrowth [9, 10]. Additionally, a recent study demonstrated that mGluR5 coupled with PrPC mediated the cellular toxicity of soluble β-amyloid oligomers [10]. Further, in the APPswe/PS1dE9 Alzheimer’s disease mouse model, altered PrPC processing and a selective increase in cortical mGluR1 expression has been reported. The authors hypothesized that complex processing of PrPC in connection with mGluR1 over-expression is triggered by β-amyloid peptides [11]. In the setting of accumula- tion of PrPSc, our immunohistochemical assessment of a limited sample of sCJD patients found that carriers of the risk allele at rs6951643 tended to have higher mGluR8 expression in microglial cells compared to non-carriers. Ethics statement The present study was conducted according to the revised Declaration of Helsinki and Good Clinical Practice guidelines. A signed informed consent to participate in genetic research was obtained from all participants or patients’ relatives. The study was approved by Comité de Ética de la Investigación y de Bienestar Animal (CEIyBA), National Health Institute Carlos III and Comité de Ética de Ensayos clínicos de Galicia and Comite de Ética de la Fundación Pú- blica Gallega de Medicina Genómica (Servicio Gallego de Salud, SERGAS) (Spain); the Lothian Health Board, Lothian Research Ethics Committee (reference MCO/103/90) (UK); the Ethik- Kommission der Universitätsmedizin Göttingen (No. 9/6/0) (Germany); the Ethic Committee of the Istituto Superiore di Sanità (CE-ISS 09/266) (Italy); the Medical Ethics Committee of The Erasmus MC and the review board of The Netherlands Ministry of Health Welfare and Sports (The Netherlands); the Ethics Committee of the Medical University of Vienna (396/ 2011) (Austria); the Human Research Ethics Committee, based at The University of Melbourne (941450) (Australia). Discussion Although our functional prediction analysis for rs6951643 was uninformative, it is of interest that a nearby variant in partial LD (rs34182595) is located in a micro-RNA target site and could influence gene expression. In conclusion, our study has detected a GRM8 genetic variant as a suggestive marker for sCJD risk mapping outside the PRNP region. Our findings provide evidence supporting a role for glutamate receptor signalling pathways in sCJD susceptibility. The involvement of gluta- mate receptor pathway in sCJD should be addressed in future studies in order to provide new insights in its pathogenesis. Our results underscore the importance of increasing sample sizes in future studies in order to augment the likelihood of detecting additional non-PRNP genetic risk factors for sCJD. A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD Table 5. Study Design and populations tested. STAGES Table 5. Study Design and populations tested. STAGES Table 5. Study Design and populations tested. STAGES POPULATION EFFECTIVELY ASSESSED* sCJD CASES (N) CONTROLS (N) Stage 1: Discovery Germany (113) Genotyping method: Affymetrix 500k array UK (269) UK WTCCC controls (1482) Number of SNPs effectively assessed 279389 Netherlands (52) Netherlands-RS controls (457) Number of SNPs selected for replication: 23 TOTAL sCJD (434) TOTAL controls (1939) Stage 2: Replication in independent sCJD Cases Germany (284) Genotyping method: Sequenom iPLEX GOLD Netherlands (76) Number of SNPs effectively assessed: 22 Italy (292) Number of SNPs replicated: 5 Australia (48) France (150) Spain (203) Austria (56) TOTAL sCJD (1109) Stage 3: Replication in independent Controls Spain-USC (2193) Genotyping method: Sequenom iPLEX GOLD Netherlands-RS (71) Number of SNPs effectively assessed: 5 TOTAL controls (2264) Number of SNPs replicated: 2 TOTAL controls (4203) POOLED ANALYSIS TOTAL sCJD (1543) Netherlands-RS (6192) British in England and Scotland-10KG (137) Controls added in analysis stratiﬁed by country Toscani in Italy-10KG (385) Iberian populations in Spain-10KG (217) Utah residents with Northern and Western European ancestry-10KG (290) TOTAL sCJD (1543) TOTAL controls (11424) Number of samples effectively genotyped that passed quality control sCJD, sporadic Creutzfeldt-Jakob disease; WTCCC, Welcome Trust Case Control Consortium; RS, Rotterdam Study; USC, University of Santiago de Compostela; 10KG, 1000 Genome Project. Stage 3: Replication in independent Controls Genotyping method: Sequenom iPLEX GOLD Number of SNPs effectively assessed: 5 Number of SNPs replicated: 2 Controls added in analysis stratiﬁed by country Number of samples effectively genotyped that passed quality control sCJD, sporadic Creutzfeldt-Jakob disease; WTCCC, Welcome Trust Case Control Consortium; RS, Rotterdam St Compostela; 10KG, 1000 Genome Project. *Number of samples effectively genotyped that passed quality control sCJD, sporadic Creutzfeldt-Jakob disease; WTCCC, Welcome Trust Case Control Consortium; RS, Rotterdam Study; USC, University of Santiago de Compostela; 10KG, 1000 Genome Project. doi:10.1371/journal.pone.0123654.t005 Populations and study design All patients were ascertained by National CJD Surveillance Centers. Only definite or probable sCJD cases, according to accepted classification criteria, were included [12]. Table 5 summa- rizes the study design and populations included after quality control. All cases and controls were of Caucasian origin. Legal representatives gave written informed consent, and all samples were taken in accordance with the Helsinki declaration. 8 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 Statistical analysis of genetic data The statistical analyses were conducted using GenABEL [15]. For the individual SNP analysis, we excluded those SNPs: 1) with call rates <98% within either group (n = 219,182); 2) with minor allele frequency<0.01 (n = 1); 3) with controls not in Hardy-Weinberg equilibrium (False Discovery Rate for unacceptably high individual heterozygosity<1%) (n = 1,300). The quality control further included a check of the sex chromosomes against the reported sex and unexpected sample duplicates (Identity by State, IBS>95%). All quality control was performed with the ‘check.marker’ function of GenABEL. After quality control, 279,389 out of 499,872 SNPs were included in the analyses. In the discovery analyses (stage one), we conducted a one degree of freedom additive score test between cases and controls coding the presence of the minor genotype 0 for non-carriers, 1 for heterozygous and 2 for homozygous carriers. We con- trolled subpopulation structure using principal components analysis (PCA). In brief, we select- ed a random set of 10,000 SNPs and calculated a genomic kinship matrix using pair wise identity by state statistic (function 'ibs'). We then performed PCA analysis (function 'cmdscale'), and adjusted our analysis by the three main IBS matrix principal components (function 'mlreg'). We calculated the genomic inflation factor lambda (λ) for both autosomal and X chromosome SNPs. In the replication phase (stages two and three), we compared allele frequencies in cases and controls by a Chi-squared test implemented in Haploview [16]. We used a Bonferroni correction to adjust for multiple testing, setting the threshold for significance to a p-value of 0.0023 (0.05/22 SNPs successfully genotyped). We combined the gene discovery and replication series of patients for all validated SNPs and used as the criterion for genome wide significance a p-value of 5x10-8. We attempted to adjust for country of origin after stage three. We analysed the data from stage two and three with genotypes of the 1000 Genomes Proj- ect [17] and imputed genotypes from the Rotterdam Study [18]. We extracted the SNPs from the 1000 Genomes reference set excluding the children and other family members (Version: phase I v3). Additionally we imputed the same SNPs from the Rotterdam Study (imputations of the same phase I v3of the 1000 Genomes). Imputed genotypes had a very high quality score (R2 > = 0.99). Next we paired our cases and controls by country of origin (S4 Table) and calcu- lated effect estimates of the SNPs per country. Genotyping and quality control At the discovery stage (stage one) patient’s samples were characterized using the Affymetrix 500k array at the USC node of the Spanish National Genotyping Center. Out of the initially available 554 sCJD samples there was not enough DNA in 61 (36 samples from the UK and 25 from Germany), leaving 493 for genotyping. Genotypes were called first by the Dynamic Model (DM) and those samples with an overall call rate >93% were subsequently called by a Bayesian Robust linear model with Mahalanobis distance (BRLMM). Samples with BRLMM call rates <95% and with call and discrimination rates of the Modified Partitioning Around Medoids algorithm (MDR-MCR) >10% were excluded (31 patient samples out of 493 did not reach these thresholds, leaving in total 462 available for the analysis). Genotype and quality control of the WTCCC controls have been described elsewhere [13]. The other set of controls (Rotterdam Study) were genotyped at the Genetic Laboratory, Department of Internal Medi- cine at Erasmus Medical Center (Rotterdam) also using the Afymmetrix 500K array following same protocols as those used for cases. After quality control, 28 patients and 25 controls were excluded resulting in a final discovery population of 434 sCJD patients and 1,939 controls. Rep- lication was attempted in independent sCJD patients (n = 1,201) (stage two) and control series PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 9 / 14 A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD (n = 2,264) (stage three) with the Sequenom iPLEx GOLD platform. SNPs for replication were selected as follows: after excluding outliers, we chose the 10 SNPs with lowest p-values (when a cluster of SNPs was in LD tagging the same gene we selected only the one with lowest p-value) and the remainder from the top 100 when a) they were in linkage disequilibrium with other top 100 SNPs or b) agreed in direction with the previous sCJD GWAS [6], or c) were connected to pathways of interest based on our previous study [14] (phosphatidylinositol) or based on the pathway analyses performed with the current data (glutamate receptors). PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 Sequencing We sequenced the 11 exons and intronic flanking areas of the GRM8 gene in 96 sCJD patients using specific primers (S5 Table). The amplification reactions were carried out with 25 ng of genomic DNA and 0.5 units of Taq DNA Polymerase in a volume of 25 μl. The final concentra- tions of other reactants were: 1x Taq DNA Polymerase Buffer, 0.1 mM dNTPs and 0.1 μM of each primer. The final concentration of MgCl2 was 2 mM for the amplification of exons 1–7 and 10; 4 mM for exons 8 and 9a; 1.5 mM for exon 9b and 1 mM for exon 11. Additionally, the amplification reactions of exons 1 and 8 were supplied with DMSO 10% and 5%, respectively. The PCR cycling conditions were as follows: initial denaturation at 96°C for 3 min followed by 30 cycles of 96°C for 30 s, annealing temperature (see S5 Table) for 30 s and extension tempera- ture of 72°C for 1 min with a final extension at 72°C for 10 min. A 2 μl aliquot of the amplifica- tion reaction was sequenced using 0.1μM of the above primers. Statistical analysis of genetic data The effect estimates of the SNPs from the discov- ery stage (adjusted by PCAs) and the effect estimates from the replication were meta-analysed using a inverse variance weighted meta-analysis(METAL version released 2011-03-25). [19]. We performed pathway analyses using ALIGATOR, a method for studying groups of genes by testing for over-representation of members of those groups within lists of genes containing sig- nificantly associated SNPs from GWA studies [20]. This analysis used 410 SNPs with a p-value for significance of <0.001 out of 115,565 within-gene SNPs from a total of 279,389 available. We found these SNPs were associated with 94 genes from a total of 13,092 genes with GO an- notation covered in this study. Additionally we used Ingenuity Pathway Analysis (IPA) www. ingenuity.com) to determine the functional pathways in the genes tagged by our top ranked SNPs. We again selected those genes tagged by SNPs with p-value <0.001 and selected the ca- nonical pathway analysis implemented in IPA software. PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 10 / 14 A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD Functional prediction analysis Functional prediction analysis of genetic variants was performed by the use of FuncPred online software (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm) [21]. Exonic splicing enhancers were analyzed by RESCUE-ESE Web Server (http://genes.mit.edu/burgelab/rescue-ese/) [22] and PITA online software (http://genie.weizmann.ac.il/pubs/mir07/index.html) [23] was used to assess potential micro-RNA target sites. We searched for correlations between our genetic variants and eQTL in GTEX [24] and Genenetwork (http://genenetwork.nl:8080/ GeneNetwork/) [25]. Phenotypic correlations We performed an immunohistochemical study in brain samples from 48 sCJD patients. The rs6951643 genotype distribution was as follows: 13 AA, 25 AG, and 9 GG. Sections from the hippocampal region CA1 sub-region, temporal cortex and white matter were immunostained for mGluR8. The intensity of mGluR8 (1:50; polyclonal rabbit antibody, Novus Biologicals, Cambridge, UK) immunostaining was evaluated using a scale of 0–3 (0: no; 1: weak; 2: moder- ate; 3: strong staining). The frequency of mGluR8 positive cells was scored semi-quantitatively using three categories: 1, <10%; 2, 10–50%; 3, >50% and was evaluated to provide information about the relative number of mGluR8 positive cells within the tissue. The product of these two values (intensity and frequency scores) was used to give the overall scores (total scores). In ad- jacent sections microglial activation using immunostaining for HLA-DP, DQ, DR (clone CR3/ 43, 1:00, monoclonal mouse), and spongiosis were also estimated semi-quantitatively. The analysis was performed (GGK) blinded to rs6951643 genotype. Ordinal regression was em- ployed to test for association between rs6951643 genotypes and brain semi-quantitative expres- sion of mGluR8. We adjusted by disease duration, PRNP M129V genotype, gliosis and spongiosis. We correlated clinical variables (age at death and patient’s disease onset) with the presence of 0, 1 or 2 sCJD risk alleles. In order to assess rs6951643 influence on age at onset and disease duration we performed a time to event analysis using Cox regression adjusting by PRNPM129V genotype and other relevant factors like sex and country of origin. S1 Fig. Q-Q plots for autosomal and X chromosome SNPs. (TIF) Acknowledgments The author thanks Simon Mead for sharing his data in order to select SNPs for replication. We also thank the Wellcome Trust Case Control Consortium (WTCCC) for the use of their control data. UK. The national UK CJD Surveillance Unit is grateful to clinicians, patients, and family members throughout the UK for a remarkable level of cooperation with the CJD surveillance program. France We thank all members of the French National Surveillance Network for Creutzfeldt-Jakob disease and all physicians for case notification. Italy We are very grateful to neurologists and neuropathologists, patients, and family members throughout Italy for their collaboration. The Netherlands We thank Pascal Arp, Mila Jhamai, Marijn Verkerk, Lizbeth Herrera, and Marjolein Peters for their help in creating the GWAS database, and Karol Estrada and Maksim V. Struchalin for their support in creation and analysis of imputed data. The au- thors are grateful to the study participants, the staff from the Rotterdam Study, and the partici- pating general practitioners and pharmacists. Spain. The authors thank Biobanco Valdecilla and IDIVAL for its support throughout this project thanks to J.M. Polo for their critical advice, thanks to Beatriz Sobrino and María Torres (CEGEN, Spanish National Genotying Center) for all their work in the sample genotyping, and Weyma Notel for her help in editing the manu- script. Australia. The ANCJDR thanks all patients, families, clinicians and allied health person- nel for their support and cooperation to facilitate this study. S6 Table. GWA analysis results X chromosome SNPs. (ZIP) S6 Table. GWA analysis results X chromosome SNPs. (ZIP) S6 Table. GWA analysis results X chromosome SNPs. (ZIP) S7 Table. GWA analysis results autosomic SNPs. (ZIP) S7 Table. GWA analysis results autosomic SNPs. (ZIP) S7 Table. GWA analysis results autosomic SNPs. (ZIP) Supporting Information 11 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0123654 April 28, 2015 A GWA Study Links Glutamate Receptor Pathway to Sporadic CJD S2 Fig. Genotype clusters of the five SNPs studied in stage three with Sequenom iPLEx GOLD. (TIF) S3 Fig. Semi-quantitative assessments of mGluR8 expression in microglia (0 to 3) in the temporal region across the three rs6951643 genotypes in 48 sCJD patients. (TIF) S1 Table. Demographic and clinical features of cases. (DOCX) S2 Table. Top 100 SNPs after GWA analysis. (XLS) S3 Table. Pathway analysis with ALIGATOR. (XLS) S4 Table. Meta-analysis Sample population matching. (DOCX) S5 Table. Pairs of primers used for sequencing of the 11 exons and intronic flanking areas of GRM8 gene. (DOCX) S6 Table. GWA analysis results X chromosome SNPs. (ZIP) S7 Table. GWA analysis results autosomic SNPs. (ZIP) S3 Table. Pathway analysis with ALIGATOR. (XLS) S4 Table. Meta-analysis Sample population matching. (DOCX) S5 Table. Pairs of primers used for sequencing of the 11 exons and intronic flanking areas of GRM8 gene. (DOCX) References 1. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, et al. Mice devoid of PrP are resistant to scrapie. Cell. 1993; 73:1339–47. 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W1029389589.txt	http://journals.iucr.org/e/issues/2014/07/00/cv5453/cv5453.pdf	en		(<i>E</i>)-3-Chloro-<i>N</i>′-(2-fluorobenzylidene)thiophene-2-carbohydrazide	Acta crystallographica. Section E	2,014	cc-by	2,263	organic compounds Acta Crystallographica Section E Data collection Structure Reports Online ISSN 1600-5368 Bruker SMART APEX CCD areadetector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2000) Tmin = 0.776, Tmax = 0.985 (E)-3-Chloro-N0 -(2-fluorobenzylidene)thiophene-2-carbohydrazide Refinement Sadia Sultan,a,b Muhammad Taha,c,b Syed Adnan Ali Shah,a,b Bohari M. Yamind,b and Hamizah Mohd Zakic,b* a Faculty of Pharmacy, University Teknologi Mara (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E., Malaysia, bAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E., Malaysia, cFaculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor D.E., Malaysia, and dSchool of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor D.E., Malaysia Correspondence e-mail: miiza73@yahoo.com Received 23 April 2014; accepted 19 May 2014 Key indicators: single-crystal X-ray study; T = 302 K; mean (C–C) = 0.002 Å; R factor = 0.023; wR factor = 0.065; data-to-parameter ratio = 13.4. The title compound, C12H8ClFN2OS, is a hydrazide derivative adopting an E conformation with an azomethine N C double bond length of 1.272 (2) Å. The molecular skeleton is approximately planar; the terminal five- and six-membered rings form a dihedral angle of 5.47 (9) . In the crystal, molecules are linked by N—H O and C—H O hydrogen bonds into zigzag chains propagating in [100]. Related literature For the applications and biological activity of hydrazones, see: Taha et al. (2013); Musharraf et al. (2012); Melnyk et al. (2006); Terzioglu & Gursoy (2003). For the crystal structures of related compounds, see: Alanazi et al. (2012a,b). Experimental Crystal data C12H8ClFN2OS Mr = 282.71 Orthorhombic, P21 21 21 a = 5.6833 (3) Å b = 13.0817 (6) Å c = 16.4001 (8) Å Acta Cryst. (2014). E70, o751 V = 1219.30 (10) Å3 Z=4 Mo K radiation = 0.48 mm1 T = 302 K 0.55 0.46 0.03 mm 47474 measured reflections 2255 independent reflections 2210 reflections with I > 2(I) Rint = 0.028 max = 0.15 e Å3 min = 0.12 e Å3 Absolute structure: Flack (1983), 916 Friedel pairs Absolute structure parameter: 0.02 (5) R[F 2 > 2(F 2)] = 0.023 wR(F 2) = 0.065 S = 1.09 2255 reflections 168 parameters H atoms treated by a mixture of independent and constrained refinement Table 1 Hydrogen-bond geometry (Å, ). D—H A D—H H A D A D—H A N1—H1A O1i C7—H7A O1i 0.86 0.93 2.12 2.41 2.9552 (18) 3.2268 (19) 163 147 Symmetry code: (i) x þ 12; y þ 32; z. Data collection: SMART (Bruker, 2000); cell refinement: SAINT (Bruker, 2000); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to refine structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL and PLATON (Spek, 2009). SS acknowledges the Principal Investigator Support Initiative Grant Scheme ERGS Phase 600-RMI/DANA 5/3/PSI (236/2013) UiTM and Dana Kecemerlangan 5/3 RIF (39/2012) (UiTM, Malaysia) for financial support. Supporting information for this paper is available from the IUCr electronic archives (Reference: CV5453). References Alanazi, A. M., Lahsasni, S., El-Emam, A. A. & Ng, S. W. (2012a). Acta Cryst. E68, o314. Alanazi, A. M., Kadi, A. A., El-Emam, A. A. & Ng, S. W. (2012b). Acta Cryst. E68, o315. Bruker (2000). SADABS, SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Flack, H. D. (1983). Acta Cryst. A39, 876–881. Melnyk, P., Leroux, V., Sergheraert, C. & Grellier, P. (2006). Bioorg. Med. Chem. Lett. 16, 31–35. Musharraf, S. G., Bibi, A., Shahid, N., Najam-ul-Haq, M., Khan, M., Taha, M., Mughal, U. R. & Khan, K. M. (2012). Am. J. Anal. Chem. 3, 779–789. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Taha, M., Baharudin, M. S., Ismail, N. H., Khan, K. M., Jaafar, F. M., Samreen, Siddiqui, S. & Choudhary, M. I. (2013). Bioorg. Med. Chem. Lett. 23, 3463– 3466. Terzioglu, N. & Gursoy, A. (2003). Eur. J. Med. Chem. 38, 781–786. doi:10.1107/S1600536814011568 Sultan et al. o751 supporting information supporting information Acta Cryst. (2014). E70, o751 [https://doi.org/10.1107/S1600536814011568] (E)-3-Chloro-N′-(2-fluorobenzylidene)thiophene-2-carbohydrazide Sadia Sultan, Muhammad Taha, Syed Adnan Ali Shah, Bohari M. Yamin and Hamizah Mohd Zaki S1. Comment Hydrazone derivatives are known as good ligands for complexation reactions. They have also displayed a wide spectrum of biological activities including antileishamanial (Taha et al., 2013), antimalarial (Melnyk et al., 2006) and anti-cancer (Terzioglu et al., 2003) properties. Recently the hydrazones are reported to be used as UV-LDI Matrices for measuring the mass of macromolecules (Musharraf et al., 2012) . The title compound, (I) (Fig. 1), is similar to that of previously reported N′-[(1E)-(2,6-difluorophenyl)methylidene]thiophene-2-carbohydrazide (Alanazi et al., 2012a) and N′-[(1E)-(4-fluorophenyl)methylidene]- thiophene-2-carbohydrazide (Alanazi et al., 2012b) except the thiophene ring is substituted with fluorine atom. The whole molecule is appearently planar with maximum deviation of 0.181 (1)Å for F1 atom from the least square plane. The chlorothiophenecarbonyl O1/C8/S1/(C9-C12)/Cl fragment is trans to the fluorobenzyl, F1/(C1-C7), group across the N1-N2 bond. The bond lengths and angles in (I) are normal and comparable to those in the analogs (Alanazi et al., 2012a,b). The crystal is stablized by N—H···O and C—H···O intermolecular hydrogen bonds (Table 1) to form zigzag chains of molecules extended along the a axis (Fig. 2). S2. Experimental The title compound (I) was synthesized by refluxing in methanol a mixture (0.352 g, 2 mmol) of 3-chlorothiophene- 2carbohydrazide and (0.248 g, 2 mmol) of 2 florobenzaldehyde along with a catalytical amount of acetic acid for 3 h. The progress of reaction was monitored by TLC. After completion of reaction, the solvent was evaporated by vacuum to afford crude material which was purified by repeated recrystallized in methanol to obtain needle like crytals (0.495 g, ° yielded 88). All chemicals (methyl 3-chlorothiophene-2-carboxylate 99%,2-florobenzaldehyde 98%) were purchased from sigma Aldrich. S3. Refinement All H atoms except H12A were positioned geometrically (C—H = 0.93 Å and N—H 0.86 Å) and constrained to ride on their parent atoms with Uiso(H) = 1.2Ueq(C, N). Atom H12A attached to C12 was located on a Fourier map and isotropically refined. Acta Cryst. (2014). E70, o751 sup-1 supporting information Figure 1 The molecular structure of (I) with displacement ellipsoids drawn at the 50% probability level. Figure 2 A portion of the crystal packing viewed down the a axis. Dashed lines denote hydrogen bonds. (E)-3-Chloro-N′-(2-fluorobenzylidene)thiophene-2-carbohydrazide Crystal data C12H8ClFN2OS Mr = 282.71 Orthorhombic, P212121 Hall symbol: P 2ac 2ab a = 5.6833 (3) Å b = 13.0817 (6) Å Acta Cryst. (2014). E70, o751 c = 16.4001 (8) Å V = 1219.30 (10) Å3 Z=4 F(000) = 576 Dx = 1.540 Mg m−3 Mo Kα radiation, λ = 0.71073 Å sup-2 supporting information Cell parameters from 9699 reflections θ = 3.1–25.5° µ = 0.48 mm−1 T = 302 K Slab, colourless 0.55 × 0.46 × 0.03 mm Data collection Bruker SMART APEX CCD area-detector diffractometer Radiation source: fine-focus sealed tube Graphite monochromator Detector resolution: 83.66 pixels mm-1 ω scan Absorption correction: multi-scan (SADABS; Bruker, 2000) Tmin = 0.776, Tmax = 0.985 47474 measured reflections 2255 independent reflections 2210 reflections with I > 2σ(I) Rint = 0.028 θmax = 25.5°, θmin = 3.1° h = −6→6 k = −15→15 l = −19→19 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.023 wR(F2) = 0.065 S = 1.09 2255 reflections 168 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.0388P)2 + 0.1642P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.15 e Å−3 Δρmin = −0.12 e Å−3 Extinction correction: SHELXTL (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.021 (2) Absolute structure: Flack (1983), 916 Friedel pairs Absolute structure parameter: 0.02 (5) Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) S1 Cl1 F1 O1 N1 H1A N2 C1 H1B x y z Uiso/Ueq 0.17368 (8) −0.35177 (8) 0.9968 (2) −0.0135 (3) 0.2895 (2) 0.3269 0.4234 (2) 0.7057 (3) 0.5728 0.52165 (3) 0.75148 (4) 0.52634 (9) 0.75985 (10) 0.65589 (9) 0.6912 0.57273 (10) 0.39194 (12) 0.3967 0.22226 (3) 0.21939 (3) −0.05506 (7) 0.08685 (8) 0.06578 (7) 0.0235 0.08483 (8) 0.10909 (10) 0.1420 0.05051 (13) 0.06171 (15) 0.0641 (3) 0.0592 (3) 0.0424 (3) 0.051* 0.0402 (3) 0.0498 (4) 0.060* Acta Cryst. (2014). E70, o751 sup-3 supporting information C2 H2B C3 H3A C4 H4A C5 C6 C7 H7A C8 C9 C10 C11 H11A C12 H12A 0.8577 (4) 0.8273 1.0558 (4) 1.1577 1.1029 (3) 1.2357 0.9492 (3) 0.7486 (3) 0.5958 (3) 0.6247 0.1010 (3) 0.0325 (3) −0.1608 (3) −0.1957 (4) −0.3197 −0.0282 (4) −0.005 (5) 0.31121 (13) 0.2620 0.30280 (15) 0.2479 0.37504 (14) 0.3698 0.45490 (13) 0.46727 (12) 0.55497 (12) 0.5989 0.68555 (11) 0.62923 (11) 0.65037 (12) 0.58121 (15) 0.5852 0.50870 (17) 0.4586 (17) 0.11894 (13) 0.1584 0.07046 (15) 0.0776 0.01183 (13) −0.0210 0.00305 (10) 0.04998 (9) 0.03748 (9) −0.0059 0.11008 (9) 0.18418 (9) 0.23145 (10) 0.29608 (10) 0.3330 0.29804 (12) 0.3352 (16) 0.0602 (5) 0.072* 0.0652 (5) 0.078* 0.0618 (5) 0.074* 0.0484 (4) 0.0428 (3) 0.0430 (4) 0.052* 0.0396 (3) 0.0392 (3) 0.0459 (3) 0.0599 (5) 0.072* 0.0654 (5) 0.086 (7)* Atomic displacement parameters (Å2) S1 Cl1 F1 O1 N1 N2 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 U11 U22 U33 U12 U13 U23 0.0573 (2) 0.0550 (2) 0.0649 (6) 0.0655 (8) 0.0472 (7) 0.0439 (7) 0.0503 (9) 0.0631 (11) 0.0560 (11) 0.0455 (10) 0.0493 (9) 0.0420 (8) 0.0474 (8) 0.0437 (8) 0.0430 (8) 0.0476 (8) 0.0673 (11) 0.0806 (14) 0.0498 (2) 0.0632 (3) 0.0732 (7) 0.0571 (7) 0.0429 (6) 0.0378 (6) 0.0459 (8) 0.0470 (9) 0.0507 (10) 0.0655 (11) 0.0503 (9) 0.0443 (8) 0.0436 (8) 0.0395 (7) 0.0401 (7) 0.0497 (8) 0.0673 (11) 0.0708 (12) 0.0445 (2) 0.0670 (3) 0.0542 (6) 0.0551 (7) 0.0370 (6) 0.0389 (6) 0.0533 (9) 0.0706 (11) 0.0889 (15) 0.0746 (12) 0.0457 (8) 0.0421 (7) 0.0380 (7) 0.0355 (7) 0.0345 (7) 0.0406 (8) 0.0452 (9) 0.0447 (9) 0.00003 (18) 0.0061 (2) −0.0086 (6) 0.0185 (6) 0.0018 (6) −0.0010 (5) 0.0002 (7) 0.0036 (8) 0.0117 (8) 0.0037 (8) −0.0072 (7) −0.0034 (6) −0.0045 (6) −0.0019 (6) −0.0050 (6) −0.0089 (7) −0.0139 (10) −0.0108 (11) −0.00151 (19) 0.0052 (2) 0.0104 (5) 0.0050 (6) 0.0005 (6) −0.0040 (5) −0.0053 (8) −0.0161 (10) −0.0153 (11) −0.0020 (9) −0.0041 (7) −0.0059 (6) −0.0026 (6) −0.0057 (6) −0.0064 (6) −0.0018 (7) 0.0113 (8) 0.0056 (9) 0.01644 (17) −0.0115 (2) −0.0038 (5) 0.0202 (6) 0.0104 (5) 0.0050 (5) 0.0012 (7) 0.0002 (8) −0.0148 (10) −0.0256 (10) −0.0125 (7) −0.0059 (6) 0.0026 (6) 0.0051 (6) 0.0026 (6) −0.0049 (6) 0.0027 (8) 0.0191 (9) Geometric parameters (Å, º) S1—C12 S1—C9 Cl1—C10 F1—C5 O1—C8 N1—C8 Acta Cryst. (2014). E70, o751 1.700 (2) 1.7362 (15) 1.7224 (18) 1.362 (2) 1.2304 (19) 1.351 (2) C3—C4 C3—H3A C4—C5 C4—H4A C5—C6 C6—C7 1.375 (3) 0.9300 1.369 (3) 0.9300 1.385 (2) 1.453 (2) sup-4 supporting information N1—N2 N1—H1A N2—C7 C1—C2 C1—C6 C1—H1B C2—C3 C2—H2B 1.3639 (17) 0.8600 1.272 (2) 1.374 (2) 1.404 (2) 0.9300 1.382 (3) 0.9300 C7—H7A C8—C9 C9—C10 C10—C11 C11—C12 C11—H11A C12—H12A 0.9300 1.474 (2) 1.373 (2) 1.408 (2) 1.344 (3) 0.9300 0.90 (2) C12—S1—C9 C8—N1—N2 C8—N1—H1A N2—N1—H1A C7—N2—N1 C2—C1—C6 C2—C1—H1B C6—C1—H1B C1—C2—C3 C1—C2—H2B C3—C2—H2B C4—C3—C2 C4—C3—H3A C2—C3—H3A C5—C4—C3 C5—C4—H4A C3—C4—H4A F1—C5—C4 F1—C5—C6 C4—C5—C6 C5—C6—C1 91.82 (10) 123.24 (12) 118.4 118.4 115.83 (13) 120.76 (17) 119.6 119.6 120.37 (18) 119.8 119.8 120.41 (18) 119.8 119.8 118.29 (18) 120.9 120.9 118.06 (17) 118.18 (16) 123.76 (18) 116.41 (16) C5—C6—C7 C1—C6—C7 N2—C7—C6 N2—C7—H7A C6—C7—H7A O1—C8—N1 O1—C8—C9 N1—C8—C9 C10—C9—C8 C10—C9—S1 C8—C9—S1 C9—C10—C11 C9—C10—Cl1 C11—C10—Cl1 C12—C11—C10 C12—C11—H11A C10—C11—H11A C11—C12—S1 C11—C12—H12A S1—C12—H12A 120.37 (15) 123.21 (15) 121.23 (14) 119.4 119.4 118.68 (14) 120.69 (14) 120.63 (13) 125.21 (14) 109.27 (11) 125.43 (12) 114.11 (16) 126.46 (13) 119.42 (14) 111.83 (17) 124.1 124.1 112.96 (14) 129.0 (18) 117.9 (18) C8—N1—N2—C7 C6—C1—C2—C3 C1—C2—C3—C4 C2—C3—C4—C5 C3—C4—C5—F1 C3—C4—C5—C6 F1—C5—C6—C1 C4—C5—C6—C1 F1—C5—C6—C7 C4—C5—C6—C7 C2—C1—C6—C5 C2—C1—C6—C7 N1—N2—C7—C6 C5—C6—C7—N2 C1—C6—C7—N2 N2—N1—C8—O1 −179.62 (14) −0.2 (3) 0.0 (3) 0.1 (3) 179.92 (17) 0.1 (3) 179.91 (14) −0.2 (2) −0.6 (2) 179.24 (15) 0.3 (2) −179.19 (15) −178.51 (13) −173.37 (14) 6.1 (2) −177.85 (14) N2—N1—C8—C9 O1—C8—C9—C10 N1—C8—C9—C10 O1—C8—C9—S1 N1—C8—C9—S1 C12—S1—C9—C10 C12—S1—C9—C8 C8—C9—C10—C11 S1—C9—C10—C11 C8—C9—C10—Cl1 S1—C9—C10—Cl1 C9—C10—C11—C12 Cl1—C10—C11—C12 C10—C11—C12—S1 C9—S1—C12—C11 1.4 (2) 2.1 (2) −177.17 (14) 178.35 (13) −0.9 (2) 0.39 (13) −176.37 (14) 176.15 (14) −0.62 (18) −5.0 (2) 178.22 (10) 0.6 (2) −178.35 (14) −0.3 (2) −0.07 (17) Acta Cryst. (2014). E70, o751 sup-5 supporting information Hydrogen-bond geometry (Å, º) D—H···A i N1—H1A···O1 C7—H7A···O1i D—H H···A D···A D—H···A 0.86 0.93 2.12 2.41 2.9552 (18) 3.2268 (19) 163 147 Symmetry code: (i) x+1/2, −y+3/2, −z. Acta Cryst. (2014). E70, o751 sup-6
https://openalex.org/W3200867402	http://seer.tupa.unesp.br/index.php/BIOENG/article/download/201/199	English	null	EFEITO DA ADIÇÃO DE FÓSFORO SOBRE A BIODISPONIBILIDADE DE CHUMBO E MERCÚRIO EM SOLO CONTAMINADO	Revista Brasileira de Engenharia de Biossistemas	2,014	cc-by	3,363	ABSTRACT The soils enriched with elements potentially toxic (EPT) have limited use for agricultural purposes. In this case, the corresponding site should be isolated and then procedures for decontamination or stabilization of EPT in the soils must be applied. The objective of this study was to evaluate the influence of adding different rates of phosphate on the bioavailability of mercury (Hg) and lead (Pb) in a contaminated soil, using lettuce grown on this soil. For this, different phosphorus rates were used to control bioavailability of Hg and Pb, in lettuce plants. The element concentrations were analyzed by instrumental neutron activation analysis (INAA), and the results were compared to control treatment to verify the reduction of absorption of Hg and Pb. The Hg concentration in shoots decreased with the application of 250 mg kg-1 of P and Pb with the application of 250 to 1000 mg kg-1 of P. The accumulation of Hg and Pb in shoots of lettuce increased as a function of P demonstrating the inefficiency of application of P in the absorption of these elements. Keywords: contamination, lettuce, imobilization Keywords: contamination, lettuce, imobilization EFFECTS OF PHOSPHORUS ADDITIONS ON LEAD AND MERCURIUM BIOAVAILABILITIES IN A CONTAMINATED SOIL M. J. A. Armelin1, A. R. Trevizam1, M. L. S. Silva2, M. Saiki1, V. A. Maihara1 IPEN-CNEN - Instituto de Pesquisas Energéticas e Nucleares, São Paulo, SP, Brasil. 2 UFLA - Univ Federal de Lavras, Lavras, MG, Brasil. M. J. A. Armelin1, A. R. Trevizam1, M. L. S. Silva2, M. Saiki1, V. A. Maihara1 IPEN-CNEN - Instituto de Pesquisas Energéticas e Nucleares, São Paulo, SP, Brasil. 2 UFLA - Univ Federal de Lavras, Lavras, MG, Brasil. Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 Palavras-chave: contaminação, alface, imobilização INTRODUCTION formation and/or precipitation (MCGOWEN, 2001). Phosphates, limestone, Fe or Mn oxides, organic materials and zeolites are the chemicals used for the reduction and bioavailability. formation and/or precipitation (MCGOWEN, 2001). Phosphates, limestone, Fe or Mn oxides, organic materials and zeolites are the chemicals used for the reduction and bioavailability. In most cases, the concentration of microelements and toxic elements found in soil does not pose a risk to the environment. However, in recent decades the mining, industrial process, the use of agricultural inputs such as fertilizers, limestone, pesticides has greatly contributed to the enrichment of inorganic elements in areas close to events (ALLOWAY, 1995; SILVA et al., 2007). It is known that Pb phosphates are very stable forms of Pb in the environment. From the reaction of lead with soluble phosphate (P) various pyromorphite minerals insoluble are formed, thus immobilizing Pb in soil and in consequence reducing its absorption by plants (BOLAN et al., 2003; ZWONITZER et al., 2003). Cadmium, lead, mercury and arsenic, classified as elements potentially toxic (EPT) are a huge problem for the public health. When they are present in the soil may persist due to their long life-time in soils, and could be readily available for plants, especially in acid soils, and being transferred to the human food chain. (KPONBLEKOU & TABATABAI, 1994; CAMELO, 1997). Thus, soils enriched with EPT have limited use for agricultural purposes. In this case, the corresponding site should be isolated and then procedures for decontamination or stabilization of EPT in the soils must be applied (TREVIZAM et al., 2010). The uptake of Hg by lettuce, under the conditions of this study, was evaluated because of Hg to be an important EPT, although not known very stable compounds Hg phosphate. Despite the presence of Hg in the soil, there is evidence that almost no Hg from the soil is taken up into the shoots. Experiments carried out in some plant species using culture solutions has shown that 95 to 99% of Hg has remained in the root (LINDQVIST et. al, 1991). The objective of this study was to evaluate the influence of adding different doses of phosphate on the bioavailabilities of Hg and Pb in a contaminated soil, using lettuce grown on this soil. INTRODUCTION The addition of chemicals to contaminated soil is one of the practices used for immobilization of EPT through reducing the solubility and bioavailability of these elements, due to the complex MATERIAL AND METHODS Soil sampling and treatment for the experiment soil were transferred to pots where plants were sown. A 1 kg subsample soil was taken for chemical and physical characterizations (pH; available P, Ca, Mg and K; total acidity and organic matter) (RAIJ et al, 2001). In the Table 1 show the chemical attributes of soil. The soil was collected from a site of 22.000 m2, located in Piracicaba, SP-Brazil. This site is under receivership of the Companhia de Tecnologia e Saneamento Ambiental (CETESB) because it has high level of contamination by potentially toxic elements. RESUMO Os solos enriquecidos com elementos potencialmente tóxicos (EPT) têm seu uso limitado para fins agrícolas. Neste caso, o local deve ser isolado e, em seguida, os procedimentos para a descontaminação ou estabilização dos EPT nos solos devem ser aplicados. O objetivo deste estudo foi avaliar a influência da adição de fósforo sobre a biodisponibilidade de mercúrio (Hg) e chumbo (Pb), em solo contaminado, cultivando alface neste solo. Para isso, diferentes taxas de fósforo foram usadas para controlar a biodisponibilidade de Hg e Pb, em plantas de alface. As concentrações dos elementos foram analisados por análise instrumental por ativação de neutrônica (INAA), e os resultados foram comparados para controlar o tratamento para verificar a redução de absorção de Hg e Pb. A concentração de Hg na parte aérea diminuiu com a aplicação de 250 mg kg-1 de P e Pb com a aplicação de 250 a 1000 mg kg-1 de P. O acúmulo de Hg e Pb na parte aérea da alface aumentou em função de P demonstrando a ineficiência da aplicação de P na absorção desses elementos. Palavras-chave: contaminação, alface, imobilização Palavras-chave: contaminação, alface, imobilização * aanrt@hotmail.com 255 Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 Installation of the experiment with lettuce To assess the effect of phosphorus in reducing and the availability of EPT in soil, lettuce plants (Lactuca sativa L.) were grown in pots containing 2 kg of soil. The trial was performed at the green house with A sample of 50 kg soil was collected for this study in an area of 2 x 3 m from 0-20 cm depth, passed through 4 mm mesh sieve and then homogenized. Subsamples of 2 kg 256 Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 Elmer® FIMS), where it was used stannous chloride as reducing agent. Eletrothermal atomic absorption spectroscopy (ET AAS, Perkin Elmer® Analyst 800 spectrometer with Zeeman background correction) was employed to quantify Pb. Elmer® FIMS), where it was used stannous chloride as reducing agent. Eletrothermal atomic absorption spectroscopy (ET AAS, Perkin Elmer® Analyst 800 spectrometer with Zeeman background correction) was employed to quantify Pb. ventilation and humidification system at the CENA. The experimental design was a randomized block, with 6 treatments (rates of phosphorus): 0, 250, 500, 1000, 2000 and 4000 mg kg-1 of P, with 3 replicates of each treatment, totaling 18 pots. The P source used was Ca(H2PO4)2. Aliquots of about 40 mg for lettuce and 200 mg for Mixed Polish Herbs (INCT- MPH-2), a certified reference materials (SRM) were digested with 4 ml of concentrated HNO3 (Merck®) and left standing for a period of 8 h, after 1 ml of 30% H2O2 was added. The flasks were stirred and left again for about 15 h. To finalize the sample digestion, the closed flasks were placed in an aluminum block at 90°C, for 3 h. The reference material was analyzed to control the analytical results. The soils of the pots, after receiving the P fertilizer, were incubated for 15 days under 60% moisture content. At the end of the incubation period six seedlings of lettuce were transplanted in each pot. After 7 days, the plants were thinned to two plants per pot. Soil moisture was maintained at 70% by daily watering with deionized water. As additional fertilizer, nitrogen was applied as ammonium nitrate at rates of 100 mg N per pot in four applications. Ten days after transplanting, 0.2 mg of boron as boric acid and 0.25 mg of molybdenum in the form of ammonium molybdate were applied in all pots. Analytical method used y The quantification of Hg was performed with cold vapour atomic absorption spectroscopy (CV AAS, Perkin Installation of the experiment with lettuce For the construction of calibration curves were used solutions whose concentrations ranged from 0.5 to 2.5 µg/L in the case of Hg, and from 5 to 25 µg/L for Pb. During the analysis and construction of calibration curves, the following parameters were kept fixed, 1.) Hg: injection volume in 500 µL, flow charger (HCl P.A. 3% v/v – Merck) in 10 mL min-1, flow reducer in 6 mL min-1 and flow of argon (carrier gas) in 50 mL min-1; 2.) Pb: wave-length in 283.3 nm, slit in 0.7 nm, current lamp in 440 mA, volume of sample injection in 20 µL and injection volume of the chemical modifier in 10 µL. The lettuce leaves were collected at 70 days after transplanting, rinsed with deionized water, oven dried (at 65ºC), weighed and ground in agate mortar for quantification of Hg and Pb. RESULTS AND DISCUSSION The results obtained for Hg and Pb in Mixed Polish Herbs (INCT-MPH-2) are shown in Table 2. The agreement between the values of Hg and Pb concentrations obtained and certified, around 12%, for Mixed Polish Herbs showed that the analytical procedure was appropriate to determine these elements in lettuce. matter production of shoots when treatments are compared to the control treatment, at doses between 250-2000 mg kg-1 of P, demonstrating that the addition of P does not compromise their development. The increased production of dry matter demonstrates that possibly occurred a decrease the toxic effect of Hg and Pb, however its greatest development may result in greater absorption of these elements occurred. The lettuce crop is used as an indicator of the availability of heavy metals Regarding the development of lettuce in the contaminated soil, especially in relation to adverse soil characteristics (Table 1). The lettuce presented increase of dry 257 Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 in contaminated soils and thus entry into the food chain (BASTA et al., 2001; MACHADO et al., 2008). the absorption by lettuce, however found an increase of Pb content in shoots of lettuce in soil containing 397 mg kg-1, and other soil containing 2450 mg kg-1 of Pb presented in lettuce at the same concentration of the control treatment. The authors reported that by presenting concentration below 6 mg kg-1 in lettuce resulted in difficulties to determine the element. The Hg concentration in the shoots of lettuce (Figure 1) ranged 17-168 μg kg-1. The values found are above 6.9 μg kg-1 encountered by STERTZ et al. (2005) in lettuce collected in production fields. The objective of immobilize of Hg with the application of phosphorus in the soil was only observed with the application of 250 mg kg-1 P, where there was a reduction of 15.3% compared to the control treatment. In this sense the application of high rates above 250 mg kg-1, promoted an increase in dry matter production of shoots and consequently greater absorption of Hg. The Pb concentrations in the shoots of lettuce can be quite different depending on the cropping system and the type of soil that is contaminated or not. COSTA et al. (2001) found maximum concentration of 4.95 mg kg-1 in three varieties of lettuce grown with urban waste compost and COSTA et al. RESULTS AND DISCUSSION (1994), applying organic compost, found concentration of 6.33 mg kg- 1 dry matter of lettuce leaves. SANTOS et al. (1998) found concentrations ranging from 6.53 to 23.48 mg kg-1 in ten cultivars of lettuce grown with urban waste compost. AGBENIN et al. (2009) found in Nigeria Pb concentration in lettuce from 0.65 to 4.8 mg kg-1 on fresh weight. ZHAO & WANG (2010) verified the availability of Hg contained in calcium superphosphate (5.1 mg kg-1) to maize. The authors concluded that the fertilizer may decrease the toxicity of Hg in maize, inhibiting Hg uptake and translocation from the root to the shoots of culture. The Hg concentration in lettuce shoots may be related to their greater binding to cell walls of roots or its smallest transport through the flow of transpiration in plants (DU et al., 2005). This peculiarity of the Hg may significantly influence the results obtained in this study. The accumulation of Pb in the shoots of lettuce increased as a function of P applied to the soil (Figure 3). The increased accumulation of P in the shoots of lettuce demonstrates the inefficiency of application of P in reducing the availability of Pb in the soil, especially when compared to the control treatment. When the development of lettuce associated with Hg concentration in shoots it was verified that the accumulation of Hg in shoots increased with the addition of P to the soil, and this increase of 530% compared to the control treatment (Figure 1). The increased accumulation of Hg demonstrates the inefficiency of P on the absorption of Hg, verified by the accumulation of Hg in shoots of lettuce. In relation the addition of P in soils contaminated was verified the reduction of 51.9% of Pb in Brassica oleracea L. var. acephala and in 65.5% in Brassica campetris L. var. communis (ZHU et al., 2004), from 32.6 to 57.9% in Sorghum bicolour L. (CHEN et al., 2007) and in Sorghum vulgare L. Moench and Beta vulgaris L. Koch (Hettiarachchi & Pierzynski 2002). Although these studies verify the effectiveness of the reduced availability of Pb to crops, in none of these work has verified the accumulation of Pb, which is of fundamental importance since this parameter takes into account the development of culture. The Pb concentrations in the shoots of lettuce ranged 1069-5501 μg kg-1 (Figure 2). CONCLUSIONS The accumulation of Hg and Pb in shoots of lettuce increased as a function of P, demonstrating the inefficiency of application of P in the absorption of these elements. The Hg concentration in shoots of lettuce decreased with the application of 250 mg kg-1 of P and Pb with the application of 250 to 1000 mg kg-1 of P. RESULTS AND DISCUSSION P addition to soil promoted a reduction of Pb in the shoots of lettuce at 250-1000 mg kg-1 rates of P. BASTA et al. (2001) working with lettuce, the phosphate rock used to mitigate 258 Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 FIGURE 1 – Dry Matter of lettuce in function of rates of phosphorus FIGURE 1 – Dry Matter of lettuce in function of rates of phosphorus FIGURE 2 – Mercury concentration and accumulation in lettuce FIGURE 2 – Mercury concentration and accumulation in lettuce 259 259 Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 FIGURE 3 – Lead concentration and accumulation in lettuce FIGURE 3 – Lead concentration and accumulation in lettuce TABLE 1 - Chemical characteristics of soil used in the experiment Characteristics Solo pH (CaCl2) 7.06 Organic Matter (g dm-3) 28 Phosphorus (mg dm-3) 75 Potassium (mmolc dm-3) 7.0 Calcium (mmolc dm-3) 400 Magnesium (mmolc dm-3) 92.0 H + Al (mmolc dm-3) 9.0 Sum of bases (mmolc dm-3) 499 CTC (mmolc dm-3) 508 V (%) 98 Mercury total (µg kg-1) 204±52 Lead total (µg kg-1) 891±151 Sand (g kg-1) 650 Silt (g kg-1) 80 Clay (g kg-1) 270 Texture medium clay TABLE 1 - Chemical characteristics of soil used in the experiment Ch i i S l TABLE 1 - Chemical characteristics of soil used in the experiment Characteristics Solo Table 2. Hg and Pb Concentrations obtained for Mixed Polish Herbs Element Obtained Certified* Hg (µg kg-1) 19.7 ± 1.4 17.6 ± 1.6 Pb (mg kg-1) 2.44 ± 0.22 2.16 ± 0.23 *Certified values with uncertainties reported by the producer 260 Brazilian Journal of Biosystems Engineering v. 8(3): 255-262, 2014 REFERÊNCIAS BIBLIOGRÁFICAS AGBENIN, J.O.; DANKOA, M.; WELP, G. Soil and vegetable compositional relationships of eight potentially toxic metals in urban garden fields from northern Nigeria. COSTA, C.A.; CASALI, V.W.D.; RUIZ, H.A.; JORDÃO, C.P.; CECON, P.R. Teor de metais pesados e produção de alface adubada com composto de lixo urbano. Horticultura Brasileira, v. 19 n. 1, p. 10-16, 2001. Journal of the Science of Food and Agriculture, v. 89, p. 49–54, 2009. COSTA, C.A.; CASALI, V.W.D.; LOURES, E.G. Teor de metais pesados em alface (Lactuca sativa L.) adubada com composto orgânico de lixo urbano. Revista Ceres, v. 41, p. 629-640, 1994. ALLOWAY, B.J. Heavy metals in soils. 2. ed. 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REFERÊNCIAS BIBLIOGRÁFICAS 1, p. 63-70, 2008. ZHU, Y.G.; CHEN, S.B.; YANG, J.C. Effects of soil amendments on lead uptake by two vegetable crops from a lead- contaminated soil from Anhui, China. Environmental International, v. 30, p.:351-356, 2004. ZHU, Y.G.; CHEN, S.B.; YANG, J.C. Effects of soil amendments on lead uptake by two vegetable crops from a lead- contaminated soil from Anhui, China. Environmental International, v. 30, p.:351-356, 2004. ZWONITZWER, J.C.; PIERZYNSKI, G.M.; HETTIARACHCHI, G.M. Effects of phosphorus additions on lead, cadmium, and zinc bioavailabilities in metal-contaminated soil. Water, Air, and Soil Pollution, v. 143, n. 1-4, p. 193-209, 2003. McGOWEN, S.L.; BASTA, N.T.; BROWN, G.O. Use of diammonnium phosphate to reduce heavy metal solubility and transport in smelter-contaminated soil. Journal Environmental Quality, v. 30, p. 493-500, 2001. SANTOS, I.C.; CASALI, V.N.D.; MIRANDA, G.V. 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https://openalex.org/W3010791465	https://cyberleninka.ru/article/n/ispolzovanie-algoritmov-optimizatsii-s-samoobucheniem-dlya-upravleniya-dinamicheski-izmenyayuschimisya-sistemami/pdf	Russian	null	Использование алгоритмов оптимизации с самообучением для управления динамически изменяющимися системами	Programmnye produkty i sistemy	2,020	cc-by	4,870	1 Московский государственный университет им. М.В. Ломоносова, г. Москва, 119991, Россия 1 Московский государственный университет им. М.В. Ломоносова, г. Москва, 119991, Россия В статье предлагается подход к управлению динамически изменяющимися системами. В ходе ра- боты они могут изменять свое состояние: состав оборудования, нагрузку и выполняемые функции. Необходимо выбирать значения управляющих параметров в зависимости от состояния системы таким образом, чтобы обеспечить требуемые значения характеристик ее работы. Для решения этой задачи авторы предлагают использовать оптимизационные алгоритмы с самообу- чением: направленного случайного поиска с самообучением и муравьиные. Эти алгоритмы, действуя методом проб и ошибок, позволяют настраиваться на текущее состояние системы за счет введения в алгоритмы памяти об удачном и неудачном выполнении предыдущих шагов. Основная идея применения алгоритмов с самообучением для управления системой заключается в том, что задача управления рассматривается как задача безусловной оптимизации. Элементами вектора оптимизируемых переменных являются управляющие параметры системы. Шаги алгоритмов рассмат- риваются как возможные действия по управлению системой (операции изменения значений управляю- щих параметров). Целевая функция может быть задана одним из двух способов: как суммарное среднее отклонение характеристик работы системы от требуемых (важность характеристик можно учесть с по- мощью весов при суммировании отклонений) и как максимальное отклонение характеристик работы системы от требуемых. р у Предложенный подход к управлению допускает нестабильное поведение окружающей среды, огра- ниченность информации об управляемой системе и позволяет учитывать наличие многих характери- стик работы системы, значения которых требуется поддерживать в заданных пределах. Если отказ эле- ментов системы не приводит к отказу системы в целом, а лишь ухудшает значения характеристик ра- боты системы, то при использовании данного подхода отклонения значений характеристик от требуемых будут минимизироваться. Ограничением данного подхода является допустимость переходного процесса при смене состояния системы. При изменении состояния системы алгоритму потребуется ряд шагов для переобучения. Не- которые шаги могут приводить к нарушению требуемых характеристик работы системы. Ключевые слова: динамическая система, управление, математическое программирование, мура- вьиные алгоритмы. рудования, работающего в данный момент в здании. В качестве характеристик работы си- стемы для здания с автоматическим управле- нием климатом выступают внутренние темпе- ратура, влажность, количество углекислого газа, а для компьютерной сети – задержка пе- редачи пакетов, количество потерянных паке- тов. Примером управляющих параметров для здания с автоматическим управлением клима- том являются температура обогревателей, сте- пень открытия форточек, обороты двигателей вентиляционной системы. Динамически изменяющаяся система харак- теризуется состоянием (состав оборудования, нагрузка на систему, поведение окружающей среды, выполняемые системой функции), зна- чениями характеристик работы системы и дан- ными управляющих параметров. Состояние системы меняется, поэтому тре- буется выбирать управляющие параметры та- ким образом, чтобы обеспечить требуемые зна- чения характеристик ее работы. 1 (33) 2020 1 (33) 2020 Программные продукты и системы / Software & Systems Дата подачи статьи: 05.09.19 2020. Т. 33. № 1. С. 020–026 Дата подачи статьи: 05.09.19 2020. Т. 33. № 1. С. 020–026 В.А. Костенко 1, к.т.н., доцент факультета ВМК, kostmsu@gmail.com В.А. Костенко 1, к.т.н., доцент факультета ВМК, kostmsu@gmail.com 1 Московский государственный университет им. М.В. Ломоносова, г. Москва, 119991, Россия 1 Московский государственный университет им. М.В. Ломоносова, г. Москва, 119991, Россия Примерами таких систем являются техноло- гические процессы, компьютерные сети, пти- цефабрики, здания с автоматическим управле- нием климатом. Так, нагрузка на компьютер- ную сеть задается трафиком, на систему автоматического управления климатом – коли- чеством людей, рассеиваемой мощностью обо- Проблемой применения известных в теории автоматического управления методов [1] для управления динамически изменяющимися си- стемами является то, что состояние системы меняется со временем и плохо прогнозируется 20 1 (33) 2020 Программные продукты и системы / Software & Systems F(Y(t), X(t), S(t)) и ее аргумент S(t) неизвестны, оценка значения Δ может быть получена только экспериментально, если она не зависит от S(t). В противном случае Δ необходимо рас- сматривать как оптимизируемый параметр, значение которого должно изменяться динами- чески в ходе работы алгоритма. (возможна большая ошибка прогноза) или во- обще не прогнозируется. Проблемой примене- ния методов, использующих обучающую вы- борку (например, нейросетей [2–4], метода опорных векторов [5], аксиоматического под- хода [6–9]) является то, что обучение ведется на одной системе, а полученные алгоритмы применяются к другой. (возможна большая ошибка прогноза) или во- обще не прогнозируется. Проблемой примене- ния методов, использующих обучающую вы- борку (например, нейросетей [2–4], метода опорных векторов [5], аксиоматического под- хода [6–9]) является то, что обучение ведется на одной системе, а полученные алгоритмы применяются к другой. Пусть Ck ∙τ – время работы алгоритма изме- нения управляющих параметров, тогда должно выполняться условие Δ ≥ Ck ∙τ, где Ck – число итераций алгоритма; τ – время выполнения од- ной итерации. Для решения этой задачи предполагается использовать оптимизационные алгоритмы с самообучением: алгоритмы направленного случайного поиска с самообучением и муравь- иные алгоритмы. Эти алгоритмы, действуя ме- тодом проб и ошибок, позволяют настраи- ваться на текущее состояние системы за счет введения памяти об удачном и неудачном вы- полнении предыдущих шагов. Задачу управления изменяющимися дина- мическими системами можно сформулировать как задачу безусловной оптимизации. Элементами вектора оптимизируемых пара- метров являются управляющие параметры си- стемы. Основная идея применения алгоритмов с са- мообучением для управления системой заклю- чается в том, что задача управления рассматри- вается как задача оптимизации, а пробные шаги алгоритмов – как возможные действия управления системой (операции изменения значений управляющих параметров). Целевую функцию f(X) можно определить одним из следующих способов. • Суммарное среднее отклонение характе- ристик работы системы от требуемых: * 1 min k i i Y i y y = −  . Важность характеристик можно учесть с по- мощью весов при суммировании отклонений. Задача управления как задача безусловной оптимизации • Максимальное отклонение характери- стик работы системы от требуемых: * min (max ) i i Y y y − . • Максимальное отклонение характери- стик работы системы от требуемых: Задача безусловной оптимизации заключа- ется [10] в нахождении компонентов вектора X = (x1, …, xn) (оптимизируемых параметров), минимизирующих целевую функцию f(X). * min (max ) i i Y y y − . Алгоритм направленного случайного поиска с самообучением Введем следующие обозначения: S(t) – со- стояние системы; Y(t) = (y1(t), …, yk(t)) – вектор значений характеристик работы системы; X(t) = (x1(t), …, xn(t)) – вектор значений управ- ляющих параметров; Y(t) = (y1(t), …, yk*(t)) – вектор требуемых значений характеристик ра- боты системы. Рассмотрим основные принципы построе- ния алгоритмов направленного случайного по- иска с самообучением [11]. Основой методов направленного случай- ного поиска служит итерационный процесс: Значения характеристик работы системы изменяются со временем в зависимости от со- стояния системы, значений управляющих па- раметров и текущих характеристик: Y(t + Δ) = = F(Y(t), X(t), S(t)). 1 , 0,1, , k k k X X k +  = +  =   где k – величина шага;  = (1, , n) – неко- торая реализация n-мерного случайного век- тора . Здесь 1/Δ – частота изменения управляю- щих параметров для коррекции работы си- стемы. Значение Δ определяется инерционно- стью системы. Здесь 1/Δ – частота изменения управляю- щих параметров для коррекции работы си- стемы. Значение Δ определяется инерционно- стью системы. Алгоритмы случайного направленного по- иска с самообучением заключаются в пере- стройке вероятностных характеристик поиска, то есть в определенном целенаправленном воз- действии на случайный вектор . Информация о состоянии системы S(t) недо- ступна или ограничена. Как следствие – функ- ция F(Y(t), X(t), S(t)) неизвестна. Это достигается введением вектора памяти 1 2 ( , , , ) k k k k n P p p p = , где k jp – вероятность вы- бора положительного направления по j-й коор- динате на k-м шаге. Под инерционностью системы будем пони- мать скорость изменения значений характери- стик работы системы при изменении значений управляющих параметров. Поскольку функция 21 1 (33) 2020 Программные продукты и системы / Software & Systems Идея муравьиных алгоритмов [12, 13] осно- вана на моделировании поведения муравьев при нахождении кратчайшего пути от муравей- ника к источнику пищи. Муравьи при переме- щении оставляют особое вещество – феромон, который используется в дальнейшем другими муравьями при выборе пути. Чем выше кон- центрация феромона на том или ином пути, тем больше вероятность того, что муравьи выберут для движения именно этот путь. Алгоритм направленного случайного по- иска с самообучением определяется тремя эле- ментами: − алгоритмом выбора пробных состояний на текущем шаге; − решающим правилом, по которому на каждом шаге выбирается новое текущее при- ближение решения; − алгоритмом самообучения, корректиру- ющим вектор памяти с точки зрения информа- ции, полученной на текущем шаге. − при невыполнении условия останова возврат к построению пути. 1 при 0, 2 ( ) 1 1 при 0; 2 aw aw e w p w e w −    =  −   ( 1) ( ) ( ) ( ) ( ) i i i i j j j w w sign x f + = −   Операция построения муравьем пути. Му- равей строит путь, переходя из одной вершины в другую. Пройденные муравьем вершины до- бавляются в табу-список (память муравья), чтобы избежать их повторного посещения. Ве- роятность перехода муравья из i-й вершины в j-ю зависит от количества феромона на данном ребре, значения локальной целевой функции на ребре и состояния табу-списка. Вероятность перехода k-го муравья из i-й вершины в j-ю на t-й итерации алгоритма рассчитывается по сле- дующей формуле: где  – шаг, определяющий скорость обучения. где  – шаг, определяющий скорость обучения. р р у Таким образом, алгоритм в ходе своей ра- боты осуществляет перестройку вероятност- ных характеристик поиска путем целенаправ- ленного воздействия на случайный вектор . Он уже перестает быть равновероятным и в ре- зультате самообучения приобретает опреде- ленное преимущество в направлениях наилуч- ших шагов. Это достигается введением вектора памяти. Алгоритм рекуррентно корректирует значения компонентов этого вектора на каждой итерации в зависимости от того, насколько удачным/неудачным (изменилось значение це- левой функции) был сделанный шаг. Таким образом, алгоритм в ходе своей ра- боты осуществляет перестройку вероятност- ных характеристик поиска путем целенаправ- ленного воздействия на случайный вектор . д у р  Он уже перестает быть равновероятным и в ре- зультате самообучения приобретает опреде- ленное преимущество в направлениях наилуч- ших шагов. Это достигается введением вектора памяти. Алгоритм рекуррентно корректирует значения компонентов этого вектора на каждой итерации в зависимости от того, насколько удачным/неудачным (изменилось значение це- левой функции) был сделанный шаг. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , , , 0, . k ij ij k il il ij k l J k t t j L t t P t j L              =       Здесь ij(t) – количество феромона на ребре (i, j); ij(t) – значение локальной целевой функ- ции на ребре (i, j);   0 и   0 – параметры алгоритма, определяющие важность феромон- ного следа и локальной целевой функции; Lk – множество вершин, включенных в табу-список муравья k. Линейная зависимость: 0 при -1, 1 ( ) (1 ) при -1 1, 2 1 при 1 . w p w w w w    = −       − построение муравьями пути (каждый му- равей строит путь независимо от остальных); − обновление количества феромона на ре- брах; Алгоритм направленного случайного поиска с самообучением Для применения муравьиных алгоритмов задачу надо свести к нахождению в графе за- мкнутого пути минимальной длины, в который каждая вершина входит однократно (задача коммивояжера). Общую схему работы муравь- иных алгоритмов можно представить следую- щим образом: Рассмотрим алгоритм самообучения с про- извольным законом изменения вероятнос- ти [11]. Пусть ( ) ( ) ( ) i i j j p p w = . Вид функции может быть различным, но она должна быть монотон- ной и неубывающей. − задание начального количества феро- мона на ребрах графа, количества и начального положения муравьев; − задание начального количества феро- мона на ребрах графа, количества и начального положения муравьев; Линейная зависимость: Муравьиный алгоритм Муравьиные алгоритмы позволяют автома- тически настраиваться на пример задачи (за- даны конкретные значения исходных данных) путем дополнительной разметки исходных дан- ных, которая используется для построения ре- шения на каждой итерации алгоритма и уточня- ется по мере увеличения числа итераций. Операция обновления количества феромона на ребрах. После того, как все муравьи завер- шили построение путей, обновляется количе- ство феромона на ребрах: 22 Программные продукты и системы / Software & Systems 1 (33) 2020 проблемы можно, обучаясь на модели, а не на показаниях датчиков системы. ( ) ( ) ( ) ( ) , 1 1 1 m ij ij ij k k t p t t =  + = −  +   , ( ) ( ) ( ) ( ) ( ) ( ) ( ) , , , , 0, , . k k ij k k F T t i j T t t i j T t     =    Также эта проблема может быть ослаблена заданием условий применения операций изме- нения управляющих параметров. Например, если температура в здании выше требуемой, то применение операции увеличения темпера- туры нагревателей надо запрещать. Здесь Tk(t) – путь, построенный k-м му- равьем; F(T) – целевая функция, определяю- щая качество пути; m – количество муравьев; p  [0, 1] – коэффициент испарения феромонов. Испарение феромонов вводится во избежание попадания алгоритма в локальный оптимум, когда первый найденный путь с относительно хорошим значением целевой функции стано- вится единственно значимым. Если требуемые значения характеристик ра- боты системы задаются в виде интервала, то можно попытаться настроить алгоритм так, чтобы во время переобучения значения харак- теристик находились в заданном интервале. Для алгоритмов случайного поиска подбира- ется значение шага, определяющего скорость обучения. Для муравьиных алгоритмов подби- раются значения коэффициента испарения фе- ромонов и значения параметров, определяю- щие важность феромонного следа и локальной целевой функции. Рассмотрим построение муравьиных алго- ритмов для задачи минимизации суммарного запаздывания работ [14]. По такому же прин- ципу может быть построен муравьиный алго- ритм для управления динамически изменяю- щимися системами. Имеется множество работ T = {T1, …, Tn}, для каждой из которых заданы время выполне- ния ti и директивные сроки выполнения [0, fi). Программная система и требования к аппаратной платформе Требуется для каждой работы определить время начала выполнения. Критерий оптималь- ности – суммарное время запаздывания 1 ( ) max(0, ( ) ) NW j j j F c f = −  . Программная система управления динами- чески изменяющимися системами включает следующие основные модули: ( ) max(0, ( ) ) j j F c f = −  . − модуль ввода информации от датчиков и исполнительных устройств; Вводятся два класса вершин: вершины ра- боты и вершины позиции в упорядоченном списке работ. Табу-список – размещенные работы и занятые позиции (каждая вершина присутствует в маршруте только один раз). Пе- реход к следующей позиции в списке осу- ществляется последовательно. Расписание од- нозначно определяется последовательностью работ в упорядоченным списке, построенном муравьиным алгоритмом. В качестве локаль- ных целевых функций на ребрах графа могут быть использованы: h1ij = 1/fj – минимальный директивный срок завершения, h2ij = 1/tj – ми- нимальное время выполнения, h3ij = 1/max(S + + tj, fj) – выбор минимального запаздывания работы (S – время завершения всех размещен- ных работ). − вычислительный модуль; − вычислительный модуль; − модуль передачи информации исполни- тельным устройствам; − модуль задания параметров алгоритма; − модуль диагностики датчиков и испол- нительных устройств. Модуль ввода информации от датчиков и исполнительных устройств выполняет: Модуль ввода информации от датчиков и исполнительных устройств выполняет: − первичную обработку информации, по- лучаемой от датчиков: нормализацию данных и фильтрацию; − формирование данных для вычислитель- ного модуля; − получение данных от вычислительного модуля и формирование сообщений для кон- троллеров исполнительных устройств. В вычислительном модуле запускаются ал- горитмы выработки управляющих параметров для исполнительных устройств. − коммутации; − коммутации; − коммутации; − центрального процессора (на базе про- цессора Intel i7, 4 ядра); − реконфигурируемого процессора (на базе ПЛИС Xilinx Virtex 6) с возможностью расширения функциональных возможностей путем установки мезонинных модулей АЦП/ ЦАП; − графического процессора (на базе NVi- dia Quadro K2100M); − процессора «Эльбрус» (на базе процес- сора «Эльбрус-4С»); Работа выполнена при финансовой поддержке РФФИ, грант № 19-07-00614. Работа выполнена при финансовой поддержке РФФИ, грант № 19-07-00614. Модуль диагностики датчиков и исполни- тельных устройств осуществляет контроль их исправности. Модуль диагностики датчиков и исполни- тельных устройств осуществляет контроль их исправности. − процессора «Байкал» (на базе процес- сора «Байкал-Т1»); − интерфейсный оптический PCIe; Отличительными требованиями, предъяв- ляемыми к аппаратной платформе, являются: − носителя SSD диска (интерфейс SATA3). − носителя SSD диска (интерфейс SATA3). В зависимости от требований к условиям эксплуатации ГВП доступна в трех исполне- ниях: В зависимости от требований к условиям эксплуатации ГВП доступна в трех исполне- ниях: − наличие модулей аналого-цифровых (АЦП) и цифро-аналоговых преобразовате- лей (ЦАП); − с вентиляторным охлаждением (предна- значена для эксплуатации в отапливаемых по- мещениях, температурный диапазон – от 0 до +40 °С, электропитание – однофазная сеть пе- ременного тока, напряжение питающей сети – от 187 до 242 В); − наличие широкой номенклатуры интер- фейсов связи; − ориентация вычислительной системы на эксплуатацию в промышленных условиях; − отсутствие регулярного системного ад- министрирования вычислительной системы. − с кондуктивным отводом тепла (для экс- плуатации во встроенных системах, темпера- турный диапазон – от –55 до +55 °С, электро- питание – сеть постоянного тока, напряжение питающей сети – от 18 до 36 В); Первые два требования обусловлены широ- кой номенклатурой датчиков и исполнитель- ных устройств, используемых на управляемом объекте, следующие два – длительной авто- номной работой системы управления в жест- ких условиях эксплуатации. − с гибридным отводом тепла (темпера- турный диапазон – от –55 до +55 °С, электро- питание – сеть постоянного тока, напряжение питающей сети – от 18 до 36 В). − с гибридным отводом тепла (темпера- турный диапазон – от –55 до +55 °С, электро- питание – сеть постоянного тока, напряжение питающей сети – от 18 до 36 В). Наиболее полно всем этим требованиям удовлетворяет гетерогенная вычислительная платформа (ГВП), разработанная в НИИ ВК им. М.А. Карцева [15, 16]. Заключение В состав ГВП могут входить функциональ- ные модули: Предложенный подход к управлению до- пускает нестабильное поведение окружающей среды, ограниченность информации об управ- ляемой системе и позволяет учитывать нали- чие многих характеристик работы системы, значения которых требуется поддерживать в заданных пределах. Если отказ элементов си- стемы не приводит к отказу системы в целом, а только ухудшает значения характеристик ра- боты системы, то при использовании данного подхода отклонения значений характеристик от требуемых будут минимизироваться. Программные продукты и системы / Software & Systems Программные продукты и системы / Software & Systems Литература 1. Ким Д.П. Алгебраические методы синтеза систем автоматического управления. М.: Физматлит, 2014. 192 с. Работа выполнена при финансовой поддержке РФФИ, грант № 19-07-00614. Проблема переходного процесса Модуль задания параметров алгоритмов позволяет: Модуль задания параметров алгоритмов позволяет: При изменении состояния системы алго- ритму необходим ряд пробных шагов для пере- обучения. Некоторые пробные шаги могут приводить к нарушению требуемых характери- стик работы системы. − выбирать алгоритм; − задавать параметры алгоритма; − задавать допустимый диапазон изменения значений каждого управляющего параметра. Если есть модель системы, избежать этой 23 1 (33) 2020 5. Berndt D.J., Clifford J. Using dynamic time warping to find patterns in time series. Proc. AAAI Te al Report WS KDD-94, 1994, pp. 229–248. 7. Kostenko V.A., Shcherbinin V.V. Training methods and algorithms for recognition of nonlinearly dis- torted phase trajectories of dynamic systems. Optical Memory and Neural Networks, 2013, vol. 22, no. 1, pp. 8–20. 8. Костенко В.А., Коваленко Д.С. 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Algorithms for recognizing abnormal behavior of dynamic systems that are resistant to non-linear distortions of the system phase trajectories. Proc. Int. Sc. and Pract. Conf. AITA. Moscow, 2011, pp. 897–905 (in Russ.). 9. Kovalenko D.S., Kostenko V.A., Shcherbinin V.V. Parametric family of recognition algorithms for nonlinearly distorted phase trajectories of dynamic systems. Proc. XIV Conf. Neuroinformatics-2012. Moscow, 2012, part 1, pp. 266–276 (in Russ.). p pp 10. Minu M. Mathematical Programming. Theory and Algorithms. Moscow, Nauka Publ., 1990, 488 p. (in Russ.). 11. Rastrigin L.A. Statistical Search Methods. Moscow, Nauka Publ., 1968, 398 p. 12. Dorigo M. Optimization, Learning and Natural Algorithms. PhD thesis, Milano, 1992, 140 p. g p 13. Shtovba S.D. Ant algorithms: theory and application. Programming and Computer Software. 2005, no. 4, pp. 3–18 (in Russ.). 14. Gafarov E.R. Hybrid algorithm for solving the problem of minimizing the total delay for a single device. Information Technologies. 2007, no. 1, pp. 30–37 (in Russ.). 15. Barybin A.K., Lobanov V.N., Cheldiev M.I., Chuchkalov P.B. Configurable computer platform with heterogeneous architecture. References Issues of Radio Electronics. 2016, vol. 2, no. 7, pp. 70–77 (in Russ.). 16. Baranov L.D., Lobanov V.N., Cheldiev M.I. Use of multiprocessor computing platform with heteroge- neous architecture for solving the problems of hydroacoustics and radiolocation. Issues of Radio Electronics. 2018, no. 5, pp. 7–16 (in Russ.). Using self-learning optimization algorithms to manage dynamically changing system V.A. Kostenko 1, Ph.D. (Engineering), Associate Professor, kostmsu@gmail.com V.A. Kostenko 1, Ph.D. (Engineering), Associate Professor, kostmsu@gmail.com 1 Lomonosov Moscow State University (MSU), Moscow, 119991, Russian Federation Abstract. The paper proposes an approach to the management of dynamically changing systems. Such systems has a response that during the system operation their state may: the list of the equipment, the load of the system and the functions performed by the system. It is required to choose the values of control parameters depending on the system state in such a way as to provide the required values of the characteristics of the system. To solve this problem, the authors plan to use optimization algorithms with self-learning: directional ran- dom search algorithms with self-learning and ant algorithms. These algorithms, operating by trial and error method, allow you to tune in to the current state of the system. The introducing into the algorithms the memory of successes and failures on the previous steps helps to achieve it. The main idea of using self-learning algorithms to control the system is that we will consider the control problem as an unconditional optimization problem. The elements of the vector of optimized variables are the control parameters of the system. The authors consider the algorithm steps as possible actions of system control (operations of changing control parameter values). The objective function can be set in one of two ways: by the calculation of the total mean probable characteristics error of the system from the required ones. The weights help take into account the characteristics importance by summing deviations; the researching of the maximum characteristics deviation of the system operation from the required ones. The proposed management approach allows the unstable environmental behavior, limited managed system information and allows you to take into account the presence of many system characteristics, their values must be maintained within specified limits. If the system elements failure does not lead to the whole system failure, but only worsens the system characteristics values, this approach will minimize the characteristics values de- viations from the required. 25 1 (33) 2020 Программные продукты и системы / Software & Systems A limitation of this approach is the transition process allow ability by changing the system state. In this way, the algorithm will require a number of steps for retraining. Some steps cause disturbance the required system performance. y p Keywords: dynamic system, control, mathematical programming, ant colony algorithms. For citation Kostenko V.A. Using self-learning optimization algorithms to manage dynamically changing systems. Software & Systems. 2020, vol. 33, no. 1, pp. 020–026 (in Russ.). DOI: 10.15827/0236- 235X.129.020-026. 26
https://openalex.org/W2619736730	https://europepmc.org/articles/pmc5442032?pdf=render	English	null	Photoluminescence Properties of Polymorphic Modifications of Low Molecular Weight Poly(3-hexylthiophene)	Nanoscale research letters	2,017	cc-by	6,012	Backgrounds l ( lk l h studies; actual form II samples usually contain significant fractions of form I modifications as well as amorphous backbones. Poly(3-alkylthiophene)s (P3ATs), which are representa- tive π-conjugated polymers, are known to occur in more than two different crystalline structures depending on the processing conditions [1–15]. High molecular weight (MW) P3ATs usually form a lamellae π-stacking struc- ture (form I) where fully planar backbones stack face to face with a stacking distance of 3.8 Å [3, 6, 9, 11, 16]. Because of such a short distance, charge states in form I are delocalized over several backbones [16–18]. On the other hand, solid-state samples of low MW P3ATs often exhibit also a different packing structure (form II) [3, 6, 9], in which the distance between the nearest-neighbor back- bones increases up to 4.4 Å due to tilted and interdigitated alkyl chains [2, 3, 12–14]. Such differences in the crystal- line structure are naturally expected to alter the optoelec- tronic properties. However, the difference in the optical properties, in particular the photoluminescence (PL), be- tween the form I and II modifications has not yet been re- vealed. This may be due to the difficulty in preparation of form II samples whose quality is high enough for PL Recently, Lu et al. have found that formation of form II of poly(3-butylthiophene) (P3BT) is promoted by slowly evaporating a disulfide solvent or exposing the sample to a disulfide vapor (a vapor treatment) [7, 8]. Using the fact that form II of P3ATs is converted into form I by thermal annealing [2, 3, 9, 15], Lu et al. have demonstrated the reversible transformation between form I and II modifications of P3BT. Interestingly, such behavior is very similar to the phase transition of poly(9,9-dioctylfluorene) (F8) [19–24]; the crystalline phase of F8 is prepared by thermal annealing whereas β- phase F8 appears after exposure of the samples to a vapor of a good solvent. The reversible transformation between crystalline and β phases has also been con- firmed [23, 24]. In the case of F8, high-quality β-phase thin films are prepared by dropping a dilute solution onto a substrate and waiting for a few hours to evapor- ate the solvent (drop-casting) [22]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. NANO EXPRESS Open Access Kobayashi et al. Nanoscale Research Letters (2017) 12:368 DOI 10.1186/s11671-017-2134-5 Abstract The structural and photoluminescence (PL) properties of thin films of poly(3-hexylthophene) (P3HT) with molecular weights (MWs) of 3000 and 13,300 have been investigated. Although high MW P3HT always self-organizes into one packing structure (form I), low MW P3HT forms two different packing structures (forms I and II) depending on the fabrication conditions. In this work, several fabrication techniques have been examined to obtain form II samples with little inclusion of a form I component. It is found that drop-cast thin films of low MW P3HT (form II) exhibit a PL spectrum that is different from that of form I and does not contain the form I component. The PL spectrum can thus be attributed to form II. The differences in PL properties between forms I and II can be understood in terms of weakened interchain interactions due to the longer interchain distance in form II. rds: P3HT, Molecular weight, Polymorph, Photoluminescence, Intermolecular interactions Keywords: P3HT, Molecular weight, Polymorph, Photoluminescence, Intermolecular interactions Photoluminescence Properties of Polymorphic Modifications of Low Molecular Weight Poly(3-hexylthiophene) Takashi Kobayashi1,2* , Keita Kinoshita1, Akitsugu Niwa1, Takashi Nagase1,2 and Hiroyoshi Naito * Correspondence: tkobaya@pe.osakafu-u.ac.jp; naito@pe.osakafu-u.ac.jp 1Department of Physics and Electronics, Osaka Prefecture University, 1-1 Gakuencho, Nakaku, Sakai, Osaka 599-8531, Japan Full list of author information is available at the end of the article Methods Regioregular P3HTs with different molecular weights were purchased and used as received. Their average MW and polydispersity index (PDI) were determined by gel permeation chromatography referred to poly- styrene standards. Among those P3HTs, here, we re- port the results of P3HTs with MW = 3000 (PDI = 1.3) and MW = 13,300 (PDI = 1.3), and we hereafter refer them to low and high MW P3HTs, respectively. Note that a single P3HT chain with MW = 3000 consists of nearly 20 thiophene rings. (a) (b) Fig. 1 Out-of-plane XRD patterns of a high and b low MW P3HTs. The arrow indicates the small reflection around 20.2°. S, D, and P mean the samples prepared by spin-coating, drop-casting, and precipitation, respectively. For more details of fabrication methods see the text. The extremely broad peak centered on 22° is a halo of quartz substrates. The patterns are vertically offset for clarity (a) (b) (a) y g Thin films were fabricated by spin-coating or drop- casting from chloroform solutions onto quartz sub- strates, which were simply ultrasonically cleaned in several organic solvents. The P3HT concentrations of the solutions were controlled so that the resultant film thickness is in a range from 80 to 120 nm. To remove residual solvents, all the thin films were dried in a vac- uum for 30 min. For some of the thin films, thermal annealing at 155 °C for 30 min was carried out in a vacuum. Vapor treatment was performed by exposing some of the thin films to a saturated atmosphere of chloroform vapor for 15 h. For XRD studies, in addition to those thin films, we prepared a precipitate of low MW P3HT that was obtained by adding a large amount of poor solvent, i.e., methanol, into the chloro- form solution and this was then dried on a Si substrate. The absorption spectra of the thin films were mea- sured at 6 K with an optical multichannel analyzer equipped with a calibrated CCD detector and a Xenon lamp. The PL spectra were measured at 6 K with the optical multichannel analyzer and a green diode laser (532 nm). For the measurements of the excitation spectra, we used a double monochromator and a high- power Xenon lamp instead of the green diode laser. During the absorption and PL measurements, the sam- ples were maintained in a vacuum with a closed cycle Fig. Backgrounds l ( lk l h Since there are many similarities between P3BT and F8 in spite of their en- tirely different backbone structures, it may be expected on the analogy with F8 that better quality form II thin films of P3ATs can be prepared by drop-casting. * Correspondence: tkobaya@pe.osakafu-u.ac.jp; naito@pe.osakafu-u.ac.jp 1Department of Physics and Electronics, Osaka Prefecture University, 1-1 Gakuencho, Nakaku, Sakai, Osaka 599-8531, Japan Full list of author information is available at the end of the article Kobayashi et al. Nanoscale Research Letters (2017) 12:368 Page 2 of 7 Kobayashi et al. Nanoscale Research Letters (2017) 12:368 In this work, we have fabricated thin films of poly(3- hexylthiophene) (P3HT) with several MWs by using several techniques, including drop-casting, and investi- gated their structural and optical properties. We have chosen P3HT in this work because, compared to P3BT, more data for P3HT is available in the literature. Among form II modifications obtained in this work using P3HT with MW = 3000, the ones prepared by drop-casting are the most suitable for PL measure- ments as we expected; the PL component of the other form is largely suppressed in the observed PL spectrum. We also discuss the mechanisms of the formation of the form II modifications and of their PL spectral differences. He cryostat. The out-of-plane XRD measurements were carried out at ambient atmosphere with a dif- fractometer using Cu Kα radiation. Results and Discussion Figure 1a shows the out-of-plane XRD patterns of thin films of high MW P3HT. The observed patterns are typ- ical of thin films of form I, in which the first and some- times higher-order diffractions due to the separation between π stacks are observed [1–4, 6, 9]. The lack of a diffraction around 22° corresponding to the stacking dis- tance of 3.8 Å indicates that, in those thin films, the stacking direction is parallel to the substrate. In the case of high MW P3HT, the packing structure is independent of the fabrication method used. As shown in Fig. 1b, spin-coated and annealed thin films of low MW P3HT also exhibit XRD patterns Methods The number of alkyl chains attached to the single backbone is approximately proportional to its MW, and consequently, the stabilization due to the crystallization of alkyl chains increases in step with the MW. On the other hand, nonbonded attractions be- tween the polymer backbones are not proportional to the chain length. The van der Waals force is considered to increase with a chain length but this proportionality is valid only for a short-chain regime. In a longer chain regime, the van der Waals force gradually becomes less MW dependent as the chain length increases and finally approaches the particular value of an infinite chain. This can be confirmed from the relationship between a melt- ing point of polyethylene and its MW [31]. Therefore, although attractions between the polymer backbones and between alkyl chains compete with each other in low MW P3HT, high MW P3HT always form a packing structure where attractions between alkyl chains are pre- ferred (form I). What would be expected for P3ATs with shorter alkyl chains, such as P3BT? Nonbonded attrac- tions between butyl chains are weaker than those be- tween hexyl chains. Thus, the two types of attractions would be balanced over a range of even longer MW. This explains why Lu et al. have obtained form II sam- ples of P3BT with relatively large MW [8]. Before showing their optical properties, we discuss a possible mechanism of the polymorphic behavior of P3ATs. The existence of the polymorphic modifica- tions indicates that the energetic stabilities of the two packing structures are very similar. Since the poly- thiophene backbones and alkyl chains adopt fully pla- nar and all-trans conformations, respectively, at room temperature [25, 26], the stability of a packing struc- ture is determined by nonbonded attractions between polymer backbones and between alkyl chains [27–29]. The ones between a backbone and an alkyl chain are minor so that they are usually ignored [28, 29]. From the observations, it seems to be reasonable to con- sider that in form I and II modifications, a different type of those attractions largely contribute to the stabilization. Let us consider the formation of form II during a drop-casting process. Chloroform is a good solvent for alkyl chains but polythiophene backbones are intrinsically insoluble in organic solvents. Methods 1 Out-of-plane XRD patterns of a high and b low MW P3HTs. The arrow indicates the small reflection around 20.2°. S, D, and P mean the samples prepared by spin-coating, drop-casting, and precipitation, respectively. For more details of fabrication methods see the text. The extremely broad peak centered on 22° is a halo of quartz substrates. The patterns are vertically offset for clarity Kobayashi et al. Nanoscale Research Letters (2017) 12:368 Page 3 of 7 Page 3 of 7 thermodynamically most favored packing structure (form I) [13, 30]. characteristic of form I. On the other hand, an additional series of diffractions at 7.35° and 14.7° emerges in the XRD patterns of drop-cast thin films of low MW P3HT. Those diffraction angles are in good agreement with the values reported for the form II modification of P3HT [6, 9]. The mixing ratio of forms I and II is sensitive to the processing conditions (see D1 and D2 in Fig. 1b). As the evaporation of the solvent is slowed down, the relative intensity of the diffraction features corresponding to form II increases. Form II samples of low MW P3HT can also be prepared by exposing form I samples, e.g., spin-coated thin films, to a chloroform vapor. This result indicates that the re- versible transformation between the form I and II modifications is possible with low MW P3HT. Note that the vapor-treated samples of low MW P3HT had rough surfaces. The resultant low coverage of the polymer on the quartz substrate accounts for the lower diffraction intensity (see S + vapor in Fig. 1b). In order to further confirm the formation of form II, we prepared a precipitate of low MW P3HT, in which the stacking direction is expected to be randomly ori- ented. Such a sample indeed shows diffraction at 20.2°, which represents the separated stacking distance of 4.4 Å in form II [2, 3, 6, 9]. This scenario consistently explains several experimen- tal observations. For example, spin-coated thin films always become form I because the time period in which only alkyl chains are dissolved is too short to allow form II to appear. As far as we examined, the formation of form II was not recognized in samples of P3HT with MWs larger than or equal to 5200. Also in the literature, the form II modifications were obtained only for low MW P3HT [6, 9]. Methods S and D mean the samples prepared by spin-coating and drop-casting, respectively 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 Absorbance (normalized) S + anneal S D S (b) (a) Low MW P3HT Photon Energy (eV) High MW P3HT D 6 K Fig. 2 Normalized absorption spectra of thin films of a high and b low MW P3HTs at 6 K. S and D mean the samples prepared by spin-coating and drop-casting, respectively 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 Absorbance (normalized) S + anneal S D S (b) (a) Low MW P3HT High MW P3HT D 6 K Photon Energy (eV) Fig. 2 Normalized absorption spectra of thin films of a high and b low MW P3HTs at 6 K. S and D mean the samples prepared by spin-coating and drop-casting, respectively largely reduced if the formation of form I is promoted by thermal annealing. This means that the true reason for the blueshift is the presence of a larger fraction of amorphous backbones in the samples [38]. On the other hand, the measured absorption spectrum of drop-cast thin films of low MW P3HT has a large baseline shift due to light scattering from the slightly rough surface. Unfortunately, from the spectrum, it is difficult to find the absorption bands specific to form II; this point will be discussed further later. largely reduced if the formation of form I is promoted by thermal annealing. This means that the true reason for the blueshift is the presence of a larger fraction of amorphous backbones in the samples [38]. On the other hand, the measured absorption spectrum of drop-cast thin films of low MW P3HT has a large baseline shift due to light scattering from the slightly rough surface. Unfortunately, from the spectrum, it is difficult to find the absorption bands specific to form II; this point will be discussed further later. Fig. 3 Normalized PL spectra of a high and b, c low MW P3HTs at 6 K. S and D mean the samples prepared by spin-coating and drop-casting, respectively featureless PL [35–37], the blueshifted PL is attributable to form II. In Fig. 3c, we also show the PL spectra of spin-coated thin films of low MW P3HT after thermal annealing or vapor treatment. Methods There- fore, before the chloroform evaporates completely, there exists a time period in which the backbones at- tempt to form a packing structure while alkyl chains are still dissolved. If such a time period is long enough, as in a drop-casting process, the polymer chains self-organize into a packing structure where at- tractions between the backbones are preferred to those between alkyl chains (form II). On the other hand, thermal annealing influences equally the backbones and alkyl chains, and thus results in the The abovementioned mechanism is also valid for F8. If a solvent slowly evaporates, the polymer backbones adopt the most stable, fully planar conformation [19–21]. Unlike P3ATs, F8 does not form a stacking structure because of the steric hindrance between adjacent alkyl chains. As a result, in β-phase thin films, ordered packing structures are not formed, and no clear X-ray diffraction peaks are observed [22, 32]. On the other hand, in thermally annealed thin films, crystallization of alkyl chains is pre- ferred and thus the backbones adopt less stable, twisted conformations [33]. Next, we show the absorption spectra of the prepared thin films of P3HT in Fig. 2. As shown in Fig. 2a, the ab- sorption spectra of drop-cast and spin-coated thin films of high MW P3HT are the same as those in literature [34–37]. The absorption spectrum of spin-coated thin films of low MW P3HT is slightly blueshifted with re- spect to those of high MW P3HT. This blueshift is sometimes attributed to the shorter backbones but is Kobayashi et al. Nanoscale Research Letters (2017) 12:368 Page 4 of 7 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 Absorbance (normalized) S + anneal S D S (b) (a) Low MW P3HT Photon Energy (eV) High MW P3HT D 6 K Fig. 2 Normalized absorption spectra of thin films of a high and b low MW P3HTs at 6 K. S and D mean the samples prepared by spin-coating and drop-casting, respectively (a) (b) (c) (a) (b) (c) Fig. 3 Normalized PL spectra of a high and b, c low MW P3HTs at 6 K. S and D mean the samples prepared by spin-coating and drop-casting, respectively (a) (b) (c) Fig. 3 Normalized PL spectra of a high and b, c low MW P3HTs at 6 K. Methods 3b is attrib- uted to the increase in the stacking distance from 3.8 to 4.4 Å [2, 3, 6, 9]. In addition to the blueshift, the PL spectrum of form II has the slightly larger 0–0 transition at 1.98 eV. Spano and his group have developed a theor- etical model, i.e., a weakly coupled H aggregate model, and have succeeded to explain several characteristic fea- tures of the PL of P3HT thin films (form I) such as the redshifted PL spectrum with respect to that of solution samples, the extremely low PL quantum efficiency, and the suppressed 0–0 transition [42–45]. In the past, these were believed to be caused by different factors. For in- stance, the redshift was attributed solely to the planari- zation of the backbone, the decrease in PL quantum efficiency was explained by efficient energy transfer into quench sites, and the suppressed 0–0 transition was as- cribed to the reabsorption effect. Now, the weakly coupled H aggregate model has been widely accepted but there is still lack of clear experimental evidence of the model, i.e., interchain interactions. According to the model, in form II where interchain interactions are weakened due to the longer stacking distance, the slight blueshift of PL, the recovery of the 0–0 transition, and an increase in PL quantum efficiency with respect to those of form I are naturally expected. The former two expectations can be found in Fig. 3b, and the last one can be confirmed by the fact that the PL quantum effi- ciency of form II samples was triple that of form I in our measurements. Therefore, we believe that our compari- son between the form I and II modifications could be an important evidence of interchain interactions in P3HT. other. This spectral similarity is probably a reason why the characteristic absorption spectrum of form II cannot be seen in Fig. 2b. The shift of the two excitation spectra is determined to be around 0.05 eV. This shift corresponds to half of the PL blueshift of 0.1 eV. The rest of the PL blueshift must be attributed to a decrease in the Stokes shift al- though the Stokes shift is not directly influenced by in- terchain interactions. However, the Stokes shift may depend on the strength of interchain interactions through the migration process of the excited states. Methods In annealed (vapor- treated) samples, the PL component from form I (form II) is dominant but the other form is also present. Therefore, it can be concluded that for PL studies on the form II modifications, simply drop-cast samples are more suitable than the others. PL spectra of the prepared thin films are shown in Fig. 3. As shown in Fig. 3a, the PL spectral shape of thin films of high MW P3HT is also the same as those reported for form I of other polythiophene derivatives [34, 36, 39]. Interestingly, low MW P3HT exhibits the similar PL spectrum if the samples are prepared by spin- coating (see Fig. 3b). The observed PL can thus be at- tributed to form I and indicates that the spectral shape and the peak photon energy are virtually independent of the backbone length and are mainly determined by the packing structure. In contrast to those samples, drop- cast thin films of low MW P3HT show a PL spectrum that is blueshifted by more than 0.1 eV with respect to those of form I. Since the amorphous backbones of poly- thiophene derivatives exhibit much broader and The results in Fig. 3b give us valuable information re- garding interchain interactions in π-conjugated poly- mers. Unlike small molecules [40, 41], it is not easy to obtain experimental evidence showing intermolecular (interchain) interactions in π-conjugated polymers. In the case of small molecules, intermolecular interactions can be investigated from simple comparisons between Kobayashi et al. Nanoscale Research Letters (2017) 12:368 Page 5 of 7 Fig. 4 Excitation spectra of spin-coated and drop-cast thin films measured at 6 K solid-state and solution samples. For example, the shift of the lowest excited state due to intermolecular interac- tions can be determined from the difference in the onset of PL. On the other hand, in π-conjugated polymers, the polymer backbones adopt different conformations in solid-state and solution samples, and the planarization of the polymer backbones also result in a redshift of PL [35–37]. As a result, the observed redshift of PL is not a direct evidence showing interchain interactions. In con- trast, since the polymer backbones adopt fully planar conformations in form I and II modifications, compari- sons between them allow us to focus on interchain interactions. Since a π stack is distant from the adjacent one by more than 10 Å, the blueshift of PL in Fig. Methods Solid-state samples of π-conjugated polymers are not single crystals and can be regarded as ensembles of sites and crystalline domains with various energy levels. Thus, the excited states tend to migrate into sites and domains with lower energy levels prior to PL emission [46–48]. As a result, the observed Stokes shift depends on the distribution of the energy levels. In form I samples, the distribution of the energy levels is more greatly expanded by stronger interchain interac- tions compared to form II samples. It is thus reason- able to expect that the migration process within such the large energy level distribution results in the larger Stokes shift. Competing interests The authors declare that they have no competing interests. 19. Grell M, Bradley DDC, Ungar G, Hill J, Whitehead KS (1999) Interplay of physical structure and photophysics for a liquid crystalline polyfluorene. Macromolecules 32:5810–7 19. Grell M, Bradley DDC, Ungar G, Hill J, Whitehead KS (1999) Interplay of physical structure and photophysics for a liquid crystalline polyfluorene. Macromolecules 32:5810–7 Abbreviations 14. Rahimi K, Botiz I, Stingelin N, Kayunkid N, Sommer M, Koch FPV, Nguyen H, Coulembier O, Dubois P, Brinkmann M, Reiter G (2012) Controllable processes for generating large single crystals of poly(3-hexylthiophene). Angew Chem Int Ed 51:11131–5 14. Rahimi K, Botiz I, Stingelin N, Kayunkid N, Sommer M, Koch FPV, Nguyen H, Coulembier O, Dubois P, Brinkmann M, Reiter G (2012) Controllable processes for generating large single crystals of poly(3-hexylthiophene). Angew Chem Int Ed 51:11131–5 F8: Poly(9,9-dioctylfluorene); MW: Molecular weight; P3AT: Poly(3- alkylthiophene); P3BT: Poly(3-butylthiophene); P3HT: Poly(3-hexylthiophene); PDI: Polydispersity index; PL: Photoluminescence; XRD: X-ray diffraction 15. Poelking C, Andrienko D (2013) Effect of polymorphism, regioregularity and paracrystallinity on charge transport in poly(3-hexylthiophene) [P3HT] nanofibers. Macromolecules 46:8941–56 15. Poelking C, Andrienko D (2013) Effect of polymorphism, regioregularity and paracrystallinity on charge transport in poly(3-hexylthiophene) [P3HT] nanofibers. Macromolecules 46:8941–56 Conclusions Finally, we show the excitation spectra of forms I and II at 6 K in Fig. 4. These excitation spectra were ob- tained by measuring PL intensities at 1.7 and 1.8 eV for form I and II, respectively. Although the excitation spectrum is not necessarily consistent with the absorp- tion spectrum, in particular in the case of the sample consisting of several crystalline and amorphous compo- nents, the excitation spectra in Fig. 4 suggest that the absorption spectra of forms I and II are similar to each In this work, we have prepared thin films of low and high MW P3HT using several fabrication techniques and compared their X-ray diffraction patterns and PL spectra. It has been found that simple drop-cast thin films of low MW P3HT exhibit the PL spectrum attrib- utable to the form II modifications, having less inclusion of the other PL components. Since the polymer back- bones adopt fully planar conformations in both form I Kobayashi et al. Nanoscale Research Letters (2017) 12:368 Page 6 of 7 Page 6 of 7 and II modifications, the differences in PL properties between them can be attributed to the difference in the stacking distance. Therefore, the comparison between these PL spectra shows how interchain interactions influence the PL properties of P3HT in the solid state. 11. Kayunkid N, Uttiya S, Brinkmann M (2010) Structural model of regioregular poly(3-hexylthiophene) obtained by electron diffraction analysis. Macromolecules 43:4961–7 12. Liu J, Sun Y, Gao X, Xing R, Zheng L, Wu S, Geng Y, Han Y (2011) Oriented poly(3-hexylthiophene) nanofibril with the π-π stacking growth direction by solvent directional evaporation. Langmuir 27:4212–9 13. Brinkmann M (2011) Structure and morphology control in thin films of regioregular poly(3-hexylthiophene). J Polym Sci Polym Phys 49:1218–33 13. Brinkmann M (2011) Structure and morphology control in thin films of regioregular poly(3-hexylthiophene). J Polym Sci Polym Phys 49:1218–33 Acknowledgements g This work is supported in part by JSPS KAKENHI Grant Numbers 15H03883 and 15J12038 and by a Grant-in-Aid for Scientific Research on Innovative Areas “New Polymeric Materials Based on Element-Blocks (No. 2401)” (No. JP24102011). This work is supported in part by JSPS KAKENHI Grant Numbers 15H03883 and 15J12038 and by a Grant-in-Aid for Scientific Research on Innovative Areas “New Polymeric Materials Based on Element-Blocks (No. 2401)” (No. JP24102011). 16. Prosa TJ, Winokur MJ, Moulton J, Smith P, Heeger AJ (1992) X-ray structural studies of poly(3-alkylthiophenes): an example of an inverse comb. Macromolecules 25:4364–72 16. Prosa TJ, Winokur MJ, Moulton J, Smith P, Heeger AJ (1992) X-ray structural studies of poly(3-alkylthiophenes): an example of an inverse comb. Macromolecules 25:4364–72 Publisher’s Note 20. 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https://openalex.org/W2920882431	https://revistes.uab.cat/doblele/article/download/v4-garcia-ambadiang/38-pdf-es, https://ddd.uab.cat/pub/doblele/doblele_a2018v4n1/doblele_a2018v4n1p22.pdf	es		La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas	Doblele	2,018	cc-by	8,491	La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas 1 The non-mother tongues teaching in the age of migrations: the importance of linguistic biographies ISABEL GARCÍA PAREJO / THEÓPHILE AMBADIANG UNIVERSIDAD COMPLUTENSE DE MADRID / UNIVERSIDAD AUTÓNOMA DE MADRID igarcia@edu.ucm.es / theophile.ambadiang@uam.es Resumen: Aunque el concepto de ‘identidad’ ha estado en el centro del debate sobre el aprendizaje de una L2, su disociación de la capacidad de decisión de los sujetos (‘agentividad’) tiene el efecto de simplificar aquella en exceso y permite una conceptualización no problemática del proceso de enseñanza-aprendizaje de la L2. A partir de datos obtenidos de cuestionarios administrados a inmigrantes adultos procedentes de Rumanía, Marruecos, Guinea Ecuatorial y otros países del Africa subsahariana que viven en Madrid, se sugiere que el concepto de ‘agentividad’ es crucial en cualquier reflexión antropológica sobre el aprendizaje de una segunda lengua ya que subyace a la identidad y la motivación, está mediado por lo que Schiffman (2006) denomina ‘cultura lingüística’ y, además, está íntimamente asociado a la alteridad. Palabras clave: segunda lengua, agentividad, identidad, cultura lingüística, adultos inmigrantes Abstract: Although a concept such as ‘identity’ has been at the centre of the debate on second language learning, its dissociation from agency has the effect of oversimplifying it, and allows for an excessively unproblematic conceptualization of the process of second language learning/teaching. Based on data obtained from questionnaires administered to adult immigrants living in Madrid who come from Romania, Morocco, Equatorial Guinea and other subSaharan countries, it is suggested that agency is crucial in any anthropological reflection on language learning, as it underlies identity and motivation, is mediated by what Schiffman (2006) calls ‘linguistic culture’ and, moreover, is intimately associated to alterity. Key words: second language, agency, identity, linguistic culture, adult migrants 1 Este trabajo se enmarca dentro de los estudios sobre identidad y variación discursiva desarrollados en los proyectos de investigación FFI2012-31702 y FI2008-04221/FILO. ISSN: 2014-1130 n.º 4 \| Diciembre 2018 \| 22-40 DOI: https://doi.org/105565/rev/doblele.38 Recibido: 02/10/2018 Aprobado: 31/10/2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas 1. Introducción Este artículo discute algunos de los problemas que el uso de conceptos relacionados con la antropología puede plantear para la enseñanza de una segunda lengua (L2). Sostenemos que aunque uno de los conceptos más importantes, el de ‘identidad’, ha estado en el centro del debate sobre el aprendizaje de las segundas lenguas, su disociación de la agentividad tiene el efecto de simplificar excesivamente el proceso de enseñanzaaprendizaje de las L2, al inducir a perder de vista los muchos sesgos existentes en su conceptualización (aprendizaje formal, hablante nativo, contexto monolingüe, dimensión cultural etc.). Desde nuestro punto de vista, el concepto de ‘agentividad’ es crucial en cualquier reflexión antropológica sobre el aprendizaje de lenguas, ya que subyace a la identidad, la motivación y la inversión (Norton, 2008), además de estar mediada por lo que Schiffman (2006) denomina cultura lingüística (linguistic culture). Esto es particularmente cierto en un contexto que, como es el caso hoy en día, se caracteriza por la complejidad de las interacciones, debido en parte al contacto de lenguas y culturas asociado a la migración masiva y al turismo. Nuestra discusión sobre la noción de ‘agentividad’ se basa en datos obtenidos de cuestionarios administrados a inmigrantes adultos africanos y de Europa del Este que viven en Madrid, cuyos resultados se ofrecen aquí con un análisis descriptivo. Los datos sugieren la existencia de un continuo en uno de cuyos extremos se presentan perfiles de comportamiento lingüístico específicos que pueden resultar útiles para una caracterización de la agentividad, en asociación íntima con la alteridad. Antes de presentar y analizar los datos, la sección 2 de nuestro artículo se centra en la relación entre la identidad y la cultura lingüística, así como en los efectos de la agentividad en el aprendizaje de lenguas. En la sección 3 describimos los datos procedentes del cuestionario. En la sección 4 discutimos los mismos centrándonos en la forma en que la agentividad puede influir en el proceso de aprendizaje de una L2. Finalmente, la sección 5 concluye el trabajo subrayando las ideas principales de la discusión. 2. Cultura lingüística, agentividad e identidad 2.1 Creencias y valores de una comunidad lingüística Schiffman (1996) utiliza el término 'cultura lingüística' en referencia a las características de la lengua-cultura que residen en algún lugar de la conciencia de las comunidades lingüísticas, pero no pueden ser localizadas o ‘ser parte de’ la lengua entendida como código o gramática. La propuesta de Schiffman resulta ser muy interesante desde un punto de vista conceptual y teórico, ya que se refiere a un tipo de conocimiento que no se puede subsumir con conocimientos lingüísticos ni culturales. Por un lado, el concepto de ‘cultura’ plantea problemas tanto en sus conceptualizaciones 23 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang esencialistas como emergentes, similares a los observados con otros constructos tales como la ‘identidad’. Por otro lado, la definición que propone Schiffman de ‘cultura lingüística’ incluye aspectos tan diversos que resulta bastante difícil de concebir la realidad que se describe en términos de una entidad coherente. Para Schiffman, existe una asociación específica entre una determinada comunidad de hablantes y una cultura lingüística en particular. Esta última es definida por él como “(the) sum totality of ideas, values, beliefs, attitudes, prejudices, myths, religious structures, and all other cultural “baggage” that speakers bring to their dealings with language from their culture” (Schiffman 2006: 112). A pesar de su naturaleza cambiante, la cultura lingüística funciona como un "filtro interpretativo" y, como tal, tiene relevancia en la planificación lingüística de la comunidad. 2 Los hechos que discute Schiffman (1996, 2006) y los datos que aduce en apoyo de sus afirmaciones a propósito de la existencia de un conocimiento específico que no puede ser reducido solo a la lengua o a la cultura son consistentes con la idea de que existe una correlación entre, por un lado, las prácticas lingüísticas típicas de diferentes espacios generalmente asociados a lenguas específicas y, por otra parte, la prevalencia en cada uno de estos espacios de un ethos lingüístico distinto. 3 La relevancia de estas diferencias para la discusión teórica hace necesaria la búsqueda de un término menos problemático que el de ‘cultura lingüística’, al menos desde un punto de vista analítico. Proponemos aquí un término mucho menos ambicioso, ‘ethos lingüístico’, que nos permite evitar las connotaciones de un metaconcepto como el de ‘cultura’, así como la complejidad implícita en la definición de Schiffman relativa a “suma total de valores, creencias, actitudes” acerca de la lengua que el hablante porta. Con el término propuesto, somos capaces de captar la dimensión variable a la vez que colectiva de los modos de comportamiento de un grupo, así como la agentividad de los hablantes individuales. Además, a través de este término referimos a la noción de prácticas comunicativas como representadas a través de las prácticas lingüísticas propias de los miembros de una determinada comunidad.4 Por otra parte, las prácticas lingüísticas y comunicativas pueden cambiar a través del tiempo tanto a nivel individual como colectivo, además de tener fuertes 2 Sin embargo, aunque Schiffman señala que la cultura lingüística tiene implicaciones en “the formulation of language policy, the specification of language planning measures as well as the responses of the community to policies and language planning activities” (2006:112), parece que el determinismo implícito en esta observación desaparece en páginas posteriores cuando afirma que la cultura lingüística manifiesta tanto aspectos abiertos como ocultos, “since if one simply takes at face value what decision-makers and the “power elite” in a society say about language and language policy, the true picture of what is happening will not emerge” (2006: 116). 3 La discusión de Schiffman (1996) se centra en Francia, Alemania y Estados Unidos. 4 En este sentido, mucho de lo que Schiffman incluye en la cultura lingüística no viene dado, sino que más bien puede ser concebido como representaciones producidas indirectamente a través de ciertas prácticas asociadas al modo en que los hablantes se relacionan con su lengua; cf. Fairclough (2006: 6) y Canut (1996). 24 Doblele \| n.º 4 \| Diciembre 2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas implicaciones para la relación entre la lengua y la cultura, la manera en que se concibe el aprendizaje y uso de una segunda lengua o el estatuto de un aprendiente de idiomas. Dos de esas concepciones que resultan de mayor interés para nuestra discusión tienen que ver con el valor simbólico y el valor instrumental que los sujetos de una comunidad otorgan a la(s) lengua(s). En el primer caso una lengua se concibe principalmente como un código, y la relación con sus hablantes está mediada a través de diferentes instancias que implican la autoridad (Estado, escuela, por ejemplo). La función comunicativa del lenguaje es privilegiada en el segundo caso, ya que se tiende a reducir la importancia de la mediación y permite una postura más agentiva para el aprendiente / hablante de la L2. Así, a pesar de las diferencias evidenciadas en los repertorios lingüísticos de los miembros de una comunidad comunicativa dada (reflejo de su biografía lingüística), sus prácticas lingüísticas tienden a presentar unas características y un perfil idénticos. 2.2. Identidad, agentividad y prácticas lingüísticas Según Brubaker y Cooper (2000), la ‘identidad’ ha sido definida, básicamente, de dos maneras que plantean problemas tanto desde el punto de conceptual como operativo. Puede significar mucho, como cuando se entiende en un sentido fuerte (identidad estática, esencialista), muy poco, si se interpreta en sentido débil (identidad dinámica, emergente), o incluso nada en absoluto si tenemos en cuenta su ambigüedad o las contradicciones inherentes a la primera definición. Además, en ambos usos, fuerte y débil, las connotaciones cosificadoras son ineludibles. Por otra parte, subyace la necesidad de diferenciar las concepciones teórica y práctica de la identidad, a pesar de las complejidades asociadas a la combinación de categorías locales y globales (Augé y Colleyn 2005: 26, 121; Brubaker y Cooper 2000: 17, 20), y se proponen tres subconjuntos de términos que pueden sustituir provechosamente el de ‘identidad’: (i) ‘identificación’ y ‘categorización’, (ii) ‘auto-comprensión’ y ‘ubicación social’, y (iii) ‘comunidad’, ‘conectividad’ y ‘agrupación’.5 De éstos, los conceptos de ‘identificación’, ‘autocomprensión’ y ‘comunidad’ son de mayor importancia para nuestra discusión en este trabajo. La ‘auto-comprensión’ refiere a "la propia comprensión de lo que uno es" y, por lo tanto, se subsume bajo el concepto de ‘identificación’ y, de manera más general, en el de ‘categorización’. Por su parte, el concepto de ‘comunidad’ tiene que ver con el hecho de compartir algún atributo común, en tanto que el de ‘conectividad’ denota los lazos relacionales que vinculan a las personas. Mientras que los atributos subsumidos en esa ‘comunidad’ tienen el efecto de indicar la pertenencia de un individuo a un grupo, los procesos 5 Los términos propuestos por Brubaker y Cooper (2000: 17) son: (i) identification and categorization, (ii) self-understanding and social location, and (iii) commonality, connectedness and groupness. 25 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang asociados a la ‘identificación’ y a la ‘categorización’ evidencian la agentividad del individuo, particularmente en contextos de diversidad cultural y lingüística. En ambos casos, sin embargo, la representación resultante se produce en función del peso relativo de la ‘autocomprensión’ comparada con la categorización externa. Desde este punto de vista, existe una correlación entre la primacía dada a la autoidentificación o auto-representación y la agentividad, en contraste con los casos en los que la auto-comprensión es anulada por una categorización externa extremadamente coercitiva (Brubaker y Cooper, 2000: 17). Concebida como la capacidad de los individuos para actuar en una relación dialéctica con las estructuras sociales y políticas que forman y, al mismo tiempo, son transformadas por ellos, la ‘agentividad’ puede manifestarse de diferentes maneras en las prácticas comunicativas y lingüísticas. Por otra parte, puede ser descrita en función del estatus que el hablante se asigna a sí mismo y en función del estatus que le asignen sus interlocutores en el proceso comunicativo. Los dos casos siguientes ilustran la importancia de la categorización del hablante en relación a su agentividad en las prácticas lingüísticas. El primer caso describe dinámicas sociolingüísticas típicas de Malí, en especial las que caracterizan a los peuls en relación con el bambara, la lengua mayoritaria en Bamako. Según Canut (1996 : 72-73 ) “Très souvent, les Peuls, les Songhay (plutôt âgés) se montrent très peu compétents dans la langue de Bamako. Leurs énoncés restent, en apparence, et même au bout de 10 ou 30 ans de résidence dans la capitale, très approximatifs et parfois mélangés au français pour les francophones. Il ne s’agit en fait rarement d’un problème de compétence, mais plutôt de performance : la plupart connaissent la langue mais la “bredouillent”, selon leurs dires, volontairement. Ce procédé s’exerce à plusieurs niveaux : “Je garde mon accent, comme ça on sait que je suis peul. Je n’ai jamais appris cette langue, je n’en ai pas besoin. Je ne connais que les noms des condiments pour faire mon marché ”. Lo que es importante, como señala Canut (pag. 73), más que la elección de ese uso aproximado, es que el juego de habilidades lingüísticas que manifiesta el hablante es un desafío para el propio código, para sus límites y su validación como una forma significativa. En otras palabras, los hablantes peul de bambara actúan estratégicamente sobre esta lengua con el fin de hacer visible la forma en que se representan a sí mismos con respecto a ella y a sus hablantes, ya sean nativos o no nativos. Esto puede interpretarse como una manifestación de su agentividad (por ejemplo, cuando manifiestan “Je garde mon accent”). El otro caso se basa en un experimento que trata de la interacción de hablantes nativos (americano) de inglés y un grupo específico de los hablantes no nativos de inglés, coreanos, con el fin de evaluar la correlación que pueda existir entre las actitudes de los primeros con 26 Doblele \| n.º 4 \| Diciembre 2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas respecto al inglés de los coreanos y el éxito de las interacciones en las que los miembros de ambos grupos estaban involucrados. (Lindeman 2002). Lindeman centró su discusión en los nativos americanos y más concretamente sobre la forma en que estos clasificaron a sus interlocutores como hablantes de L2 (competentes o incompetentes). Puesto que los sujetos coreanos no tenían voz y por lo tanto no podían mostrar la representación que tenían de sí mismos como hablantes de inglés, puede decirse que la categorización externa que les fue asignada por sus interlocutores estadounidenses prevalece sobre cualquier forma de auto-representación que podrían haber ejercitado. La representación que los peul tienen de sí mismos como hablantes de bambara tiene consecuencias interesantes. Por un lado, en la medida en que están satisfechos con la manera en que combinan su competencia y sus estrategias comunicativas, esperan (de hecho, requieren) la colaboración de los otros hablantes nativos o no nativos, que tiene que compensar sus fallos comunicativos, sean intencionados o no. Por otro lado, tal representación cuestiona implícitamente la posición de poder asociada a la condición de ‘hablante nativo del bambara'. La auto-categorización o la auto-comprensión tiene aún más relevancia cuando se considera en relación a los aprendientes de una L2, ya que existe una clara correlación entre la forma en que se clasifican y / o representan a sí mismos y su actitud hacia el proceso de aprendizaje y sus objetivos. En este sentido, en el siguiente apartado se pretende mostrar cómo algunos aprendientes de lenguas tienden a privilegiar o no su autorepresentación, en contraste con aquellos sujetos que ceden ante procesos de categorización impuestos a ellos por otros, generalmente a partir de una posición de poder asociada con el estatus de hablante nativo. Así, se puede observar que, debido a su biografía lingüística, los sujetos pueden situarse a lo largo de un continuo en cuyos extremos prevalece, por un lado, la representación que tienen de sí mismos como hablantes de una L2 y, por otro, la categorización externa que pueda imponerse como aprendientes de L2. 3. Datos Para desarrollar nuestra reflexión, nos apoyamos en un conjunto de datos que, además de ser obtenidos en momentos ligeramente diferentes, se han discutido por separado en artículos anteriores (Ambadiang 2003, 2010, García Parejo 2014, Ambadiang y García Parejo 2008). Todos ellos toman como base un cuestionario administrado a adultos africanos subsaharianos que viven en Madrid (España), cuyo objetivo ha sido explorar la relación de este colectivo con el idioma español (cf. Ambadiang 2003). Posteriormente, el cuestionario se ha utilizado para estudiar el valor simbólico que otorgan a la lengua española otros colectivos que estudian español (cf. García Parejo 2014). En este trabajo (véase tabla I), vamos a centrarnos en datos relativos a 34 adultos de origen marroquí, 41 adultos de origen rumano y 51 adultos subsaharianos de diferentes nacionalidades, de los cuales 17 27 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang son ecuatoguineanos que están en contacto con la lengua y cultura española desde su país de origen (Guinea Ecuatorial).6 Grupos Subsaharia Ecuatoguinea Marroquí nos nos es Rumanos N % N % N % N 16-20 0 0% 1 5,8% 3 20-25 4 11,8% 4 23,5% 4 5 1 2 25-30 8 23,5% 7 41,1% 4 8,8% 11,8 % 11,8 % 30-35 11 32,3% 2 11,7% 7 20,5% 2 11,7% 40-45 1 2,9% 0 0% 6 45-50 0 0% 0 0% 4 5,9% 29,4 % 17,6 % 11,8 % 4 35-40 2 1 0 Más de 50 0 0% 1 5,8% 1 2,9% NS/NC 2 5,9% 0 0% Total 34 100% 17 10%0 Hombre 27 79,4% 9 52,9% Mujer 6 17,6% 8 47% Total Formaci ón Estudios primarios 34 100% 17 100% 1 2,9% 1 5,8% Estudios secundarios Estudios de formación profesional 13 38,2% 9 52,9% 0 0% 0 0% 0 0 0 0 3 100,0 4 100,0 4 % 1 % 1 41,2 1 46,3 4 % 9 % 2 58,8 2 53,7 0 % 2 % 3 100,0 4 100,0 4 % 1 % 1 41,2 14,6 4 % 6 % 1 29,4 2 56,1 0 % 3 % 17,6 19,5 6 % 8 % Estudios superiores 18 52,9% 7 41,1% 1 NS/NC 1 2,9% 0 0% Total 34 100% 17 100% 3 8,8% 0 0,0% 3 100,0 4 100,0 4 % 1 % Edad Sexo 2,9% 6 % 12,2 % 29,3 % 14,6 % 7 9,8% 17,1 % 4 9,8% 2 4,9% 1 2,4% 4 9,8% Tabla I. Configuración de la muestra Además de datos sobre los sujetos (país, género), las preguntas centrales del cuestionario permiten recoger información acerca de dos cuestiones fundamentales: sus opiniones acerca del interés que tiene el aprendizaje y uso del español y sus actitudes positiva/ negativa hacia la lengua-cultura 6 De la configuración de la muestra interesa resaltar que, entre los marroquíes, se da el menor nivel de estudios y el mayor porcentaje de mayores de 40 años. Para más detalles sobre el cuestionario y el desarrollo de estas investigaciones véase García Parejo (2014). 28 Doblele \| n.º 4 \| Diciembre 2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas meta y sus hablantes. Las preguntas que nos interesan para este estudio son aquellas que exploran de manera directa o indirecta (i) cómo considera el sujeto que es su nivel de español; (ii) cómo podría describirse a sí mismo a ese respeto a través de los ojos de sus interlocutores; (iii) el grado en que le preocupa lo que piensan los españoles y otras personas sobre su manera de hablar español y (iv) la importancia dada a la lengua española ya sea para fines profesionales, sociales o comunicativos. Las preguntas del cuestionario se han elaborado siguiendo el modelo de escala de Likert o de actitud, donde se provoca la respuesta subjetiva del informante ante una afirmación (cf. Larsen–Freeman y Long 1991:35). La escala se ha organizado alrededor de 4 puntos (de “nada importante” a “muy importante”), salvo en aquellas preguntas en las que se ha solicitado la valoración del sujeto a propósito de sus resultados sobre el aprendizaje de la L2, donde se ha añadido una quinta opción del tipo “solo solo me preocupa que me entiendan”. Una vez recogidos los cuestionarios, se han volcado los datos en una tabla Excel y se ha procedido a realizar una descripción estadística.7 A partir del análisis de resultados esperamos tener una idea lo más precisa posible en relación a la manera en que diferentes grupos procedentes del continente africano y del este de Europa se representan a sí mismos como aprendientes (y hablantes) de la lengua española. Esperamos poder evaluar las diferencias que puedan existir entre estos grupos de sujetos y el grado en que se correlacionan con la caracterización de ellos que aquí se proponen. 3.1 Representaciones de los sujetos sobre su nivel de español Los resultados registrados en la Tabla II corresponden a los grupos de sujetos presentados en la tabla I. Estos pueden agruparse en dos grandes subgrupos: a) adultos procedentes del continente africano y b) adultos procedentes del Este de Europa, concretamente de Rumania, por lo que sus hablantes pueden sentir cierta familiaridad con la lengua española al compartir la L1 y la L2 origen románico. Entre los sujetos del grupo a), los presentados en el segundo lugar proceden de Guinea Ecuatorial y, debido a la historia de este país, han estado en contacto con el idioma español mucho antes de llegar a España. El resto de los sujetos vienen de otros países del África subsahariana, principalmente Camerún y Nigeria (columna 1) y de Marruecos (columna 3). La principal diferencia entre estos grupos tiene que ver con el hecho de que el español es idioma oficial de Guinea Ecuatorial, se utiliza en el sistema educativo y los medios de comunicación, así como en la calle (en mezcla con otras lenguas locales). En los otros 7 Además del estudio descriptivo de frecuencias como el que mostramos aquí, el análisis de datos del cuestionario más actual (García Parejo 2014) aplicado a 141 sujetos incluye una prueba (chi-cuadrado) para medir la significatividad estadística de las frecuencias. Comentaremos alguno de estos resultados al hilo de nuestra discusión. 29 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang países, el español es sólo una asignatura escolar y, como tal, se enseña en la escuela secundaria y en la universidad. Como consecuencia, las rutinas comunicativas relacionadas con la lengua española son aprendidas por los colectivos de los otros grupos solamente a su llegada a España. Sin embargo, mientras que el colectivo ecuatoguineano (ECG) considera en un 37,5% que su nivel de español es suficiente para cubrir sus necesidades, el resto de grupos africanos considera que su nivel es bueno (SSH 30,3%; MARC 26,5%) o suficiente para la comunicación (SSH 24,2%, MARC 20,6%). En contraste, el colectivo rumano (RUM) prefiere puntuar su competencia de español y, al igual que los otros colectivos, considera que su nivel de español es muy bueno (22%), bastante bueno (34,1%) o bueno (26,8%). En términos más generales, más del 60% de cada grupo valoran su competencia en español de manera positiva, incluso entre aquellos que consideran que es suficiente para sus necesidades comunicativas. Grupo Tu nivel de español te parece: Muy bueno Bastante bueno Bueno Suficiente para cubrir mis necesidades Nada bueno NS/NC Total Subsaharianos Ecuatoguineanos Marroquíes Rumanos N % N % N % N 7 6 10 21,2% 18,2% 30,3% 2 7 1 12,5% 43,8% 6,3% 7 8 9 20,6% 9 23,5% 14 26,5% 11 22,0% 34,1% 26,8% 8 2 1 34 24,2% 6,1% 2,9% 100,0% 6 0 1 17 37,5% 0,0% 6,3% 100,0% 7 3 0 34 20,6% 7 8,8% 0 0,0% 0 100,0% 41 17,1% 0,0% 0,0% 100,0% % Tabla II. Nivel de lengua española desde la perspectiva del sujeto Mientras que la tabla II muestra la percepción que nuestros sujetos tienen de sí mismos como hablantes de español, la tabla III muestra lo que creen acerca de la forma en que sus interlocutores (compatriotas y otros) evalúan su nivel en esta lengua. En la tabla III, apartado a, los datos revelan una asimetría interesante entre los subgrupos de sujetos. Por una parte, los africanos se sitúan en la horquilla intermedia (‘bastante bueno' y ‘bueno’) referido a sus compatriotas, frente a los rumanos que puntúan con un 41,5% en el extremo ‘muy bueno’. Ninguno de los sujetos cree que sus compatriotas piensan que su nivel sea malo y ninguno de los subsaharianos cree que lo valoren como ‘suficiente’. Esto no es de extrañar si tenemos en cuenta las diferencias evidenciadas en la relación de todos estos subgrupos de sujetos con la lengua española. Desde otro punto de vista, estas diferencias también pueden tener que ver con la asimetría que existe en los repertorios lingüísticos de estos colectivos.8 Por otra parte, ni 8 En el estudio de variables con Chi-cuadrado realizado por García Parejo (2014) se halla que existe una relación significativa entre la variable ‘grupo-origen’ y las preguntas (i) 30 Doblele \| n.º 4 \| Diciembre 2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas subsaharianos ni ecuatoguineanos creen que otros interlocutores consideren su nivel de lengua bajo, tampoco los rumanos. Sin embargo, más del 70% de los dos primeros colectivos, frente a más del 60% de los rumanos y el 50% de los marroquíes, cree que los otros valoran bastante bien o muy bien su nivel de español. Grupo Subsaharianos Ecuatoguineanos Marroquíes a. b. Rumanos Crees que a tus compatriotas les parece: Muy bueno Bastante bueno Bueno Suficiente para entenderte Nada bueno NS/NC Total Crees que a otras personas les parece Muy bueno Bastante bueno Bueno Suficiente para entenderte Nada bueno NS/NC Total N 10 9 13 0 0 2 34 % 31,3% 28,1% 40,6% 0,0% 0,0% 6,1% 100,0% N 4 5 5 1 0 2 17 % 26,7% 33,3% 33,3% 6,7% 0,0% 12,5% 100,0% N % N % 9 26,5% 17 41,5% 12 35,3% 10 24,4% 9 26,5% 9 22,0% 4 11,8% 5 12,2% 0 0,0% 0 0,0% 0 0,0% 0 0,0% 34 100,0% 41 100,0% 7 7 16 2 0 2 34 21,9% 21,9% 50,0% 6,3% 0,0% 6,1% 100,0% 4 7 5 0 0 1 17 25,0% 43,8% 31,3% 0,0% 0,0% 6,3% 100,0% 9 26,5% 13 31,7% 8 23,5% 14 34,1% 8 23,5% 8 19,5% 6 17,6% 6 14,6% 1 2,9% 0 0,0% 2 5,9% 0 0,0% 34 100,0% 41 100,0% Tabla III. Nivel de lengua española desde la perspectiva de los interlocutores imaginados por los sujetos 3.2 Actitudes de los sujetos hacia su nivel de español Por su parte, la Tabla IV refleja la importancia que nuestros sujetos dan a la opinión y valoración que sus interlocutores, nativos y no nativos, puedan emitir acerca de su nivel de español. Los porcentajes registrados en la Tabla IV no sólo inducen a mantener los subgrupos que se presentan en las Tablas II y III, sino que también parecen confirmar la necesidad de tratar separadamente interlocutores españoles y no españoles, ya que esta diferencia tiene alguna relevancia en los resultados obtenidos. Aquí, la comparación de los datos ofrecidos por los ecuatoguineanos con los de los otros sujetos del continente africano muestra algunas diferencias “lees otras lenguas” (0,039), destacando que un 31,7% de las personas del grupo Rumanos no lee en otras lenguas; (ii) “primera lengua elegida para hablar” (0,011), destacando que las personas del grupo Marroquíes son las que en menor porcentaje usan el español como primera opción; (iii) “en qué lenguas te comunicas habitualmente con amigos” (0,010), destacando el porcentaje más alto de uso del español por parte del grupo Rumanos; y (iv) “en qué lenguas te comunicas habitualmente con tus hijos” (0,001), destacando el porcentaje más alto de uso del español por parte del grupo Marroquíes. 31 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang interesantes. Por ejemplo, los ecuatoguineanos no están preocupados por la opinión de sus compatriotas (75%) ni por la de los españoles (46,7%). Los marroquíes admiten estar un poco preocupados por la opinión de los españoles (44,1%), al igual que los subsaharianos (32,3%). Por su parte, la respuesta de los rumanos está más repartida al respecto. En general, a los subsaharianos y, sobre todo a los marroquíes, no les preocupa en exceso la opinión de otros interlocutores (‘un poco’ a los SSH, 21,2% y a los MARC, nada 55,9%), sin embargo, a más del 58% de los rumanos les preocupa en alguna medida. A nivel general, podría señalarse que los resultados de la Tabla IV indican que los sujetos no llegan a representar a sus interlocutores, sean ‘hablantes nativos’ o ‘hablantes no nativos’ como si estos fuesen bloques homogéneos que pudieran describirse en términos de un conjunto de características estables y constantes que sirviesen de referencia para todos los colectivos que aprenden una segunda lengua, en este caso, la lengua española. Parece que las diferencias observadas entre los subgrupos son sólo parciales (preocupación de los rumanos por lo que otros opinen, nativos y no nativos vs. menos preocupación por la opinión del interlocutor por parte de los colectivos africanos), pero pueden resultar de interés para el proceso de enseñanza-aprendizaje de la lengua.9 Grupo Subsaharianos Ecuatoguineanos Marroquíes N % Te preocupa la opinión de los españoles Mucho 4 11,8% Bastante 5 15,2% Un poco 11 32,3 Nada 9 28,1% Sólo me preocupa que me entiendan 4 11,8% NS/NC 1 2,9% Total 34 100,0% Te preocupa la opinión de los no españoles Mucho 3 9,1% Bastante 5 15,2% Un poco 7 21,2% Nada 13 39,4% Sólo me preocupa que me entiendan 5 15,2% NS/NC 1 2,9% Total 34 100,0% Rumanos N % N % N 1 4 2 7 6,7% 26,7% 13,3% 46,7% 2 6 15 8 5,9% 10 17,6% 11 44,1% 7 23,5% 8 1 2 17 6,7% 12,5% 100,0% 3 8,8% 5 12,2% 0 0,0% 0 0,0% 34 100,0% 41 100,0% 0 1 0 12 0,0% 6,3% 0,0% 75,0% 1 2 8 19 3 1 17 18,8% 6,3% 100,0% 4 11,8% 4 9,8% 0 100,0% 0 100,0% 34 100,0% 41 100,0% 2,9% 7 5,9% 8 23,5% 9 55,9% 13 % 24,4% 26,8% 17,1% 19,5% 17,1% 19,5% 22,0% 31,7% Tabla IV. Relevancia que tiene para los sujetos lo que creen los interlocutores acerca de su nivel de lengua española 9 En el estudio estadístico realizado por García Parejo (2014) se halla que existe una relación significativa entre la preocupación por la opinión de los interlocutores no españoles y la variable ‘nivel de formación’ (0,006). Recordemos que en la muestra, el grupo de personas marroquíes era el que menor nivel de estudios presentaba. 32 Doblele \| n.º 4 \| Diciembre 2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas 3.3 Actitudes de los sujetos hacia la lengua y cultura española El último tipo de datos que analizamos se refiere al valor, ya sea práctico o simbólico, que nuestros sujetos otorgan a la lengua española. La tabla V, donde se registran los porcentajes correspondientes, es la que muestra las asimetrías más interesantes entre los grupos analizados. Para los rumanos (3,72) y guineanos (3,47), las dos comunidades más cercanas a la lengua española, se da prioridad al uso del español para comunicarse con los españoles. En el caso de los guineanos, eso puntúa más que el valor que pueda tener la lengua para conseguir un trabajo (3,29). Por su parte, son los subsaharianos y rumanos los que consideran que esta L2 tiene valor alto para la formación académica (3,7 y 3,69 respectivamente), pero los primeros no lo consideran tan importante para conseguir un trabajo (3,32). Los porcentajes más bajos se relacionan con elementos como la ‘promoción y mejora social’, sobre todo entre los subsaharianos, así como el valor de la lengua para la ‘integración’, sobre todo por parte de los ecuatoguineanos. Por razones obvias, los porcentajes más bajos también están relacionados con elementos como el valor que tiene la lengua española para ‘la comunicación con otros’, aunque tienden a ser mayores en el caso de los sujetos de Rumania y Guinea. El valor que pueda tener el español para la vida práctica obtiene porcentajes más bajos entre los subsaharianos y ecuatoguineanos (2,6), frente al porcentaje que ha alcanzado este ítem entre marroquíes (3,15) y rumanos (3,30). 10 Grupo Subsaharianos Ecuatoguineanos Marroquíes Rumanos Rango 1-4 Media Media Media Media . Formación 3,7 3,52 3,44 3,69 . Trabajo 3,32 3,29 3,47 3,72 . Integración en la sociedad 3,38 2,94 3,24 3,62 . Promoción y mejora social 2,7 2,82 3,06 3,33 . Comunicación con los españoles 3,26 3,47 3,29 3,72 . Comunicación con otras personas no españolas 2 2,41 2,09 2,64 . Vivir en general 2,6 2,6 3,15 3,30 Tabla V. Valor práctico y/ o simbólico que otorgan los sujetos a la lengua española 10 En el estudio de García Parejo (2014) se hallan diferencias significativas entre las variables ‘grupo-origen’ y el grado de importancia otorgado al español para “Conseguir trabajo” (0,040). Los rumanos (junto con otros colectivos de Europa del Este) son los que otorgan mayor importancia al aprendizaje de la lengua para alcanzar este fin. Según la edad, se dan diferencias significativas en la importancia que se otorga al español “para participar en actividades sociales” (0,048, más valor entre los sujetos del grupo 40- 45 años) y según la formación, se dan diferencias significativas en la importancia que se otorga al español “para la formación cultural” (0,045, menos valor entre los sujetos que solo tienen estudios primarios). 33 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang Aunque los cuadros anteriores se refieren a muy diferentes tipos de datos, los resultados registrados en ellos sugieren que nuestros sujetos tienden a mostrar una marcada tendencia a construir su propia identidad en tanto que individuos que estudian, hablan y utilizan el español. Así, si comparamos los datos recogidos en la tabla II con los datos de las tablas III y IV podremos observar la coherencia que existe entre la percepción que nuestros sujetos tienen acerca de su conocimiento de la lengua española por un lado y, por otro, la manera en que perciben la opinión de diferentes interlocutores al respecto. Esa tendencia a construir una identidad como hablante de L2, que es consistente con las observaciones registradas en muchas encuestas sociolingüísticas llevadas a cabo en el África subsahariana (cf. observaciones de Canut 1996, anteriormente, entre otros), puede contrastar con respuestas registradas en estudios que solicitan la auto-representación del sujeto como hablante de la L2 al participar, por ejemplo, en tareas escolares. En estos casos, la imagen que de sí mismos proyectan los individuos puede resultar menos positiva (cf. Ambadiang et al. 2009). Sugerimos que tal contraste tiene que ver no sólo con la identidad o las auto-representaciones tal como se construyen a partir de un punto de vista ideológico o discursivo, sino que tiene que ver también con la identidad conformada a través de la participación del sujeto en múltiples prácticas comunicativas y lingüísticas (cf. Hastings y Manning, 2004). La discusión que sigue tiene por objeto determinar el grado de coherencia entre las respuestas obtenidas y los datos presentados anteriormente, antes de comentar brevemente las implicaciones de todo ello para la enseñanza-aprendizaje del español L2. 4. Discusión de resultados 4.1. La (ir)relevancia del hablante nativo Los hechos discutidos anteriormente y los datos presentados en la sección anterior son bastante difíciles de explicar en un contexto en el que la enseñanza-aprendizaje de lenguas se concibe exclusivamente con referencia a la instrucción formal y no tiene en cuenta las biografías lingüísticas de los sujetos. Esto se debe, en parte, a que las instituciones educativas asociadas con este tipo de instrucción tienen ciertas representaciones acerca del lenguaje y de los sujetos que aprenden lenguas que son aceptadas, incluso legitimadas, mientras que otras quedan descartadas o excluidas. A través de los datos hemos intentado sugerir que los sujetos tienden a construir de tal manera a sus interlocutores, que la condición de estos, por ejemplo, como ‘hablante nativo’ (HN) puede perder relevancia y especificidad. Esta interpretación tiene muchas consecuencias interesantes. Por un lado, nuestros sujetos, sobre todo los subsaharianos, se perciben a sí mismos y a los hablantes nativos, principalmente, como interlocutores (participantes en un intercambio y no tanto como jueces que evalúan su nivel de español) y, como consecuencia, tienden a nivelar o 34 Doblele \| n.º 4 \| Diciembre 2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas cancelar la diferencia de estatus implícita en términos tales como ‘hablante nativo’ o ‘lengua materna’ (cf. Ambadiang 2010). Las prácticas lingüísticas típicamente asociadas con esta actitud se pueden concebir como "actos de alteridad" en el sentido de que les permiten afirmar su identidad sobre la base de la diferencia o la especificidad (Hastings & Manning, 2004). En términos más generales, los sujetos cuestionan el estatus 'subalterno' impuesto implícitamente al estudiante de la L2, cuando subrayan, por ejemplo, que se contentan con tener una competencia comunicativa que les permita cubrir sus necesidades. Además, dado que suponen que su conocimiento de la L2 que aprenden es parcial e imperfecto, esperan (de hecho, requieren) la colaboración de sus interlocutores, sean hablantes nativos o no, a fin de compensar sus fallos comunicativos. En la medida en que se puede discernir la agentividad en esta actitud, se puede decir que está fuertemente asociada no solo con la alteridad, con la otredad y con la disposición u obligación moral de los interlocutores a cooperar, lo que Abdallah-Pretceille (1999) llama "éthique de l'altérité", sino también con la resistencia y la subversión (Canagarajah, 2004). 4.2. Implicaciones para la enseñanza-aprendizaje de segundas lenguas Hemos visto que la actitud de los subsaharianos difiere parcialmente de la observada con los sujetos de Europa del Este, aunque en su mayor parte los miembros de ambos grupos han asistido a algún tipo de institución educativa (véase tabla I). El grado en el que se puede discernir la agentividad en el discurso de estos sujetos no debe hacernos perder de vista los diferentes contextos en los que sus estrategias comunicativas son implementadas: por ejemplo, el bambara se aprende en un entorno que es característicamente informal, mientras que el español se aprende y se enseña en la escuela secundaria. Por lo tanto, el aspecto más relevante, en este caso, no tiene tanto que ver con la naturaleza formal o informal del proceso de aprendizaje como con el grado en que el aprendizaje informal (o el entorno informal, de manera general) incide en ese proceso. Lo que se quiere decir aquí es que, contrariamente a la conclusión que puede extraerse del análisis de Canagarajah (2004), es decir, que las actitudes subversivas son esperadas de cualquier estudiante de una L2, incluso en contextos educativos formales, la resistencia subsumida en estas actitudes se manifiesta en el mejor de los casos con ciertos grupos de alumnos más que con otros y, en el peor, se evidencia solo con algunos de esos grupos. Además, como sugieren los casos aducidos por el propio Canagarajah, tales actitudes subversivas se correlacionan con la posición característicamente marginal o estigmatizada de estos estudiantes. Las diferencias observadas entre los grupos de sujetos, sugerimos, pueden explicarse sobre la base de tales correlaciones. Así, como estudiantes de lenguas, los africanos tienden a oponer sus autorepresentaciones a las categorías que se les atribuyen, generalmente, desde instancias externas de poder. El resultado es la irrupción de una 35 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang concepción diferente acerca del lenguaje (y de su relación con la cultura), así como del proceso de aprendizaje. De acuerdo con esta concepción, al menos en el caso de nuestros sujetos, una lengua puede separarse de la cultura generalmente asociada a ella, y por tanto aprenderse como un nuevo recurso comunicativo y práctico. Además, si este aspecto tiende a privilegiarse, se espera un uso del lenguaje ligado a la negociación y la colaboración. Finalmente, se supone que las prácticas lingüísticas manifiestan ineludiblemente casos de mezcla de código y cambio de código en contradicción con conceptos tales como la pureza del lenguaje, por ejemplo, que están típicamente asociados al sesgo monolingüe. Los sujetos europeos, por su parte, responden a una representación en la que parece prevalecer la hetero-categorización. Nótese, sin embargo, que esto es aparentemente así porque estos estudiantes asumen una concepción acerca de la lengua asociada al hablante nativo. Este sesgo formal en el proceso de enseñanza-aprendizaje de la lengua tiene que ver particularmente con la tendencia a combinar todos los procesos y contextos de aprendizaje con una instrucción formal del lenguaje. El sesgo formal también tiene el efecto de hacer que las interacciones que típicamente ocurren en el aula sean mucho menos naturales, ya que las relaciones de poder asimétricas subsumidas en ellas tienden a reforzarlo. Una consecuencia interesante es que la L2 tiende a aprenderse (y a enseñarse) como un sistema o conjunto de reglas. Tal interpretación parece ser consistente con el uso que Norton (2008) propone para el concepto de 'inversión'. De acuerdo con Norton (2008: 47), If learners invest in the target language, they do so with the understanding that they will acquire a wider range of symbolic and material resources, which will in turn increase the value of their cultural capital. Aunque Norton reconoce que los procesos de comprensión o producción están mediados tanto por la inversión del estudiante como por su identidad sociocultural, las fuertes implicaciones que supone que la "inversión" se conciba en términos de ‘riqueza simbólica y material’ pueden sugerir una comprensión estática del concepto de ‘inversión’, lo que es incompatible con la actitud de los africanos subsaharianos, por ejemplo. De modo general, no queda claro cómo se produce esa mediación, en parte porque no se discute la interacción entre la inversión fuertemente ligada a los intereses del sujeto- y la relación construida social e históricamente, de los alumnos con la lengua meta, a la que Norton relaciona con la ‘identidad sociocultural’. Como consecuencia, no sabemos de qué manera la inversión de los estudiantes puede variar en relación a su identidad sociocultural y las cambiantes relaciones de poder en las que están involucrados. Los datos discutidos en la sección anterior sugieren que tal correlación puede ser tenida en cuenta a lo largo del proceso de aprendizaje y no solo en un momento dado, como se puede inferir de Norton (2008). Visto de esta manera, la inversión puede concebirse en 36 Doblele \| n.º 4 \| Diciembre 2018 La enseñanza de lenguas no maternas en la era de las migraciones: la importancia de las biografías lingüísticas función de los intereses de los estudiantes, su (auto)representación y la concepción que tengan acerca de la lengua. 5. A modo de conclusión La discusión anterior sugiere que el proceso de aprendizaje de las lenguas puede variar según el contexto, la concepción sobre el lenguaje que el aprendiente posee, así como sus necesidades y objetivos. En este sentido, la posición que adopta cada aprendiente de una L2 puede ser vista como un punto de convergencia de la concepción lingüística típica del grupo al que pertenece y la representación que de sí tiene el sujeto, basada en las experiencias de socialización por las que ha pasado a lo largo de su vida (su biografía sociolingüística). Desde este punto de vista, propuestas basadas en conceptos tales como el de ‘pedagogía centrada en el alumno’, o nociones tales como la de ‘inversión’, no dan cuenta de todo lo que está subsumido en la agentividad de un sujeto que aprende. Ni los maestros, ni los aprendientes de una L2 están exentos de las restricciones asociadas al modo en que se concibe el lenguaje ni al modo en que se clasifican los individuos, lo que influye en gran medida en el proceso de enseñanza y aprendizaje. Por tanto, existe la necesidad de observar las interacciones que se producen entre los hablantes, así como las que se producen entre los profesores y los aprendientes, en toda su complejidad. Esto supone examinar no sólo el proceso de aprendizaje, sino también las rutinas y estrategias utilizadas por los aprendientes de la L2 en la producción de sus mensajes (por ejemplo, si tienden a utilizar expresiones idiomáticas o prefieren sus equivalentes analíticos), y a las que sus interlocutores tienden a recurrir en el proceso de recepción. Como se sugirió anteriormente, la agentividad se manifiesta a lo largo de un continuo entre los dos polos del proceso comunicativo, los interlocutores. Es decir, por un lado, la agentividad se evidencia en los actos más o menos subversivos de la alteridad integrados en las prácticas comunicativas y lingüísticas típicas de los hablantes de lenguas no nativas (mezcla de códigos, cambio de códigos, etc.). Por otro lado, la alteridad, entendida como un imperativo moral (Abdallah-Pretceille, 1999), requiere que los hablantes nativos estén dispuestos a colaborar con los hablantes no nativos. Desde este punto de vista, el empoderamiento de los aprendientes de una L2 en contextos formales implicará que, al menos, las prácticas lingüísticas típicamente asociadas con actos de alteridad sean reconocidas incluso en el contexto de la instrucción formal (cf. Ambadiang 2014, 2017, Ambadiang y García Parejo, 2006; García Parejo 2017). Esta manera de acercarse al proceso de enseñanza-aprendizaje de la L2 tiene muchas ventajas. Además de favorecer la comunicación y la informalidad en la interacción, exige mucha más flexibilidad del sistema de enseñanza, ya que tiene que adaptarse a cada alumno considerado en toda su complejidad. Por otra parte, la correlación entre agentividad y resistencia permite a los aprendientes de la L2 comprar un poco de espacio para el mantenimiento de las otras lenguas que forman parte de su 37 Doblele \| vol.º 4 \| Diciembre 2018 \| 22-40 Isabel García Parejo y Theóphile Ambadiang repertorio y biografía lingüística y, de manera más general, para la preservación de la diversidad lingüística. Desde esta perspectiva, cada clase de lengua, y cada proceso comunicativo que implica hablantes nativos y no nativos, tiene mucho de un diálogo entre diferentes concepciones acerca del lenguaje, los modos de categorización e, incluso, las diferentes lenguas que se hablan, en las que la alteridad y la agentividad son especialmente relevantes. La investigación en didáctica de segundas lenguas puede beneficiarse de la observación de la forma en que las interacciones se desarrollan en los nuevos contextos de comunicación multilingües, ya que estas pueden sugerir estrategias para acomodar los desajustes que existen a propósito de los conocimientos lingüísticos y de las habilidades de acomodación comunicativa entre hablantes nativos y no nativos. Como señala Parekh (2007: 134), al igual que una lengua se puede hablar con diferentes acentos, la cultura debe dar cabida a la pluralidad y permitir a las personas actuar según sus propias maneras diferentes, siempre –se puede añadir– contando con la colaboración del otro en el intercambio comunicativo. BIBLIOGRAFÍA ABDALLAH-PRETCEILLE, Martine (1999), “Éthique de l’altérité”, en Le français dans le monde (January), pp. 28-38. 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TRUDELL, Barbara (2006), “Local agency in the development of minority languages: three language committees in Northwest Cameroon”, en Journal of Multilingual and multicultural development, nº 27, pp. 196210. https://doi.org/10.1080/01434630608668775 40 Doblele \| n.º 4 \| Diciembre 2018
https://openalex.org/W4323980262	http://www.jneb.org/article/S149940462300026X/pdf	English	null	Wealth and Sedentary Time Are Associated With Dietary Patterns Among Preadolescents in Nairobi City, Kenya	Journal of nutrition education and behavior	2,023	cc-by	8,530	ABSTRACT Objective: The study aimed to compare dietary patterns in preadolescents in urban areas with different physical activity and socioeconomic proﬁles in Nairobi, Kenya. Design: Cross sectional Participants: Preadolescents aged 9−14 years (n = 149) living in low- or middle-income areas in Nairobi. Variables Measured: Sociodemographic characteristics were collected using a validated questionnaire. Weight and height were measured. Diet was assessed using a food frequency questionnaire and physical activity by accelerometer. Analysis: Dietary patterns (DP) were formed through principal component analysis. Associations of age, sex, parental education, wealth, body mass index, physical activity, and sedentary time with DPs were ana- lyzed with linear regression. Results: Three DPs explained 36% of the total variance in food consumption: (1) snacks, fast food, and meat; (2) dairy products and plant protein; and (3) vegetables and reﬁned grains. Higher wealth was associ- ated with higher scores of the ﬁrst DP (P < 0.05). Conclusions and Implications: Consumption of foods often deemed unhealthy (eg, snacks and fast food) was more frequent among preadolescents whose families were wealthier. Interventions that seek ways to promote healthy lifestyles among families residing in urban areas of Kenya are warranted. TagedPKey Words: dietary patterns, body mass index, physical activity, sedentary time, preadolescentsTagedEnd (J Nutr Educ Behav. 2023;55:322−330.) Accepted February 6, 2023. Published online March 12, 2023. Accepted February 6, 2023. Published online March 12, 2023. Research Article TagedH1Wealth and Sedentary Time Are Associated With Dietary Patterns Among Preadolescents in Nairobi City, Kenya nerva, PhD1; Lucy Joy Wachira, PhD2; Noora Uusi-Ranta, MS1; TagedPNoora Kanerva, PhD1; Lucy Joy Wachira, PhD2; Noora Uusi-Ranta, MS1; Esther L. Anono, BS3; Hanna M. Walsh1; Maijaliisa Erkkola, PhD1; Sophie Ochola, PhD3; Nils Swindell, PhD4; Jatta Salmela, PhD5; Henna Veps€al€ainen, PhD1; Gareth Stratton, PhD6; Vincent Onywera, PhD2; Mikael Fogelholm, PhD1TagedEnd TagedEnd1Department of Food and Nutrition, University of Helsinki, Helsinki, Finland TagedEnd2Department of Physical Education, Exercise and Sports Science, Kenyatta University, Nai- robi, Kenya TagedEnd3Department of Food, Nutrition and Dietetics, Kenyatta University, Nairobi, Kenya TagedEnd4Department of Sport Sciences, Swansea University, Swansea, United Kingdom TagedEnd5Department of Public Health, University of Helsinki, Helsinki, Finland TagedEnd6Department of Paediatric Exercise Science, Sport and Exercise Sciences, Swansea University, Swansea, United Kingdom TagedEndTagedEndConﬂict of Interest Disclosure: The authors have not stated any conﬂicts of interest. TagedEndAddress for correspondence: Noora Kanerva, PhD, Department of Food and Nutrition, Uni- versity of Helsinki, Agnes Sj€obergin katu 2, Helsinki 00014, Finland; E-mail: noora. kanerva@helsinki.ﬁ 2023 The Authors. Published by Elsevier Inc. on behalf of Society for Nutrition Education and Behavior. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/) https://doi.org/10.1016/j.jneb.2023.02.001 Journal of Nutrition Education and Behavior Volume 55, Number 5, 2023 TagedEnd1Department of Food and Nutrition, University of Helsinki, Helsinki, Finland TagedEnd2Department of Physical Education, Exercise and Sports Science, Kenyatta University, Nai robi, Kenya 3 Journal of Nutrition Education and Behavior Volume 55, Number 5, 2023 TAGEDH1INTRODUCTIONTAGEDEND The ﬁnal number of partici- pating households was 72 for Embakasi and 77 for Langata.TagedEnd changing from traditional staple foods toward reﬁned grains, espe- cially in urban environments7 and in women.8,9 However, unhealthy die- tary habits are still more common among women with high SES. In cross-sectional studies conducted in Nairobi, the diets of children aged 8 −11 years have been generally healthy,6 but the reported consump- tion of fast foods and sweetened bev- erages have been higher than recommended.10 Signs of lifestyle transition can also be seen in physi- cal activity. A comparison of physical activity in rural and urban children showed that rural children were more active and engaged signiﬁ- cantly less in playing screen games.11 The studies above suggest that Kenya has not reached a developmental position in which the lifestyle transi- tion would have reached the low-SES groups. However, the data were mostly collected earlier than 2010; thus, there is a need for more recent evidence on the situation.TagedEnd changing from traditional staple foods toward reﬁned grains, espe- cially in urban environments7 and in women.8,9 However, unhealthy die- tary habits are still more common among women with high SES. In cross-sectional studies conducted in Nairobi, the diets of children aged 8 −11 years have been generally healthy,6 but the reported consump- tion of fast foods and sweetened bev- erages have been higher than recommended.10 Signs of lifestyle transition can also be seen in physi- cal activity. A comparison of physical activity in rural and urban children showed that rural children were more active and engaged signiﬁ- cantly less in playing screen games.11 The studies above suggest that Kenya has not reached a developmental position in which the lifestyle transi- tion would have reached the low-SES groups. However, the data were mostly collected earlier than 2010; thus, there is a need for more recent evidence on the situation.TagedEnd y TagedPEthical clearance was sought and received after a full board review from Kenyatta University’s Ethical Review Committee (Ethical clearance no. PKU/946/I1002), and a research permit was received from the National Commission for Science, Technology and Innovation. Eligible preadolescents and their guardians were given an informed consent form to sign to show a willingness to participate in the study. TAGEDH1INTRODUCTIONTAGEDEND The purpose of the study, the interviews to be done, the voluntary nature of partici- pation, and the right to refuse to par- ticipate in any part of the study were also explained orally. All question- naires were available in both English and Swahili.TagedEnd TagedPThe data were collected using a digital mobile data collection plat- form (version 2021.2.4, ODK Collect, Open Data Kit).13 To determine the sociodemographic characteristics of the participating households, we used parts of a researcher-adminis- tered structured questionnaire previ- ously used and validated in the Kenya Demographic and Health Sur- vey.14 On the questionnaire, multi- ple sociodemographic characteristics were asked, but the questions used in this study are self-reported sex (male or female; for both adolescents and guardians), date of birth (used for cal- culating age in years), and education. The question regarding the guardi- an’s education included originally 9 answer categories which were con- densed into 6 categories as follows (original categories given in paren- thesis): (1) none, (2) not completed primary (adult education, not com- pleted primary), (3) completed pri- mary, (4) not completed secondary, (5) completed secondary, and (6) ter- tiary education (certiﬁcate training, diploma, degree).TagedEnd TagedPThe cross-sectional data was col- lected in 2019 from 2 subcounties in Nairobi: Embakasi Central, which represents a low-SES area and is partly an informal settlement, and Langata, which represents a middle-SES area. These 2 areas were chosen to give a wide variation of SES, with mainly the very wealthy population not included. The inclusion criteria were a household with a 9−14-year-old preadolescent who had lived in either area for at least 6 months before the study and whose care- givers provided consent to partici- pate. The exclusion criteria were the documentation of chronic disease conditions impacting diet or any sig- niﬁcant illnesses preventing partici- pation (eg, tuberculosis).TagedEnd TagedPWe hypothesize that if the life- style transition has moved forward in urban Kenya (ie, Nairobi) during the last 10 years, we would see much smaller (if any) differences between households with higher and lower SES or even an unhealthier lifestyle in the lower SES population in a cross-sectional design. Thus, in this study, we ﬁrst analyzed dietary pat- terns (DPs) among preadolescents aged 9−14 years residing in urban areas with different SES proﬁles in Nairobi. Second, we investigated the association of sociodemographic characteristics and physical activity with these patterns. The design is cross-sectional, with a single time- point assessment. TAGEDH1INTRODUCTIONTAGEDEND The results are used to compare to earlier studies and make indirect hypotheses about the lifestyle transition.TagedEnd TagedPOne of the main outcomes of the study was preadolescents with over- weight/obesity, which was used as the basis for power calculation. Ac- cording to Broyles et al,12 expected difference in child BMI z-score between low- and middle-SES in low- income countries is 0.5. When we use 1.0 as the SD, a of 0.05, and power of 80%, the total 2-group sam- ple size is n = 126 (n = 63 per group). To allow for nonresponse, we aimed to invite 160 households. The ﬁnal study population comprised 149 households (93% of those invited).TagedEnd p g TagedPFurthermore, the questionnaire included questions on living condi- tions and the materials from which the household is constructed, such as drinking water sources, latrine facili- ties, house materials (eg, ﬂoor and wall materials), and ownership of as- sets in the house and other goods (eg, television, radio, bicycle, and car). These items were used to form a wealth index that is a composite measure of a household’s cumulative TAGEDH1INTRODUCTIONTAGEDEND transition affects different popula- tion groups depending on the country’s economic status.2 When a country’s economic status (in terms of Gross National Product) improves, and with increasing urbanization, the unhealthy lifestyle leading to obesity shifts from high to low socio- economic status (SES).TagedEnd traditional diets toward an unhealthy Western dietary pattern that follows economic development and a reduc- tion of occupational, domestic and transportation activity as a result of increased mechanization.1 Lifestyle TagedPThe increase in obesity in lower-mid- dle-income countries (LMICs) is largely thought to be affected by life- style transition: the change from TagedPIn Kenya, the prevalence of over- weight (body mass index [BMI], 25 −29.9) and obesity (BMI, > 30) in adult women was 18% and 8%, respectively, in 2008.3 However, the prevalence of women with obesity has been observed to be much higher in urban settings4 reaching up to 24%.5 Children living in Nairobi were reported to have a prevalence of overweight or obese of 21%, with the majority of the children classiﬁed as overweight/obese being female.6 Die- tary habits of people in Kenya are TagedEndAddress for correspondence: Noora Kanerva, PhD, Department of Food and Nutrition, Uni- versity of Helsinki, Agnes Sj€obergin katu 2, Helsinki 00014, Finland; E-mail: noora. kanerva@helsinki.ﬁ @ 2023 The Authors. Published by Elsevier Inc. on behalf of Society for Nutrition Education and Behavior. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/) https://doi org/10 1016/j jneb 2023 02 001 Journal of Nutrition Education and Behavior Volume 55, Number 5, 2023 322 TagedEndJournal of Nutrition Education and Behavior Volume 55, Number 5, 2023 323 Kanerva et al NCD-related lifestyles and risk factors in Kenya.TagedEnd low SES (partly an informal settle- ment), and Langata was selected to represent the middle SES. Next, 5 vil- lages were selected randomly from Kayole North Central Ward (from Embakasi) and 12 estates from Nai- robi West Ward (from Langata). The ﬁnal stage of sampling involved the enumeration of households with a child aged 9−14 years from the selected villages and estates with the assistance of Community Health Vol- unteers (CHVs). In total, 223 house- holds were enumerated in Embakasi and 173 in Langata. A simple random sampling technique was used to select 80 households from both sub- counties. TAGEDH1METHODSTAGEDEND The details of forming the patterns are given in the section on statistical analysisTagedEnd TagedPPreadolescents’ height was mea- sured with a portable stadiometer with 0.1 cm accuracy and weight with a portable electric scale to 0.1 kg accuracy in a standardized way. Age- and sex-adjusted z-scores for BMI were calculated using World Health Organization’s 2007 growth refer- ence data and World Health Organ- ization’s macro package for R- software.16TagedEnd TagedPThe dietary intake data were col- lected using a 7-day food frequency questionnaire (FFQ), modiﬁed from a previously validated FFQ for adults in Nairobi.17 The FFQ consisted of cul- tural and urban-speciﬁc foods and foods commonly consumed on the basis of a market survey and pretest of the FFQ in the study area. We added locally available foods that are more commonly used among preado- lescents (such as noodles, pancakes, scones and other sweet bakery prod- ucts, colored ice, pizza, etc) and re- placed the adult portion sizes with portion sizes suitable for preadoles- cents. In the FFQ, the participant was asked to indicate for each food item how many times on average they consumed it over the last 7 days. The preadolescents answered the ques- tions themselves, but the caregivers were allowed to help when needed. The FFQ included 174 food items. From the following analyses, we excluded food items used by none (n = 27) or only 1 participant (n = 10), as well as items we considered unreli- ably reported (n = 3) (Supplementary Table 1). Hence, we included 135 food items, which were divided into 19 groups (Supplementary Table 2): reﬁned grains, whole grains, por- ridges, side dishes (eg, rice, tradi- tional maize porridge called ugali), roots and tubers (eg, cassava, arrow- root and plantain), vegetables (eg, spinach, kales and traditional vegeta- bles), beans, nuts and seeds, fruits, dairy products, meat and eggs, ﬁsh and seafood, oils and margarine, spreads and sauces, sweets, savory snacks, sodas and juices, and fast y TagedPPhysical activity was measured by an accelerometer (GT3X+, ActiGraph). Accelerometer measure- ments have been previously vali- dated among Kenyan children.18 At least 4 valid days (≥10 h/d of waking wear time), including 1 weekend day, was required for the participant’s data to be included in the accelerom- eter analysis (104 participants ful- ﬁlled this requirement). TAGEDH1METHODSTAGEDEND TagedPThe Kenya-Finland Education and Research Alliance aims to improve education and teaching capacity on noncommunicable diseases (NCDs)- related health behaviors at Kenyatta University, Nairobi. The research part of this project consisted of an in- depth study that aimed at collecting novel and important new data on TagedPA multistage sampling technique was conducted to identify the house- holds in which data would be col- lected. In the ﬁrst stage, Embakasi was purposively selected to represent Journal of Nutrition Education and Behavior Volume 55, Number 5, 2023 TagedEnd324 Kanerva et al living standard. It has been widely used in large household surveys con- ducted by the Demographic and Health Surveys14 and World Food Programme Surveys.15 The calcula- tion of the index is explained in the section on statistical analyses.TagedEnd food (eg, samosa, hot dog, kebab). In addition, coffee and tea were included in the analysis as a group of their own.TagedEnd mean and SD (continuous variables) or frequencies and percentages (cate- gorical variables).TagedEnd g ) TagedPOur study used principal compo- nent analysis (PCA) to create the wealth index and form the DPs. Prin- cipal component analysis is a method for reducing dimensions in data (ie, compress the information of multiple variables into fewer varia- bles). It typically includes several iter- ation rounds that could be described in the following 3 steps: (1) an unre- stricted number of components and inspection of the scree plot and Ei- genvalues and the Kaiser-Meyer-Ol- kin test value, (2) restriction of the number of components, and (3) describing the factors by rotated fac- tor loadings. Rotation turns the ini- tial factors into ones that are easier to interpret. Varimax rotation is one of the most used ones. It maximizes the sum of the variance of the squared loadings resulting in a small number of important variables with high fac- tor loadings.TagedEnd TagedPIn addition to single food groups, we made a whole-diet dietary pattern analysis. The dietary pattern analysis simpliﬁes data and reduces the num- ber of analyses needed and, there- fore, helps to control for multiple statistical testing (3 patterns vs 19 food groups). The dietary pattern approach gives additional insight into the diet and helps to identify a bigger picture. Moreover, we com- pared our results to the most recent larger study, including adolescent participants in Kenya (International Study of Childhood Obesity, Lifestyle and the Environment,) which used dietary pattern analysis. TAGEDH1METHODSTAGEDEND The data was downloaded onto a computer using an analysis software package (version 6.13, ActiGraph), reviewed for com- pleteness and analyzed using Kine- soft. Using a validated algorithm,19 nocturnal sleep episodes were removed from the data and nonwear periods were deﬁned as any sequence of at least 20 consecutive minutes of zero activity counts. Evenson cutpoints20 were used to deﬁne time spent in sedentary behavior, light, moderate, and vigorous physical activity.TagedEnd TagedPWe created the household wealth index following the World Food Pro- gram’s guidelines15 using PCA (SPSS command FACTOR, Method: Princi- pal components, Varimax rotation). The ﬁnal wealth index had a Kaiser- Mayer-Olkin value of 0.87, and it ex- plained 40% of the total variation and included: sanitation, ﬂoor mate- rial, television, refrigerator, chair, cupboard, wall clock, microwave, DVD player, electric or gas stove, ker- osene stove, bicycle and car or truck. We grouped the wealth index in quintiles for the analyses. The associ- ation between wealth index quintiles and each food group was tested with linear regression. P < 0.05 were con- sidered statistically signiﬁcant, and Bonferroni corrected a for the linear trend test was 0.05/17 = 0.003.TagedEnd TagedPPrincipal component analysis is one of the main empirical methods for compressing the food consump- tion information into pattern scores, which tells how much each partici- pant adheres to the derived DPs. In other words, each participant has a diet that includes stronger or weaker associations with the derived DPs. Initially, we used the 19 food groups (consumption frequencies) in our analysis, which resulted in 3 patterns having at least 5-factor loadings > 0.3 TagedH2Statistical AnalysisTagedEnd TagedPAll analyses were conducted using SPSS (version 25, IBM, 2017) and R statistical software (version 3.6.3, R Foundation for Statistical Computing Platform, 2020). Participants’ socio- demographic characteristics, BMI, moderate-to-vigorous physical activ- ity (MVPA) and sedentary time, and consumption frequencies of food groups have been described with TagedEndJournal of Nutrition Education and Behavior Volume 55, Number 5, 2023 325 Kanerva et al wealth index in 9 food groups out of 19 (Table 2). After the Bonferroni cor- rection, the trend remained statisti- cally signiﬁcant for 6 food groups (P < 0.003). All of these indicated higher consumption frequency in the higher wealth index quintiles. The largest proportional difference was seen for sodas and juices (includ- ing both sweetened and unsweet- ened) consumed 8 times more often among adolescents in the highest wealth index quintile compared with the lowest.TagedEnd and reaching the Kaiser-Meyer-Olkin test value of 0.66. The percentage of the total variance in explaining food consumption was 36%. We calcu- lated standardized principal compo- nent scores for these 3 patterns and each of the preadolescents by assign- ing weights to their frequency of use of each food. A higher score indicates a stronger adherence to the empiri- cally derived dietary pattern.TagedEnd and reﬁned grains pattern, was less diverse than the other 2 and charac- terized by the intake of rather cheap food items like vegetables, reﬁned grains, oils, and tea, explaining 8.5% of the variance in food con- sumption.TagedEnd TagedPIn the univariate linear regression analysis model for the snacks, fast food, and meat pattern and the dairy and plant protein pattern, parental education and wealth index were positively associated with higher scores (Table 4). In univariate mod- els, none of the examined factors were associated with the vegetables and reﬁned grains pattern scores. In the multivariate analyses, higher wealth and lower sedentary time were associated with higher snacks, fast food and meat pattern scores. After Bonferroni correction, only the association between the wealth index and snacks, fast food and meat pat- tern remained statistically signiﬁcant (P < 0.006).TagedEnd TagedPWe used linear regression to examine the association between so- ciodemographic (age, sex, parental education, wealth index, BMI z- score) and lifestyle variables (MVPA and sedentary time) with the dietary pattern scores, and P < 0.05 were considered statistically signiﬁcant. TAGEDH1RESULTSTAGEDEND TagedPTable 1 shows that participants in Langata had higher education and wealth than Embakasi Central. We found a statistically signiﬁcant trend in the consumption frequency by the TagedH2Statistical AnalysisTagedEnd Bonferroni corrected a for the associ- ation between sociodemographic and lifestyle variables with dietary pattern scores was 0.05/8 = 0.006.TagedEnd TagedPIn the PCA, 3 DPs were found (Table 3). The ﬁrst pattern was char- acterized by food groups often deemed unhealthy, such as snacks, fast food, sodas and juices, and sweets and was thus named the snacks, fast food, and meat pattern. This pattern explained 19% of the variance in food consumption. The second pattern included food groups often considered healthy, such as beans and nuts, dairy, roots and tubers, ﬁsh and seafood, whole- grain, and fruits and was named the dairy and plant protein pattern; this explained 8.8% of the variance. The third pattern, named the vegetables TAGEDH1DISCUSSIONTAGEDEND TagedPConsumption of reﬁned cereals, dairy, fruits, meat, spreads, and Table 1. Characteristics of Preadolescents Residing in Embakasi Central and Langata, in Nairobi City, Kenya ents Residing in Embakasi Central and Langata, in Nairobi City, Kenya acteristics of Preadolescents Residing in Embakasi Central and Langata, in Nairobi City, Keny Table 1. Characteristics of Preadolescents Residing in Embakasi Central and Langata, in Na Table 1. Characteristics of Preadolescents Residing in Embakasi Central and Langata, in Nairobi City, Kenya Characteristics All (n = 149) Embakasi Central (n = 72)a Langata (n = 77)b Pc Mean age, y 11.1 § 1.50 11.2 § 1.15 11.2 § 1.15 0.99 Girls 78 (52) 39 (54.1) 39 (50.1) 0.79 Parental education < 0.001 No formal education/incomplete primary school 20 (13) 16 (22) 4 (5) Primary school 31 (21) 26 (36) 5 (7) Secondary school 48 (32) 26 (36) 22 (29) Certiﬁcate training or adult education 33 (22) 4 (6) 29 (38) Tertiary education 17 (11) 0 (0) 17 (22) Wealth index ﬁfths, < 0.001 First (lowest) 30 (20) 30 (42) 0 (0) Second 28 (19) 25 (35) 3 (4) Third 31 (21) 14 (19) 17 (22) Fourth 30 (20) 3 (4) 27 (35) Fifth (highest) 30 (20) 0 (0) 30 (39) Mean BMI z-score 0.08 § 1.49 0.43 § 1.25 0.24 § 1.64 0.007 Mean time spent on MVPA, min/dd 71.3 § 27.7 79.7 § 30.3 61.2 § 20.1 < 0.001 Mean time spent on SED, min/dd 607.7 § 78.5 601.0 § 76.1 615.9 § 81.3 0.34 BMI indicates body mass index; MVPA, moderate-to-vigorous physical activity; SED, sedentary behavior. aEmbakasi Central = low socioeconomic status (SES) area in Nairobi City County; bLangata = mid-SES area in Nairobi City County; cDifference between areas was tested with analysis of variance for continuous variables and with a chi-square test for categorical variables, P < 0.05 were considered statistically signiﬁcant; dAdolescents who wore the accelerometer for at least 4 valid days (≥10 h/d of waking wear time), including 1 weekend day (n = 104): Embakasi Central (n = 57) and Langata (n = 47). Note: Values are presented as mean § SD or n (%). Characteristics BMI indicates body mass index; MVPA, moderate-to-vigorous physical activity; SED, sedentary beha b aEmbakasi Central = low socioeconomic status (SES) area in Nairobi City County; bLangata = mid-SES area in Nairobi City County; cDifference between areas was tested with analysis of variance for continuous variables and with a chi-square test for categorical variables, P < 0.05 were considered statistically signiﬁcant; dAdolescents who wore the accelerometer for at least 4 valid days (≥10 h/d of waking wear time), including 1 weekend day (n = 104): Embakasi Central (n = 57) and Langata (n = 47). Note: Values are presented as mean § SD or n (%). Journal of Nutrition Education and Behavior Volume 55, Number 5, 2023 TagedEnd326 Kanerva et al TagedEnd326 TagedEnd Table 2. Characteristics Frequency of Food Consumption (Times/wk) by Wealth Index Quintiles Among Preadolescents Residing in Nairobi City, Kenya (n = 149) Wealth Index Quintiles Food Groups Lowest (n = 30) Second (n = 28) Third (n = 31) Fourth (n = 30) Highest (n = 30) Pa,b White cereals 4.67 § 2.52 2.96 § 3.12 4.61 § 3.95 5.83 § 2.79 8.17 § 5.24 < 0.001c Whole grains 0.67 § 1.99 0.46 § 1.40 1.52 § 1.84 1.57 § 3.11 1.93 § 2.18 0.003 Porridges 1.13 § 2.01 1.93 § 2.58 1.61 § 2.40 1.10 § 1.94 0.83 § 2.67 0.29 Pasta and rice 7.73 § 5.51 6.54 § 4.66 7.74 § 3.78 7.67 § 3.45 7.67 § 3.91 0.71 Roots 0.80 § 1.75 1.25 § 2.15 1.06 § 1.31 1.50 § 1.50 0.87 § 1.50 0.63 Beans and nuts 3.97 § 3.62 3.89 § 2.88 4.90 § 3.54 4.67 § 2.60 4.73 § 3.34 0.20 Vegetables 14.00 § 11.50 11.57 § 11.30 14.00 § 15.83 9.30 § 8.02 15.23 § 13.98 0.92 Fruits 4.17 § 5.93 4.50 § 4.22 5.23 § 4.60 8.67 § 7.18 8.73 § 6.98 < 0.001c Dairy 1.07 § 1.70 2.25 § 2.80 2.03 § 2.81 3.60 § 4.45 4.63 § 3.51 < 0.001c Meat 2.97 § 2.71 2.46 § 2.96 3.61 § 2.79 5.47 § 3.49 6.53 § 2.21 < 0.001c Fish 1.13 § 1.28 1.11 § 1.29 0.87 § 0.96 0.83 § 1.29 1.10 § 2.14 0.63 Oils 10.87 § 6.61 9.39 § 6.30 11.71 § 6.92 11.07 § 6.96 13.63 § 7.54 0.07 Spreads 0.93 § 1.70 0.86 § 2.07 2.81 § 3.53 2.27 § 2.63 4.60 § 3.36 < 0.001c Sweets 7.00 § 4.80 6.89 § 5.44 5.71 § 3.69 6.17 § 6.90 5.33 § 4.11 0.18 Snacks 1.07 § 2.38 0.79 § 1.64 1.16 § 2.05 2.53 § 3.84 2.40 § 3.31 0.004 Sugar-sweetened beveragesd 0.37 § 0.76 0.64 § 1.45 1.39 § 1.93 3.47 § 4.26 3.23 § 3.45 < 0.001c Fast foods 1.80 § 2.92 2.04 § 3.10 1.45 § 1.84 2.43 § 2.49 3.90 § 3.10 0.004 Tea 8.37 § 3.96 6.18 § 4.18 8.06 § 3.08 8.57 § 4.61 7.83 § 4.64 0.65 Coffee < 0.01 § < 0.01 0.07 § 0.38 < 0.01 § < 0.01 < 0.01 § < 0.01 0.47 § 1.78 0.08 aTrend between wealth index quintiles and consumption of food groups tested with linear regression; bLinear regression analy- sis for trend was adjusted for participant’s age and sex; cSigniﬁcant after Bonferroni correction (P < 0.003); dBoth sweetened and unsweetened beverages are included. Characteristics S TagedEnd Table 2. Frequency of Food Consumption (Times/wk) by Wealth Index Quintiles Among Preadolescents Residing in Nairobi City, Kenya (n = 149) aTrend between wealth index quintiles and consumption of food groups tested with linear regression; bLinear regression analy- sis for trend was adjusted for participant’s age and sex; cSigniﬁcant after Bonferroni correction (P < 0.003); dBoth sweetened and unsweetened beverages are included. g Note: Values are presented as mean § SD. TagedEnd Table 3. Factor Loadings for Food Groups by Dietary Patterns Identiﬁed Through Principal Component Analysis ings for Food Groups by Dietary Patterns Identiﬁed Through Principal Component Analysis Dietary Patterns TagedEnd Table 3. Factor Loadings for Food Groups by Dietary Patterns Identiﬁed Through Principal Component Analysis Dietary Patterns Food Group Snacks, Fast Food, and Meat Dairy and Plant Protein Vegetables and Reﬁned Grains Savory snacks 0.738a 0.039 0.107 Fast food 0.725a 0.094 0.209 Meat and eggs 0.657a 0.317a 0.236 Sodas and juices 0.644a 0.099 0.018 Spreads and sauces 0.525a 0.344a 0.118 Sweets 0.510a 0.071 0.085 Coffee 0.330a 0.051 0.258 Dairy products 0.231 0.649a 0.256 Beans and nuts 0.048 0.593a 0.199 Roots and tubers 0.133 0.572a 0.084 Whole grains 0.068 0.461a 0.003 Side dishes 0.018 0.430a 0.104 Fish and seafood 0.080 0.416a 0.363a Fruits 0.401a 0.408a 0.165 Vegetables 0.036 0.051 0.664a Tea 0.176 0.032 0.542a Reﬁned grains 0.437a 0.075 0.500a Oils and margarine 0.102 0.309a 0.424a Porridges 0.110 0.005 0.176 aFactor loadings > 0.3. Table 3. Factor Loadings for Food Groups by Dietary Patterns Identiﬁed Through Principal Component Analysis Dietary Patterns ournal of Nutrition Education and Behavior Volume 55, Number 5, 2023 Kanerva et al 327 TagedEnd Table 4. Association Between Sociodemographic Characteristics and Lifestyle With Dietary Pattern Scores Table 4. Association Between Sociodemographic Characteristics and Lifestyle With Dietary Pattern Scores een Sociodemographic Characteristics and Lifestyle With Dietary Pattern Scores ble 4. Association Between Sociodemographic Characteristics and Lifestyle With Dietary Pa Table 4. Characteristics To get more insight into the underlying die- tary habits, we used PCA to reduce the number of dietary variables by creating linear combinations of varia- bles with strong correlations. Three DPs were identiﬁed, and they ex- plained 36% of the total variance in preadolescents’ food consumption: (1) a pattern high in foods regarded as unhealthy, such as snacks, fast food, sodas and juices, and sweets (snacks, fast food, and meat), (2) a pattern high in foods regarded as healthy, such as beans and nuts, dairy, roots and tubers, ﬁsh and sea- food, wholegrain, and fruits (dairy and plant protein), and (3) a pattern high in rather cheap foods com- monly used in urban areas such as vegetables, reﬁned grains (products made from reﬁned wheat, maize, etc), oils, and tea (vegetables and reﬁned grains). We found higher wealth associated with higher snacks, fast foods, and meat pattern scores.TagedEnd with higher scores of both the snacks, fast food, and meat pattern and the dairy and plant protein pattern in univariate models, these associations disappeared after controlling for wealth index, indicating that wealth probably explains most of the associ- ation between parental education and diet. It should also be noted that the above-mentioned unadjusted correlations were found for both unhealthy and healthy patterns. Thus, the ﬁndings suggest that the association between education, wealth and diet was more quantita- tive (participants with higher educa- tion consume all foods more frequently) than health-related (par- ticipants with higher education would consume only healthy foods more frequently). In middle-SES fam- ilies, income level allows spending money on beverages, dairy products, nuts and seeds, and meat. Compared with families with the lowest wealth index quintile, the average consump- tion frequency of all these food groups and variance in consumption was much higher among preadoles- cents in the highest wealth index Lifestyle and the Environment research project identiﬁed 2 DPs in children aged 9−11 years living in Nairobi: an unhealthy and healthy diet pattern.21 These patterns resembled the snacks, fast food, and meat pattern and dairy and plant protein pattern found in this study, although there were some differ- ences; for example, dairy product and ﬁsh consumption were lower among our participants. Characteristics Association Between Sociodemographic Characteristics and Lifestyle With Dietary Pattern Scores Snacks, Fast Food, and Meat Dairy and Plant Protein Vegetables and Reﬁned Grains Characteristics ß 95% CI Pa ß 95% CI Pa ß 95% CI Pa Univariate model Age, y 0.046 0.15 to 0.06 0.39 0.043 0.15 to 0.06 0.42 0.015 0.12 to 0.09 0.77 Sex (reference female) 0.132 0.45 to 0.19 0.42 0.243 0.56 to 0.08 0.14 0.094 0.42 to 0.23 0.57 Parental education 0.179 0.05−0.31 0.009 0.151 0.02−0.28 0.027 0.023 0.11 to 0.16 0.74 Wealth index 0.398 0.25−0.54 < 0.001b 0.207 0.05−0.36 0.011 0.039 0.12 to 0.20 0.64 BMI z-score 0.065 0.05 to 0.13 0.25 0.014 0.11 to 0.08 0.78 0.02 0.09 to 0.13 0.72 MVPA, min/dc 0.006 0.01 to < 0.01 0.05 0.004 0.01 to < 0.01 0.29 0.004 < 0.01 to 0.01 0.26 SED, min/dc 0.001 < 0.01 to < 0.01 0.37 <0.001 < 0.01 to < 0.01 0.72 0.002 < 0.01 to < 0.01 0.08 Multivariate modelc,d Age, y 0.015 0.13 to 0.10 0.80 0.058 0.19 to 0.08 0.40 0.012 0.12 to 0.15 0.87 Sex (reference female) 0.207 0.53 to 0.12 0.22 0.252 0.64 to 0.13 0.20 0.250 0.65 to 0.15 0.22 Parental education 0.115 0.28 to 0.05 0.18 0.112 0.08 to 0.31 0.27 0.006 0.19 to 0.21 0.95 Wealth index 0.392 0.20−0.58 < 0.001b 0.056 0.17 to 0.28 0.64 0.087 0.15 to 0.32 0.47 BMI z-score 0.018 0.10 to 0.14 0.76 0.013 0.13 to 0.15 0.85 0.039 0.10 to 0.18 0.60 MVPA, min/d 0.006 0.01 to < 0.01 0.14 0.002 0.01 to 0.01 0.70 0.003 0.01 to 0.01 0.50 SED, min/d 0.003 0.01 to < 0.01 0.04 0.001 < 0.01 to < 0.01 0.43 0.002 < 0.01 to < 0.01 0.19 BMI indicates body mass index; CI, conﬁdence interval; MVPA, moderate-to-vigorous physical activity; SED, sedentary b h i aAssociation between sociodemographic and lifestyle variables was analyzed with linear regression; bStatistically signiﬁcant after Bonferroni correction (P < 0.006); cSample size in the analysis was n = 104 because of adolescents who wore an acceler- ometer at least 4 valid days (≥10 h/d of waking wear time), including 1 weekend day. Otherwise, the sample size in the analysis was n = 149; dAll variables simultaneously in the model. sugar-sweetened beverages was more frequent among preadolescents whose families were wealthier. behavior. aAssociation between sociodemographic and lifestyle variables was analyzed with linear regression; bStatistically signiﬁcant after Bonferroni correction (P < 0.006); cSample size in the analysis was n = 104 because of adolescents who wore an acceler- ometer at least 4 valid days (≥10 h/d of waking wear time), including 1 weekend day. Otherwise, the sample size in the analysis was n = 149; dAll variables simultaneously in the model. Characteristics In summary, despite a marked economic development in Kenya, our results support the evi- dence that Nairobi, representing an urban area of a new LMIC, would still be in a situation in which higher wealth would be associated with un- healthier DPs.2,26TagedEnd warranted to investigate what works for promoting a healthy lifestyle in the context of low- and middle- income countries.33−35 Furthermore, whether interventions promoting healthy lifestyles reduce social in- equalities in health in low- and mid- dle-income countries remains uncertain.36,37 As these countries are still in an early phase of their lifestyle transition, the expansion of obesity to epidemic proportions may be pre- vented if the correct actions are known and taken shortly.TagedEnd TagedPAlthough we acknowledge limita- tions, there were also strengths to this work. For example, the selec- tion of 2 study areas that differed by the wealth index provided novel in- sights into the understanding of lifestyle behaviors of boys and girls in low- and middle-SES families in an urban setting in a lower middle- income economy. The background questionnaire used for assessing par- ticipants’ sociodemographic back- ground has been validated in the Kenyan context, and we followed standardized methods for construct- ing the wealth index. The FFQ used was extensive and thus likely to cap- ture the true food consumption pat- terns. In addition, FFQ makes it possible to assess the habitual intake of foods, which reduces the day-to- day variation in data.32 For physical activity data, device-based measure- ment is not prone to self-report bias and allows the detection of different intensities and sedentary time. As research on the association between sociodemographic and lifestyle factors and diet is lacking in Africa and LMICs, this study provided ini- tial indicators of the associations between wealth status, geographic location, and lifestyle behaviors in Kenyan adolescents.TagedEnd TagedPModerate-to-vigorous physical acti- vity was not associated with any DPs, but surprisingly, lower sedentary time was associated with a higher snack, fast food, and meat pattern. However, correction for multiple testing indi- cated that this association must be considered cautiously. Our results do not indicate that healthy thinking would simultaneously drive food con- sumption and physical activity pat- terns. Characteristics Similar unhealthy and healthy DPs have been identiﬁed in other LMIC settings.22 As a part of the lifestyle transition, the availability of snacks and fast foods had increased dramatically since 1990 when super- market growth started in Kenya.23 In addition to high availability, unhealthy foods, such as sugar-sweet- ened beverages, are among the most advertised foods.24 This was reﬂected in our data as the snacks, fast food, and meat pattern explained most of the variance in food consumption fre- quency.TagedEnd sugar-sweetened beverages was more frequent among preadolescents whose families were wealthier. To get more insight into the underlying die- tary habits, we used PCA to reduce the number of dietary variables by creating linear combinations of varia- bles with strong correlations. Three DPs were identiﬁed, and they ex- plained 36% of the total variance in preadolescents’ food consumption: (1) a pattern high in foods regarded as unhealthy, such as snacks, fast food, sodas and juices, and sweets (snacks, fast food, and meat), (2) a pattern high in foods regarded as healthy, such as beans and nuts, dairy, roots and tubers, ﬁsh and sea- food, wholegrain, and fruits (dairy and plant protein), and (3) a pattern high in rather cheap foods com- monly used in urban areas such as vegetables, reﬁned grains (products made from reﬁned wheat, maize, etc), oils, and tea (vegetables and reﬁned grains). We found higher wealth associated with higher snacks, fast foods, and meat pattern scores.TagedEnd TagedPIn general, higher parental educa- tion has been associated with healthy DPs in children in many countries.25 Although we observed that higher parental education was associated TagedPPreviously, the international Inter- national Study of Childhood Obesity, Journal of Nutrition Education and Behavior Volume 55, Number 5, 2023 TagedEnd328 Kanerva et al into the validity of the FFQ and prompt greater adherence to wear time for accelerometers.TagedEnd quintile. Characteristics Along with CHVs, Kenya provides a great opportunity to use mHealth: mobile phone coverage has increased rapidly and is currently > 100% per 100 in- habitants and 82% in Africa.42 Mobile Health has been widely ex- ploited in Kenya as the country has one of the highest reported mHealth projects globally.40TagedEnd y TagedPThis study includes limitations. As already mentioned, the sample size was limited (n = 149). Analyses, including accelerometer data, suf- fered more than the other analyses (multivariate analysis in Table 4; n = 104) because of the high preva- lence of unacceptable data collection and missing data. The main reason for the limited sample size is that the whole project includes an in-depth analysis of the participants, with numerous analyses and question- naires. We aimed to get a deep under- standing of a speciﬁc population, not a superﬁcial overview of a larger group.TagedEnd Mobile Health interventions are rap- idly gaining popularity for their potential to improve public health, and many low and LMICs see them as an important resource for frontline health workers. Most CHV and mHealth programs in developing countries have focused on reproduc- tive health (including family plan- ning), maternal child health (promoting breastfeeding), and infec- tious and vectorborne diseases.40,41 Characteristics Previously, unhealthy diets and high screen time were clustered in Nai- robian preadolescents.27 Studies con- ducted in high-income countries and some in Latin America have observed greater time spent on MVPA to be associated with lower adherence to unhealthy diet patterns.28,29 Similarly, many studies have also found greater time spent on MVPA associated with healthy diets.30,31TagedEnd TagedPA useful approach for health pro- motion through social support in an LMIC setting is the use of peer coun- selors or lay health educators, also known as CHVs.38 The CHVs are an established network of community members to whom other community members turn for care, advice, infor- mation, and support. In Kenya, the naturally occurring social network of CHVs is indigenous to the commu- nity and offers culturally relevant and effective social support. CHVs belong to community health units responsible for making weekly home visits to households within desig- nated geographical areas.38 The Mobile Health (mHealth) approach has the potential to bridge systemic gaps needed to improve access to and use of health services, particularly among underserved populations.39,40 TagedPA useful approach for health pro- motion through social support in an LMIC setting is the use of peer coun- selors or lay health educators, also known as CHVs.38 The CHVs are an established network of community members to whom other community members turn for care, advice, infor- mation, and support. In Kenya, the naturally occurring social network of CHVs is indigenous to the commu- nity and offers culturally relevant and effective social support. CHVs belong to community health units responsible for making weekly home visits to households within desig- nated geographical areas.38 The Mobile Health (mHealth) approach has the potential to bridge systemic gaps needed to improve access to and use of health services, particularly among underserved populations.39,40 Mobile Health interventions are rap- idly gaining popularity for their potential to improve public health, and many low and LMICs see them as an important resource for frontline health workers. Most CHV and mHealth programs in developing countries have focused on reproduc- tive health (including family plan- ning), maternal child health (promoting breastfeeding), and infec- tious and vectorborne diseases.40,41 However, the potential of using CHVs and mHealth to facilitate change in lifestyle behaviors in this context could be examined. TAGEDH1ACKNOWLEDGMENTSTAGEDEND 2014. Kenya National Bureau of Sta- tistics; 2015. http://publications.uni- versalhealth2030.org/uploads/kenya_- demographic_and_health_sur- vey_2014.pdf. Accessed October 13, 2022.TagedEnd 2014. Kenya National Bureau of Sta- tistics; 2015. http://publications.uni- versalhealth2030.org/uploads/kenya_- demographic_and_health_sur- vey_2014.pdf. Accessed October 13, 2022.TagedEnd settings in Nairobi, Kenya. PLoS One. 2015;10:e0129943.TagedEnd TagedPThe Kenya-Finland Education and Research Alliance project is funded by the Finnish Ministry of Foreign Af- fairs through the Higher Education Institutions Institutional Coopera- tion Instrument (Higher Education Institutions Institutional Coopera- tion Instrument; grant no. HEL7M0453-82). The authors would like to thank Elisa S€arkilahti (Univer- sity of Helsinki), who did her mas- ter’s thesis on the DPs within this project. The authors also want to thank Victor Okoth Odhiambo (Ken- yatta University), the Kenya-Finland Education and Research Alliance project coordinator, who gave a lot of general support during the study to the students from Kenyatta Uni- versity who participated in data col- lection. Our warmest thanks also go to the health ofﬁcers and CHVs who helped collect data. Finally, the au- thors want to thank the families who took part in our study and the com- munities of Langata and Embakasi Central for their acceptance and con- tribution.TagedEnd 5. Christensen DL, Eis J, Hansen AW, et al. Obesity and regional fat distribu- tion in Kenyan populations: impact of ethnicity and urbanization. Ann Hum Biol. 2008;35:232–249.TagedEnd vey_2014.pdf. Accessed October 13, 2022.TagedEnd TagedP15. Hjelm L, Miller D, Wadhwa A. VAM Guidance Paper. Creation of Wealth Index. World Food Programme; 2017. https://docs.wfp.org/api/documents/ WFP-0000022418/download/. Ac- cessed October 13, 2022.TagedEnd 6. Wachira LJ, Muthuri S, Ochola S, Onywera V, Tremblay M. Association between dietary behaviours and weight status of school children: results from the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE) - Kenya. Child and Adolescent Obesity. 2021;4:1–22.TagedEnd TagedP16. de Onis M, Onyango AW, Borghi E, Siyam A, Nishida C, Siekmann J. Development of a WHO growth refer- ence for school-aged children and ado- lescents. Bull World Health Organ. 2007;85:660–667.TagedEnd 7. Oniang’o RK, Mutuku JM, Malaba SJ. Contemporary African food habits and their nutritional and health implica- tions. Asia Pac J Clin Nutr. 2003;12:331–336.TagedEnd TagedP17. Vila-Real C, Pimenta-Martins A, Magu JS, et al. A culture-sensitive semi-quantitative FFQ for use among the adult population in Nairobi, Kenya: development, validity and reproducibil- ity. Public Health Nutr. 2021;24:834– 844.TagedEnd 8. Steyn NP, Nel JH, Parker WA, Ayah R, Mbithe D. Dietary, social, and environ- mental determinants of obesity in Ken- yan women. Scand J Public Health. TAGEDH1SUPPLEMENTARY DATATAGEDEND TagedPSupplementary data related to this article can be found at https://doi. org/10.1016/j.jneb.2023.02.001.TagedEnd TagedP11. Onywera VO, Adamo KB, Sheel AW, Waudo JN, Boit MK, Tremblay M. Emerging evidence of the physical activity transition in Kenya. J Phys Act Health. 2012;9:554–562.TagedEnd TAGEDH1ACKNOWLEDGMENTSTAGEDEND 2011;39:88–97.TagedEnd TagedP18. Barreira TV, Schuna JM, Tudor-Locke C, et al. Reliability of accelerometer- determined physical activity and seden- tary behavior in school-aged children: a 12-country study. Int J Obes Suppl. 2015;5(suppl 2):S29–S35.TagedEnd 9. Mbochi RW, Kuria E, Kimiywe J, Ochola S, Steyn NP. Predictors of over- weight and obesity in adult women in Nairobi Province, Kenya. BMC Public Health. 2012;12:823.TagedEnd TagedP10. Kigaru DMD, Loechl C, Moleah T, Macharia-Mutie CW, Ndungu ZW. Nutrition knowledge, attitude and practices among urban primary school children in Nairobi City, Kenya: a KAP study. BMC Nutr. 2015;1:44.TagedEnd TagedP19. Tudor-Locke C, Barreira TV, Schuna JM, Mire EF, Katzmarzyk PT. Fully automated waist-worn accelerometer algorithm for detecting children’s sleep-period time separate from 24-h physical activity or sedentary behaviors. Appl Physiol Nutr Metab. 2014;39:53– 57.TagedEnd TAGEDH1IMPLICATIONS FOR RESEARCH AND PRACTICETAGEDEND TagedPWealth showed the strongest asso- ciation with a dietary pattern char- acterized by food groups often deemed unhealthy, such as sodas, juices, and sweets. Based on our observation, the dietary habits of preadolescents living in the urban environment of Nairobi, Kenya— which was upgraded as an LMIC < 10 years ago—do not yet resemble high-income countries in which preadolescents from less wealthy families would have unhealthier di- ets compared with preadolescents from wealthier families.TagedEnd g p TagedPThe cross-sectional design of the study does not allow inferences of causality, and without proper previ- ous reference data, no valid assess- ment of change can be made. We conducted multiple exploratory anal- yses using various variables, and thus, the results found may, to some degree, be attributed to chance. How- ever, correction for multiple testing did not change the interpretation of the results remarkably. Furthermore, methodological reﬁnement would help provide more detailed insight However, the potential of using CHVs and mHealth to facilitate change in lifestyle behaviors in this context could be examined. Along with CHVs, Kenya provides a great opportunity to use mHealth: mobile phone coverage has increased rapidly and is currently > 100% per 100 in- habitants and 82% in Africa.42 Mobile Health has been widely ex- ploited in Kenya as the country has one of the highest reported mHealth projects globally.40TagedEnd TagedPAs > 90% of existing epidemiologi- cal evidence and almost all interven- tions have been carried out in high- income countries, actions are Kanerva et al Volume 55, Number 5, 2023 329 TagedEndJournal of Nutrition Education and Behavior d settings in Nairobi, Kenya. PLoS One. 2015;10:e0129943.TagedEnd ehavior Volume 55, Number 5, 2023 TAGEDH1REFERENCESTAGEDEND TagedP20. Trost SG, Loprinzi PD, Moore R, Pfeiffer KA. Comparison of accelerom- eter cut points for predicting activity intensity in youth. Med Sci Sports Exerc. 2011;43:1360–1368.TagedEnd TagedP 1. Jaacks LM, Vandevijvere S, Pan A, et al. The obesity transition: stages of the global epidemic. 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https://openalex.org/W3017371359	https://oskar-bordeaux.fr/bitstream/20.500.12278/11424/3/BMC_AnaesthCritCarePainMed_2020_Gruel.pdf	English	null	Diagnosis and management of heparin-induced thrombocytopenia	Anaesthesia critical care & pain medicine	2,020	cc-by	19,297	Anaesth Crit Care Pain Med 39 (2020) 291–310 Anaesth Crit Care Pain Med 39 (2020) 291–310 Diagnosis and management of heparin-induced thrombocytopenia§ Diagnosis and management of heparin-induced thrombocytopenia§ Yves Gruel a, Emmanuel De Maistre b, Claire Pouplard c, Franc¸ois Mullier d, Sophie Susen e,f, Ste´phanie Roullet g,h,i, Normand Blais j, Gre´goire Le Gal k,l, Andre´ Vincentelli m, Dominique Lasne n,o, Thomas Lecompte p, Pierre Albaladejo q, Anne Godier r,, Members of the French Working Group on Perioperative Haemostasis (Groupe d’inte´reˆt en he´mostase pe´riope´ratoire [GIHP]) a Laboratoire d’He´matologie-He´mostase, CHRU de Tours, Hoˆpital Trousseau, avenue de la Re´publique, 37044 Tours cedex 9, France b Service d’He´matologie Biologique, unite´ d’he´mostase, CHU Dijon, 21000 Dijon, France g g q j j c Service d’He´matologie-He´mostase, Centre Re´gional de Traitement de l’He´mophilie, GICC EA 7501, Hoˆpital Trousseau, CHRU de Tours, avenue de la Re´publique, 37170 Chambray-le`s-Tours, France p q y d Laboratoire d’he´matologie-he´mostase, Universite´ catholique de Louvain, CHU UCL Namur, rue Dr-Gaston-Therasse 1, 5530 Yvoir, Belgium e CHU Lille, Hematology Transfusion, Lille, France gy f f INSERM, U1011, University Lille, U1011-EGID, Institut Pasteur de Lille, Lille, France g U it ´ d’A th ´ i ´ i ti l i t t l t ti ´ l CHU d B f INSERM, U1011, University Lille, U1011-EGID, Institut Pasteur de Lille, Lille, France g Unite´ d’Anesthe´sie-re´animation uro-vasculaire et transplantation re´nale, CHU de Bordeaux, place Ame´lie-Raba-Le´on, 33076 Bordeaux, Fra h Service d’Anesthe´sie-Re´animation Tripode, CHU de Bordeaux, place Ame´lie-Raba-Le´on, 33076 Bordeaux, France i INSERM U 1034, Biologie des Maladies Cardiovasculaires, Universite´ de Bordeaux, 1, avenue Magellan, 33600 Pessac, France j Service d’he´matologie et d’oncologie me´dicale, de´partement de me´decine, CHUM, 1000, rue Saint-Denis, H2X 0C1 Montre´al, Canada k Division d’he´matologie, De´partement de me´decine, Universite´ d’Ottawa, 501, rue Smyth, boıˆte 201A, Ottawa, ON K1H 8L6, Canada l Institut de recherche, Hoˆpital d’Ottawa, 501 Smyth Rd, boıˆte 201A, Ottawa, ON K1H 8L6, Canada m Service de Chirurgie cardiaque, Institut Cœur Poumon, CHU de Lille, boulevard Pr-Leclercq, 59037 Lille, France n Laboratoire d’he´matologie ge´ne´rale Hoˆpital Necker AP–HP Paris France , , y , , , , g Unite´ d’Anesthe´sie-re´animation uro-vasculaire et transplantation re´nale, CHU de Bordeaux, place Ame´lie-Raba-Le´on, 33076 Bordeaux, F h Service d’Anesthe´sie-Re´animation Tripode, CHU de Bordeaux, place Ame´lie-Raba-Le´on, 33076 Bordeaux, France i INSERM U 1034, Biologie des Maladies Cardiovasculaires, Universite´ de Bordeaux, 1, avenue Magellan, 33600 Pessac, France j Service d’he´matologie et d’oncologie me´dicale, de´partement de me´decine, CHUM, 1000, rue Saint-Denis, H2X 0C1 Montre´al, Canada k Division d’he´matologie, De´partement de me´decine, Universite´ d’Ottawa, 501, rue Smyth, boıˆte 201A, Ottawa, ON K1H 8L6, Canada l Institut de recherche Hoˆpital d’Ottawa 501 Smyth Rd boıˆte 201A Ottawa ON K1H 8L6 Canada l Institut de recherche, Hoˆpital d’Ottawa, 501 Smyth Rd, boıˆte 201A, Ottawa, ON K1H 8L6, Canada ervice de Chirurgie cardiaque, Institut Cœur Poumon, CHU de Lille, boulevard Pr-Leclercq, 59037 Lille, France o Universite´ Paris Sud Paris Saclay, Inserm U1176, Le Kremlin-Biceˆtre, France o Universite´ Paris Sud Paris Saclay, Inserm U1176, Le Kremlin-Biceˆtre, France ˆ p Hospital and academic departments of medicine, Hoˆpitaux Universitaires de Gene`ve, rue Gabrielle-Perret-Gentil 4, 1205 Gene`ve, Switzerland q Poˆle d’anesthe´sie-re´animation, CS10217, CHU Grenoble-Alpes, 38043 Grenoble cedex 9, France , , p , , imation, hoˆpital europe´en Georges-Pompidou, INSERM UMRS-1140, universite´ Paris Descartes, 25, rue Leblanc, 75015 Paris, France rvice d’anesthe´sie-re´animation, hoˆpital europe´en Georges-Pompidou, INSERM UMRS-1140, universite´ Paris Descartes, 25, rue Leblanc, 7501 Medicine (Socie´te´ franc¸aise d’anesthe´sie et de re´animation [SFAR]) to deﬁne updated proposals for the diagnosis and care of HIT. § Proposal from the French Working on Perioperative Haemostasis (Groupe d’inte´reˆt en he´mostase pe´riope´ratoire [GIHP]) and the French Study Group on Thrombosis and Haemostasis (Groupe d’e´tude sur l’he´mostase et la thrombose [GFHT]), in collaboration with the French Society of Anaesthesia and Intensive Care Medicine (Socie´te´ franc¸aise d’anesthe´sie et de re´animation [SFAR]). Corresponding author. E-mail address: anne.godier@aphp.fr (A. Godier). p // g/ /j p 2352-5568/ C 2020 The Author(s). Published by Elsevier Masson SAS on behalf of Socie´te´ franc¸aise d’anesthe´sie et de re´animation (Sfar). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.accpm.2020.03.012 2352-5568/ C 2020 The Author(s). Published by Elsevier Masson SAS on behalf of Socie´te´ franc¸aise d’anesthe´sie et de re´animation (Sfar). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ed by Elsevier Masson SAS on behalf of Socie´te´ franc¸aise d’anesthe´sie et de re´animation (Sfar). This is an open access article under s.org/licenses/by/4.0/). https://doi.org/10.1016/j.accpm.2020.03.012 2352-5568/ C 2020 The Author(s). Published by Elsevier Masson SAS on behalf of Socie´te´ franc¸a the CC BY license (http://creativecommons.org/licenses/by/4.0/). § Proposal from the French Working on Perioperative Haemostasis (Groupe d’inte´reˆt en he´mostase pe´riope´ratoire [GIHP]) and the French Study Group on Thrombosis and Haemostasis (Groupe d’e´tude sur l’he´mostase et la thrombose [GFHT]), in collaboration with the French Society of Anaesthesia and Intensive Care Medicine (Socie´te´ franc¸aise d’anesthe´sie et de re´animation [SFAR]). Introduction Heparin-induced thrombocytopenia (HIT) is a rare, iatrogenic disease characterised by its potential severity, mainly related to thrombosis, and by difﬁculties regarding its diagnosis and management of affected patients. In 2002, a conference of experts mobilised by the French Society of Anaesthesia and Intensive Care Medicine (Socie´te´ franc¸aise d’anesthe´sie et de re´animation [SFAR]) drafted recommendations for the management of HIT [1]. Since then, the drugs available to treat patients have evolved: lepirudin has disappeared, the prescription of fondaparinux and direct oral anticoagulants has increased, and ﬁnally the biological tests required for diagnosis are more effective. These developments have led the French Working on Perioperative Haemostasis (Groupe d’inte´reˆt en he´mostase pe´riope´ratoire [GIHP]) and the French Study Group on Thrombosis and Haemostasis (Groupe d’e´tude sur l’he´mostase et la thrombose [GFHT]), in collaboration with the French Society of Anaesthesia and Intensive Care Since 2002, many articles have been published, but with few large patient series and even fewer randomised trials comparing available treatments. The level of evidence of the available studies remains uncertain and the level of recommendation is therefore quite low, explaining that our group has decided, as in 2002, not to assign a grade to the proposals issued. We have adopted a different attitude from that of the American College of Chest Physicians (ACCP) [2] or the British [3], which in 2012 proposed strong recommendations. The methodology used to develop these proposals was as follows: the issues addressed were assigned to several working groups, composed of members of the GIHP and/or the GFHT. A ﬁrst text incorporating updated data from the literature and recommendations or guidelines from UK [3], ACCP [2] and more recently issued by ASH [4] was written based on the 2002 expert conference [1]. This ﬁrst text was then reviewed, discussed and modiﬁed by the other groups, and then submitted for critical analysis by other GIHP and GFHT members. Finally, these proposals were validated by a vote (n = 32 participants), thus determining the strength of each proposal. To retain a proposal, at least 50% of members had to express their agreement (for a strong agreement, the threshold was set at 70%), while less than 20% of them could express their opposition. In the absence of agreement, the proposals were reformulated and put to a new vote in order to obtain a better agreement. These proposals were developed in collaboration with the SFAR Clinical Referential Committee. Y. General information It is proposed to deﬁne the level of risk of HIT during heparin administration as: Two types of thrombocytopenia may occur in patients treated with heparin, unfractionated heparin (UFH) or low molecular weight heparin (LMWH): low (less than 0.1%) during treatments with low molecular weight heparin (LMWH) in medicine (except cancer), obstetrics (except surgery including caesarean section), or in the course of minor trauma; during all fondaparinux treatments; during isolated non-fraction- ated heparin (UFH) injection for endovascular procedure or simple surgery; during any treatment by UFH or LMWH lasting beyond one month; a benign thrombocytopenia (type I), of non-immune origin and early onset, without thrombotic complications and regresses despite continued heparin therapy; a more severe thrombocytopenia (type II), which is most often moderate, of delayed onset, immune and potentially very severe since associated with thrombosis. It alone can be qualiﬁed as HIT and is the subject of these proposals. intermediate (between 0.1 and 1%) in case of prophy- lactic treatment with UFH in medicine/obstetrics, or with LMWH in a cancer patient, or a severe trauma patient, or a patient treated with LMWH in postopera- tive care (including cardiac surgery); HIT is a clinical-biological syndrome induced by IgG isotype antibodies, which almost always recognise heparin-modiﬁed platelet factor 4 (PF4/H) [5], with intense platelet activation associated with explosive thrombin generation that can lead to venous and/or arterial thrombosis [6,7]. Thrombocytopenia results from massive activation of platelets and from their elimination by the mononuclear phagocyte system, the cells being sensitised by PF4/heparin/IgG complexes. Thrombosis is the consequence of a multi-cellular activation involving platelets with the release of procoagulant microparticles, endothelial cells, neutrophils and especially monocytes that express tissue factor contributing to the hypercoagulability of patients [8]. high (above 1%) in the case of prophylactic treatment with UFH in surgery, (including circulatory assistance) or for renal replacement therapy; in the case of all curative treatments with UFH in medicine/surgery/ obstetrics. (STRONG AGREEMENT) Rationale Introduction Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 292 Finally, our text addresses the following 12 questions and presents 40 proposals useful for clinical practice: The thrombotic risk is considered high during the ﬁrst month after diagnosis [9], the antibody titre and their ability to activate platelets and haemostasis decreasing thereafter, to no longer be detectable in good standing beyond three months [10]. What are the different stages and levels of risk of HIT? Which platelet count monitoring for HIT detection during heparin therapy? Question 1: What are the different stages and level of risk of TIH? What are the circumstances that should be considered when diagnosing HIT? Apart from HIT, what are the other possible aetiologies of thrombocytopenia in a patient treated with heparin? Proposal # 1 It is proposed to distinguish three different stages of HIT according to its ancientness: It is proposed to distinguish three different stages of HIT according to its ancientness: Which biological tests are needed to conﬁrm the diagnosis of HIT? acute HIT, which is a recent HIT, diagnosed within the last month, during which time antiplatelet factor 4 (PF4) activating antibodies are most often present with a high thrombotic risk; What is the practical approach for the diagnosis and initial management of HIT in the acute phase? What are the alternative anticoagulants that can be used in HIT after stopping heparin? Is there a place for other treatments in a patient with suspected HIT? subacute HIT, which corresponds to HIT diagnosed 1 to 3 months ago, during which time anti-PF4 antibodies are often present with a low titre; Which treatment should be proposed for HIT in surgical settings outside cardiac surgery? previous history of HIT, which corresponds to an old HIT of more than 3 months, and at this stage, anti-PF4 antibodies are most often undetectable. Which treatment should be proposed for cardiac surgery with and without extracorporeal circulation (ECC) in the case of HIT? Which treatment should be proposed for HIT in a medical setting? antibodies are most often undetectable. (STRONG AGREEMENT) Which prevention can be proposed to avoid the occurrence of HIT or recurrence? Rationale The management of patients depends on the age of HIT and the persistence or otherwise of circulating anti-PF4 antibodies. Three different stages can be individualised (Proposal No. 1). The diagnosis of acute HIT is difﬁcult since there are often other potential causes of thrombocytopenia, particularly in the postop- erative period or in ICU patients, and it must integrate clinical circumstances and associated treatments. It is important not to neglect this diagnosis, or conversely not to wrongly conclude that HIT is present because the systematic discontinuation of heparin in the presence of any thrombocytopenia poses therapeutic problems and may expose the patient to complications. The risk of HIT varies according to the type of heparin administered (UFH or LMWH), the underlying conditions, and the duration of treatment (Table 1). In 2012, the ACCP had deﬁned its proposals taking into account only two levels of risk, higher or lower than 1% [2]. In 2018, the ASH proposed three levels of risk, high (> 1%), intermediate (between 0.1 and 1%) and low (< 0.1%) [4], a position we chose to adopt, as it better responds to the different situations with which the clinician is most frequently confronted. Biological conﬁrmation of the diagnosis is necessary in all cases but often takes several days. However, biological assays, looking for anti-PF4 antibodies, must never delay the stopping of heparin and the prescription of an alternative anticoagulant. Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 293 Table 1 Risk of HIT by context and type of heparin administered. Context Dose Level of risk Low Intermediate High UFH Surgery including caesarean section Prophylactic or Curative + Medical/ Obstetrical Curative + Renal and cardiac assistance (CPB, ECMO. . .) + Medical/ Obstetrical Prophylactic + LMWH Surgery including caesarean section Prophylactic or Curative + Cancer + Medical/ Obstetrical Prophylactic or Curative + HIT: heparin-induced thrombocytopenia; UFH: unfractionated heparin; LMWH: low molecular weight heparin; CPB: cardiopulmonary bypass; ECMO: extracorporeal membrane oxygenation. [2], and this result was not conﬁrmed [22], which is why we considered the risk of HIT in this context as intermediate. The risk of HIT is assessed as low during all medical treatments with LMWH (except cancer), and during pregnancy outside the surgical context. This risk is also very low or even non-existent under fondaparinux (which is not strictly speaking a heparin) whether prescribed with preventive or curative doses [11]. Rationale A high risk of HIT (greater than 1%) exists for the majority of patients treated with UFH, whether they receive a preventive or curative dose, particularly in orthopaedic surgery [23], or after cardiac surgery with CPB [24]. In medicine, a prospective study in 2005 found an incidence of HIT equal to 0.8% under LMWH at preventive or curative doses [12], but this result has not been conﬁrmed. Another study in 2011, involving more than 25,000 patients, found a much lower frequency of HIT ( 0.2%) in medical patients treated with a preventive dose of LMWH [13]. We therefore do not propose any monitoring of platelets in medical patients under LMWH (question 2), in accordance with the most recent British [3] and North American [4] recommendations. In medical patients, the risk of UFH-related HIT is probably lower but remains close to 1% [13,25,26] and higher during curative intravenous treatments [13,27]. Patients on extracorporeal membrane oxygenation (ECMO) require curative anticoagulant treatment with intravenous UFH and therefore have a high risk of HIT [28], although still poorly deﬁned [29,30]. In addition, a patient receiving a single bolus of UFH (especially for endovascular examination) is also at low risk of HIT, unless he or she has recently (within the previous 3 months) been exposed to heparin for several days in a row. Question 2: Which platelet count monitoring for HIT detection in heparin-treated patients? In obstetrics, a systematic review of more than 2700 pregnan- cies under LMWH conﬁrmed a very low risk of HIT, less than 0.1% [14], also justifying the lack of platelet monitoring in this context. Proposal #3 It is proposed that all patients treated with heparin, whether unfractionated or low molecular weight, should have a sys- tematic platelet count before initiation of treatment (or alter- natively as soon as possible after the ﬁrst injection, before D4). (STRONG AGREEMENT) [ ] j y g p g Finally, the risk of HIT is very low in all patients after one month of heparin treatment, regardless of the molecule administered (UFH or LMWH) and the dosage. The risk of HIT is intermediate (between 0.1 and 1%) in many clinical situations. This is particularly the case for patients treated for prophylaxis with LMWH in a surgical setting, and for whom the risk of HIT is estimated to be almost 10 times lower than with UFH [15,16]. Proposal #4 It is proposed not to monitor platelet count in patients at low risk of HIT. ( ) However, it varies greatly depending on the context and type of surgery, as evidenced by this wide range of incidence, from 0.1 to 1%, and which does not necessarily implicate the same monitoring. Thus, the risk of HIT considered as low in case of minor trauma [4] appears to be higher under LMWH after surgery for severe trauma [17], being estimated at 0.36% [18]. However, it is much lower after scheduled orthopaedic surgery, such as hip or knee replacement [19]. In France, minor surgery is not an indication in itself for drug prophylaxis, and we have therefore considered any surgical procedure treated with LMWH to be at intermediate risk of HIT. risk of HIT. (STRONG AGREEMENT) Proposal #5 p For patients at intermediate risk of HIT, it is proposed to monitor platelet counts once to twice a week from day 4 to day 14 of treatment, and then once a week for one month if heparin therapy is continued. (STRONG AGREEMENT) (STRONG AGREEMENT) In cardiovascular surgery, despite a bolus of UFH performed intraoperatively, particularly in the case of cardiopulmonary bypass (CPB) surgery, the risk of HIT is reduced if LMWH is prescribed postoperatively (0.4% versus 2.5% under UFH) [20,21]. The risk of HIT in cardiovascular surgery patients under LMWH is therefore considered to be at intermediate risk. The monitoring then depends on the level of HIT risk The monitoring then depends on the level of HIT risk The monitoring then depends on the level of HIT risk In all situations where the risk of HIT is low, no monitoring of PC is required (Proposal #4), but a blood count will be performed during any unusual or unexpected event (see question #3, Proposal #7). Control of PC is necessary for other patients, with a more sustained rhythm for those with a high risk (2 or 3 times a week). It should be noted that the ASH proposes in this case a PC check every 48 hours [4], which may be preferred if the risk is very high, as for example during cardiac surgery in a patient with a previous history of HIT. Arterial thrombosis is the most typical event, although less frequent. All territories can be affected, but more frequently abdominal aorta and its branches. Venous gangrene in the limbs is very rare, and can complicate HIT when treatment with a vitamin K antagonist (VKA) has been initiated without being combined with another effective anti- thrombotic agent [34], or if HIT is diagnosed at the time of a heparin/VKA relay. However, after cardiac surgery with CPB, repeated monitoring of the PC allows a careful analysis of the postoperative evolution and the identiﬁcation of a possible ‘‘biphasic’’ proﬁle, characterised by a decrease in the PC in the days following a phase of total or partial correction, and which is highly predictive of an HIT [21,31]. The classic heparin resistance with an extension of the initial thrombotic process is also a possible but rare discovery circumstance. Neurological complications occur in approximately 10% of patients, with ischemic stroke, cerebrovascular thrombosis, confounding conditions or transient amnesia occurring in decreas- ing order of frequency. A monitoring window the day 4 to day 14 of treatment is proposed, as the vast majority of HIT occur during this period [10]. However, a few cases have been reported after 15 days of treatment, particularly with LMWH [32], but never after one - month. It is therefore logical to maintain monitoring of platelets for one month, but with a lower frequency after 15 days of treatment. Beyond that, the risk of HIT becomes very low and no control of PC is necessary. The monitoring then depends on the level of HIT risk Other clinical manifestations that may lead to the suspicion of HIT are rare: cutaneous necrosis at heparin injection sites is most typical [11,35,36,111] and can be inaugural, preceding the drop in platelet count; cutaneous erythema is also possible, but not deﬁnitely related to HIT, particularly with LMWH [37], is exceptional. Rationale Rationale Two particularities characterise HIT: the chronology of thrombocytopenia in relation to heparin administration; the rarity of haemorrhagic manifestations and the frequency of venous and/or arterial thrombotic events. (STRONG AGREEMENT) Thrombocytopenia or a sharp and sudden decrease in platelet count typically occurs between days 5 and 14 of heparin therapy. However, this delay may be shorter (before 5 days), or even as early as the ﬁrst day of treatment in patients who have been recently exposed to heparin in the previous 3 months [10]. It may also be longer, especially with LMWH, and may exceed 3 weeks [32]. Rationale Two particularities characterise HIT: Rationale Two particularities characterise HIT: Rationale Question 3: What are the circumstances that should evoke the diagnosis of HIT? Haemorrhagic complications are also rare, as well as haemor- rhagic necrosis of the adrenals, which are promoted by DIC and associated with higher mortality. Proposal # 7 Whatever the risk of HIT, it is proposed to systematically monitor the platelet count of any patient treated with heparin in the event of an unexpected clinical event: onset or aggravation of venous or arterial thrombosis, skin necrosis or unusual reaction after heparin injection (chills, low blood pressure, dyspnea, amnesia. . .). (STRONG AGREEMENT) Proposal # 7 Whatever the risk of HIT, it is proposed to systematically monitor the platelet count of any patient treated with heparin in the event of an unexpected clinical event: onset or aggravation of venous or arterial thrombosis, skin necrosis or unusual reaction after heparin injection (chills, low blood pressure, dyspnea, amnesia. . .). An unusual reaction after injection of heparin has sometimes been reported, particularly in the context of haemodialysis; hypotension, chills, anaphylactic reaction. All clinical manifestations and data on the evolution of platelet count can be analysed to deﬁne a pre-test diagnostic probability score, which can help in the prescription of biological analyses, and 4T is the most widely used in clinical practice (question # 4). (STRONG AGREEMENT) (STRONG AGREEMENT) Question 4: What are the other possible causes of thrombocytopenia in a patient on heparin and how to deﬁne the clinical probability of HIT? Question 4: What are the other possible causes of thrombocytopenia in a patient on heparin and how to deﬁne the clinical probability of HIT? Question 4: What are the other possible causes of thrombocytopenia in a patient on heparin and how to deﬁne the clinical probability of HIT? Proposal # 8 In case of suspicion of HIT, it is proposed to deﬁne the clinical probability of HIT using the 4T score, outside a cardiac surgery context. (STRONG AGREEMENT) Proposal #6 For patients at high risk of HIT, it is proposed to monitor platelet counts two to three times a week from day 4 to day 14 of treatment, and then once a week for one month if heparin therapy is continued. In medical cancer patients, only one study reported a high incidence > 1% of HIT [12], but its methodology was controversial (STRONG AGREEMENT) Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 294 beginning of treatment. Thrombocytopenia is typically moderate, ranging from 30 to 70 G.L1 in 80% of patients. Rationale The early diagnosis of HIT Consumption coagulopathy (DIC) is also possible and does not exclude the diagnosis of HIT. It actually aggravates thrombocyto- penia. In intensive care patients or in the postoperative period, the coexistence of other pathologies (sepsis, haemorrhages, massive transfusions, DIC. . .) can also lead to deeper thrombocytopenia. The early diagnosis of HIT depends primarily on monitoring platelet count (NP). A ﬁrst platelet count (PC) is recommended before starting any heparin treatment [2,3], or alternatively as soon as possible (before the 4th day of treatment), the result obtained serving as a reference (Proposal No. 3). More rarely, thrombotic complications may occur in the absence of thrombocytopenia, but there is almost always a relative decrease in platelet count. The existence of venous and/or arterial thrombotic events under heparin is very suggestive of HIT. Deep vein thrombosis affects up to 50% of patients, which justiﬁes for some clinicians their systematic search by a Doppler ultrasound examination of the lower limbs in case of suspicion [33]; pulmonary embolism occurs in 10 to 25% of cases. Rationale Heparin thrombocytopenia is often due to potential causes other than HIT that should be identiﬁed The diagnosis should be made in the presence of a platelet count < 100 G.L1 and/or a decrease in the platelet count > 50% compared to a previous value, most often obtained at the 1. Under UFH, early and moderate thrombocytopenia may occur within the ﬁrst two days of treatment, resulting from a direct pro- Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 295 The 4th T depends on whether or not there is another potential cause of thrombocytopenia (other) aggregating effect of UFH. However, it may also exceptionally reﬂect early HIT after reintroduction of heparin in a patient sensitised by heparin therapy within the previous 3 months. This aspect is the most difﬁcult to evaluate because patients often have associated pathologies (sepsis, liver disease. . .) and treatments (chemotherapy, antibiotics, diuretics. . .) that are potentially a cause of thrombopenia. Another acute haematological pathology must always be sought and rigorous analysis of the blood count is therefore necessary. Antiplatelet alloimmunisation should also be considered in the case of recent administration of labile blood products. However, non-heparin-related transfusional or drug- related thrombocytopenia is often more severe than that associated with HIT, and complicated by bleeding and not thrombosis. y p py p 2. Other causes of isolated thrombocytopenia are possible, particularly in the intensive care patient [38] or after surgery: perioperative haemodilution and platelet consumption in extracorporeal circuits or counterpulsion with an intra-aortic balloon are clinical circumstances that are most often easily identiﬁed; consumption thrombocytopenia is also common at the end of cardiac surgery, in the case of ventricular assistance, or extrarenal epuration, all of which are at signiﬁcant risk of HIT; Each item in the 4T being scored from 0 to 2, the total score may reﬂect a low probability of HIT if it is 3, intermediate if it is equal to 4 or 5 or high if it is 6. post-transfusion purpura, linked to alloimmunisation, should also sometimes be considered (typically major and sudden decrease in platelets and haemorrhagic context). Rationale Its diagnosis is urgent given the risk of severe bleeding and the need for speciﬁc treatment; In patients who have undergone cardiac surgery with CPB, this score is more difﬁcult to apply and the analysis of the post- operative evolution proﬁle of platelet count is more efﬁcient. Indeed, a ‘‘biphasic’’ evolution proﬁle of platelet count is equi- valent to a 4T score 6, with a high probability of HIT [21,42]. In intensive care patients with multiple pathologies, the 4T score is also difﬁcult to assess due to co-morbidities and multiple treatments, and can be thus compromised [43]. the use of GPIIb-IIIa glycoprotein inhibitors in acute coronary syndromes is potentially complicated by early and often profound thrombocytopenia; the imputability of other drugs potentially responsible for immune thrombocytopenia in intensive care [39] or certain antimitotic chemotherapies is also to be mentioned. Rationale Rationale Question 5: What are the biological tests to be performed when there is a suspicion of HIT? 3. When thrombocytopenia is associated with venous or arterial thrombotic complications, other aetiologies than HIT should be considered. These are mainly antiphospholipid syndrome [40], thrombotic thrombocytopenic purpura and DIC. Thrombocytope- nia and thrombosis can also be observed in patients with cancer, with a clinical picture of pseudo-HIT. Proposal # 9 In case of suspicion, it is proposed to look for anti-PF4 antibodies as soon as possible if the clinical probability of HIT is intermediate or high. (STRONG AGREEMENT) Potential causes of thrombocytopenia other than HIT will therefore be considered in assessing the clinical probability of HIT. (STRONG AGREEMENT) The 1st T depends on the number of platelets at the time of suspicion (thrombocytopenia) The 1st T depends on the number of platelets at the time of suspicion (thrombocytopenia) Thrombocytopenia is generally never profound (often between 30 and 70 G/L) and remains > 20 G/L. In addition, an authentic HIT can be observed without actual thrombocytopenia, but a decrease in platelet count of at least 50% from the highest count before suspicion is highly predictive (2 points). On the contrary, a drop of less than 30% in the PC or a PC < 10 G/L is not in favour of HIT (0 point). Two categories of speciﬁc tests can be used to identify antibodies associated with HIT [44], each with advantages and disadvantages. The clinical probability of HIT is assessed by the 4T score The discovery of thrombocytopenia in a patient treated with heparin should be systematically controlled by examining the sample tube for a clot and the smear to exclude the presence of aggregates. A new citrate sample often ruled out false thrombocy- topenia on EDTA. The clinical probability of HIT is assessed by the 4T score [2,41], based on four major criteria and 0, 1 or 2 points are assigned for each of them (Fig. 1). Simple haemostasis tests (PT, aPTT, ﬁbrinogen, D-dimers or ﬁbrin monomers) should also be prescribed in order to look for DIC, possible in some severe HIT and therefore not excluding this diagnosis. Rationale The management of suspected HIT is based on a concerted and rapid clinical and biological approach, which is critical to the management of antithrombotic therapy and to the patient’s prognosis. p [ ] These tests can be performed on platelet-rich plasma, such as platelet aggregation tests (PAT), or after washing platelets, which sensitises the detection of activating antibodies. In the latter case, a distinction is made between the radiolabelled serotonin release test or SRA, which is considered a ‘‘gold standard’’ test, and the Heparin- Induced Platelet Activation (HIPA) test, which is rarely used in France. The search for heparin-dependent antibodies can also be performed on whole blood using an impedance technique [51]. Nevertheless, tests carried out on washed platelets are still considered as the most sensitive methods with speciﬁcity close to 100%. The use of platelets from several controls and/or selected controls increases the perfor- mance of functional tests, including platelet aggregation tests (PAT). These tests can be performed on platelet-rich plasma, such as platelet aggregation tests (PAT), or after washing platelets, which sensitises the detection of activating antibodies. In the latter case, a distinction is made between the radiolabelled serotonin release test or SRA, which is considered a ‘‘gold standard’’ test, and the Heparin- Induced Platelet Activation (HIPA) test, which is rarely used in France. The ﬁrst step is to deﬁne the clinical probability of HIT, especially with the 4T score, and if it is low, another aetiology of thrombocytopenia should be investigated without speciﬁc biolog- ical analyses in accordance with the British [3] and ASH [4] proposals. However, a testing for anti-PF4 antibodies may be discussed for patients where the 4T is difﬁcult to deﬁne, especially in the case of missing data. The search for heparin-dependent antibodies can also be performed on whole blood using an impedance technique [51]. Nevertheless, tests carried out on washed platelets are still considered as the most sensitive methods with speciﬁcity close to 100%. The use of platelets from several controls and/or selected controls increases the perfor- mance of functional tests, including platelet aggregation tests (PAT). In other cases (intermediate or high clinical probability), a blood sample for the detection of anti-PF4 antibodies should be collected as soon as possible. The SRA (requiring the use of carbon 14) and HIPA tests are long and delicate, and are reserved for a few expert laboratories. Proposal #14 If the clinical probability is intermediate or high and a signiﬁcant titre of anti-PF4 antibodies is detected, a functional test should be performed. If positive, the diagnosis of HIT is conﬁrmed. Rationale Other approaches such as ﬂow cytometry have been adapted for the biological diagnosis of HIT [50] but have been little used and have yet to be validated. Essential point: the decision to discontinue heparin and replace it with another immediate antithrombotic should not be delayed by waiting for the laboratory results. Immunological tests are performed as ﬁrst-line tests and a functional test is performed only if the detection of anti-PF4 antibodies is positive, especially in cases where the pre-test probability is intermediate. Functional or platelet activation tests (STRONG AGREEMENT) Functional or platelet activation tests show the presence in the patient’s plasma or serum of IgG isotype antibodies capable of activating the platelets of a control subject in the presence of heparin [50]. Rationale Question 6: What is the initial management of a patient with suspected TIH? In case of a high pre-test probability, associated with the presence of a relatively high titre of anti-PF4 antibodies in ELISA (e.g. OD greater than 2), the diagnosis of HIT can be conﬁrmed without the need for a functional test [4,52]. However, this approach is only applicable if anti-PF4 antibodies are tested by quantitative immunological tests. When 4T is high, a functional test is recommended if the immunological search for anti-PF4 antibodies is negative, as rare HIT cases with anti-IL8 antibodies have been reported [53]. Proposal #10 If the pre-test probability is low (4T 3), diagnosis of HIT can be excluded and treatment with heparin can be continued without speciﬁc bioassays. The search for an aetiology of thrombocytopenia with close monitoring of platelet count must be carried out. (STRONG AGREEMENT) When the clinical probability is intermediate and the laboratory can perform an immunological or functional test with a short response time (< 3 hours after sampling), the clinician may consider waiting for the results before modifying the anticoagulant treatment (Fig. 1). Proposal #11 If the pre-test probability is intermediate (4T = 4 or 5) or high (4T 6), biological tests must systematically be performed to detect anti-PF4 antibodies. For any suspicion of HIT, it is important to achieve a clear diagnostic conclusion that takes into account all the information in the ﬁle and it is mandatory to report each case to the Regional Pharmacovigilance Centre. detect anti-PF4 antibodies. (STRONG AGREEMENT) In conclusion, the diagnosis of HIT requires a concerted effort between clinicians and biologists, particularly in view of the immediate (choice of antithrombotic treatment) and secondary (possibility of a subsequent prescription of an antithrombotic treatment) issues. Proposal #12 If the clinical probability is intermediate and the search for anti-PF4 antibodies is negative, the diagnosis of HIT is excluded and heparin therapy can be continued or resumed, with close monitoring of platelet count. Question 7: What are the alternative anticoagulants that can be used in HIT after stopping heparin? How to prescribe them in the acute phase? (STRONG AGREEMENT) Immunological tests Immunological tests detect antibodies of the IgG, IgM, IgA isotype speciﬁcally directed against modiﬁed PF4. They are semi- quantitative (ELISA or chemiluminescent tests) or qualitative (agglutination, immunoﬁltration, immunoturbidimetry. . .), have excellent sensitivity and are easy to carry out. These tests should be performed as soon as possible to quickly rule out the diagnosis of HIT and guide the clinician towards ﬁnding another aetiology of thrombocytopenia. Their negative predictive value is excellent [45], but their speciﬁcity is less good since anti-PF4 antibodies can appear without being associated with HIT, particularly after cardiac surgery, after which they are present in nearly one out of two patients. The speciﬁcity and especially the positive predictive value (PPV) of immunoassay can however be improved by using a method that speciﬁcally detects IgG antibodies and expresses the result quantitatively, most often by specifying the measured absorbance value [46]. Another approach is to test whether a high The 2nd T depends on the time of onset of thrombocytopenia (Timing) In typical cases, a decrease in platelet count is observed 5 to 10 days after initiation of heparin therapy (2 points) or earlier if heparin therapy has been administered within the previous 3 months (1 point). The 3rd T depends on the presence or absence of venous and arterial thromboembolic events or other clinical events associated with thrombocytopenia (thrombosis) Thrombotic complications are sometimes obvious, but some thromboses may be asymptomatic. Some practitioners have therefore suggested that they should be systematically researched by a Doppler ultrasound examination of the lower limbs [4]. 296 Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 Fig. 1. Algorithm of clinical and biological diagnosis of heparin-induced thrombocytopenia (HIT). Fig. 1. Algorithm of clinical and biological diagnosis of heparin-induced thrombocytopenia (HIT). Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 297 concentration of heparin decreases the absorbance measured in ELISA, this result being speciﬁc for anti-PF4 antibodies associated with HIT [47]. However, this procedure, which is recommended for low levels of antibodies (OD < 1) [48], is rarely applied and its usefulness is controversial [49]. Proposal # 20 p It is proposed that the initial dose of argatroban be 1 mg/kg/ min and reduced to 0.5 mg/kg/min in patients with resuscitation, cardiac surgery and moderate hepatic failure (Child-Pugh B). (STRONG AGREEMENT) Biological monitoring of argatroban treatment should be applied daily. It may be based on aPTT (if normal before treatment) to maintain between 2 and 3 times the control value, or preferably on other more speciﬁc assays such as diluted thrombin time or ecarin test (proposed therapeutic window = 0.5 to 1.5 mg/mL). (STRONG AGREEMENT) [ ] 2. Danaparoid sodium can be administered subcutaneously (2 or 3 injections/day) or continuously intravenously, usually preceded by a bolus. The dosage varies according to the clinical, medical or surgical situation and protocols adapted to each situation have been established. [ ] 2. Danaparoid sodium can be administered subcutaneously (2 or 3 injections/day) or continuously intravenously, usually preceded by a bolus. The dosage varies according to the clinical, medical or surgical situation and protocols adapted to each situation have been established. Proposal # 22 It is recommended that AVK be prescribed for the acute phase of HIT only when platelet count is corrected (> 150 G/L) as a supplement to parenteral treatment. (STRONG AGREEMENT) When used intravenously with curative doses, danaparoid is prescribed with an intravenous loading dose that varies by weight (1250 U IV if weight 55 kg; 2500 U IV if 55 < weight 90 kg; 3750 U IV if > 90 kg) and a continuous intravenous maintenance dose of 400 U/h for 4 h, 300 U/h for the next 4 h, then 150 to 200 U/h for the duration of treatment to be adjusted according to the anti-Xa plasma activity (0.5–0.8 U/mL, assay with speciﬁc calibration). (STRONG AGREEMENT) 1. In a stable patient with no severe renal or hepatic im- pairment and no risk of bleeding (absence of comorbidity or recent or expected invasive procedure in the short-term), all available non-heparin anticoagulants can be prescribed, but the simplicity of use of fondaparinux and DOACs (without speciﬁc biological monitoring) justiﬁes that they can be pro- posed as ﬁrst-line alternative treatments, of after the use of danaparoid or argatroban. In paediatrics, the initial dose of danaparoid for a thrombosis is 30 U/kg (IV bolus) followed by a maintenance dose of 1.2 to 2.0 U kg/h. Proposal # 19 It is proposed to use argatroban as a priority for the treat- ment of HIT in severe renal failure. This anticoagulant is contraindicated in severe liver failure (Child-Pugh C). It must be used in a specialised structure. (STRONG AGREEMENT) Proposal #13 When the clinical probability of HIT is high (4T 6 or ECC with biphasic platelet count progression proﬁle), heparin ther- apy should be stopped immediately and replaced by non- heparin anticoagulant therapy with curative doses, without waiting for the results of biological tests. Proposal # 15 Anticoagulants for use in the acute phase of HIT include argatroban, bivalirudin, danaparoid, and fondaparinux, and direct oral anticoagulants. ( ) (STRONG AGREEMENT) Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 298 prescribe as a priority an injectable anticoagulant of ½ short life, argatroban or bivalirudin, combined with strict biological monitoring. Proposal # 16 Danaparoid is not recommended as a ﬁrst-line treatment for HIT in severe renal failure. 3. In a patient with severe HIT (massive PE, extensive or arterial thrombosis, venous gangrene, coagulopathy of con- sumption), it is proposed to prescribe argatroban or bivalirudin as a priority injectable treatment combined with strict biologi- cal monitoring. (STRONG AGREEMENT) 4. In a patient with severe renal impairment (creatinine clearance < 30 mL/min), only argatroban can be used. Prophylactic doses of danaparoid are not recommended for the treatment of acute HIT. Curative IV doses are more effective and necessitate the monitoring of anti-Xa activity (with a speciﬁc calibration curve). 5. In a patient with severe hepatic impairment (Child-Pugh C), bivalirudin, danaparoid or fondaparinux may be used. y (STRONG AGREEMENT) (STRONG AGREEMENT) Rationale Danaparoid sodium 1. Danaparoid sodium is an extraction heparinoid (derived like several LMWH from pig intestine) that contains heparan sulfate, dermatan sulfate and chondroitin sulfate. Its anticoagulant activity is mainly related to its anti-Xa activity, associated with a low anti- IIa activity. It does not prolong prothrombin time and does not usually increase the aPTT. The elimination half-life of the danaparoid’s anti-Xa activity is long, about 25 hours. The anti-Ia activity is shorter, about 7 hours, but it is longer in case of renal failure, a situation where argatroban is preferable. The labelled indications of danaparoid include the prophylactic and curative treatment of thromboembolic events in patients with HIT or a documented history of HIT. This drug is the oldest used in the treatment of HIT [47] although it has been evaluated in only one randomised trial [54] and two retrospective historical cohorts [55,56]. Rationale Danaparoid sodium has long been the most commonly prescribed antithrombotic agent to relay heparin in cases of suspected HIT. Then, it was possible to use lepirudin, a direct antithrombin molecule, but this drug is no longer available in France since 2012. In recent years, argatroban, another synthetic antithrombin agent, fondaparinux and, more recently, direct oral anticoagulants (DOACs) have ﬂourished. The use of the latter two drugs, although not ofﬁcially authorised in HIT, is increasingly being proposed [4]. Bivalirudin, which has also been widely used in several countries, is no longer available in France today, but generics are expected to be available soon. Finally, vitamin K antagonists (VKAs) are potentially dangerous in the acute phase of HIT. Proposal # 18 If the platelet count is not corrected, or if a thrombosis under danaparoid appears or spreads, it should be replaced by another anticoagulant. (STRONG AGREEMENT) Proposal # 20 The IV route is preferable, but if the subcutaneous route is preferred with curative doses, the maintenance dose of danaparoid varies according to weight: 1500 U SC 2 times/day if weight 55 kg; 2000 U SC 2 times/day if 55 < weight 90 kg; 1750 U SC 3 times/day if weight > 90 kg. 2. In an unstable patient, or a patient at risk of bleeding (comorbidity or recent or planned invasive procedure in the short-term), or in intensive care units, it is proposed to Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 299 free or thrombus-related. It has a short onset of action and a short elimination half-life of less than one hour (52 16 min). In continuous IV infusion, plasma levels reach steady state within 1 to 3 hours (faster if bolus injection). Importantly, the analysis of 294 patients treated with danaparoid in the acute phase of HIT concluded that prophylactic doses are less effective than higher doses administered by continuous IV [55]. The efﬁcacy and safety of argatroban in HIT were initially evaluated in two prospective multicentre studies (ARG-911 and ARG-915) involving 882 patients with HIT with or without thrombosis, and compared to historical controls [59,60], and more recently in a French study [61]. Danaparoid can be prescribed in preventive doses in a patient with a previous history of HIT at the following doses: 750 U SC 2 times/day if the weight 90 kg; 1250 U SC 2 times/day if the weight > 90 kg. The summary of product characteristics (SPC) details the protocols adapted to different clinical situations (cardiac cathe- terisation, coronary angioplasty, arterial embolectomy, peripheral vascular bypass, cardiopulmonary surgery, periodic haemodialy- sis, daily dialysis, continuous haemoﬁltration), some of which are discussed in question 11. The metabolism of argatroban is mainly hepatic. Argatroban is therefore contraindicated in cases of severe liver failure (Child- Pugh score C). In patients with moderate hepatic insufﬁciency (Child-Pugh B score), its clearance can be reduced by a factor of 4 and its half-life multiplied by 3 [62], necessitating a much lower initial dose. 3. Monitoring of treatment is necessary in the majority of cases, especially in cases of bleeding risk or renal failure, since danaparoid is mainly eliminated by the kidney. It is also recommended in cases of cachexia and in patients with a weight > 90 kg. Proposal # 20 However, prolonged aPTT before treatment (common in intensive care, after cardiac surgery or in cases of liver failure) sometimes makes it impossible to use this assay for monitoring argatroban. In addition, the effect of argatroban on aPTT depends on the reagent and coagulometer used, and a plateau effect is observed at the upper end of the therapeutic range. Therefore, aPTT is not ideal for the follow-up of a thrombin inhibitor [66] in an unstable clinical context such as HIT, and a target value well below 100 s must be 3. Argatroban prolongs routine coagulation tests such as the prothrombin time (PT) (and therefore increases INR) and aPTT. The latter can be used as a ﬁrst-line treatment-monitoring assay [57], but it must be measured before treatment is started to check its ‘‘normality’’. A stable anticoagulant effect is usually achieved only 1 to 3 hours later. The ﬁrst check should therefore be performed 2 to 3 hours after the start of the infusion. The recommended target value is 1.5 to 3 times the initial value, but not more than 100 seconds, and aPTT should be measured at least once a day; it has the advantage of being available in all laboratories. However, prolonged aPTT before treatment (common in intensive care, after cardiac surgery or in cases of liver failure) sometimes makes it impossible to use this assay for monitoring argatroban. In addition, the effect of argatroban on aPTT depends on the reagent and coagulometer used, and a plateau effect is observed at the upper end of the therapeutic range. Therefore, aPTT is not ideal for the follow-up of a thrombin inhibitor [66] in an unstable clinical context such as HIT, and a target value well below 100 s must be 4. In case of overdose, transient discontinuation of danaparoid is proposed in combination with monitoring of speciﬁc anti-Xa activity. In cases of severe bleeding, the use of protamine is not proposed by the danaparoid SPC, although it partially neutralises its anticoagulant activity. Plasmapheresis may be considered in the event of uncontrollable bleeding. 5. The danaparoid sodium-VKA switch is only initiated when the thromboembolic risk is well controlled (usually after 5 to 7 days of treatment), and when the platelets are above 150 G/L. Proposal # 20 It is recommended to stop danaparoid only when the INR is in the therapeutic window (between 2 and 3) for two consecutive days and after a minimum duration of 72 hours of VKA treatment. Proposal # 20 It is ensured by measuring the anti-Xa activity, with an adapted dosage method calibrated with speciﬁc calibra- tions. According to the SPC, the anti-Xa activity should be maintained between 0.5 and 0.8 U/mL for the treatment of acute HIT [3]. Argatroban is not eliminated by the kidney unlike sodium danaparoid and is therefore preferred in cases of renal failure. Argatroban contains ethanol. A 70 kg patient in whom the maximum recommended dose (10 micrograms/kg/min) is admin- istered therefore receives a dose of approximately 4 g ethanol per day. 2. In acute HIT with or without thrombosis, the initial dosage recommended in the SPC is most often too high (2 mg/kg/min) and associated with marked prolongation of aPTT and bleeding complications [61]. In patients with impaired liver function, such as after cardiac surgery and in intensive care, the initial dose should be signiﬁcantly reduced and most often close or equal to 0.5 mg/kg/min. The dosage may be adjusted in patients with multi- visceral failure according to APACHE II, SOFA or SAPS [63] severity scores (Table 2) [64]. In obese patients, the initial dose is calculated on the actual weight [65]. A risk of cross-reactivity in vitro of danaparoid with antibodies present in HIT patients exists in 5 to 10% of cases, but the clinical consequences that may result are rare [57]. Danaparoid therapy can therefore be initiated without waiting for the results of an in vitro cross-reactivity test, which is not mandatory, but platelet counts should be monitored daily until normalised and then twice weekly for the ﬁrst two weeks of treatment [58]. In the latter case (thrombosis or extension), a control of the anti-Xa activity allows to verify that it is not below 0.5 U/mL. 3. Argatroban prolongs routine coagulation tests such as the prothrombin time (PT) (and therefore increases INR) and aPTT. The latter can be used as a ﬁrst-line treatment-monitoring assay [57], but it must be measured before treatment is started to check its ‘‘normality’’. A stable anticoagulant effect is usually achieved only 1 to 3 hours later. The ﬁrst check should therefore be performed 2 to 3 hours after the start of the infusion. The recommended target value is 1.5 to 3 times the initial value, but not more than 100 seconds, and aPTT should be measured at least once a day; it has the advantage of being available in all laboratories. Argatroban 1. Argatroban (Arganova1 in France) is a synthetic anticoagu- lant, which speciﬁcally and directly inhibits thrombin, whether Bivalirudin In 2012, Krauel et al. demonstrated that both dabigatran and rivaroxaban had no effect on the interactions between PF4 or PF4/ heparin complexes and platelets [73]. Then, several articles reported isolated cases or small series of patients with HIT, treated with dabigatran, rivaroxaban and more rarely apixaban. In 2015, Shariﬁ et al. reported 22 patients initially treated with low doses of argatroban and then with dabigatran (n = 6), rivaroxaban (n = 11) or apixaban (n = 5). Five patients presented a new thrombotic episode, suggesting a very relative efﬁcacy of DOACs, but this retrospective study was questionable because the diagnosis of HIT was not always very well documented (no bioassay in 2 cases) and the initial treatment with argatroban was not optimal [74]. Bivalirudin, a synthetic molecule, is a direct thrombin inhibitor [68]. It also prolongs the prothrombin time and its elimination half-life is short, about 25 min if renal function is normal. Its elimination is mainly enzymatic (80%) and renal (20%). Therapeutic indications include percutaneous coronary inter- vention and cardiac surgery in patients with heparin-induced thrombocytopenia or high risk of bleeding. Bivalirudin is the most studied drug in patients with HIT and requiring cardiac surgery or a transluminal angioplasty (see questions #10 and #11). Bivalirudin is currently no longer available in France. However, generics may be prescribed in other European countries. In 2016, Linkins et al. presented the results of the only prospective study that evaluated a DOAC in HIT [75]. This drug was rivaroxaban, but the strict inclusion criteria allowed only 22 patients to be included, with only 12 HIT cases conﬁrmed by the SRA. All patients were treated with rivaroxaban 15 mg bid, continued in case of conﬁrmed HIT until thrombocytopenia was corrected or until D21 in case of thrombosis. Then, the dosage was reduced to 20 mg per day (2 10) until D30. Of the 12 cases of HIT, 6 had thrombosis and/or adrenal haemorrhagic necrosis. It should also be noted that 6 patients had received fondaparinux before inclusion for 2 or 3 days. The evolution of platelet count was favourable in 9 of the 10 cases with initial thrombocytopenia after a delay varying between 3 and 29 days. The 10th patient died on D21 of metastatic gastric cancer. Only one patient had a thrombotic recurrence with an extension of venous thrombosis of the upper limbs. Direct oral anticoagulants 4. The argatroban-AVK switch is delicate because this antithrombin molecule prolongs the prothrombin time. After the introduction of VKA (coumadin), argatroban should only be stopped when INR value is at least equal to 4 according to the algorithm below (Fig. 3). 4. The argatroban-AVK switch is delicate because this antithrombin molecule prolongs the prothrombin time. After the introduction of VKA (coumadin), argatroban should only be stopped when INR value is at least equal to 4 according to the algorithm below (Fig. 3). Direct oral anticoagulants (DOAs) currently include a thrombin inhibitor (dabigatran or Pradaxa1) and several Xa inhibitors, rivaroxaban (Xarelto1), apixaban (Eliquis1) and edoxaban (Lixiana1) more recently approved in France. DOACs are widely prescribed in atrial ﬁbrillation but also in venous thromboembolic disease, particularly Xabans. These reasons explain why they were logically proposed to treat HIT. Table 2 Table 2 Adjustment of argatroban dosage according to Acute Physiology And Chronic Health Evaluation (APACHE II), Sequential Organ Failure Assessment (SOFA) and Simpliﬁed Acute Physiology Score (SAPS) scores. According to Alatri et al. [55]. Score APACHE II Argatroban (mg/kg/min) Score SOFA Argatroban (mg/kg/min) SAPS Argatroban (mg/kg/min) 15 1.25 10 1.28 30 1.16 16 1.19 11 1.19 32 1.10 17 1.13 12 1.10 34 1.04 18 1.07 13 1.01 36 0.98 19 1.01 14 0.92 38 0.92 20 0.95 15 0.83 40 0.86 21 0.89 16 0.74 42 0.82 23 0.77 17 0.65 44 0.74 25 0.65 18 0.56 46 0.68 27 0.53 19 0.47 50 0.56 29 0.41 20 0.38 55 0.41 32 0.23 21 0.29 60 0.26 ording to Acute Physiology And Chronic Health Evaluation (APACHE II), Sequential Organ Failure Assessment (SOFA) and Simpliﬁe According to Alatri et al. [55]. ment of argatroban dosage according to Acute Physiology And Chronic Health Evaluation (APACHE II), Sequential Organ Failure As hysiology Score (SAPS) scores. According to Alatri et al. [55]. Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 300 maintained to reduce the risk of bleeding. However, the level of anticoagulation may not be optimal in some cases with pre- therapeutic prolongation of aPTT [67]. treatment with fondaparinux is lower than that of danaparoid or argatroban [71]. Fondaparinux is eliminated exclusively by the kidney and haemorrhages associated with its use in renal failure have been reported, particularly after cardiac surgery [72]. This drug should therefore not be used in cases of severe renal failure and should be avoided if the patient’s clinical condition is unstable. Other more speciﬁc tests with a linear dose-response relation- ship are available [61]: ecarin clotting time (ECT) and diluted thrombin time (TTd), which both allow the accurate measurement of circulating argatroban levels, with an ideal target value between 0.25 and 1.5 mg/mL [61]. A prescription and monitoring algorithm using aPTT and TTd, potentially useful in clinical practice, was proposed by Rozec et al. in 2014 [64] (Fig. 2). Direct oral anticoagulants Bivalirudin Another study involving 9 patients with HIT complicated by thrombosis also reported an efﬁcacy of rivaroxa- ban since in all cases the clinical and biological evolution was favourable [76]. Bivalirudin is administered exclusively intravenously, has no antidote, and is partially haemodialysable (25%). Fondaparinux Fondaparinux Fondaparinux (Arixtra1 in France), is a synthetic pentasac- charide speciﬁcally inhibiting factor Xa, and has been used for many years in the treatment of HIT, although it was not licensed in this indication, and few cases have been published without any controlled studies. In 2012, the British guidelines suggested that fondaparinux could be used to treat HIT but only with curative doses [3], taking into account the patient weight (5 mg if < 50 kg, 7.5 mg if 50– 100 kg, and 10 mg if > 100 kg), age and kidney function. Since then, other data have supported the use of fondaparinux. A retrospective study compared 133 patients treated with fondapa- rinux with matched control patients using a propensity score. The efﬁcacy and safety of fondaparinux was considered comparable to that of argatroban or danaparoid administered to patients in the control group [69]. Analysis of a German registry of 195 HIT patients also showed that nearly half of them (n = 83, 43.1%) were treated with fondaparinux despite not licensed in this indication, without complication or death, while 11.7% of cases treated with an approved anticoagulant (sodium danaparoid, and argatroban in particular) had complications (thrombosis, skin necrosis, ampu- tations) with an intra-hospital mortality of 14.4% [70]. These data explain why the ASH Expert Group proposed fondaparinux as an acceptable therapeutic option for the treatment of HIT in 2018 [4], but preferably in a stable patient (Proposal # 24). In 2017, Warkentin et al. reviewed the literature according to whether patients were treated by a DOAC in the acute or subacute phase of HIT as 1st or 2nd line after at least one dose of fondaparinux, danaparoid sodium, argatroban or bivalirudin. They concluded that DOACs are a possible option for the treatment of HIT. Based on this analysis and data from an additional study [77] (Table 3), ASH experts [4] proposed rivaroxaban to treat patients who do not have life-threatening and/or functional thrombosis, while injectable anticoagulants were preferred in more severe cases. Apixaban, which is also an anti-Xa with a good beneﬁt/risk ratio, is probably also an option in the same way as rivaroxaban. Apixaban, which is also an anti-Xa with a good beneﬁt/risk ratio, is probably also an option in the same way as rivaroxaban. Fig. 2. Algorithm for prescribing and monitoring argatroban in heparin-induced thrombocytopenia (HIT). Table 3 Proposal # 25 It is recommended not to prescribe an oral antiplatelet agent to treat acute HIT. ( ) In addition, the British and ASH proposed that VKA should be stopped in patients with acute or subacute HIT previously treated, and vitamin K injected before starting the non-heparin anticoagu- lant [3,4]. Fondaparinux Our proposal is therefore that DOACs may in some cases be an option for the treatment of HIT (proposals #15 and #24), but their prescription should not lead to neglecting the necessary approach to the diagnosis of HIT, and in particular the prescription of conﬁrmatory biological tests. There are also several arguments in favour of using fondapa- rinux in HIT: it has no cross-reactivity with anti-PF4 antibodies, unlike sodium danaparoid; it is easy to administer (one daily subcutaneous injection), and requires no dosage adjustment or speciﬁc bioassay; it has no effect on aPTT, whose prolongation thus shows more reliably an underlying coagulopathy, nor on Quick time and INR, which facilitates a relay by AVK; ﬁnally, the cost of Our proposal is therefore that DOACs may in some cases be an option for the treatment of HIT (proposals #15 and #24), but their prescription should not lead to neglecting the necessary approach to the diagnosis of HIT, and in particular the prescription of conﬁrmatory biological tests. Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 301 Fig. 2. Algorithm for prescribing and monitoring argatroban in heparin-induced thrombocytopenia (HIT). Fig. 2. Algorithm for prescribing and monitoring argatroban in heparin-induced thrombocytopenia (HIT). Fig. 3. Modalities of the argatroban-vitamin K antagonist (VKA) switch according to Rozec et al. [64]. Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 302 Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 s of the argatroban-vitamin K antagonist (VKA) switch according Fig. 3. Modalities of the argatroban-vitamin K antagonist (VKA) switch according to Rozec et al. [64]. Table 3 Table 3 Main results obtained with anti-Xa DOACs in the treatment of HIT; according to Warkentin et al. [77], Davis and Davis [112] and Cuker et al. [4]. DOAC n HIT with thrombosis DOAC as a 1st treatment Thrombosis Major bleeds Rivaroxaban 49 31 (63%) 25 (51%) 1/49 0/49 Apixaban 21 8 (38%) 7 (33%) 0/21 0/21 Dabigatran 11 6 (55%) 3 (27%) 1/11 0/11 HIT: heparin-induced thrombocytopenia. treatment of HIT; according to Warkentin et al. [77], Davis and Davis [112] and Cuker et al. [4]. Main results obtained with anti-Xa DOACs in the treatment of HIT; according to Warkentin et al. [77], Davis and Davis [ that will guide the choice. The duration of non-heparin anticoagu- lant therapy is at least 4 weeks in patients with isolated thrombocytopenia, and at least 3 to 6 months in other cases depending on the site and severity of associated thrombosis [3,4]. Rivaroxaban with curative doses (15 mg 2/day until D21 or complete and stable correction of thrombocytopenia), then 20 mg/ day for at least one month, whether or not there are thrombotic complications, is preferred as the most evaluated DOAC in this situation. Question 8: Is there a place for other treatments in a patient with suspected HIT? Proposal # 24 It is recommended not to transfuse platelets in the acute phase of HIT in the absence of life-threatening or functional bleeding. (STRONG AGREEMENT) Proposal # 25 It is recommended not to prescribe an oral antiplatelet agent to treat acute HIT. (STRONG AGREEMENT) Proposal # 26 It is proposed not to prescribe IV immunoglobulins as a ﬁrst- line treatment for the acute phase of HIT. (STRONG AGREEMENT) Proposal # 27 It is proposed not to insert an inferior vena cava ﬁlter in the acute phase of HIT. (STRONG AGREEMENT) Question 8: Is there a place for other treatments in a patient with suspected HIT? Vitamin K antagonists (VKA) VKA should never be used alone in the acute phase of HIT as they can promote the spread of venous thrombosis, their progression to gangrene or the development of skin necrosis at this stage [7]. VKA are administered only under the cover of effective parenteral anticoagulant therapy (danaparoid sodium or argatroban). Coumadin is preferred and administered as soon as possible, when platelet re-ascension is conﬁrmed (platelets > 150 G/L). The danaparoid sodium-coumadin and argatroban- coumadin switches require precautions as described above. Proposal # 29 Proposal # 29 In case of surgery in a patient treated with an oral anticoag- ulant and having acute HIT (less than 1 month), it is proposed to stop this anticoagulant and discuss a preoperative bridging with argatroban or bivalirudin, with stopping the infusion 4 hours before the procedure for argatroban and 2 hours for bivalirudin. 1. Polyvalent intravenous immunoglobulins have been used in a few cases of HIT [78–80] but their efﬁcacy remains poorly documented. They cannot be recommended for the treatment of acute HIT. However, they have recently been proposed for severe and very rare so-called ‘‘autoimmune’’ HIT [81]. (STRONG AGREEMENT) (STRONG AGREEMENT) 2. Plasmapheresis or plasma exchanges have also been exceptionally used in acute HIT and mainly in the context of urgent cardiovascular surgery [82] (see question 10). However, their relative beneﬁt compared to powerful antiplatelet drugs such as ilomedine or tiroﬁban has not been evaluated. Rationale In patients receiving long-term APA therapy for atheromatous disease (e.g. coronary artery disease, lower limb arteriopathy) with HIT, the decision to continue APA is made taking into account the risk of bleeding and vascular risk. In all cases, the diagnosis of HIT (4T score, biological results, thrombotic complication) should be conﬁrmed and the referring haemostasis team in the region should be contacted. In case of acute HIT, it is preferable to postpone the surgical procedure for at least 1 month, especially if the patient has had HIT with thrombosis. GPIIb-IIIa receptor antagonists have been used successfully in rare cases of acute coronary occlusion after angioplasty during HIT. Tiroﬁban (Agrastat1) is an option to be discussed only in case of cardiac surgery (see question 10). Iloprost (Ilome´dine1), a prostacyclin analogue, induces risks of severe hypotension, and is rarely used today (see question 10). However, it remains available as a therapeutic option by ASH in the event of emergency cardiac surgery [4]. In case of procedures usually performed under heparin (vascular surgery, coronary angioplasty, endovascular procedure), it is recommended to look for anti-PF4/heparin antibodies (ELISA test). If they are undetectable, short-term treatment with unfractionated heparin is possible during the procedure. 4. LMWH are contraindicated in UFH-HIT patients because they often result in in vivo platelet activation associated with a risk of persistent thrombocytopenia with thrombosis. Preoperatively 5. Thrombolytics can exceptionally be discussed for the management of serious thrombotic complications sometimes observed during HIT. 5. Thrombolytics can exceptionally be discussed for the management of serious thrombotic complications sometimes observed during HIT. 1. If the patient is treated for recent HIT with a parenteral anticoagulant (danaparoid, argatroban, bivalirudin, or fondapari- nux), the surgical procedure should be deferred if possible. Otherwise, the discontinuation of the anticoagulant will be decided on a case-by-case basis, taking into account its elimination half-life and the haemorrhagic risk associated with the surgical procedure. Given the long half-life of danaparoid and fondapari- nux, a relay with argatroban or bivalirudin may be proposed if the risk is considered too high. Biological assays, if available, are helpful in the management (Table 4). 6. VKA should never be used alone as they expose the patient to an increased risk of thrombosis and skin necrosis (see question 7). 6. VKA should never be used alone as they expose the patient to an increased risk of thrombosis and skin necrosis (see question 7). 7. Platelet transfusion is not recommended because it can promote the onset of thrombosis [83] or the process of consumption, and is often ineffective. Platelet transfusions are therefore only considered in cases of severe bleeding. 8. The insertion of a vena cava ﬁlter should only be discussed in cases of severe pulmonary embolism with a high risk of bleeding and transient contraindication of anticoagulants, as this procedure is associated with a risk of acute thrombotic obliteration. 2. If the patient is treated with an oral anticoagulant (VKA or DOAC), discontinuation of treatment is managed according to usual recommendations [83,84]. Preoperative relaying of the oral anticoagulant with a non- heparinic injectable anticoagulant will only be discussed in situations of high thrombotic risk (HIT < 1 month, thromboem- bolic event < 3 months or recurrent, mechanical heart valve, atrial ﬁbrillation with embolic history). In this case, the proposed anticoagulant is either argatroban or bivalirudin due to their short half-life [63]. 9. Surgical thrombectomy is performed exceptionally when ischemia threatens the functional prognosis of the limb(s) and/or the vital prognosis. Proposal # 30 In postoperative care, if prolonged anticoagulation is needed and the risk of bleeding is controlled, it is proposed to treat the patient preferentially with fondaparinux or an oral anticoagu- lant (VKA or DOAC), taking into account contraindications and precautions of use. 3. Antiplatelet agents (APA) cannot be used alone to treat HIT. The value of combining APA with a non-heparin anticoagulant may be discussed in some cases of HIT with severe arterial thrombotic complications. However, this combination in- creases the risk of bleeding and its effectiveness has not been validated. (STRONG AGREEMENT) Rationale Practical modalities for the use of non-heparin anticoagulants in the acute phase of HIT Practical modalities for the use of non-heparin anticoagulants in the acute phase of HIT Proposal # 26 It is proposed not to prescribe IV immunoglobulins as a ﬁrst- line treatment for the acute phase of HIT. (STRONG AGREEMENT) Proposal # 26 It is proposed not to prescribe IV immunoglobulins as a ﬁrst- line treatment for the acute phase of HIT. (STRONG AGREEMENT) The choice of treatment (argatroban, bivalirudin, danaparoid, anti-Xa DOACs or fondaparinux) will be inﬂuenced by several factors, some of which are speciﬁc to the usable drugs (availability, biological monitoring modalities, route of administration, ½ elimination life, cost) and others are more patient-related (renal and hepatic functions, clinical condition, severity of associated thrombosis, risk of spontaneous bleeding or caused by a procedure). The clinician’s experience will also be an element Proposal # 27 It is proposed not to insert an inferior vena cava ﬁlter in the acute phase of HIT. (STRONG AGREEMENT) Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 303 Rationale Proposal # 29 I f i ti t t t d ith l ti Apart from anticoagulants, other therapeutic approaches can be discussed but their place is very limited in practice. Table 4 Last injection > 36 h before surgery Stop infusion or last SC injection > 36 h before surgery Stop infusion 4 h before surgery Stop infusion 2 h before surgery HIT: heparin-induced thrombocytopenia. Question 10: Which treatment should be offered for cardiac surgery with and without cardiopulmonary bypass in the case of HIT? 8 hours after stopping the infusion; for DOACs, last dose at D-5 or plasma concentration < 30 ng/mL; for danaparoid and fondapari- nux, the objective is to obtain levels < detection threshold i.e. < 0.1 U anti-Xa/mL for danaparoid, and < 0.1 mg/mL for fondaparinux. A stop of more than 48 hours is probably necessary in most cases. Proposal # 31 Before any cardiac surgery in a patient with a documented history of HIT, it is proposed to systematically perform an ELISA for anti-PF4 antibodies. (STRONG AGREEMENT) However, in the case of acute HIT (< 1 month), the relatively long stopping times of DOACs, danaparoid, and fondaparinux expose the patient to a high thrombotic risk. If they are only justiﬁed by a neuraxial procedure, another type of anaesthesia should be considered. Proposal # 32 Before any cardiac surgery with cardiopulmonary bypass in a patient with acute or subacute HIT (< 3 months), it is pro- posed to deﬁne the perioperative anticoagulation protocol in a multidisciplinary consultation. This part only concerns procedures that require anticoagulant treatment during the surgery. If time permits, a search for anti-PF4/ heparin antibodies (ELISA test) is recommended; if the antibodies are no longer detectable, exposure to heparin is possible during the procedure. (STRONG AGREEMENT) p If anti-PF4/heparin antibodies are present, it is proposed to discuss the administration of danaparoid, argatroban or bivaliru- din according to the available protocols, renal status and liver status of the patient (see question 6). Rationale the experience in the service and availability of biological surveillance tests (measurement of anti-Xa activity – danapa- roid range, argatroban assay rather than aPTT, with the help of a haemostasis specialist); Unscheduled cardiac surgery in a patient with acute HIT is one of the most difﬁcult situations to manage, as unfractionated heparin remains the anticoagulant of choice for extracorporeal circulation, and as the available alternatives do not offer the same beneﬁt/risk ratio. if renal failure: no accumulation with argatroban, close follow- up of anticoagulation with danaparoid (anti-Xa activity – danaparoid range); In any case, it is appropriate (Fig. 4): if liver failure: calculation of the Child-Pugh score, with in the case of a value > 6, an elimination half-life of argatroban that increases from 50 to 152 minutes [62]. to have conﬁrmed the diagnosis of HIT (4T score, biological results, thrombotic complication); to have conﬁrmed the diagnosis of HIT (4T score, biological results, thrombotic complication); to contact the leading haemostasis team in the region; to postpone the procedure if possible for more than 3 months after HIT, or at least 1 month after a possible thrombotic complication; In case of HIT < 1 month, danaparoid should preferably be prescribed by IV route and with curative doses, adjusting the dosage on anti-Xa activity maintained between 0.5 and 0.8 U/mL (danaparoid range) (see question 7). to perform an ELISA test for anti-PF4/heparin antibodies: if it is negative, the procedure is identical to that to be followed for an old HIT (see question 11) and short-term re-exposure to heparin is possible, particularly for cardiopulmonary bypass. Argatroban IV will be prescribed at a low initial dose (0.5 mg/kg/ minute), then adjusted to aPTT or better plasma concentration, with a target value between 0.5 and 1.5 mg/mL [61]. If prolonged anticoagulant therapy is required, and considering that danaparoid and argatroban are subject to hospital prescrip- tion, it is proposed to prescribe fondaparinux or preferably oral anticoagulant, AVK or AOD (rivaroxaban) in the event of long-term anticoagulant therapy. Postoperatively Anticoagulant treatment can be resumed from the 6th postoperative hour, after assessment of the risk of bleeding. The choice of the drug to be prescribed is based on: In case of urgent surgery, it is proposed to favour an association antiplatelet IV + UFH. (STRONG AGREEMENT) the elimination half-life in case of risk of haemorrhage and/or surgical resumption: 50 minutes for argatroban, 24 hours (anti- Xa activity) for danaparoid, 17 hours for fondaparinux; Rationale Proposal # 33 In a patient with acute or subacute HIT with a signiﬁcant titre of anti-PF4 antibody (ELISA with DO > 1) and requiring cardiac surgery with cardiopulmonary bypass, possible strategies for intraoperative anticoagulation are to combine an IV antiplatelet agent (tiroﬁban or cangrelor) and UFH, or to administer a direct antithrombin agent (bivalirudin or argatroban) with close biological monitoring. Local and regional anaesthesia The performance of neuraxial procedures is contraindicated under anticoagulant. These procedures include diagnostic or therapeutic lumbar punctures, spinal anaesthesia and therapeutic spinal injections, whether or not guided by radio, as well as epidurals with or without catheters. Proposal # 28 In a patient with acute HIT (less than 1 month), it is proposed to postpone any surgery beyond the ﬁrst month following the diagnosis of HIT if this does not generate a major vital or functional risk for the patient, and to deﬁne the modalities during a multidisciplinary consultation. A neural gesture can be performed after the following stopping times: for argatroban: 8 hours after stopping the infusion and if a plasma dose conﬁrms a level < 0.1 mg/mL [62]; for bivalirudin: (STRONG AGREEMENT) (STRONG AGREEMENT) Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 304 Table 4 Management of anticoagulants used in HIT before surgery. Anticoagulant Half-life Proposed management Fondaparinux About 17 h (anti-Xa) Last injection > 36 h before surgery Danaparoid About 24 h (anti-Xa) and 7 h (anti-Iia) Stop infusion or last SC injection > 36 h before surgery Argatroban About 50 minutes Stop infusion 4 h before surgery Bivalirudin About 20–30 minutes Stop infusion 2 h before surgery HIT: heparin-induced thrombocytopenia. Fig. 4. Therapeutic strategies for cardiac surgery in heparin-induced thrombocytopenia (HIT) patients. Fig. 4. Therapeutic strategies for cardiac surgery in heparin-induced thrombocytopenia (HIT) patients. into account the team’s experience, the available drugs and the biological tests performed [86,87]. and re-exposure to unfractionated heparin without particular caution is therefore a priori contraindicated, as it exposes the patient to a recurrence of thrombocytopenia or even thrombosis. In the preoperative period However, this position is discussed because the ASH recom- mendations distinguish two types of patients with subacute HIT (with normal platelet count) and residual anti-PF4 antibodies. The ﬁrst are those for whom a platelet activation test (mainly SRA) is positive. In these cases, HIT is considered to be subacute type A and the administration of heparin for cardiac surgery should be avoided. The second group is patients with a negative SRA, and considered to have subacute HIT type B, and for ASH experts heparin use during cardiac surgery is possible [4]. The proposals for anticoagulant treatment are identical to those for conventional surgery (see question 9). Plasma exchanges that are intended to minimise the level of circulating antibodies are also possible. They can be performed preoperatively or in the operating theatre with a volume of FFP equal to the theoretical plasma volume 1.3 and before heparin is administered during the procedure. However, data on this strategy are limited [82,88,89]. In addition, the objective to be achieved is poorly deﬁned (negative ELISA? negativation of the SRA?) and in most cases, a signiﬁcant titre of anti-PF4/heparin antibodies persists in ELISA [89]. This distinction and the resulting position are, in our opinion, highly questionable because SRA may not be sensitive enough to formally eliminate the presence of potentially pathogenic anti- bodies in the patient’s plasma during cardiac surgery. This is all the more true since it has been shown that the addition of exogenous PF4 allows SRA to be positive [85], and cardiopulmonary bypass induces a signiﬁcant and rapid increase in plasma PF4 concentra- tion in operated patients. If HIT is acute or subacute (< 3 months) In this situation, the patient often has a residual titre of anti-PF4 antibodies, especially if one is very close to HIT (within a month) Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 305 The second uses a thrombin inhibitor anticoagulant: bivalirudin or argatroban The second uses a thrombin inhibitor anticoagulant: bivalirudin or argatroban Intraoperatively Two strategies are possible. The anti-PF4 antibody titre estimated in ELISA is also important to deﬁne because, when elevated with an OD greater than 1.5, the clinical risk is likely to be higher after re-exposure to heparin, compared to a patient with a lower antibody concentration (with an OD < 1). The ﬁrst combines an intravenous antiplatelet agent and unfractionated heparin The ﬁrst combines an intravenous antiplatelet agent and unfractionated heparin This strategy consists of inhibiting platelet aggregation before administering heparin so that IgG-PF4-heparin complexes cannot induce platelet activation or thrombus formation. Heparin is injected as a bolus, monitored with ACT and neutralised by protamine on the end of surgery as usual. This strategy is limited to It is therefore essential that in all cases, the anticoagulation protocol be deﬁned within the framework of multidisciplinary consultation (anaesthetist, surgeon, haemostasis specialist), taking Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 306 the cardiac surgery operating room, and any other administration of heparin is strictly contraindicated (heparin lock, catheter ﬂush, routine administration). Its use involves a bolus of 100 mg/kg followed by a continuous IV infusion at 5 mg/kg/min with an ACT evaluation > 400 seconds to start (ECC), then every 15 minutes with a maintenance between 500 and 600 seconds during the procedure. Very prolonged ACTs were observed, complicating the adjustment of the infusion rate [64,93]. Two antiplatelet agents can be used: tiroﬁban, a GPIIbIIIa platelet receptor inhibitor, is administered as an IV bolus (10 mg/ kg) followed 15 minutes later by the usual injection of heparin, then a continuous IV infusion (0.15 mg/kg per minute) is initiated, stopped 1 hour before the end of cardiopulmonary bypass, at the time of aortic declamping [90]. However, its prolonged effect exposes the patient to bleeding at the end of the bypass and in immediate postoperative care because correction of platelet function is obtained only 8 hours after stopping the tiroﬁban infusion. Renal failure extends this time. In addition, early discontinuation of the infusion is a problem in the event of a resumption of cardiac surgery. Bivalirudin is also a possible option for emergency cardiac surgery in the acute phase of HIT [4,94], although Angiox1 has no longer been marketed in France since the end of 2017, but generics should soon be available. Bivalirudin is prescribed and monitored as deﬁned in Table 5. If ACT < 300 seconds, increase infusion rate by 0.25 mg/kg per hour If ACT < 2.5 basic ACT, additional bolus from 0.1 to 0.5 mg/kg Possible measurement of bivalirudin level in whole blood (expert opinion) In postoperative care Anticoagulant treatment can be resumed from the 6th postoperative hour, after assessment of the risk of bleeding: as a preventive dose with danaparoid or fondaparinux, or with curative doses of argatroban rather than danaparoid, considering its short half-life (50 minutes) starting with an initial dose of 0.5 mg/kg per minute, or deﬁned according to APACHE II, SAPS 2 or SOFA (see question 7) [63]. In cases of severe liver failure (Child-Pugh C score), danaparoid is preferred. Cangrelor, a P2Y12 platelet receptor inhibitor, has the advan- tage of an immediate effect, obtained in 2 min and a short half-life of 3 to 6 min. The platelet inhibition it induces is stable during infusion and not affected by renal or hepatic failure or blood stagnation. However, its use in the case of HIT is still poorly documented. The proposed protocol is as follows: intravenous bolus of 30 mg/kg 10 minutes before heparin administration according to the usual regimen, immediately followed by an IV infusion of 4 mg/kg/min, interrupted 5 min before the CPB is stopped [91,92]. Monitoring of platelet function has been proposed using VerifyNow1, but target values are not deﬁned. When ECMO is used, the possible options are argatroban and danaparoid, the former being easier to manage given its short half-life. However, patients’ organ failures alter the pharmacokinetics of argatroban and require dosage reductions, often below 0.5 mg/kg/min (see question 7) at the outset of treatment. Frequent biological monitoring is necessary to adapt the doses to the patient’s evolution and failures. Diluted thrombin time or ecarin time is preferable to aPTT, which is often already prolonged. Danaparoid, with its long half-life, is difﬁcult to use in this context of high risk of bleeding and invasive procedures. Iloprost (Ilome´dine1) is still sometimes used. This prostacyclin analogue has a half-life of 15 to 30 min. It can be administered as an infusion of 6 to 12 ng/kg/min, stopped 20 minutes before protamine. However, it does expose to episodes of severe low blood pressure. If ACT < 300 seconds, increase infusion rate by 0.25 mg/kg per hour If ACT < 2.5 basic ACT, additional bolus from 0.1 to 0.5 mg/kg Possible measurement of bivalirudin level in whole blood (expert opinion) HIT: heparin-induced thrombocytopenia. In the case of surgery with HIT in remission (> 3 months) Argatroban is an option but experience is limited and bleeding and/or thrombotic complications (presence of clots in the cardiotomy reservoir or pericardium) have been reported at the time of the bypass procedure. It was not proposed in the 2012 ACCP recommendations that favoured bivalirudin, and it remains poorly or not recommended by some experts in this context [4,87]. Cardiac surgery with or without CPB may be performed under heparin, according to the usual protocol. However, it is recom- mended to test for anti-PF4/heparin antibodies, as the persistence of pathogenic antibodies for several months or even for a prolonged period has been reported in rare cases. Table 5 Prescription of bivalirudin in HIT (contraindication in severe renal failure: creatinine clearance < 30 mL/min) Dosage Monitoring Medical treatment of HIT (few data) Start IV infusion at 0.15–0.25 mg/kg per hour aPTT target: 1.5 to 2.5 control time Coronary angioplasty IV bolus of 0.75 mg/kg then IV infusion at 1.75 mg/kg per hour during the procedure and 4 hours maximum after the procedure (if creatinine clearance between 30 and 59 mL/min: start infusion at 1.4 mg/kg per hour) If ACT after IV bolus < 225 seconds, 2nd bolus of 0.3 mg/kg Cardiac surgery Without CPB IV bolus of 0.75 mg/kg and IV infusion at 1.75 mg/kg per hour during the procedure If ACT < 300 seconds, increase infusion rate by 0.25 mg/kg per hour With CPB IV bolus of 1 mg/kg + 50 mg in pump priming ﬂuid then IV infusion at 2.5 mg/kg per hour during the procedure Stopping the infusion 15 minutes before the announced end of the CPB (if still in CPB 20 minutes, bolus IV 0.5 mg/ kg and restart the IV infusion at 2.5 mg/kg per hour) Caution should be exercised when considering the enzymatic degradation of bivalirudin (cleavage by thrombin): avoid pericardial aspiration, avoid blood stasis If ACT < 2.5 basic ACT, additional bolus from 0.1 to 0.5 mg/kg Possible measurement of bivalirudin level in whole blood (expert opinion) HIT: heparin-induced thrombocytopenia. The patient is not treated with argatroban The patient is not treated with argatroban The patient is not treated with argatroban Teams mastering this technique can consider the realisation of RRT with citrate. Argatroban can also be the anticoagulant used for circuit anticoagulation. It is then administered as a bolus (100 mg/ kg for continuous RRT or 250 mg/kg for intermittent haemo- dialysis) followed by a continuous infusion according to its conventional regimen (see question 7). The infusion is stopped one hour before the end of the session [63]. Proposal # 36 In case of HIT during pregnancy, it is proposed to treat patients preferentially with danaparoid or if this drug is not available, with fondaparinux. Danaparoid accumulates in renal failure. Its use for continuous RRT is delicate, with very high doses proposed in this context of risk of bleeding. In case of intermittent haemodialysis, the danaparoid SPC proposes the following protocol: bolus of 3750 U (2500 U if weight < 55 kg) before the ﬁrst two sessions and 3000 U (2000 U if weight < 55 kg) [88]. Proposal # 37 It is proposed that the procedures for monitoring platelet counts in children treated with heparin should be the same as those recommended for adults. ( ) Rationale Rationale The general principles of HIT treatment in medical settings do not differ from those applied to surgical patients. Nevertheless, we will address several speciﬁc situations: acute coronary syndrome, renal failure, pregnancy and childhood. In cardiac surgery, the development of anti-PF4 heparin antibodies in postoperative care can affect 3 to more than 50% of operated children, with the incidence of immunisation increasing with the number of procedures [104]. But, as in adults, HIT will affect only a small proportion of immunised children with a long overestimated risk, but which has recently been assessed as intermediate, equal to 0.33% [105]. Therefore, as in adults, platelet count monitoring should be maintained in all children treated with UFH regardless of age and underlying context. Proposal # 38 Proposal # 38 It is proposed that treatment of HIT in children should be provided by sodium danaparoid or argatroban, with rigorous dose adjustment based on weight and bioassay results. HIT and severe renal failure HIT and severe renal failure Proposal # 34 In the case of acute HIT, it is proposed to treat patients requiring transluminal angioplasty for acute coronary syn- drome, preferably with bivalirudin or an analogue, or failing that with argatroban. In the case of renal replacement therapy (RRT), two situations can be distinguished. In the case of surgery with HIT in remission (> 3 months) Prescription of bivalirudin in HIT (contraindication in severe renal failure: creatinine clearance < 30 mL/min) IV bolus of 0.75 mg/kg and IV infusion at 1.75 mg/kg per hour during the procedure IV bolus of 1 mg/kg + 50 mg in pump priming ﬂuid then IV infusion at 2.5 mg/kg per hour during the procedure Stopping the infusion 15 minutes before the announced end of the CPB (if still in CPB 20 minutes, bolus IV 0.5 mg/ kg and restart the IV infusion at 2.5 mg/kg per hour) Caution should be exercised when considering the enzymatic degradation of bivalirudin (cleavage by thrombin): avoid pericardial aspiration, avoid blood stasis Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 307 Question 11: Which treatments are available for HIT in medicine, obstetrics or paediatrics? Proposal # 34 In the case of acute HIT, it is proposed to treat patients requiring transluminal angioplasty for acute coronary syn- drome, preferably with bivalirudin or an analogue, or failing that with argatroban. (STRONG AGREEMENT) Proposal # 35 In the case of HIT, it is proposed for renal replacement therapy to use citrate or argatroban as the preferred treatment for circuit anticoagulation. (STRONG AGREEMENT) Proposal # 36 In case of HIT during pregnancy, it is proposed to treat patients preferentially with danaparoid or if this drug is not available, with fondaparinux. (STRONG AGREEMENT) Proposal # 37 It is proposed that the procedures for monitoring platelet counts in children treated with heparin should be the same as those recommended for adults. (STRONG AGREEMENT) Proposal # 38 It is proposed that treatment of HIT in children should be provided by sodium danaparoid or argatroban, with rigorous dose adjustment based on weight and bioassay results. (STRONG AGREEMENT) Question 11: Which treatments are available for HIT in medicine, obstetrics or paediatrics? The removal of the desilet will be performed two or four hours after stopping bivalirudin or argatroban, respectively. The patient is being treated with argatroban The patient is being treated with argatroban (STRONG AGREEMENT) Renal failure has little inﬂuence on the pharmacokinetics of argatroban, as does RRT. RRT is started without an argatroban bolus, continuing infusion and biological monitoring. Proposal # 35 In the case of HIT, it is proposed for renal replacement therapy to use citrate or argatroban as the preferred treatment for circuit anticoagulation. HIT in children (STRONG AGREEMENT) The risk of HIT is a priori considered to be lower in children with lower circulating levels of PF4 [102]. For example, a recent study found an HIT prevalence of 0.058% in a single paediatric centre [103]. The same study identiﬁed 12 children with suspected HIT, most of whom were hospitalised in surgery and treated with UFH. HIT and pregnancy Pregnancy is very rarely complicated by HIT. Danaparoid sodium, which does not cross the placenta, has been the most commonly used anticoagulant in this context [99] and is therefore recommended as a ﬁrst-line treatment, particularly by the British [3]. Argatroban is contraindicated, as are DOACs. (STRONG AGREEMENT) If danaparoid is not available, fondaparinux is an option that can also be proposed [3] although there is little data [100,101]. The secondary prevention, and therefore of a recurrence of HIT, is based on three measures: [9] Warkentin TE, Kelton JG. A 14-year study of heparin-induced thrombocyto- penia. Am J Med 1996;101(5):502–7. establish for each patient who has had an HIT a medical certiﬁcate or card attesting to the reality of this history and the results of the biological tests that led to the diagnosis. Permanent wearing of this document by the patient is particularly important within 3 months of HIT because the risk of recurrence is higher during this period if there is new exposure to heparin, although it is not constant [10]; [10] Warkentin TE, Kelton JG. Temporal aspects of heparin-induced thrombocy- topenia. N Engl J Med 2001;344(17):1286–92. p g J ( ) [11] Warkentin TE. Fondaparinux: does it cause HIT? Can it treat HIT? Expert Rev Hematol 2010;3(5):567–81. [12] Prandoni P, Siragusa S, Girolami B, Fabris F. The incidence of heparin-induced thrombocytopenia in medical patients treated with low-molecular-weight heparin: a prospective cohort study. Blood 2005;106(9):3049–54. [13] Kato S, Takahashi K, Ayabe K, Samad R, Fukaya E, Friedmann P, et al. Heparin- induced thrombocytopenia: analysis of risk factors in medical inpatients. Br J Haematol 2011;154(3):373–7. perform a sensitive biological test (ELISA) to test the absence or persistence of PF4-speciﬁc antibodies in the blood before any further exposure to heparin, as a high titre exposes the patient to a risk of HIT recurrence [109]; [14] Greer IA, Nelson-Piercy C. Low-molecular-weight heparins for thrombopro- phylaxis and treatment of venous thromboembolism in pregnancy: a sys- tematic review of safety and efﬁcacy. Blood 2005;106(2):401–7. preferably prescribe an oral anticoagulant (VKA or DOAC, dabigatran), or fondaparinux in case of a history of HIT, respecting the validated indications. Failing this in very speciﬁc situations for which no other option is possible, a non-heparinic injectable anticoagulant, danaparoid sodium or argatroban, will be used. It should be noted, however, that the risk of HIT recurrence after re-exposure of the patient to heparin is uncertain or even considered quite low, especially if LMWH is administered. It is higher if the patient is exposed to UFH for more than 5 days [110] and in the context of cardiac surgery [109]. [15] Martel N, Lee J, Wells PS. Risk for heparin-induced thrombocytopenia with unfractionated and low-molecular-weight heparin thromboprophylaxis: a meta-analysis. Blood 2005;106(8):2710–5. y ( ) [16] Junqueira DR, Zorzela LM, Perini E. Unfractionated heparin versus low molecular weight heparins for avoiding heparin-induced thrombocytopenia in postoperative patients. Cochrane Database Syst Rev 2017;4:CD007557. Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 Y. Gruel et al. / Anaesth Crit Care Pain Med 39 (2020) 291–310 308 Question 12: What prevention can be used to avoid the occurrence of HIT or recurrence? Proposal # 39 It is proposed that a haemostasis consultation be performed within 3 months of the diagnosis of HIT and that a card attesting this complication, specifying the results of the bio- logical tests and recommending the exclusion of all heparin treatment be given to the patient. (STRONG AGREEMENT) Proposal # 40 In case of a history of HIT, it is proposed to prescribe an oral anticoagulant (VKA or DOAC) or fondaparinux when a prophy- lactic or curative anticoagulation is indicated. Argatroban, biva- lirudin and danaparoid should only be considered in cases where oral anticoagulants and fondaparinux are contraindicated. (STRONG AGREEMENT) Question 12: What prevention can be used to avoid the occurrence of HIT or recurrence? Question 12: What prevention can be used to avoid the occurrence of HIT or recurrence? A. Godier has received consulting fees and non-ﬁnancial support from Bayer Healthcare, BMS/Pﬁzer, Boehringer-Ingelheim, Sanoﬁ-Adventis, LFB, CSL-Behring, and Octapharma. Y. Gruel reports consulting and/or travel fees from Aguettant, LFB, and Sanoﬁ; grant from Stago. G. Le Gal is co-investigator on grants received from Portola Pharma- ceuticals, Boehringer-Ingelheim, Pﬁzer, Bristol-Myers Squibb, LEO Pharma, Daiichi Sankyo, and Bayer. He has received speaker honoraria (not taken as salary) from Bayer, Pﬁzer, LEO Pharma, Sanoﬁ, and bioMe´rieux. G. Le Gal is co-investigator on grants received from Portola Pharma- ceuticals, Boehringer-Ingelheim, Pﬁzer, Bristol-Myers Squibb, LEO Pharma, Daiichi Sankyo, and Bayer. He has received speaker honoraria (not taken as salary) from Bayer, Pﬁzer, LEO Pharma, Sanoﬁ, and bioMe´rieux. E. de Maistre reports conﬂicts of interest with Stago, Boehringer- Ingelheim, Pﬁzer, BMS, and Bayer (advisory boards). E. de Maistre reports conﬂicts of interest with Stago, Boehringer- Ingelheim, Pﬁzer, BMS, and Bayer (advisory boards). (STRONG AGREEMENT) F. Mullier reports institutional fees from Stago, Werfen, Nodia, Sysmex and Bayer. He also reports speaker fees from Boehringer-Ingelheim, Bayer Healthcare, Bristol-Myers Squibb-Pﬁzer, Stago, Werfen and Aspen all outside the submitted work. C. Pouplard reports a grant from Stago. p In case of a history of HIT, it is proposed to prescribe an oral anticoagulant (VKA or DOAC) or fondaparinux when a prophy- lactic or curative anticoagulation is indicated. Argatroban, biva- lirudin and danaparoid should only be considered in cases where oral anticoagulants and fondaparinux are contraindicated. The secondary prevention, and therefore of a recurrence of HIT, is based on three measures: [17] Lubenow N, Hinz P, Thomaschewski S, Lietz T, Vogler M, Ladwig A, et al. The severity of trauma determines the immune response to PF4/heparin and the frequency of heparin-induced thrombocytopenia. Blood 2010;115(9):1797– 803. [18] Bloemen A, Testroote MJ, Janssen-Heijnen ML, Janzing HM. Incidence and diagnosis of heparin-induced thrombocytopenia (HIT) in patients with trau- matic injuries treated with unfractioned or low-molecular-weight heparin: a literature review. Injury 2012;43(5):548–52. [19] Craik JD, Cobb AG. Heparin-induced thrombocytopenia following hip and knee arthroplasty. Br J Haematol 2013;161(2):255–61. [20] Pouplard C, May MA, Iochmann S, Amiral J, Vissac AM, Marchand M, et al. Antibodies to platelet factor 4 – Heparin after cardiopulmonary bypass in patients anticoagulated with unfractionated heparin or a low-molecular- weight heparin – Clinical implications for heparin-induced thrombocytope- nia. Circulation 1999;99(19):2530–6. References [1] Socie´te´ franc¸aise d’anesthe´sie et de re´animation. Thrombope´nie induite par l’he´parine. Ann Fr Anesth Reanim 2003;22:150–9. [2] Linkins LA, Dans AL, Moores LK, Bona R, Davidson BL, Schulman S, et al. Treatment and prevention of heparin-induced thrombocytopenia: anti- thrombotic therapy and prevention of thrombosis, 9th ed: American College f h h d d l l d l h [2] Linkins LA, Dans AL, Moores LK, Bona R, Davidson BL, Schulman S, et al. Treatment and prevention of heparin-induced thrombocytopenia: anti- thrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141(2 Suppl.). e495S-e530S. (STRONG AGREEMENT) of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141(2 Suppl.). e495S-e530S. Rationale [3] Watson H, Davidson S, Keeling D. Guidelines on the diagnosis and manage- ment of heparin-induced thrombocytopenia: second edition. Br J Haematol 2012;159(5):528–40. The primary prevention of HIT is mainly ensured by three types of measures: [4] Cuker A, Arepally GM, Chong BH, Cines DB, Greinacher A, Gruel Y, et al. American Society of Hematology 2018. Guidelines for management of venous thromboembolism: heparin-induced thrombocytopenia. Blood Adv 2018;2(22):3360–92. prescribe heparins only in validated indications; prescribe heparins only in validated indications; preferably prescribe oral anticoagulants, or as a second-line choice LMWH, in validated indications, avoiding as much as possible the use of UFH; [5] Amiral J, Bridey F, Dreyfus M, Vissac A, Fressinaud E, Wolf M, et al. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia. Thromb Haemost 1992;68(1):95–6. [6] Greinacher A. Clinical practice. Heparin-induced thrombocytopenia. N Engl J Med 2015;373(3):252–61. limit the duration of heparin treatment to the shortest possible time (< 4–5 days) with early relay with oral anticoagulant. [7] Arepally GM. Heparin-induced thrombocytopenia. Blood 2017;129(21): 2864–72. [8] Pouplard C, Iochmann S, Renard B, Herault O, Colombat P, Amiral J, et al. Induction of monocyte tissue factor expression by antibodies to heparin- platelet factor 4 complexes developed in heparin-induced thrombocytopenia. Blood 2001;97(10):3300–2. The secondary prevention, and therefore of a recurrence of HIT, is based on three measures: HIT and acute coronary syndrome No randomised studies have compared non-heparin anti- coagulants in patients with HIT and acute coronary syndrome (ACS) requiring emergency transluminal angioplasty. Bivalirudin [95] and argatroban [96,97] were successfully administered in small groups of patients. A study conducted in 19,772 patients without HIT showed that bivalirudin induces a lower risk of bleeding [98]. The experience of danaparoid sodium in this situation (ACS and HIT) is patchy [57] and the longer elimina- tion half-life of this drug does not encourage its selection as a priority. Regarding the diagnosis, clinical data with the 4T score and biological analyses must be combined to exclude or afﬁrm HIT. For the treatment, danaparoid sodium and argatroban can be used, taking into account, as in adults, hepatic and renal functions to select the drug with the lowest risk of overdose [106–108]. A small series reported the history of 4 infants aged 3 to 7 months with HIT following cardiac surgery, and treated with low doses of argatroban, monitored with TCA and ACT [106]. Large variations in doses and test effects were noted and 3 out of 4 children survived. The recommended regimen for bivalirudin is a bolus of 0.75 mg/kg followed by an intravenous infusion of 1.75 mg/kg/ hour. For argatroban, a bolus of 350 mg/kg is proposed followed by an intravenous infusion of 25 mg/kg/min adapted to maintain an ACT between 300 and 450 seconds. Disclosure of interest P. Albaladejo reports conﬂicts of interest with Bayer, Pﬁzer-BMS, Sanoﬁ, Aspen, and Portola. N. Blais, D. Lasne, T. Lecompte, S. Roullet, S. Susen, A. Vincentelli declare that they have no competing interest. 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W4283314571.txt	https://www.detstom.ru/jour/article/download/492/341	en		Abnormal wear patterns in different types of malocclusions	Stomatologiâ detskogo vozrasta i profilaktika	2,022	cc-by	6,494	DOI: 10.33925/1683-3031-2022-22-2-111-121 Оригинальная статья І Original article Паттерны повышенной стираемости зубов при разных видах зубочелюстных аномалий А.А. Смирнова, О.А. Гаврилова, К.В. Федорова, Л.Н. Соколова, Д.В. Бобров Тверской государственный медицинский университет, Тверь, Российская Федерация АННОТАЦИЯ Актуальность. Повышенная стираемость зубов – заболевание, характеризующееся чрезмерной убылью твердых тканей прорезавшихся зубов, не связанное с физиологическим процессом. Отмечается рост распространенности этого заболевания в последние десятилетия. Причиной повышенной стираемости зубов в литературе часто указывают зубочелюстные аномалии, при этом редко отмечают влияние вида аномалии на отдельные зубы. Цель исследования: установление взаимосвязи между видом зубочелюстной аномалии и степенью и характером повышенной стираемости зубов у детей в разных периодах формирования прикуса. Материалы и методы. Были проанализированы результаты обследования 266 пациентов в возрасте от 8 до 18 лет, не проходивших когда-либо ранее ортодонтического лечения. Ортодонтический диагноз ставился по классификации МКБ-10. Пациенты были разделены на группы в зависимости от периода формирования прикуса. Оценка повышенной стираемости проводилась по индексу TWI (только постоянные зубы). Степень повышенного стирания зубов определялась по максимальному баллу индекса TWI. Статистическая обработка данных проводилась в программе StatTech v. 2.5.6. Сравнения выполнялись с помощью критерия Краскела – Уоллиса (ANOVA), с помощью критерия хи-квадрат Пирсона, апостериорные сравнения – с помощью критерия Данна с поправкой Холма. Прогностическая модель разрабатывалась с помощью метода линейной регрессии. Результаты. У детей и подростков наблюдалась достаточно высокая распространенность повышенной стираемости зубов (у 69,2% был хотя бы один зуб с фасеткой стирания), степень стираемости увеличивалась с возрастом (коэффициент корреляции rxy = 0,674, p < 0,001). Установлено, что частота развития повышенной стираемости зубов при дистальной и глубокой окклюзии увеличивалась у резцов и клыков (p < 0,001), при мезиальной окклюзии – моляров и премоляров, при открытом прикусе – моляров (p < 0,001). Сочетание дистальной и глубокой окклюзии приводило к тяжелым формам повышенной стираемости зубов. Выводы. Результаты исследования показали, что при разных зубочелюстных аномалиях у пациентов отличаются паттерны стирания. Полученные данные помогут в разработке эффективных мер профилактики повышенной стираемости зубов. Ключевые слова: зубочелюстные аномалии, повышенная стираемость зубов, подростки, детский возраст, твердые ткани зубов. Для цитирования: Смирнова АА, Гаврилова ОА, Федорова КВ, Соколова ЛН, Бобров ДВ. Паттерны повышенной стираемости зубов при разных видах зубочелюстных аномалий. Стоматология детского возраста и профилактика. 2022;22(2):111-121. DOI: 10.33925/1683-3031-2022-22-2-111-121. Abnormal wear patterns in different types of malocclusions А.A. Smirnova, O.A. Gavrilova, K.V. Fedorova, L.N. Sokolova, D.V. Bobrov Tver State Medical University, Tver, Russian Federation ABSTRACT Relevance. Abnormal tooth wear is a disease characterized by excessive loss of hard tissues of the erupted teeth, unrelated to the physiological process. This disease prevalence appears to have increased in recent decades. The literature often indicates malocclusion as the cause of abnormal tooth wear, though it rarely notes the impact of abnormality type on individual teeth. Purpose. The study aimed to establish the relationship between the abnormality type and the degree and nature of abnormal tooth wear in children at different stages of dentition development. Materials and methods. The study analyzed the examination results of 266 patients aged 8 to 18 years without a history of previous orthodontic treatment. The orthodontic diagnosis was made based on the ICD-10. The patients 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 111 Оригинальная статья І Original article formed the groups according to the stage of dentition development. The tooth wear index (TWI) (only permanent teeth) evaluated the abnormal tooth wear. The maximum TWI score determined the grade of abnormal tooth wear. The study statistically analyzed the data by StatTech v. 2.5.6, compared the data using the Kruskal – Wallis test (ANOVA) and Pearson chi-square, and made post hoc comparisons using Dunn’s test with Holm adjustment. The prognostic model was developed using the linear regression method. Results. The children and adolescents showed a sufficiently high prevalence of abnormal tooth wear (69.2% had at least one tooth with a tooth wear facet), tooth-wear grade increased with age (correlation coefficient rxy = 0.674, p < 0.001). The risk of abnormal tooth wear in distal and deep occlusion appeared to increase at incisors and canines (p < 0.001), in mesial occlusion - molars and premolars, in open bite – molars (p < 0.001). The combination of distal and deep occlusion led to severe abnormal tooth wear. Conclusion. The study results showed that wear patterns differ in various types of malocclusions. The received data will help to develop effective measures to prevent abnormal tooth wear. Key words: malocclusion, abnormal tooth wear, adolescents, child age, hard dental tissues. For citation: Smirnova AA, Gavrilova OA, Fedorova KV, Sokolova LN, Bobrov DV Abnormal wear patterns in different types of malocclusions. Pediatric dentistry and dental prophylaxis. 2022;22(2):111-121 (In Russ.). DOI: 10.33925/16833031-2022-22-2-111-121. ВВЕДЕНИЕ Повышенная стираемость зубов (ПСЗ) – это многофакторное заболевание, приводящее к чрезмерной потере твердых тканей зубов. В зависимости от предполагаемой этиологии повышенную стираемость зубов разделяют на аттриции (механический износ, вызванный контактом зуба с зубом), эрозии (химический износ, вызванный кислотами небактериального происхождения) и абразии (механический износ, вызванный контактом зуба с каким-либо материалом) [1]. Также выделяют абфракцию – теорию окклюзионной перегрузки зубов, которая приводит к потере твердых тканей пришеечной области зубов [2]. Среди причин ПСЗ выделяют местные и общие факторы риска [3]. К местным относят: нарушения окклюзии, кислую диету, дневной и ночной бруксизм, возраст и др. [4]. Трудно идентифицировать ПСЗ, основываясь исключительно на клиническом виде, так как этиология поражения ПСЗ, как правило, многофакторна из-за сочетания эрозий, аттриций и абразий [5, 6]. Van't Spijker и соавт. (2007) в систематическом обзоре рассматривает аттриции как прямой результат действия окклюзионных факторов, однако не было обнаружено убедительных доказательств применения определенных протоколов лечения аттриции при разных видах окклюзионных взаимоотношений [7]. Данные Щербенко А. О. (2017) подтверждают, что у молодых людей (обследовали студентов 1-5 курсов) одной из самых частых причин ПСЗ является патология прикуса [8]. Автор утверждает, что на процесс стираемости зубов оказывает влияние вид зубочелюстных аномалий (ЗЧА). Гайворонский И. В. с соавт. (2007) изучали зубочелюстной аппарат взрослых (30-60 лет). Исследование показало, что при ортогнатическом прикусе зубы меньше всего подвержены стираемости и она отмечается во всех группах зубов. При открытом прикусе наблюдали значительную убыль твердых тканей премоляров и моляров. Авторы отмечали повышенную стираемость зубов при прямом прикусе [9]. Также по- 112 вышенная стираемость усиливается при частичной потере зубов, так как оставшиеся воспринимают повышенную и не свойственную им функциональную нагрузку, в результате чего наблюдается повышенное стирание антагонирующих пар зубов [10, 11]. Agnani S и соавт. (2019) изучали стираемость при дистальной окклюзии (II класс 1 и 2 подкласс по Энглю). По мнению ученых, в группе с дистальной окклюзией наблюдалась статистически более значимая стираемость вестибулярных поверхностей боковых резцов, премоляров и первых моляров нижней челюсти, окклюзионных поверхностей верхних и нижних премоляров и верхних первых моляров, небных поверхностей вторых премоляров верхней челюсти, чем в группе с нормальной окклюзией [12]. Стираемость зубов можно считать физиологическим процессом с ожидаемой годовой скоростью износа приблизительно 11 мкм [13]. Соответственно, в возрастной группе до 18 лет видимых глазом изменений быть не должно. В то же время в клинической практике часто наблюдается выраженная убыль твердых тканей зубов, особенно у пациентов с глубоким прикусом. Raj A. и соавт. (2021) исследовали влияние чрезмерного вертикального и горизонтального перекрытия на дентальный статус пациентов и пришли к выводу, что это важный этиологический фактор развития аттриционного типа стираемости. Согласно данным авторов, горизонтальное перекрытие влияет сильнее вертикального на стираемость зубов [14]. Лазарева О. В. с соавт. (2018) считают, что повышенная стираемость зубов при глубоком резцовом перекрытии является адаптивным механизмом к смещению нижней челюсти [15]. В связи с растущей тенденцией к неправильному прикусу у детей необходимо определить причину износа зубов (то есть физиологическую или патологическую), чтобы ортодонт мог соответствующим образом лечить пациента и проводить эффективную профилактику. Для этого целесообразно подтвердить характер износа при различных ЗЧА [16]. Исследования по данной проблеме у детей и подростков в России не проводились. 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Оригинальная статья І Original article В связи с вышеизложенным, целью исследования стало установление взаимосвязи между видом зубочелюстной аномалии и степенью и характером повышенной стираемости зубов у детей в разных периодах формирования прикуса. МАТЕРИАЛЫ И МЕТОДЫ На базе отделения стоматологии детского возраста и ортодонтии Тверского ГМУ в период с мая 2019-го по июнь 2021 года было обследовано 266 первичных ортодонтических пациентов (123 лица мужского пола и 143 лица женского пола). В исследование были включены дети и подростки в возрасте от 8 до 18 лет, не проходившие (в анамнезе и в настоящее время) ортодонтическое лечение. В младшую возрастную группу включали детей с полным прорезыванием всех первых постоянных моляров и у них исследовали зубы с полным прорезыванием или те, которые контактируют с временными зубами-антагонистами. У пациентов учитывались даже незначительные изменения твердых тканей зубов ввиду молодого возраста. Письменное информированное добровольное согласие было получено от всех участников старше 15 лет и от законных представителей участников, не достигших этого возраста. Критерии исключения – пациенты с тяжелыми хроническими заболеваниями, инвалидностью (5 группа здоровья), дети с признаками острых вирусных заболеваний (насморк, кашель), отказ от исследования. Пациенты, принявшие участие в исследовании, направлялись на консультацию ортодонта после профилактического осмотра детским стоматологом. Ортодонтический диагноз ставился по классификации зубочелюстных аномалий МКБ-10. В исследовании не рассматривались функциональные нарушения (группа К.07.5 по МКБ-10). В группу 1 (период раннего сменного прикуса) были отнесены пациенты до начала прорезывания премоляров. Соответственно, в группу 2 (период позднего сменного прикуса) были включены дети, у которых начал прорезываться хотя бы один премоляр и до момента выпадения последнего временного зуба. В группу 3 (период формирования постоянного прикуса) относились оставшиеся пациенты. Повышенная стираемость зубов оценивалась с помощью индекса TWI (tooth wear index), где 0 баллов – нет стираемости, 1 балл – убыль в пределах эмали, 2 балла – убыль с обнажением дентина до 1/3 поверхности зуба, 3 балла – убыль с обнажением более 1/3, 4 балла – убыль с обнажением пульпы [17]. На наличие стираемости обследовались только постоянные зубы. Пациентов распределяли по степени ПСЗ, которая ставилась по максимальному баллу индекса TWI (0 – норма, хотя бы у одного зуба 1 балл – 1 степень, 2 балла – 2 степень, 3 балла – 3 степень, 4 балла по индексу хотя бы у одного зуба – 4 степень ПСЗ). Всего было обследовано 5319 зубов. Статистический анализ проводился с использованием программы StatTech v. 2.5.6 (разработ- Таблица 1. Структура зубочелюстных аномалий, выявленных у детей и подростков Table 1. Malocclusions detected in children and adolescents Код МКБ-10 / ICD-10 Code Вид прикуса / Bite Type n % K07.2 Аномалии соотношений зубных дуг: Anomalies of dental arch relationship: 172 70,6 K07.20 Дистальный прикус Disto-occlusion 108 40,9 K07.21 Мезиальный прикус Mesio-occlusion 29 10,9 K07.22 Чрезмерное горизонтальное перекрытие Excessive overjet (horizontal overbite) 53 19,9 K07.23 Чрезмерное вертикальное перекрытие Excessive overjet (vertical overbite) 85 31,9 K07.24 Вертикальная дизокклюзия Openbites 11 4,1 K07.25 Перекрестный прикус (передний, задний) Crossbite (anterior, posterior) 13 4,8 K07.26 Смещение зубных дуг от средней линии Midline deviation 7 2,6 Сочетанная патология (сочетание двух и более патологий) Combined pathology (Combination of two or more pathologies) 94 35,3 Нейтральный прикус Neutral bite 94 29,3 Всего / Total 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 266 113 Оригинальная статья І Original article чик – ООО «Статтех», Россия). Количественные показатели оценивали на предмет соответствия нормальному распределению с помощью критерия Шапиро – Уилка (при числе исследуемых менее 50) или критерия Колмогорова – Смирнова (при числе исследуемых более 50). Количественные показатели, имеющие нормальное распределение, описывались с помощью средних арифметических величин (M) и стандартных отклонений (SD), границ 95% доверительного интервала (95% ДИ). Категориальные – с указанием абсолютных значений и процентных долей. Сравнение трех и более групп по количественному показателю, распределение которого отличалось от нормального, выполнялось с помощью критерия Краскела – Уоллиса, апостериорные сравнения – с помощью критерия Данна с поправкой Холма. Сравнение процентных долей при анализе многопольных таблиц сопряженности выполнялось с помощью критерия хи-квадрат Пирсона. Прогностическая модель, характеризующая зависимость количественной переменной от факторов, разрабатывалась с помощью метода линейной регрессии. РЕЗУЛЬТАТЫ Из 266 пациентов, участвовавших в обследовании, у 70,6% были выявлены различные виды зубочелюстных аномалий, структура которых представлена в таблице 1. При сравнении показателя «пол» в зависимости от показателя «ПСЗ» не удалось выявить статистически значимых различий (p = 0,512, рис. 1). Средняя степень стираемости составила 0,902 ± 0,811 балла. 3 и 4 степень стиремости (3 и 4 балла хотя бы у одного зуба, соответственно) наблюдались у 12 пациентов (4,5%), 2 степень – у 32 (12,0%), 1 степень – у 140 (52,6%), отсутствие каких-либо признаков стираемости – у 82 пациентов (30,8 %). Согласно полученным данным, частота обнаружения наибольшей степени стираемость была отмечена в группе 3 (период формирования постоянного прикуса), и в этой группе средний балл индекса TWI выше, чем в других группах (табл. 2). При дистальной окклюзии отмечена более высокая распространенность ПСЗ, чем при нейтральном прикусе, причем чаще стиранию подвергались нижние и верхние резцы, нижние клыки и первые моляры (p < 0,001) (табл. 3). При глубокой окклюзии (чрезмерном вертикальном перекрытии) повышенная стираемость зубов максимально выражена на верхних и нижних резцах. Причем при сочетании дистальной и глубокой окклюзии была выявлена ПСЗ в 4 балла на нижних резцах. При мезиальной окклюзии стиранию чаще подвержены верхние и нижние моляры, верхние и нижние премоляры. Дети с вертикальной дизокклюзией имели меньшую распространенность ПСЗ, чем их сверстники, при этом чаще поражались верхние и нижние моляры и премоляры. При перекрестной окклюзии нам не удалось выявить статистически значимые различия в регистрации стираемости твердых тканей зубов по сравнению с пациентами с нейтральным прикусом (табл. 3). Отмечено прогрессирование ПСЗ у детей с возрастом (p < 0,001, критерий Краскела – Уоллиса) (рис. 2). Таблица 2. Встречаемость повышенной стираемости зубов в зависимости от периода формирования прикуса Table 2. Abnormal tooth wear vs dentition development stage Повышенная стираемость зубов n (%) / Abnormal tooth wear n (%) Группа Group Нет 1 2 3 4 Всего стираемости степень степень степень степень Total No wear Grade 1 Grade 2 Grade 3 Grade 4 Группа 1 (ранний сменный прикус) Group 1 (early mixed dentition) 23 (8,6) 40 (15,0) 5 (1,9) 0 (0,0) 0 (0,0) 68 Группа 2 (поздний сменный прикус) Group 2 (late mixed dentition) 34 (12,8) 55 (20,7) 10 (3,7) 0 (0,0) 0 (0,0) 99 Группа 3 (формирующийся постоянный прикус) Group 3 (permanent dentition development) 25 (9,4) 45 (16,9) 17 (6,4) 8 (3) 4 (1,5) 98 Всего n (%) Total, n (%) 82 (30,8) 140 (52,6) 32 (12) 8 (3) 4 (1,5) 266 (100) p < 0,001* p нет стираемости – 3 степень = 0,005* p 1 степень – 3 степень = 0,005* n – количество пациентов / n – number of patients 114 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Средний балл TWI Mean TWI score (M ± SD) 0,721 ± 0,569 0,765 ± 0,623 1,15 ± 0,973 0,902 ± 0,811 Оригинальная статья І Original article Таблица 3. Анализ зависимости частоты встречаемости повышенной стираемости отдельных зубов и разных видов зубочелюстных аномалий Table 3. Analysis of abnormal tooth wear prevalence in different teeth versus malocclusion type. ЗЧА / Malocclusion Верхние премоляры / Upper premolars (n = 710) Верхние моляры / Upper molars (n = 729) Зубы Teeth n = 5319 Зуб 1.7 Tooth 1.7 n = 105 Зуб 1.6 Tooth 1.6 n = 257 Зуб 2.6 Tooth 2.6 n = 258 Зуб 2.7 Tooth 2.7 n = 109 Зуб 1.5 Tooth 1.5 n = 159 Зуб 1.4 Tooth 1.4 n = 191 Зуб 2.4 Tooth 2.4 n = 193 Зуб 2.5 Tooth 2.5 n = 167 Чрезмерное Чрезмерное горизонтальное вертикальное Вертикальная Перекрестный перекрытие перекрытие дизокклюзия прикус Excessive Excessive Openbite Crossbite overjet overbite (K07.24) (K07.25) (K07.22) (K07.23) Дистальная окклюзия Distal occlusion (K07.20) Мезиальная окклюзия Mesial occlusion (K07.21) Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No 0 38 48 1 85 15 71 25 61 0 86 6 80 I 3 14 11 6 1 16 2 15 5 12 0 17 II 0 2 2 0 0 2 0 2 2 0 0 2 ПСЗ ATW p < 0.001* 0.064 0 71 128 0.400 p < 0.001* 0.231 0.495 10 189 28 171 55 144 2 197 11 188 I 27 10 6 31 19 18 22 15 2 35 0 37 II 3 14 10 7 2 15 3 14 5 12 1 16 III 0 4 3 1 0 4 0 4 2 2 0 4 p p < 0.001* p < 0.001* p < 0.001* p < 0.001* p < 0.001* 0 70 128 10 188 34 164 54 144 2 196 10 188 I 31 14 9 36 16 29 25 20 2 43 2 43 0.495 II 2 9 6 5 0 11 1 10 5 6 0 11 III 0 4 3 1 0 4 0 4 2 2 0 4 p p < 0.001* p < 0.001* p < 0.001* p < 0.001* p = 0.005* 0 36 57 7 86 14 79 26 67 1 92 6 87 I 3 11 8 6 1 13 2 12 5 9 0 14 II 0 2 2 0 0 2 0 2 0 2 0 2 p p < 0.001* 0.268 0.624 0.848 p < 0.001* 0.398 0.583 0 45 81 7 119 17 109 37 89 2 124 7 119 I 14 10 7 17 10 14 10 14 3 21 0 24 II 0 9 6 3 0 9 0 8 3 6 0 9 p 0.084 p < 0.001* p = 0.108 p < 0.001* 0.209 0.386 0 50 103 10 143 19 134 43 110 2 151 7 146 I 19 15 8 26 14 20 13 21 5 29 1 33 II 1 4 3 2 0 5 1 4 2 3 0 5 p p = 0.081 p < 0.001* p < 0.001* p = 0.461 p < 0.001* p = 0.812 0 52 99 10 141 22 129 40 111 3 148 6 145 I 21 19 10 30 13 27 14 26 5 35 2 38 II 1 1 1 1 0 2 1 1 1 1 0 2 p p = 0.106 p < 0.001* p = 0.026 p = 0.453 p < 0.001* p = 0.918 0 50 85 9 126 21 114 39 96 2 133 6 129 I 14 13 8 19 8 19 10 17 4 23 1 26 II 0 5 4 1 0 5 0 5 2 3 0 5 p p = 0.071 p < 0.001* p = 0.123 p = 0.248 p < 0.001* p = 0.880 Продолжение / Сontinuation 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 115 Оригинальная статья І Original article Нижние резцы / Lower incisors (n = 1051) Верхние резцы / Upper incisors (n = 1037) Верхние клыки / Upper canines (n = 236) Зубы Teeth n = 5319 Зуб 1.3 Tooth 1.3 n = 118 Зуб 2.3 Tooth 2.3 n = 118 Зуб 2.2 Tooth 2.2 n = 254 Зуб 1.1 Tooth 1.1 n = 264 Зуб 2.1 Tooth 2.1 n = 264 Зуб 2.2 Tooth 2.2 n = 255 Зуб 3.2 Tooth 3.2 n = 262 Зуб 3.1 Tooth 3.1 n = 264 ЗЧА / Malocclusion Чрезмерное Чрезмерное горизонтальное вертикальное Вертикальная Перекрестный перекрытие перекрытие дизокклюзия прикус Excessive Excessive Openbite Crossbite overjet overbite (K07.24) (K07.25) (K07.22) (K07.23) Есть Нет Есть Нет Есть Нет Есть Нет Yes No Yes No Yes No Yes No Дистальная окклюзия Distal occlusion (K07.20) Мезиальная окклюзия Mesial occlusion (K07.21) Есть Yes Нет No Есть Yes Нет No 0 12 54 7 59 6 60 9 57 2 64 3 63 I 22 22 11 33 7 37 14 30 4 40 2 42 II 7 1 0 8 4 4 6 2 0 8 1 7 p p < 0.001* p = 0.056 p = 0.007* p < 0.001* p = 0.291 p = 0.613 0 15 59 10 64 6 68 12 62 4 70 4 70 I 20 17 8 29 7 30 11 26 2 35 1 36 II 6 1 0 7 4 3 5 2 0 7 1 6 p p < 0.001* p = 0.273 p < 0.001* p = 0.003 p = 0.819 p = 0.432 0 I II p 0 I II p 0 I II p 0 I II 60 133 37 15 5 4 p < 0.001* 45 125 56 27 7 4 p < 0.001* 51 126 49 26 8 4 p < 0.001* 61 131 35 18 6 4 21 172 5 47 3 6 p = 0.106 12 158 14 69 3 8 p = 0.114 13 164 13 62 3 9 0.014 13 179 12 41 3 7 28 165 18 34 2 7 p = 0.004 26 144 24 59 3 8 p = 0.033 28 149 22 53 3 9 p = 0.221 31 161 17 36 2 8 48 145 29 23 4 5 p < 0.001* 31 139 47 36 6 5 p < 0.001* 38 139 41 34 6 6 p < 0.001* 51 141 28 25 4 6 11 182 0 52 0 9 p = 0.163 9 161 2 81 0 11 p = 0.436 9 168 2 73 0 12 p = 0.517 9 183 2 51 0 10 8 185 3 49 0 9 p = 0.711 9 161 3 80 0 11 p = 0.635 7 170 5 70 0 12 p = 0.474 7 185 3 50 0 10 p p < 0.001* 0.058 p = 0.035 p = 0.001* p = 0.758 p = 0.647 0 21 115 14 122 11 125 15 121 10 126 8 128 I 64 33 13 84 27 70 51 46 1 96 3 94 II 16 7 3 20 10 13 12 11 0 23 1 22 III 5 0 0 5 3 2 5 0 0 5 0 5 IV 1 0 0 1 1 0 1 0 0 1 0 1 ПСЗ ATW p p < 0.001* p = 0.793 p < 0.001* p < 0.001* p = 0.133 p = 0.860 0 21 120 13 128 13 128 15 126 10 131 7 134 I 63 29 14 78 24 68 49 43 1 91 4 88 II 17 7 3 21 11 13 16 8 0 24 1 23 III 4 0 0 4 3 1 2 2 0 4 0 4 IV 3 0 0 2 1 3 0 0 3 0 p p < 0.001* 3 0.681 p < 0.001* p < 0.001* 0.161 Продолжение / Сontinuation 116 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 3 0.982 Оригинальная статья І Original article ЗЧА / Malocclusion Нижние премоляры / Lower premolars (n = 696) Нижние клыки / Lower canines (n = 151) Нижние резцы / Lower incisors (n = 1051) Зубы Teeth n = 5319 Зуб 4.1 Tooth 4.1 n = 262 Зуб 4.2 Tooth 4.2 n = 263 Зуб 3.3 Tooth 3.3 n = 126 Зуб 4.3 Tooth 4.3 n = 125 Зуб 3.5 Tooth 3.5 n = 158 Зуб 3.4 Tooth 3.4 n = 192 Зуб 4.4 Tooth 4.4 n = 187 Зуб 4.5 Tooth 4.5 n = 159 Чрезмерное Чрезмерное горизонтальное вертикальное Вертикальная Перекрестный перекрытие перекрытие дизокклюзия прикус Excessive Excessive Openbite Crossbite overjet overbite (K07.24) (K07.25) (K07.22) (K07.23) Дистальная окклюзия Distal occlusion (K07.20) Мезиальная окклюзия Mesial occlusion (K07.21) Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No 0 22 113 13 122 11 124 13 122 10 125 5 130 ПСЗ ATW I 62 41 16 87 26 77 54 49 1 102 7 96 II 18 2 0 20 13 7 15 5 0 20 0 20 III 2 0 0 2 2 0 1 1 0 2 0 2 IV 2 0 0 2 0 2 2 0 0 2 0 2 p p < 0.001* 0 24 121 14 I 64 35 15 p < 0.001* p < 0.001* 131 12 133 15 130 10 135 8 137 84 28 71 54 45 1 98 4 95 0.253 0.127 0.636 II 13 1 0 14 7 7 10 4 0 14 0 14 III 3 0 0 3 1 2 2 1 0 3 0 3 IV 2 0 0 2 1 1 2 0 0 2 0 2 p p < 0.001* 0 15 60 9 I 22 14 II 11 4 p p < 0.001* 0 10 65 9 I 24 15 II 8 3 p p < 0.001* 0 39 81 7 I 18 16 10 II 0 4 3 p p < 0.001* p < 0.001* 66 9 66 13 62 4 71 4 71 7 29 6 30 13 23 2 34 1 35 3 12 7 8 8 7 0 15 1 14 0.136 p < 0.001* 66 6 69 11 64 4 71 3 72 9 30 7 32 13 26 2 37 2 37 1 10 5 6 7 4 0 11 1 10 0.808 p < 0.001* 113 17 103 33 87 2 118 5 115 24 8 26 12 22 4 30 1 33 1 0 4 0 4 2 2 0 5 0.284 101 7 137 18 126 38 106 I 26 21 14 33 14 33 18 II 1 0 0 1 1 0 1 p < 0.001* 0.002 0.894 0.860 p < 0.001* 0.298 43 0.061 0.663 p < 0.001* 0.413 0 p 0.866 p = 0.002 0.340 p < 0.001* 0.028 0.201 2 142 29 6 0 0 0.873 6 138 41 1 46 1 0 1 0.003 0.091 0.796 0 45 102 12 135 20 127 37 110 3 144 7 140 I 23 16 9 30 13 26 16 23 6 33 1 38 II 1 0 0 1 1 0 0 1 0 1 0 p 0.106 0.002 0.03 0.002 0.122 1 0.815 0 41 87 13 115 21 107 33 95 2 126 5 123 I 17 8 4 21 9 16 10 15 5 20 1 24 II 0 6 5 1 0 6 0 6 1 5 0 6 p 0.102 p < 0.001* p = 0.135 p = 0.108 p < 0.001* p = 0.885 Продолжение / Сontinuation 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 117 Оригинальная статья І Original article ЗЧА / Malocclusion Нижние моляры / Lower molars (n = 709) Зубы Teeth n = 5319 118 Зуб 3.7 Tooth 3.7 n = 117 Зуб 3.6 Tooth 3.6 n = 254 Зуб 4.6 Tooth 4.6 n = 224 Зуб 4.7 Tooth 4.7 n = 114 Чрезмерное Чрезмерное горизонтальное вертикальное Вертикальная Перекрестный перекрытие перекрытие дизокклюзия прикус Excessive Excessive Openbite Crossbite overjet overbite (K07.24) (K07.25) (K07.22) (K07.23) Дистальная окклюзия Distal occlusion (K07.20) Мезиальная окклюзия Mesial occlusion (K07.21) Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No Есть Yes Нет No 0 31 70 13 88 12 89 25 76 2 99 6 95 ПСЗ ATW I 8 4 2 8 4 8 3 9 3 9 0 12 II 0 4 3 4 0 4 0 4 2 2 0 4 p p = 0.160 p = 0.003* p = 0.089 p = 0.521 p < 0.001* p = 0.606 0 42 122 13 151 23 141 34 130 2 162 9 155 I 53 19 9 63 22 50 40 32 2 70 2 70 II 7 9 5 11 5 11 5 11 5 11 1 15 III 0 2 1 1 0 2 0 2 2 0 0 p p < 0.001* 0 42 131 14 159 21 152 37 136 2 171 9 164 I 51 12 6 57 25 38 37 26 2 61 1 62 II 7 12 8 11 4 15 5 14 5 14 1 18 III 0 2 1 1 0 2 0 2 2 0 0 2 0.009* < 0.001* 0.014 < 0.001* 2 0.801 p p < 0.001* p < 0.001* p = 0.336 p = 0.130 p < 0.001* 0 33 61 8 86 13 81 24 70 1 93 6 88 I 8 8 6 10 3 13 5 11 3 13 0 16 II 0 4 3 1 0 4 0 4 2 2 0 4 p 0.162 p < 0.001* 0.622 0.438 p < 0.001* 0.657 0.510 Рис. 1. Распределение пациентов по полу в зависимости от степени повышенной стираемости зубов Fig. 1. Patient allocation by sex according to the abnormal tooth wear grade Рис. 2. Возрастные характеристики пациентов в зависимости от степени повышенной стираемости зубов Fig. 2. Patients’ age characteristics based on the abnormal tooth wear grade Также была построена общая линейная модель с зависимым количественным показателем (ПСЗ) и двумя предикторами с помощью метода множественной линейной регрессии. Наблюдаемая зависимость ПСЗ от наличия аномалий соотношения зубных дуг и возраста описывается уравнением линейной регрессии: YПовышенная стираемость зубов = -0,699 + 0,081XВозраст + 1,018Xесть где YПовышенная стираемость зубов – величина показателя «повышенная стираемость зубов», Xесть – аномалии соотношения зубных дуг (K07.2) (0 – нет, 1 – есть), XВозраст – возраст 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Оригинальная статья І Original article При наличии аномалий соотношения зубных дуг можно ожидать увеличения ПСЗ на 1,018 (SD 0,077, t = 13,237) балла TWI, при увеличении возраста на 1 год следует ожидать увеличения ПСЗ на 0,081 балла TWI (SD 0,012, t = 6,680). Полученная регрессионная модель характеризуется коэффициентом корреляции rxy = 0,674, что соответствует заметной тесноте связи по шкале Чеддока. Модель была статистически значимой (p < 0,001). Полученная модель объясняет 45,5% наблюдаемой дисперсии показателя ПСЗ. ОБСУЖДЕНИЕ Установлена достаточно высокая распространенность повышенной стираемости зубов среди пациентов: у 69,2% детей наблюдался хотя бы один зуб с фасеткой стирания и износ твердых тканей увеличивался с возрастом. Полученные данные схожи с исследованием Tian Yu и соавт. (2021) [18], в котором 59,7% детей имели признаки стираемости зубов, которая также прогрессировала с возрастом (р < 0,001). У детей с дистальным прикусом (II класс по Энглю) и глубоким прикусом повышенная стираемость резцов и клыков на нижнем зубном ряду регистрировалась чаще всего, сходные результаты были получены Oltramari-Navarro PV с соавт. (2010) [12]. Важно, что настоящее исследование включает систематическую выборку, которая позволяет использовать полученные результаты у пациентов, посещающих врачей-ортодонтов. В то же время их следует с осторожностью переносить на общую группу пациентов, поскольку в выборку исследования вошли только пациенты, самостоятельно обратившиеся за стоматологической помощью, что объясняет такой высокий процент ЗЧА у обследованных пациентов. В исследовании не учитывались другие факторы риска развития ПСЗ, такие как бруксизм, диета и др. Полученные данные оправдывают раннее лечение зубочелюстных аномалий с целью профилактики ПСЗ у пациентов. Согласно результатам исследования имеются определенные паттерны стирания при разных видах ЗЧА: при дистальной и глубокой окклюзии повышенному стиранию чаще подвержены резцы и моляры, причем при сочетании этих аномалий нижние резцы страдают сильнее всего, при мезиальной окклюзии и вертикальной дизокклюзии есть тенденция к повышенному стиранию моляров и премоляров. ЗАКЛЮЧЕНИЕ Результаты исследования дают возможность сделать выводы о том, что у детей и подростков, самостоятельно обратившихся за ортодонтической помощью, наблюдалась высокая встречаемость повышенной стираемости зубов, у 69,2% наблюдался хотя бы один зуб с фасеткой стирания, степень стираемости увеличивалась с возрастом. Дистальная и глубокая окклюзия больше других зубочелюстных аномалий способствуют износу твердых тканей, больше подвержены резцы и клыки, первые моляры. При сочетании дистальной и глубокой окклюзии отмечена наиболее выраженная стираемость. Необходимо продолжить проведение исследований повышенной стираемости зубов в отдельных возрастных группах с дифференцированием на эрозии, абразии, аттриции. Понимание паттернов повышенной стираемости зубов при различных видах ЗЧА будет способствовать разработке эффективных мер профилактики. СПИСОК ЛИТЕРАТУРЫ 1. Shellis RP, Addy M. The interactions between attrition, abrasion and erosion in tooth wear. Monogr Oral Sci. 2014;25:32-45. doi: 10.1159/000359936 2. Bartlett DW, Shah P. A critical review of non-carious cervical (wear) lesions and the role of abfraction, erosion, and abrasion. J Dent Res. 2006;85(4):306-312. doi: 10.1177/154405910608500405 3. Loomans B, Opdam N, Attin T, Bartlett D, Edelhoff D, Frankenberger R и др. Severe Tooth Wear: European Consensus Statement European Consensus Statement on Management Guidelines. J Adhes Dent. 2017;19(2):111-119. doi: 10.3290/j.jad.a38102 4. Bartlett D, O'Toole S. Tooth Wear: Best Evidence Consensus Statement. J Prosthodont. 2020;30(S1):20-25 doi: 10.1111/jopr.13312 5. Bartlett D. Etiology and prevention of acid erosion. Compend Contin Educ Dent. 2009;30(9):616-620. Режим доступа: https://www.aegisdentalnetwork.com/cced/2009/12/ literature-review-etiology-and-prevention-of-acid-erosion 6. El Aidi H, Bronkhorst EM, Truin GJ. A longitudinal study of tooth erosion in adolescents. J Dent Res. 2008;87(8):731-735. doi: 10.1177/154405910808700813 7. Spijker van't A, Kreulen CM, Creugers NH. Attrition, occlusion, (dys)function, and intervention: a systematic review. Clin Oral Implants Res.2007;18 Suppl 3:117-126. doi: 10.1111/j.1600-0501.2007.01458.x 8. Щербенко АО. Изучение распространения повышеной стираемости твердых тканей зубов среди молодых людей. Восточно-европейский научный журнал. 2017;(8-1):40-43. Режим доступа: https://cyberleninka.ru/article/n/izuchenierasprostraneniya-povyshenoy-stiraemosti-tverdyhtkaney-zubov-sredi-molodyh-lyudey 9. Гайворонский ИВ, Дубова МА, Пономарев АА. Влияние формы прикуса на степень стираемости твердых тканей зубов. Вестник Санкт-Петербургского университета. Медицина. 2007;(1):98-101. Режим доступа: https://cyberleninka.ru/article/n/vliyanie-formyprikusa-na-stepen-stiraemosti-tverdyh-tkaney-zubov-1 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 119 Оригинальная статья І Original article 10. Онопа ЕН. Распространенность деформаций зубных рядов у больных с частичным отсутствием зубов при различной степени стираемости зубов. Современные проблемы науки и образования. 2012;(4):3. Режим доступа: https://science-education.ru/ru/article/view?id=6469 11. Иорданишвили АК, Янковский ВВ, Сериков АА. Возрастные особенности этиологии и клинического течения повышенной стираемостии твердых тканей зубов у взрослого человека. Курский научнопрактический вестник «Человек и его здоровье». 2014;(2):33-40. Режим доступа: https://cyberleninka.ru/article/n/vozrastnye-osobennosti-etiologii-i-klinicheskogo-techeniya-povyshennoy-stiraemostii-tvyordyh-tkaney-zubov-u-vzroslogo-cheloveka 12. Agnani S, Bajaj K, Mehta S, Pandey L. Tooth wear patterns in subjects with class II division 1 and class II division 2 malocclusion. Int J Adolesc Med Health. 2019;33(4):10. doi: 10.1515/ijamh-2018-0227 13. Pintado MR, Anderson GC, DeLong R, Douglas WH. Variation in tooth wear in young adults over a two-year period. J ProsthetDent. 1997;77(3):313–320. doi: 10.1016/s0022-3913(97)70189-6 14. Raj A, Ranjan R, Kumar A, Kumar M, Mala N, Ramesh K. Evaluation of Dental Status in Relation to Excessive Horizontal and Vertical Overlap in North Indian Population. J Pharm Bioallied Sci. 2021;13(Suppl 1):276-279. doi: 10.4103/jpbs.JPBS_731_20 15. Лазарева ОВ, Бимбас ЕС, Бриштен ВЛ. Факторы декомпенсации зубочелюстной системы у взрослых пациентов с глубоким резцовым перекрытием. Проблемы стоматологии. 2018;14(4):87-92. doi: 10.18481/2077-7566-2018-14-4-87-92 16. Oltramari-Navarro PV, Janson G, de Oliveira RB, Quaglio CL, Castanha Henriques JF, de Carvalho SalesPeres SH и др. Tooth-wear patterns in adolescents with normal occlusion and Class II Division 2 malocclusion. Am J Orthod Dentofacial Orthop. 2010;137(6):730.e1-731. doi: 10.1016/j.ajodo.2010.01.020 17. Smith BG, Knight JK. An index for measuring the wear of teeth. British Dental Journal. 1984;156:435-438. doi: 10.1038/sj.bdj.4805394 18. Yu T, Tao DY, Lu HX, Zhu JL, Xie CY, Bartlett D и др. Prevalence and Associated Factors of Tooth Wear in Shanghai. Chin J Dent Res. 2021;24(2):95-103. doi: 10.3290/j.cjdr.b1530421 REFERENCES 1. Shellis RP, Addy M. The interactions between attrition, abrasion and erosion in tooth wear. Monogr Oral Sci. 2014;25:32-45. doi: 10.1159/000359936 2. Bartlett DW, Shah P. A critical review of non-carious cervical (wear) lesions and the role of abfraction, erosion, and abrasion. J Dent Res. 2006;85(4):306-312. doi: 10.1177/154405910608500405 3. Loomans B, Opdam N, Attin T, Bartlett D, Edelhoff D, Frankenberger R и др. Severe Tooth Wear: European Consensus Statement European Consensus Statement on Management Guidelines. J Adhes Dent. 2017;19(2):111-119. doi: 10.3290/j.jad.a38102 4. Bartlett D, O'Toole S. Tooth Wear: Best Evidence Consensus Statement. J Prosthodont. 2020;30(S1):20-25. doi: 10.1111/jopr.13312 5. Bartlett D. Etiology and prevention of acid erosion. Compend Contin Educ Dent. 2009;30(9):616-620. Available from: https://www.aegisdentalnetwork.com/cced/2009/12/ literature-review-etiology-and-prevention-of-acid-erosion 6. El Aidi H, Bronkhorst EM, Truin GJ. A longitudinal study of tooth erosion in adolescents. J Dent Res. 2008;87(8):731-735. doi: 10.1177/154405910808700813 7. Spijker van't A, Kreulen CM, Creugers NH. Attrition, occlusion, (dys)function, and intervention: a systematic review. Clin Oral Implants Res. 2007;18Suppl 3:117-126. doi: 10.1111/j.1600-0501.2007.01458.x 8. Shcherbenko АО. Study of distribution of raised rub of teeth hard tissues among young people. EESJ. 120 2017;(8-1):40-43 (In Russ.). Available from: https://cyberleninka.ru/article/n/izuchenie-rasprostraneniya-povyshenoy-stiraemosti-tverdyh-tkaneyzubov-sredi-molodyh-lyudey 9. Gaivoronskiy IV, Dubova MA, Ponomarev AA. Impact of the bite form on the level of hard tooth tissues abrasion. Vestnik of Saint Petersburg university. Medicine. 2007;1:98-101. Available from: https://cyberleninka.ru/article/n/vliyanie-formy-prikusa-na-stepen-stiraemosti-tverdyh-tkaney-zubov-1 10. Onopa EN. The extent of dentition deformation in the patients with partial missing of the teeth with different degree of dental erosion. Modern Problems of Science and Education. 2012;(4):3. Available from: https://science-education.ru/ru/article/view?id=6469 11. Iordanishvili AK, Yankovsky VV, Serikov AA. Agerelated features of aetiology and clinical course of the increased abrasion of hard dental tissues in adults. Kursk scientific and practical bulletin „Man and his health”. 2014;(2):33-40. Available from: https://cyberleninka.ru/article/n/vozrastnye-osobennosti-etiologii-i-klinicheskogo-techeniya-povyshennoy-stiraemostii-tvyordyh-tkaney-zubov-u-vzroslogo-cheloveka 12. Agnani S, Bajaj K, Mehta S, Pandey L. Tooth wear patterns in subjects with class II division 1 and class II division 2 malocclusion. Int J Adolesc Med Health. 2019;33(4):10. doi: 10.1515/ijamh-2018-0227 13. Pintado MR, Anderson GC, DeLong R, Douglas WH. Variation in tooth wear in young adults over a twoyear period. J ProsthetDent. 1997;77(3):313–320. doi: 10.1016/s0022-3913(97)70189-6 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis Оригинальная статья І Original article 14. Raj A, Ranjan R, Kumar A, Kumar M, Mala N, Ramesh K. Evaluation of Dental Status in Relation to Excessive Horizontal and Vertical Overlap in North Indian Population. J Pharm Bioallied Sci. 2021;13(Suppl 1):276-279. doi: 10.4103/jpbs.JPBS_731_20 15. Lazareva OV, Bimbas ES, Brishten VL. Factors of decompensation in orofacial system in adult patients with deep overbite. Actual problems in dentistry. 2018;14(4):87-92. doi: 10.18481/2077-7566-2018-14-4-87-92 16. Oltramari-Navarro PV, Janson G, de Oliveira RB, Quaglio CL, Castanha Henriques JF, de Carvalho Sales- Peres SH et al. Tooth-wear patterns in adolescents with normal occlusion and Class II Division 2 malocclusion. Am J Orthod Dentofacial Orthop. 2010;137(6):730.e1-731. doi: 10.1016/j.ajodo.2010.01.020 17. Smith BG, Knight JK. An index for measuring the wear of teeth. British Dental Journal. 1984;156:435-438. doi: 10.1038/sj.bdj.4805394 18. Yu T, Tao DY, Lu HX, Zhu JL, Xie CY, Bartlett D et al. Prevalence and Associated Factors of Tooth Wear in Shanghai. Chin J Dent Res. 2021;24(2):95-103. doi: 10.3290/j.cjdr.b1530421 СВЕДЕНИЯ ОБ АВТОРАХ Автор, ответственный за связь с редакцией: Смирнова Анна Алексеевна, ассистент кафедры детской стоматологии и ортодонтии Тверского государственного медицинского университета, Тверь, Российская Федерация Для переписки: Annasemen-69@mail.ru ORCID: https://orcid.org/0000-0002-1881-3910 Гаврилова Ольга Анатольевна, доктор медицинских наук, профессор, заведующая кафедрой детской стоматологии и ортодонтии Тверского государственного медицинского университета, Тверь, Российская Федерация Для переписки: kafdetstom@mail.ru ORCID: https://orcid.org/0000-0001-9227-9173 Федорова Ксения Владимировна, врач-ортодонт стоматологической клиники ООО «Импульс», Тверь, Российская Федерация Для переписки: k.ksushina2018@yandex.ru ORCID: https://orcid.org/0000-0002-1613-6581 Соколова Людмила Николаевна, кандидат медицинских наук, доцент кафедры детской стоматологии и ортодонтии Тверского государственного медицинского университета, Тверь, Российская Федерация Для переписки: sokolovaln18@mail.ru ORCID: https://orcid.org/0000-0002-8226-1807 Бобров Дмитрий Васильевич, кандидат медицинских наук, доцент кафедры детской стоматологии и ортодонтии Тверского государственного медицинского университета, Тверь, Российская Федерация Для переписки: bobrov_d@tvergma.ru ORCID: https://orcid.org/0000-0001-6325-2641 INFORMATION ABOUT THE AUTHORS Corresponding author: Anna A. Smirnova, DMD, Assistant Professor, Department of Pediatric Dentistry and Orthodontics, Tver State Medical University, Tver, Russian Federation For correspondence: annasemen-69@mail.ru ORCID: https://orcid.org/0000-0002-1881-3910 Olga A. Gavrilova, DMD, PhD, DSc, Professor, Head of the Department of Pediatric Dentistry and Orthodontics, Tver State Medical University, Tver, Russian Federation For correspondence: kafdetstom@mail.ru ORCID: https://orcid.org/0000-0001-9227-9173 Kseniya V. Fedorova, DMD, Orthodontist, „Impuls” dental clinic, Tver, Russian Federation For correspondence: k.ksushina2018@yandex.ru ORCID: https://orcid.org/0000-0002-1613-6581 Ludmila N. Sokolova, DMD, PhD, Associate Professor, Department of Pediatric Dentistry and Orthodontics, Tver State Medical University, Tver, Russian Federation For correspondence: sokolovaln18@mail.ru ORCID: https://orcid.org/0000-0002-8226-1807 Dmitriy V. Bobrov, DMD, PhD, Associate Professor, Department of Pediatric Dentistry and Orthodontics, Tver State Medical University, Tver, Russian Federation For correspondence: bobrov_d@tvergma.ru ORCID: https://orcid.org/0000-0001-6325-2641 Конфликт интересов: Авторы декларируют отсутствие конфликта интересов/ Сonflict of interests: The authors declare no conflict of interests Поступила / Article received 19.01.2022 Поступила после рецензирования / Revised 13.04.2022 Принята к публикации / Accepted 15.04.2022 2022; 22(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis 121
https://openalex.org/W2990360639	https://escholarship.org/content/qt6n74k0nw/qt6n74k0nw.pdf?t=rcjfrn	English	null	Molecular subtype and growth hormone effects on dysmorphology in Prader–Willi syndrome	American journal of medical genetics. Part A	2,019	cc-by	5,737	UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ UC Irvine UC Irvine Previously Published Works Title Molecular subtype and growth hormone effects on dysmorphology in Prader–Willi syndrome Permalink https://escholarship.org/uc/item/6n74k0nw Journal American Journal of Medical Genetics Part A, 182(1) ISSN 1552-4825 Authors Oldzej, Jeannine Manazir, Javeria Gold, June‐Anne et al. Publication Date 2020 DOI 10.1002/ajmg.a.61408 Copyright Information This work is made available under the terms of a Creative Commons Attribution L availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Molecular subtype and growth hormone effects on dysmorphology in Prader–Willi syndrome Permalink https://escholarship.org/uc/item/6n74k0nw Journal American Journal of Medical Genetics Part A, 182(1) ISSN 1552-4825 Authors Oldzej, Jeannine Manazir, Javeria Gold, June‐Anne et al. Publication Date 2020 DOI 10.1002/ajmg.a.61408 Copyright Information This work is made available under the terms of a Creative Commons Attribution L availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Jeannine Oldzej1 \| Javeria Manazir1 \| June-Anne Gold1,2,3 \| Ranim Mahmoud1,4 \| Kathryn Osann5 \| Pamela Flodman1 \| Suzanne B. Cassidy1,6 \| Virginia E. Kimonis1,2 1Division of Genetics and Genomic Medicine, Department of Pediatrics, University of California, Irvine, California 2Division of Biochemical and Clinical Genetics, Children's Hospital of Orange County, Orange, California 3Division of Genetics and Metabolism, Department of Pediatrics, Loma Linda University Medical School, Loma Linda, California 4Genetics Unit, Department of Pediatrics, Faculty of Medicine, Mansoura University, Mansoura, Egypt 5Department of Medicine, University of California, Irvine, California 6Division of Medical Genetics, Department of Pediatrics, University of California, San Francisco, California Correspondence Virginia E. Kimonis, Division of Genetics and Genomic Medicine, Department of Pediatrics, University of California Irvine, 101 The City Drive South, ZC4482, Orange CA 92868. Email: vkimonis@uci.edu Funding information March of Dimes Foundation 1Division of Genetics and Genomic Medicine, Department of Pediatrics, University of California, Irvine, California 2Division of Biochemical and Clinical Genetics, Children's Hospital of Orange County, Orange, California 3Division of Genetics and Metabolism, Department of Pediatrics, Loma Linda University Medical School, Loma Linda, California 4Genetics Unit, Department of Pediatrics, Faculty of Medicine, Mansoura University, Mansoura, Egypt 5Department of Medicine, University of California, Irvine, California 6Division of Medical Genetics, Department of Pediatrics, University of California, San Francisco, California Correspondence Virginia E. Kimonis, Division of Genetics and Genomic Medicine, Department of Pediatrics, University of California Irvine, 101 The City Drive South, ZC4482, Orange CA 92868. Email: vkimonis@uci.edu Funding information March of Dimes Foundation Abstract Prader–Willi syndrome (PWS) affects 1/15,000–1/30,000 live births and is characterized by lack of expression of paternally inherited genes on 15q11.2-15q13 caused by pater- nal deletions, maternal uniparental disomy (UPD), or imprinting defects. Affected individ- uals have distinct physical features, and growth hormone (GH) deficiency occurs in some individuals with PWS. The aim of this study is to test the hypotheses that (a) individuals with deletions and UPD have different physical and dysmorphic features, (b) individuals treated with GH have different physical and dysmorphic features than those not treated, and (c) GH treatment effects are different for individuals with UPD in comparison to those with deletions. Study participants included 30 individuals with deletions or UPD, who did or did not have GH treatment. Participants’ molecular abnormalities were deter- mined by molecular and cytogenetic analysis. Clinical data were obtained by a single dysmorphologist. 1 \| INTRODUCTION hypogonadism, short stature, and hyperphagia beginning in childhood leading to morbid obesity (Prader, Labhart, & Willi, 1956b). PWS is the most common genetic syndrome associated with obesity and obesity-related morbidity and mortality (Butler, 1990; Crino, Fintini, Bocchini, & Grugni, 2018). Correspondence dysmorphology, GH, imprinting disorders, microdeletion, Prader–Willi syndrome, uniparental disomy Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org Received: 11 May 2019 Revised: 28 September 2019 Accepted: 18 October 2019 Received: 11 May 2019 Revised: 28 September 2019 Accepted: 18 October 2019 DOI: 10.1002/ajmg.a.61408 DOI: 10.1002/ajmg.a.61408 Jeannine Oldzej1 \| Javeria Manazir1 \| June-Anne Gold1,2,3 \| Ranim Mahmoud1,4 \| Kathryn Osann5 \| Pamela Flodman1 \| Suzanne B. Cassidy1,6 \| Virginia E. Kimonis1,2 Individuals with deletions were found to be heavier (p = .001), taller (p = .031), with smaller head circumferences (p = .042) and were more likely to have fair skin and hair than their family members (p = .031, .049, respectively) compared to UPD patients. Females with deletions more commonly had hypoplastic labia minora (p = .009) and clitoris (.030) in comparison to those with UPD. Individuals who received GH in both deletion and UPD groups were taller (p = .004), had larger hands (p = .011) and feet (p = .006) and a trend for a larger head circumference (p = .103). Interestingly, the GH-treated group also had a lower rate of strabismus (esotropia [p = .017] and exotropia [p = .039]). This study showed statistically significant correlations between phenotype and molecular subtypes and also between phenotype and GH treatment. O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E wileyonlinelibrary.com/journal/ajmga Molecular subtype and growth hormone effects on dysmorphology in Prader–Willi syndrome Jeannine Oldzej1 \| Javeria Manazir1 \| June-Anne Gold1,2,3 \| Ranim Mahmoud1,4 \| Kathryn Osann5 \| Pamela Flodman1 \| Suzanne B. Cassidy1,6 \| Virginia E. Kimonis1,2 170 OLDZEJ ET AL. Cassidy & Driscoll, 2009) and is equally distributed throughout all genders and ethnic groups (Whittington et al., 2001). PWS was the first recognized disorder in humans caused by an error in genomic imprinting (Reik, 1989). It is characterized by lack of expression of genes on the paternally inherited chromosome 15q11.2-15q13 (Cassidy & Driscoll, 2009; Ledbetter et al., 1981; Muscogiuri et al., 2019; Nicholls, Knoll, Butler, Karam, & Lalande, 1989). Genomic imprinting is the modification of genes based on the parent of origin, leading to differential expression of maternal and paternal genes in the zygote (Monk, 1988). The majority of the genes in this region are involved in RNA and protein processing of neuroregulators and hormones; thus disruption in this region nega- tively affects neuronal development and endocrine function (Bittel & Butler, 2005). Three types of molecular lesions are known to cause PWS, all involving chromosome 15. Paternally inherited interstitial dele- tions in chromosome 15 are found in 65–75% of affected individuals (Cassidy & Driscoll, 2009; Ledbetter et al., 1981). Maternal uniparental disomy (UPD) of chromosome 15, first described in 1989 by Nicholls et al., occurs in 20–30% of affected patients (Cassidy & Driscoll, 2009; Nicholls et al., 1989). Sporadic deletions or microduplications in chromo- some 15 imprinting centers, regions that appear to control regional methylation patterns, occur in about 2–5% of affected patients (reviewed in Cassidy & Driscoll, 2009; Buiting et al., 1995). height after 8 years of GH treatment with a mean dose of 0.22 mg/ kg/week. Significant improvement in linear growth and adult height was reported (Bakker et al., 2017). These studies, and others, demon- strate the benefits of GH treatment in patients with PWS, and prompted the U.S. Food and Drug Administration to approve inject- able somatropin (GH) as a standard of care treatment for children with PWS who growth failure (Deal et al., 2013; Heksch, Kamboj, Anglin, & Obrynba, 2017). Although prior authors demonstrate improvements in physical characteristics in PWS-affected individuals following GH therapy, none have quantified this change in terms of dysmorphic features. The goals of this study were to determine if individuals with deletions were more likely to have dysmorphic features than those with UPD, to determine the effects of GH treatment on dysmorphic features in patients with PWS, and to determine if individuals with maternal UPD versus those with deletions respond differently to GH treatment. 2.1 Physical and facial features, including continuous and categorical vari- ables, were assessed. Continuous variables include measurements of height, weight, BMI, head circumference, facial features, arm span, hand and foot length, and penile length. For statistical purpose, this data were converted into age and gender-adjusted centiles using the WHO reference tables. Categorical variables of interest included presence of facial features (eye esotropia and exotropia, narrow nasal bridge, flat philtrum, downturned upper lip, thick and hypopigmented hair), and physical characteristics including height, head circumfer- ence, and other measures. 2 \| MATERIALS AND METHODS Study participants were recruited for a genotype–phenotype study in 2000–2003 at the University of California, Irvine (HS#: 2000-1405). Participants were recruited through notices on the websites for Prader–Willi Syndrome Association, USA and the statewide Prader– Willi California Foundation. Many participants contacted the project coordinator to participate, unsolicited, via the internet. After the pro- tocol was fully explained and all questions were answered, informed consent was signed by each participant and/or a legal guardian. 1.1.1 \| Facial dysmorphology and physical features When Prader et al. first described the syndrome in 1956, they reported several distinct facial and physical features associated with PWS, including distinct eyes, small hands and feet, hypogonadism, short stature, and obesity (Prader et al., 1956b). Typical PWS facial and physical features have been since noted to include narrow bifrontal diameter, almond-shaped palpebral fissures, narrow nasal bridge, thin upper lip with downturned mouth, small hands and feet, and scoliosis (Cassidy & Driscoll, 2009). The average height of individ- uals with PWS is below the third centile of the general population height beginning around 3 years of age, and the weight for affected individuals older than 2 years of age increases significantly compared to the general population average (Wollmann, Schultz, Grauer, & Ranke, 1998) if uncontrolled externally. Clinical and genetic data were obtained by standardized measure- ment of physical variables including facial features, as well as partici- pants description by a single dysmorphologist (SBC). 1.1 \| Background Prader–Willi syndrome (PWS) was one of the first described complex neurodevelopmental disorders (Prader, Labhart, & Willi, 1956a). It is characterized by an array of symptoms including hypotonia at birth, PWS affects about 1/15,000–1/30,000 individuals (reviewed in Butler, 1990; Cassidy, Schwartz, Miller, & Driscoll, 2012; © 2019 Wiley Periodicals, Inc. Am J Med Genet. 2020;182A:169–175. wileyonlinelibrary.com/journal/ajmga wileyonlinelibrary.com/journal/ajmga 1.1.2 \| Growth hormone treatment and benefits One of the first comprehensive studies to measure benefits of growth and body composition with use of GH on individuals with PWS was completed by Lindgren et al. (1997). All 27 enrolled individuals showed an increase in height and muscle mass and a decrease in body fat percentage, regardless of time on GH. The study also suggested that GH treatment improved the adverse behavioral and psychiatric issues that are associated with PWS (Lindgren et al., 1997). 17 171 OLDZEJ ET AL. 2 \| Data analysis The data were summarized using mean and SD for continuous vari- ables, such as height, weight, head circumference. Continuous vari- ables were analyzed using a two-sample t test and categorical variables were analyzed using Pearson's chi-square tests and Fisher's Bakker et al. (2017) described the largest international cohort of 522 prepubertal children with PWS who received GH therapy for three consecutive years and 173 adolescents who reached adult p values <.05 in bold and <.01 in asterix. p values <.05 in bold and <.01 in asterix. 172 OLDZEJ ET AL. average age at time of entry into the study was 9.2 years (range 4–16 years) for those treated and 12.1 years for those not treated with GH. GH treatment was started at average age of 6.4 years and was administered for an average of 2.3 years (range = 1 month– 8 years) by the time of the study. For individuals with UPD, the aver- age age at time of entry into the study was 11.8 years (range 3–29 years) for those treated and 9.9 years for those not treated with GH by the time of the study. GH treatment was started at average age of 8.3 years and was given for an average of 2.9 years (range = 1–6 years). These individuals were still receiving growth hor- mone at the time of enrollment. exact tests. When data were compared between subgroups classified by both molecular subtype and GH treatment, categorical variables were analyzed using Mantel–Haenszel chi-square test to test for GH effect after adjusting for molecular subtypes. For continuous vari- ables, a differential effect of GH treatment would be detected by a significant interaction between the two independent variables molec- ular subtype and GH treatment within the analysis of variance test (Table 2). For categorical variables, the Mantel–Haenszel test deter- mined whether there is an overall effect for GH treatment after adjusting for any differences due to molecular subtype. Statistical sig- nificance was considered at p < .05. Phenotypic characteristics of the study population according to molecular subtype and GH treatment TABLE 1 Phenotypic characteristics of the study population according to molecular subtype and GH treatment Molecular subclass Growth hormone treatment Physical measurements UPD Deletion p-value No GH GH p-value N Mean (SD) or % frequency N Mean (SD) or % frequency N Mean (SD) or % frequency N Mean (SD) or % frequency Height %ile 30 21.1 (28.0) 34 36.7 (28.3) .031 33 19.4 (25.5) 31 40.0 (29.2) .004* Weight %ile 30 63.9 (33.1) 34 87.4 (18.2) .001* 33 74.5 (32.3) 31 78.4 (24.5) .586 Weight % for height 29 80.7 (23.6) 31 91.6 (12.6) .033 30 87.5 (22.0) 30 85.1 (16.6) .641 BMI 30 23.5 (7.6) 34 26.6 (8.0) .114 33 25.8 (8.5) 31 24.4 (7.3) .467 Head circumference %ile 30 57.0 (31.8) 32 39.9 (33.1) .042 32 41.5 (30.8) 30 55.4 (34.9) .103 Innercanthal distance %ile 30 53.23 33 48 (5.3) .904 32 47.3 (5.5) 31 54.5 (5.4) .37 Interpupillary distance %ile 30 44.4 (5.7) 33 56 (5.6) .920 32 50 (5.8) 31 51 (5.9) .832 Outercanthal distance %ile 30 5.7 (2.1) 33 12.8 (4.4) .934 32 6.6 (2.4) 31 12.4 (4.5) .242 Palpebral fissure length %ile 30 17.1 (5.1) 32 20.5 (4.5) .748 31 16.1 (3.2) 31 22.4 (6.1) .365 Right ear length %ile 28 41.7 (23.2) 32 43.5 (26.1) .784 30 43.8 (24.6) 30 41.5 (25.0) .728 Chest: internipple distance (cm) 25 20.2 (16.3) 27 17.1 (4.3) .372 24 16.9 (4.8) 28 20.0 (15.2) .315 Chest: circumference (cm) 26 72.5 (22.2) 27 77.7 (17.8) .353 24 76.0 (20.5) 29 74.5 (20.0) .797 Right hand length %ile 28 29.5 (28.0) 33 37.0 (27.5) .297 30 24.4 (28.3) 31 42.3 (24.5) .011* Right middle finger length %ile 28 16.7 (17.5) 33 29.8 (25.1) .020 30 19.3 (22.2) 31 28.1 (22.8) .129 Right middle finger as % of hand 28 18.7 (21.7) 33 30.7 (28.5) .067 30 28.3 (26.4) 31 22.1 (25.8) .360 Right foot length %ile 27 20.2 (23.7) 32 17.5 (20.6) .643 29 10.9 (17.0) 30 26.3 (24.3) .006* Right male testis size (ml) 9 2.0 (1.1) 10 1.6 (0.8) .373 9 1.5 (0.9) 10 2.1 (0.9) .134 Male: penis length %ile 13 1.7 (2.5) 15 1.6 (17.8) .057 16 4.1 (12.2) 12 10.9 (15.4) .218 Hair: color normal for family 30 96.7 30 80.0 .044 30 83.3 30 93.3 .228 Hair: hypopigmented 11 18.2 12 58.3 .049 17 35.3 6 50.0 .526 Hair: thick 14 71.4 20 80.0 .226 15 60.0 19 89.5 .009* Eyes: esotropia 28 17.9 32 9.4 .335 29 24.1 31 3.2 .017 Eyes: exotropia 29 10.3 33 3.0 .242 31 12.9 31 0.0 .039 Nose: narrow 30 30.0 32 56.2 .037 31 61.3 31 25.8 .005* Mouth: dentition carious 27 7.4 27 33.3 .018 26 26.9 28 14.3 .249 Mouth: philtrum flat 30 3.3 33 9.1 .349 32 12.5 31 0.0 .042 Mouth: upper lip downturned 30 53.3 31 51.6 .893 31 67.7 30 36.7 .015 Mouth: dentition grinding 28 7.1 30 23.3 .089 27 25.9 31 6.5 .041 Ears: normal 29 86.2 33 100.0 .027 31 93.5 31 93.5 1.000 Chest: normal 28 100.0 33 84.8 .032 30 90.0 31 93.5 .614 Genu valgus 25 12.0 29 41.4 .050 26 38.5 28 17.9 .211 Skin: fair 29 37.9 32 65.6 .031 31 64.5 30 40.0 .055 Male: scrotum rugation poor 7 43.8 2 11.8 .039 19 31.6 14 21.4 .518 Male: scrotum hypoplastic 15 53.3 17 58.8 .755 18 77.8 14 28.6 .005* Female: labia minora hypoplastic 12 33.3 13 84.6 .009* 9 66.7 16 56.2 .610 Female: clitoris hypoplastic 11 36.4 11 81.8 .030 9 77.8 13 46.2 .138 Female: clitoris normal 10 70.0 11 36.4 .123 6 16.7 15 66.7 .038 Palmar creases single 17 17.6 11 9.1 .203 11 36.4 17 0.0 .027 Neuro: Babinski 25 8.0 23 17.4 .218 25 24.0 23 0.0 .015 Neuro: muscle bulk increased 22 0.0 19 5.3 .276 19 0.0 22 4.5 .347 p values <.05 in bold and <.01 in asterix. te: All measurements are in percentiles. p values <.05 in bold and <.01 in asterix. ments are in percentiles. p values <.05 in bold and <.01 in asterix. 3.1 \| Study participants Patients with the deletion subtype when compared with the UPD sub- group were found to be taller with a mean height percentile of 36.7 ± 28.3 versus 21.1 ± 28 (p value = .031), heavier with mean weight percentile 87.4 ± 18.2 versus 63.9 ± 33.1 (p value .001), a higher weight for height percentile (91.6 ± 12.6 vs. 80.7 ± 23.6 p value = .033) and a smaller head circumference percentile (39.9 ± 33 vs. 1 57 ± 31.8, p value = .042). There was a trend for increased frequency of narrow The study included 64 participants, 34 with deletions and 30 with UPD. Fifty percent (17/34) with deletions and 47% (14/30) with UPD had received GH treatment. Among the 64 participants there were 56 Caucasian, six Hispanic, one Iranian, and one Indian. Further details on the participants’ gender, average age, molecular subtype, GH treat- ment are presented in Table 1. For individuals with deletions, the TABLE 2 Effect of GH treatment between molecular subtypes 3.3 \| Comparison between GH treated and non-GH treated groups The study participants were grouped by GH treatment (treated vs. not treated), irrespective of molecular subtype. Table 1 compares the results of continuous and categorical variables. Statistically significant differences between GH treated versus nontreated were found for height (p = .004), and foot length (p = .006), but not weight or head circumference. The prevalence of features including narrow nasal bridge (p = .005), dentition grinding (p = .041) and thickness of hair (p = .009) was higher in the GH treated group. The difference in skin fairness between the GH treated and untreated groups appeared to be higher for participants without treatment, however this difference did not reach statistical significance (p = .055). The prevalence of hypoplastic scrotum was higher in the nontreated group (p = .005). Costeff, Holm, Ruvalcaba, and Shaver (1990) have reported that poor growth and obesity in PWS are at least partly due to GH defi- ciency (Costeff et al., 1990). Additionally the body habitus including decreased lean muscle mass, increased fat mass, and short stature in individuals with PWS are more similar to those of individuals with GH deficiency than to those with simple obesity (Brambilla et al., 1997). The association between obesity and reduction in GH secretion was first described by Roth, Glick, Yalow, and Berson (1963). Lindgren et al. (1997) have shown that individuals treated with GH have increased height and muscle mass, along with decreased body fat and BMI. Eiholzer et al. (1998) supported these findings and further showed that individuals treated with GH have increased hand and foot length, arm span, and lean body mass, associated with decreased weight and skin fold thickness. Although Whitman, Myers, Carrel, and Allen (2002) allude to a normalization of the appearance of affected individuals with PWS following the use of GH treatment; no studies have been conducted to measure differences specifically for facial dysmorphic features. The lack of correlation with other studies may be due to different study population, small sample size, and limited statistical power. Alternatively, improved study design (single observer blind to molecular subtype) may be responsible for detection of differ- ences not detected in other studies. OLDZEJ ET AL. 173 bifrontal diameter. Patients with the deletion subtype were more fair skinned and fair-haired (p = .031 and p = .049, respectively) than their family members, attributed to loss of a single copy of the OCA2 albi- nism gene in the 15q11-13 region. The deletion group had a higher incidence of carious dentition 33.3% versus 7.4% (p = .018). They also had a higher incidence of hypoplastic labia minora 84.6% versus 33.3% (p = .009), and clitoris 81.8% versus 36.4% (p = .03), with a trend for hypoplastic genitalia in males with deletions but no other significant dif- ferences in scrotal or penile anomalies. explanation for why individuals with UPD were diagnosed later than those with deletions, at an average age of 9.29 years compared with 3.76 years respectively (Gunay-Aygun, Heeger, Schwartz, & Cassidy, 1997). Dobrescu, Chirita-Emandi, Andreescu, Farcas, and Puiu (2016), however reported that the delayed age at diagnosis in their cohort was not related to the genotype but was attributable to the lack of medical expertise and molecular diagnostic tests in different regions. The analyses presented here include results from between geno- type and phenotype correlation studies as assessed by a single observer blinded to molecular subtype. Because the study sample included both participants on GH treatment as well as those who are not on treatment, this was also an opportunity to investigate the effect of GH treatment on patients’ physical characteristics and the possibility of different effects of GH treatment on different molecular subtypes. Our study has shown that individuals with dele- tions are taller, weigh more, have smaller head circumferences, longer middle finger length, and a higher prevalence of narrow nasal bridges, carious dentition, genu valgus, hypopigmentation, and hypoplastic genitalia in the females. Previous studies have shown that individuals with deletions have a higher frequency of hypopigmentation (Butler, 1989), increased weight, and a higher prevalence of characteristic facial features, including narrow bifrontal diameter, almond shaped palpebral fissures, narrow nasal bridge, and downturned mouth with thin upper lip (Cassidy et al., 1997). Lin et al. also reported that individuals with deletions tend to have smaller hands and feet and increased frequency of hypoplastic genitalia in females (Lin et al. 2007). 3.4 \| Comparison of GH effect between molecular subtypes Analysis of effects of GH treatment was also undertaken for each indi- vidual molecular subtype separately (Table 2) for each variable. Based on the results of the analyses, there were no statistically significant dif- ferences by the Mantel–Haenszel chi-square tests between molecular subtypes in the effect of GH treatment on physical characteristics. In the deletions group, the individuals on GH appear to be proportionately taller (126.3 ± 23.5) than those who are on GH with UPD (115.8 ± 24) although this difference did not reach statistical significance (p = .068). TABLE 2 Effect of GH treatment between molecular subtypes yp UPD Deletion Physical measurements No GH GH p value (GH-no GH) No GH GH p value (GH-no GH) N Mean (SD) N Mean (SD) N Mean (SD) N Mean (SD) Height %ile 16 57.7 (27.9) 14 38.7 (32.5) .002* 17 32.3 (29.6) 17 41.1 (27.2) .375 Weight %ile 16 62.0 (38.4) 14 66.0 (26.6) .742 17 86.2 (19.4) 17 88.6 (17.5) .710 Weight for height %ile 15 80.1 (29.0) 14 81.4 (17.1) .879 15 94.9 (6.5) 16 88.4 (15.9) .147 BMI 16 24.3 (9.3) 14 22.5 (5.2) .511 17 27.2 (7.7) 17 25.9 (8.4) .635 Head circumference %ile 16 47.6 (31.5) 14 67.8 (29.6) .081 16 35.3 (29.9) 16 44.5 (36.4) .441 Upper/lower segment ratio 16 1.1 (0.1) 14 1.0 (0.1) .031 15 1.1 (0.1) 16 1.1 (0.1) .719 Arm/span height ratio 15 1.0 (0.03) 14 1.0 (0.1) .683 15 1.0 (0.03) 16 1.0 (0.02) .892 Innercanthal distance %ile 16 2.8 (0.4) 14 3.1 (0.3) .042 16 2.9 (0.3) 17 2.9 (0.4) .982 Interpupillary distance %ile 16 5.2 (0.7) 14 5.4 (0.5) .415 16 5.3 (0.5) 17 5.3 (0.5) .953 Outercanthal distance %ile 16 8.1 (1.0) 14 8.4 (0.7) .329 16 8.1 (0.8) 17 8.3 (0.7) .626 Palpebral fissure length %ile 16 2.6 (0.3) 14 2.7 (0.3) .156 15 2.6 (0.3) 17 2.6 (0.2) .791 Right ear length %ile 15 9.5 (22.5) 13 44.2 (24.6) .605 15 48.0 (26.5) 17 39.5 (25.9) .366 Chest: internipple distance %ile 13 17.0 (5.2) 12 23.6 (22.9) .346 11 16.8 (4.6) 16 17.4 (4.1) .772 Chest: circumference %ile 13 76.1 (24.5) 13 68.9 (20.0) .420 11 75.8 (15.8) 16 79.1 (19.4) .633 Right hand length %ile 14 17.8 (26.1) 14 41.1 (25.5) .024 16 30.2 (29.7) 17 43.3 (24.4) .180 Right middle finger length %ile 14 8.1 (10.0) 14 25.2 (19.5) .009* 16 29.0 (25.4) 17 30.5 (25.5) .864 Right middle finger as % hand 14 20.7 (21.9) 14 16.6 (22.1) .628 16 34.9 (28.9) 17 26.6 (28.3) .412 Right foot length %ile 14 9.7 (11.7) 13 31.5 (28.3) .020 15 12.1 (19.9) 17 22.3 (20.7) .165 Right male testis size 4 1.6 (1.0) 5 2.4 (1.1) .261 5 1.4 (0.9) 5 1.8 (0.7) .419 Male: penis length 9 1.0 (0.0) 4 3.2 (4.5) — 7 8.0 (18.5) 8 14.8 (17.7) .485 Note: All measurements are in percentiles. p values <.05 in bold and <.01 in asterix. 174 OLDZEJ ET AL. The results of this study suggest the possibility of a difference in height centile between individuals with UPD compared with individ- uals with deletions (p = .031) irrespective of GH treatment. Results also support a significant difference in height percentile associated with GH treatment (p = .004). These findings, together with the results for height percentile in Table 2, suggest that there may be a complex relationship between molecular subtype and GH treatment for this variable. While untreated individuals with UPD appear shorter on average than untreated individuals in the deletion group, the increase in height associated with GH treatment appears to be greater in the UPD than in the deletion group. Larger studies will be needed to confirm this suggestive finding. REFERENCES Bakker, N. E., Lindberg, A., Heissler, J., Wollmann, H. A., Camacho- Hubner, C., Hokken-Koelega, A. C., & Committee, K. S. (2017). Growth hormone treatment in children with Prader-Willi syndrome: Three years of longitudinal data in Prepubertal children and adult height data from the KIGS database. The Journal of Clinical Endocrinol- ogy and Metabolism, 102(5), 1702–1711. Bittel, D. C., & Butler, M. G. (2005). Prader-Willi syndrome: Clinical genet- ics, cytogenetics and molecular biology. Expert Reviews in Molecular Medicine, 7(14), 1–20. Brambilla, P., Bosio, L., Manzoni, P., Pietrobelli, A., Beccaria, L., & Chiumello, G. (1997). Peculiar body composition in patients with Prader-Labhart-Willi syndrome. The American Journal of Clinical Nutri- tion, 65(5), 1369–1374. Buiting, K., Saitoh, S., Gross, S., Dittrich, B., Schwartz, S., Nicholls, R. D., & Horsthemke, B. (1995). Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting Centre on human chro- mosome 15. Nature Genetics, 9(4), 395–400. 4.1 \| Study limitations Butler, M. G. (1989). 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Comparison of phenotype between patients with Prader-Willi syndrome due to deletion 15q and unipa- rental disomy 15. American Journal of Medical Genetics, 68(4), 433–440. DATA AVAILABILITY STATEMENT Gunay-Aygun, M., Heeger, S., Schwartz, S., & Cassidy, S. B. (1997). Delayed diagnosis in patients with Prader-Willi syndrome due to maternal uniparental disomy 15. American Journal of Medical Genetics, 71(1), 106–110. The data that support the findings of this study are available from the corresponding author upon reasonable request. Heksch, R., Kamboj, M., Anglin, K., & Obrynba, K. (2017). Review of Prader-Willi syndrome: The endocrine approach. Translational Pediat- rics, 6(4), 274–285. ORCID Virginia E. Kimonis https://orcid.org/0000-0003-1567-4449 4 \| DISCUSSION Our studies suggest benefits of GH therapy treatment in individuals with PWS. Individuals being treated with GH, irrespective of molecular subtype, are taller, have bigger hands and feet, thicker hair, have a lower prevalence of esotropia, exotropia, narrow nose, flat philtrum, downturned upper lip, dentition grinding, single palmar crease, fair skin, and hypoplastic scrotum. It has previously been suggested that the facial features and physical characteristics of PWS may differ in the three different molecular subtypes. Individuals with UPD were previ- ously reported to have a lower frequency of dysmorphic facial features (Cassidy et al., 1997; Gillessen-Kaesbach et al., 1995). This provided an This study examines whether the effect of GH treatment differed depending on molecular etiology. We did not identify any significantly different effects of GH treatment between the two molecular subgroups for any of the dysmorphology variables or physical measurements. 4.2 \| Conclusions Cassidy, S. B., Schwartz, S., Miller, J. L., & Driscoll, D. J. (2012). Prader-Willi syndrome. Genetics in Medicine, 14(1), 10–26. Costeff, H., Holm, V. A., Ruvalcaba, R., & Shaver, J. (1990). Growth hor- mone secretion in Prader-Willi syndrome. Acta Paediatrica Scandinavica, 79(11), 1059–1062. This study although small, shows statistically significant genotype– phenotype correlations between 34 individuals with Prader–Willi syn- drome due to paternal deletions and 30 individuals with maternal uni- parental disomy for several growth measurements and physical anomalies. The deletion group is more likely to be taller and weigh more, have smaller head circumferences, have fair skin, and hypoplas- tic genitalia compared to the UPD group. Crino, A., Fintini, D., Bocchini, S., & Grugni, G. (2018). Obesity manage- ment in Prader-Willi syndrome: Current perspectives. Diabetes, Meta- bolic Syndrome and Obesity: Targets and Therapy, 11, 579–593. Deal, C. L., Tony, M., Hoybye, C., Allen, D. B., Tauber, M., Christiansen, J. S., & Growth Hormone in Prader-Willi Syndrome Clini- cal Care Guidelines Workshop Participants. (2013). GrowthHormone research society workshop summary: Consensus guidelines for recombinant human growth hormone therapy in Prader-Willi syn- drome. The Journal of Clinical Endocrinology and Metabolism, 98(6), E1072–E1087. The study also shows significant differences between 31 individ- uals with PWS who have been treated with GH and 33 individuals who have not been treated with GH. Those individuals on GH are more likely to be taller, have larger hands and feet, and interestingly a lower frequency of esotropia and had a lower frequency of fair skin (p = .055). Finally, there were no significant findings in regards to the differential effects of GH treatment between the molecular subtypes, although it appeared that GH treatment had a larger effect for individ- ual with uniparental disomy when compared to those with deletions. Dobrescu, A. I., Chirita-Emandi, A., Andreescu, N., Farcas, S., & Puiu, M. (2016). Does the genetic Causof Prader-Willi syndrome explain the highly variable phenotype? Maedica (Buchar), 11(3), 191–197. Eiholzer, U., Gisin, R., Weinmann, C., Kriemler, S., Steinert, H., Torresani, T., … Prader, A. (1998). Treatment with human growth hor- mone in patients with Prader-Labhart-Willi syndrome reduces body fat and increases muscle mass and physical performance. European Journal of Pediatrics, 157(5), 368–377. 174 174 CONFLICT OF INTEREST Gillessen-Kaesbach, G., Robinson, W., Lohmann, D., Kaya-Westerloh, S., Passarge, E., & Horsthemke, B. (1995). Genotype-phenotype correla- tion in a series of 167 deletion and non-deletion patients with Prader- Willi syndrome. Human Genetics, 96(6), 638–643. ORCID ORCID Virginia E. Kimonis https://orcid.org/0000-0003-1567-4449 Virginia E. Kimonis https://orcid.org/0000-0003-1567-4449 OLDZEJ ET AL. 175 Ledbetter, D. H., Riccardi, V. M., Airhart, S. D., Strobel, R. 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Effects of growth hormone treat- ment on growth and body composition in Prader-Willi syndrome: A preliminary report. The Swedish National Growth Hormone Advisory Group. Acta Paediatrica. Supplement, 423, 60–62. Whittington, J. E., Holland, A. J., Webb, T., Butler, J., Clarke, D., & Boer, H. (2001). Population prevalence and estimated birth incidence and mor- tality rate for people with Prader-Willi syndrome in one UKhealth region. Journal of Medical Genetics, 38(11), 792–798. Monk, M. (1988). Genomic imprinting. Genes & Development, 2(8), 921–925. region. Journal of Medical Genetics, 38(11), 792–798. Wollmann, H. A., Schultz, U., Grauer, M. L., & Ranke, M. B. (1998). Reference values for height and weight in Prader-Willi syndrome based on 315 patients. European Journal of Pediatrics, 157(8), 634–642. Muscogiuri, G., Formoso, G., Pugliese, G., Ruggeri, R. M., Scarano, E., Colao, A., & Restare. (2019). Prader-Willi syndrome: An uptodate on endocrine and metabolic complications. Reviews in Endocrine & Meta- bolic Disorders, 20(2), 239–250. Nicholls, R. D., Knoll, J. H., Butler, M. G., Karam, S., & Lalande, M. (1989). Genetic imprinting suggested by maternal heterodisomy in non- deletion Prader-Willi syndrome. Nature, 342(6247), 281–285. Prader, A., Labhart, A., & Willi, H. (1956a). Ein Syndrom von Adipositas, Kleinwuchs, Kryptorchismus, und Oligophrnie Nach Myatonieartigem Zustand im Neugerborenenalter. Schweizerische Med Wochenschr, 8, 1260–1261. How to cite this article: Oldzej J, Manazir J, Gold J-A, et al. Molecular subtype and growth hormone effects on dysmorphology in Prader–Willi syndrome. Am J Med Genet Part A. 2020;182A:169–175. https://doi.org/10.1002/ajmg.a. 61408 How to cite this article: Oldzej J, Manazir J, Gold J-A, et al. Molecular subtype and growth hormone effects on dysmorphology in Prader–Willi syndrome. Am J Med Genet Part A. 2020;182A:169–175. https://doi.org/10.1002/ajmg.a. 61408 Prader, A., Labhart, H., & Willi, H. (1956b). 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https://openalex.org/W4280603230	https://www.researchsquare.com/article/rs-1137183/latest.pdf	English	null	Ultra-flat optical frequency comb generation based on electro-optic intensity modulator with digital driving signal	Research Square (Research Square)	2,022	cc-by	4,921	Ultra-§at optical frequency comb generation based on electro-optic intensity modulator with digital driving signal Yu Liu Shanghai University Shibao Wu (  wushibao@shu.edu.cn ) Shanghai University https://orcid.org/0000-0002-3378-7589 Xianmin Shen Shanghai University Yu Liu Shanghai University Shibao Wu (  wushibao@shu.edu.cn ) Shanghai University https://orcid.org/0000-0002-3378-7589 Xianmin Shen Shanghai University 1. Introduction Optical frequency comb has become the focus of research in recent years, and has been applied to the fields of Optical communication, photonic microwave signal processing, precise optical metrology, arbitrary waveform generation and so on[1]-[3]. In high-speed optical transmission system, flat optical frequency comb can be used as a multi wavelength light source for communication, and the flatness is an important index of the optical frequency combs in this case. At present, there are many methods to generate optical frequency comb, such as mode-locked laser method, nonlinear effect method of nonlinear medium, micro resonant cavity method and the method based on electro-optic modulator[4]- [8]. Among them, the method based on electro-optic modulator is widely used because it is easy to realize[9]. Common methods for generating optical frequency combs based on electro-optic modulators include recirculation frequency shift and direct modulation[10]-[13]. Recirculation frequency shift includes unidirectional recirculation frequency shift and bidirectional recirculation frequency shift, the seed light source of the loop can be single seed source or multiple seed source. The main disadvantage is that with the increase of the number of cycles, the noise in the system will accumulate. The direct modulation can use intensity modulator, phase modulator, polarization modulator and Mach-Zehnder modulator, and the modulators can be connected in parallel or cascaded. By changing the drive signal and direct current (DC) bias of the modulator, the comb spacing, the number of comb lines and the flatness of the generated optical frequency comb can be controlled effectively. Yan mentioned that using digital signals instead of radio frequency signals to drive modulators, and using simulated annealing algorithms to select and design the input pseudo random data sequences[14]. However, this method is fully programmed, the selected algorithm is complex. When the number of comb lines increases or the comb spacing becomes larger, the comb lines will become more and more uneven. Therefore, in this paper, we report an implementation scheme of an ultra-flat optical frequency comb generator based on the digital signal-driven intensity modulator (IM) and band-stop optical filter. It is more flexible and simpler compared with previous methods based on the radio frequency driven IM. After IM modulation, a uniform but uneven optical frequency comb can be generated, then we use a band-stop filter to adjust the amplitude of comb lines. Corresponding author E-mail address: wushibao@shu.edu.cn Abstract: We propose a new scheme to generate flat optical frequency combs (OFCs) by the electro- optic modulation method. A digital signal is used instead of the usual RF signal to drive an electro-optic intensity modulator (IM), a band-stop filter is employed to flatten the comb lines. When suitable filter bandwidth is found, the ultra-flat optical frequency comb will be generated. The scheme can generate a large number of comb lines, the number of lines is selective, and the flatness of the comb lines is less than 0.4 dB. The comb spacing of OFCs can be adjusted by controlling the bit rate of the digital signal which is simple and easy to operate. The theoretical model of the scheme is established. Two kinds of band-stop filters (Butterworth and Gaussian) are used for comparison. Keywords: Optical frequency comb; Intensity modulation; Digital driving signal; Band-stop filter Key laboratory of Specialty Fiber Optics and Optical Access Networks, Shanghai University, Baoshan District, Shanghai 200444, China Key laboratory of Specialty Fiber Optics and Optical Access Networks, Shanghai University, Baoshan District, Shanghai 200444, China Research Article Keywords: Optical frequency comb, Intensity modulation, Digital driving signal, Band-stop ¦lter Posted Date: May 11th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1137183/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Ultra-flat optical frequency comb generation based on electro- optic intensity modulator with digital driving signal Yu Liu, Shibao Wu, Xianmin Shen Key laboratory of Specialty Fiber Optics and Optical Access Networks, Shanghai University, Baoshan District, Shanghai 200444, China 1. Introduction Not only can the comb spacing be tuned in a large range, which is suitable for the optical transmission system, but also can generate lots of comb lines. The proposed scheme is simple in structure and easy to implement, and the generated optical frequency comb is very flat, the flatness is less than 0.4dB under the condition of appropriate filter bandwidth. Finally, we compared the performance of the two kinds of band-stop filters in the system. 1 2. Principle and theoretical analysis Here we present a proof-of-concept device based on IM for generating ultra-flat optical frequency combs. The device is mainly composed of pulse generator, continuous-wave seed laser, intensity modulator, band-stop filter, band-pass filter, as shown in the Fig.1. Fig. 1 Proof-of-concept setup for generating the OFC of high quality based on IM PG: pulse generator; CW: continuous-wave; IM: intensity modulator; BSF: Band-stop filter; BPF: Band-pass filter; OSA: optical l Fig. 1 Proof-of-concept setup for generating the OFC of high quality based on IM PG: pulse generator; CW: continuous-wave; IM: intensity modulator; BSF: Band-stop filter; BPF: Band-pass filter; OSA: optica spectrum analyzer PG: pulse generator; CW: continuous-wave; IM: intensity modulator; BSF: Band-stop filter; BPF: Band-pass filter; OSA: optica spectrum analyzer generator; CW: continuous-wave; IM: intensity modulator; BSF: Band-stop filter; BPF: Band-pass filter; OSA: optical l In the time domain, a waveform generator controlled by a simple binary sequence 101010… generates a periodic square-wave signal with duty ratio of 0.5. f(t) ... t 0 2 T 3 2 T 2T T 2 T  3 2 T  T  ... A f(t) ... t 0 2 T 3 2 T 2T T 2 T  3 2 T  T  ... A Fig. 2 Periodic square-wave signal waveform Fig. 2 is a waveform diagram of a periodic square-wave signal. In one period f(t) Fig. 2 Periodic square-wave signal waveform Fig. 2 is a waveform diagram of a periodic square-wave signal. In one period  0 2 0 0 2 T A t f t T t         (1) (1) Expanding ( ) f t with the Fourier series of the trigonometric Expanding ( ) f t with the Fourier series of the trigonometric Expanding ( ) f t with the Fourier series of the trigonometric    0 0 0 0 0 0 2 2 2 2 sin sin3 sin5 ... sin ... 3 5 sin n n A A A A f t t t t n t n a a n t                    (2) function: (2) Where 0 2 = T   , and T is the period of the square-wave signal. 0 0 a  , n is an odd integer, 1, 3, 5... 2. Principle and theoretical analysis IM out in in n n n in n n E E f E c n c E n A n n n                                      (4) (4) In order to obtain flat optical frequency combs, we use a band-stop filter to shape the uneven comb lines. Two kinds of band-stop filters, Gaussian filter and Butterworth filter, will be used for comparative analysis. First, we consider using Gaussian band-stop filter. The normalized power transfer function of a linea time-invariant Gaussian band-stop filter can be expressed as: 𝐻𝐺(𝜔) = 1 −𝑒𝑥𝑝[− 𝑙𝑛2⋅(𝜔−𝜔𝑐)2𝑘 2𝜔𝑓2 ] (5) (5) Where c 2 cf    , cf is central frequency of this filter. And 2 f   is the 3dB bandwidth of the filter, k is the order of the Gaussian band-stop filter. Where c 2 cf    , cf is central frequency of this filter. And 2 f   is the 3dB bandwidth of the filter, k is the order of the Gaussian band-stop filter. Thus, the final output power spectrum of the experimental device is obtained:          2 _ _ 2 2 0 c 0 2 2 2 0 0 2 ln 2 2 E 1 exp 2 ln 2 2 E 1 exp , 1, 3, 5... 2. Principle and theoretical analysis n  and ( ) f t in the frequency domain is: Where 0 2 = T   , and T is the period of the square-wave signal. 0 0 a  , n is an odd integer, 1, 3, 5... n  and ( ) f t in the frequency domain is: Where 0 2 = T   , and T is the period of the square-wave signal. 0 0 a  , n is an odd integer Where 0 2 = T   , and T is the period of the square-wave signal. 0 0 a  , n is an odd integer, 1 3 5  d ( ) f t i th f d i i Where 0 2 = T   , and T is the period of the square-wave signal. 0 0 a  , n is an odd integer, 1 3 5  d ( ) f t i th f d i i 1, 3, 5... n  and ( ) f t in the frequency domain is:    0 = 2 n n f c n         (3) (3) 2 The amplitude value of each spectral line in the complex spectrum is / 2 / n n c a A n    , the spectral line interval is 2 / T . The input optical signal of the IM can be expressed in frequency domain as    0 c =2 E in E        , where c 2 cf    , cf is central frequency of the seed light from the CW laser, and 0 E is the electric field amplitude of the optical signal. The output of the intensity modulator in the frequency domain can be expressed as:           _ 0 0 0 c 0 1 2 2 E , 1, 3, 5... 2. Principle and theoretical analysis 2 G out IM out G k c n n f k n f P E H c n n A n n                                                                           (6) (6) The same analysis can be carried out for the Butterworth band-stop filter, whose power transmission function as follows: The same analysis can be carried out for the Butterworth band-stop filter, whose power transmission function as follows: 2 1 ( ) 1 1 ( ) B k c f H        (7) (7) Thus, the final output is: 3    2 _ _ B out IM out B P E H           2 _ _ B out IM out B P E H        2 0 c 0 2 2 0 2 0 1 2 E 1 1 ( ) 2 E 1 1 , 1, 3, 5... 1 ( ) n k c n f k n f c n A n n n                                                            (8) (8) It can be seen from the equation(6) and equation(8) that when both n and 0  are determined, the magnitude of each spectral line is only related to f , the bandwidth of the filter can be changed to adjust the flatness of the OFC. 3. Experimental simulation and results analysis In this section, we discuss the generation of ultra-flat optical frequency comb based on the digital signal- driven IM and band-stop optical filter. In order to verify the feasibility of our proposed scheme, we use VPI TransmissionMaker commercial software to make a simulation, and analyze the performance of the scheme. As shown in Fig.1, one continuous-wave (CW) seed laser is used as the light source with output power of 10mW , center frequency at193.1THz and linewidth at100KHz . The MZM modulator with 40GHz bandwidth is in push-pull mode to act as an intensity modulator. In this case, we set the half- wave voltage of the MZM to 3.5V , the extinction ratio to35dB , the insertion less to 6dB and the DC bias is 0.5V. The binary sequence is 101010... with duty cycle of 0.5 . The center frequency of the band- stop filter is 193.1THz which is the same as the seed laser. The comb spacing of the generated optical frequency comb can be changed by controlling the bit rate of square-wave signal. Fig. 3 shows the output spectrum of the intensity modulator, the spectrum is a 64-line optical frequency comb with the comb spacing of 6.25GHz. It is obvious that the comb is not flat and also contains the carrier, and the comb spacing is not uniform. Therefore, a filter is added after the intensity modulator to adjust the amplitude of comb lines to generate ultra-flat optical frequency combs. The following analysis is based on the Gaussian filter. Fig. 3 The output 64-line optical comb of the IM with the comb spacing of 6.25GHz According to the above analysis in the section 2, changing the filter bandwidth will change the flatness of the comb. Therefore, in order to study the influence of filter bandwidth on the flatness of optical frequency combs, different filter bandwidths will be set for the research. Figure 4 shows the flatness of the 64-line optical frequency comb with 6.25GHz comb spacing at different filter bandwidths after the Gaussian band-stop filter. When the filter bandwidth is set to 100GHz, 232.24GHz and 400GHz, the measured flatness of the generated optical frequency comb is 5.7dB, 0.25dB and 1.8dB respectively, as shown in Fig.4 (a-c). It can be seen that there is an optimal filter bandwidth of 232.24GHz with which the generated optical frequency comb is the flattest. Fig. 3. Experimental simulation and results analysis 3 The output 64-line optical comb of the IM with the comb spacing of 6.25GHz Fig. 3 The output 64-line optical comb of the IM with the comb spacing of 6.25GHz Fig. 3 The output 64-line optical comb of the IM with the comb spacing of 6.25GHz According to the above analysis in the section 2, changing the filter bandwidth will change the flatness of the comb. Therefore, in order to study the influence of filter bandwidth on the flatness of optical frequency combs, different filter bandwidths will be set for the research. Figure 4 shows the flatness of the 64-line optical frequency comb with 6.25GHz comb spacing at different filter bandwidths after the Gaussian band-stop filter. When the filter bandwidth is set to 100GHz, 232.24GHz and 400GHz, the measured flatness of the generated optical frequency comb is 5.7dB, 0.25dB and 1.8dB respectively, as shown in Fig.4 (a-c). It can be seen that there is an optimal filter bandwidth of 232.24GHz with which the generated optical frequency comb is the flattest. According to the above analysis in the section 2, changing the filter bandwidth will change the flatness of the comb. Therefore, in order to study the influence of filter bandwidth on the flatness of optical frequency combs, different filter bandwidths will be set for the research. Figure 4 shows the flatness of the 64-line optical frequency comb with 6.25GHz comb spacing at different filter bandwidths after the Gaussian band-stop filter. When the filter bandwidth is set to 100GHz, 232.24GHz and 400GHz, the measured flatness of the generated optical frequency comb is 5.7dB, 0.25dB and 1.8dB respectively, as shown in Fig.4 (a-c). It can be seen that there is an optimal filter bandwidth of 232.24GHz with which the generated optical frequency comb is the flattest. 4 (a) (b) (c) Fig. 4 The flatness of comb lines varies with the bandwidth of Gaussian band-stop filter (a) 100 GHz, (b) 232.24 GHz, (c) 400GHz (a) 5 (a) (b) (c) Fig. 4 The flatness of comb lines varies with the bandwidth of Gaussian band-stop filter (a) 100 GHz, (b) 232.24 GHz, (c) 400GHz In the above research, the filters are all set to the first order. It can be analyzed from equation (5) and equation (6) that different order of the filter will generate different output power distribution. 3. Experimental simulation and results analysis In order to study the effect of the filter order on the filter output, different orders were set for Gaussian filter. The flattest 64-line optical frequency combs generated with different filter orders are shown in Fig. 5, the spectral line interval is 6.25GHz. As can be seen from the Fig.5, when the order is greater than 1, there will be a middle sag, which makes it impossible to form a continuous flat optical frequency comb near the center frequency. Therefore, the first order filter is selected in our research. (a) (b) (b) (c) Fig. 4 The flatness of comb lines varies with the bandwidth of Gaussian band-stop filter (a) 100 GHz, (b) 232.24 GHz, (c) 400GHz (c) Fig. 4 The flatness of comb lines varies with the bandwidth of Gaussian band-stop filter (a) 100 GHz, (b) 232.24 GHz, (c) 400GHz In the above research, the filters are all set to the first order. It can be analyzed from equation (5) and equation (6) that different order of the filter will generate different output power distribution. In order to study the effect of the filter order on the filter output, different orders were set for Gaussian filter. The flattest 64-line optical frequency combs generated with different filter orders are shown in Fig. 5, the spectral line interval is 6.25GHz. As can be seen from the Fig.5, when the order is greater than 1, there will be a middle sag, which makes it impossible to form a continuous flat optical frequency comb near the center frequency. Therefore, the first order filter is selected in our research. 5 5 5 (a) (b) Fig. 5 The flattest 64-line optical frequency combs generated with the Gaussian band-stop filter of (a) the 2nd-order, (b) the 3rd-order In order to obtain the most suitable 3dB bandwidth for generating flat optical frequency combs, we further study the relationship between the flatness and the bandwidth of the optical frequency combs with different comb lines, the comb spacing is 6.25GHz. It can be seen from the figure 6 that when more flat comb lines need to be generated, the required filter bandwidth will increase. (a) (b) Fig. 5 The flattest 64-line optical frequency combs generated with the Gaussian band-stop filter of (a) the 2nd-order, (b) the 3rd-order (a) (b) Fig. 3. Experimental simulation and results analysis 5 The flattest 64-line optical frequency combs generated with the Gaussian band-stop filter of (a) the 2nd-order, (b) the 3rd-order (a) Fig. 5 The flattest 64-line optical frequency combs generated with the Gaussian band-stop filter of (a) the 2nd-order, (b) the 3rd-order In order to obtain the most suitable 3dB bandwidth for generating flat optical frequency combs, we further study the relationship between the flatness and the bandwidth of the optical frequency combs with different comb lines, the comb spacing is 6.25GHz. It can be seen from the figure 6 that when more flat comb lines need to be generated, the required filter bandwidth will increase. Fig. 6 The comb flatness at different filter bandwidths after Gaussian band-stop filter Butterworth filter and the Gaussian filter have similar transmission characteristics, but there are still some differences. The two kinds of filters, Gaussian and Butterworth, will be compared and analyzed below The following analysis is based on the first order filters Fig. 6 The comb flatness at different filter bandwidths after Gaussian band-stop filter Butterworth filter and the Gaussian filter have similar transmission characteristics, but there are still some differences. The two kinds of filters, Gaussian and Butterworth, will be compared and analyzed below. The following analysis is based on the first order filters. Butterworth filter and the Gaussian filter have similar transmission characteristics, but there are still some differences. The two kinds of filters, Gaussian and Butterworth, will be compared and analyzed below. The following analysis is based on the first order filters. Fig. 7 shows the relationship between the comb flatness and the bandwidth of two kinds of filters when the generated optical frequency combs with 128 lines and 6.25GHz comb spacing. In the case of the same number of comb lines and the same comb spacing. Gaussian filter requires smaller 3dB bandwidth to achieve flattest comb that of Butterworth filter. Fig. 7 shows the relationship between the comb flatness and the bandwidth of two kinds of filters when the generated optical frequency combs with 128 lines and 6.25GHz comb spacing. In the case of the same number of comb lines and the same comb spacing. Gaussian filter requires smaller 3dB bandwidth to achieve flattest comb that of Butterworth filter. 6 Fi 7 Th b fl diff fil b d id h f h f fil Fig. 3. Experimental simulation and results analysis Figure 10 shows 100 comb lines selected from an optical frequency comb with 128 comb lines and 2.5GHz comb spacing. In fact, we can add a band-pass filter to select the needed number of comb lines. Figure 10 shows 100 comb lines selected from an optical frequency comb with 128 comb lines and 2.5GHz comb spacing. Fig. 10 100 comb lines selected from an optical frequency comb with 128 comb lines Fig. 10 100 comb lines selected from an optical frequency comb with 128 comb lines 4. Conclusion We propose a new scheme to generate flat optical frequency combs based on the digital signal-driven intensity modulator and band-stop optical filter. By theory analysis and experiment simulation to verify the feasibility of the proposed scheme. The scheme is more flexible and simpler compared with previous methods based on the radio frequency driven IM. The generated optical frequency combs have the characteristics such as a large number of comb lines, selectable number of comb lines and adjustable comb spacing. When a proper filter bandwidth is chosen, ultra-flat optical frequency combs can be generated. In addition, Gaussian filter and Butterworth filter have similar effect on the generation of the optical frequency combs, using Gaussian filter is relatively better. 3. Experimental simulation and results analysis 7 The comb flatness at different filter bandwidths for the two types of filters Figure 8 is the flatness of the generated flattest optical frequency combs with 6.25GHz comb spacing and different comb line numbers, respectively. As long as the appropriate filter bandwidth is selected, the comb spacing has little or no affection on the flatness of the optical frequency combs. Fig. 8 The combs flatness at different comb line numbers under two types of filters When the power of the seed carrier is determined, the average power will decrease as the number of comb lines increases. Figure 9 shows the average power of the output comb lines when the flattest combs are generated with different comb line numbers. It can be seen that the average comb line power by Gaussian filter is larger than by Butterworth filter. Fig. 8 The combs flatness at different comb line numbers under two types of filters When the power of the seed carrier is determined, the average power will decrease as the number of comb lines increases. Figure 9 shows the average power of the output comb lines when the flattest combs are generated with different comb line numbers. It can be seen that the average comb line power by Gaussian filter is larger than by Butterworth filter. When the power of the seed carrier is determined, the average power will decrease as the number of comb lines increases. Figure 9 shows the average power of the output comb lines when the flattest combs are generated with different comb line numbers. It can be seen that the average comb line power by Gaussian filter is larger than by Butterworth filter. 7 7 Fig. 9 Average comb line powers at different comb lines with 6.25GHz comb spacing In fact, we can add a band-pass filter to select the needed number of comb lines. Figure 10 shows 100 comb lines selected from an optical frequency comb with 128 comb lines and 2.5GHz comb spacing. Fig. 9 Average comb line powers at different comb lines with 6.25GHz comb spacing In fact, we can add a band-pass filter to select the needed number of comb lines. Figure 10 shows 100 comb lines selected from an optical frequency comb with 128 comb lines and 2.5GHz comb spacing. In fact, we can add a band-pass filter to select the needed number of comb lines. 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https://openalex.org/W2079687194	https://europepmc.org/articles/pmc3978244?pdf=render	English	null	Homeostatic structural plasticity increases the efficiency of small-world networks	Frontiers in synaptic neuroscience	2,014	cc-by	15,474	ORIGINAL RESEARCH ARTICLE bli h d 01 A il 2014 published: 01 April 2014 doi: 10.3389/fnsyn.2014.00007 Reviewed by: Reviewed by: Kurt Gottmann, University of Duesseldorf, Germany Michael Fauth, Georg-August Universität Göttingen, Germany Keywords: topology, development, neuronal network model, structural synaptic plasticity, homeostasis, small-world network, efﬁciency Keywords: topology, development, neuronal network model, structural synaptic plasticity, homeostasis, small-world network, efﬁciency 1. INTRODUCTION long-range connections (Watts and Strogatz, 1998). The brain seems to be optimized for maximizing cost efﬁciency of par- allel information processing (Achard and Bullmore, 2007) by widely adopting small-world topology (Sporns and Zwi, 2004; Bassett and Bullmore, 2006). Already in the infant brain, con- nectivity has small-world characteristics (Fransson et al., 2011). A recent fMRI study reported an increase in small-worldness of brain networks in the ﬁrst 2 years of life that goes along with a growing number of long-distance connections and therefore an increase in global efﬁciency (Gao et al., 2011). So far, analysis of topology has focused on neuronal networks with static connec- tivity, in which plasticity arises from changes in the strength of existing synapses rather than from the rewiring of connectivity. However, particularly during development but also in adult- hood, connectivity is not ﬁxed (Butz et al., 2009b), and synapse formation goes along with massive synapse deletion and reorga- nization of connectivity (Missler et al., 1993; Siegel and Lohmann, 2013). This observation triggered the question of this study: how does network topology develop during ontogeny when synapse long-range connections (Watts and Strogatz, 1998). The brain seems to be optimized for maximizing cost efﬁciency of par- allel information processing (Achard and Bullmore, 2007) by widely adopting small-world topology (Sporns and Zwi, 2004; Bassett and Bullmore, 2006). Already in the infant brain, con- nectivity has small-world characteristics (Fransson et al., 2011). A recent fMRI study reported an increase in small-worldness of brain networks in the ﬁrst 2 years of life that goes along with a growing number of long-distance connections and therefore an increase in global efﬁciency (Gao et al., 2011). So far, analysis of topology has focused on neuronal networks with static connec- tivity, in which plasticity arises from changes in the strength of existing synapses rather than from the rewiring of connectivity. However, particularly during development but also in adult- hood, connectivity is not ﬁxed (Butz et al., 2009b), and synapse formation goes along with massive synapse deletion and reorga- nization of connectivity (Missler et al., 1993; Siegel and Lohmann, 2013). Frontiers in Synaptic Neuroscience Homeostatic structural plasticity increases the efﬁciency of small-world networks Markus Butz 1, Ines D. Steenbuck 2 and Arjen van Ooyen 3 1 Simulation Lab Neuroscience, Bernstein Facility for Simulation and Database Technology, Institute for Advanced Simulation, Jülich Aachen Research Alliance, Forschungszentrum Jülich, Jülich, Germany 2 Student of the Medical Faculty, University of Freiburg, Freiburg, Germany 3 Department of Integrative Neurophysiology, VU University Amsterdam, Amsterdam, Netherlands In networks with small-world topology, which are characterized by a high clustering coefﬁcient and a short characteristic path length, information can be transmitted efﬁciently and at relatively low costs. The brain is composed of small-world networks, and evolution may have optimized brain connectivity for efﬁcient information processing. Despite many studies on the impact of topology on information processing in neuronal networks, little is known about the development of network topology and the emergence of efﬁcient small-world networks. We investigated how a simple growth process that favors short-range connections over long-range connections in combination with a synapse formation rule that generates homeostasis in post-synaptic ﬁring rates shapes neuronal network topology. Interestingly, we found that small-world networks beneﬁted from homeostasis by an increase in efﬁciency, deﬁned as the averaged inverse of the shortest paths through the network. Efﬁciency particularly increased as small-world networks approached the desired level of electrical activity. Ultimately, homeostatic small-world networks became almost as efﬁcient as random networks. The increase in efﬁciency was caused by the emergent property of the homeostatic growth process that neurons started forming more long-range connections, albeit at a low rate, when their electrical activity was close to the homeostatic set-point. Although global network topology continued to change when neuronal activities were around the homeostatic equilibrium, the small-world property of the network was maintained over the entire course of development. Our results may help understand how complex systems such as the brain could set up an efﬁcient network topology in a self-organizing manner. Insights from our work may also lead to novel techniques for constructing large-scale neuronal networks by self-organization. Edited by: Christian Tetzlaff, Georg-August University, Germany Reviewed by: Kurt Gottmann, University of Duesseldorf, Germany Michael Fauth, Georg-August Universität Göttingen, Germany Correspondence: Markus Butz, Simulation Lab Neuroscience, Bernstein Facility for Simulation and Database Technology, Institute for Advanced Simulation, Jülich Aachen Research Alliance, Forschungszentrum Jülich GmbH, JSC, 52425 Jülich, Germany e-mail: m.butz@fz-juelich.de SYNAPTIC NEUROSCIENCE ORIGINAL RESEARCH ARTICLE bli h d 01 A il 2014 Edited by: Edited by: Christian Tetzlaff, Georg-August University, Germany Reviewed by: Kurt Gottmann, University of Duesseldorf, Germany Michael Fauth, Georg-August Universität Göttingen, Germany *Correspondence: Markus Butz, Simulation Lab Neuroscience, Bernstein Facility for Simulation and Database Technology, Institute for Advanced Simulation, Jülich Aachen Research Alliance, Forschungszentrum Jülich GmbH, JSC, 52425 Jülich, Germany e-mail: m.butz@fz-juelich.de Christian Tetzlaff, Georg-August University, Germany Reviewed by: formation and pruning cause a constant rewiring of network connectivity? Various explanations have been proposed to account for the development of synaptic connectivity. For example, axon guid- ance molecules may form the basis of a genetically-encoded developmental scheme (Yamamoto et al., 2002; Borisyuk et al., 2008). Target neurons may secret signaling molecules that can attract or repel axons. Axons can then follow or move away from the concentration gradient. This form of chemotaxis is usually discussed in the context of the formation of global connectiv- ity. However, for the formation of local connectivity chemical cues are less suitable, since they fail to establish stable gradients over very short distances, below 0.7 mm (Kaiser et al., 2009). Synaptic adhesion molecules (e.g., neuroligins) were proposed as molecular cues for local synapse formation (Scheiffele et al., 2000; Stan et al., 2010). In addition, mechanical forces in the tissue could inﬂuence neurite outgrowth (Franze, 2013). Alternatively, the formation of local connectivity may basically be random (Braitenberg and Schüz, 1998), just depending on the acciden- tal overlap of axons and dendrites (Binzegger et al., 2004; van Pelt and van Ooyen, 2013; McAssey et al., 2014; van Ooyen et al., 2014). With random synapse formation, the chance of forming connections decreases with increasing distance between neurons (Kaiser et al., 2009). y y g y p In MSP, the local activity-dependent growth process in com- bination with a simple kernel function favoring the formation of short-range connections over long-range connections shapes the development of small-world networks. Our simulation results revealed an interesting property of homeostatic growth: as soon as most neurons approached homeostasis in electrical activity, they started forming more long-range connections than expected from the kernel function. Although the clustering coefﬁcient decreased as a result of the formation of long-range connections, the network maintained its small-world property. Furthermore, connectivity became more diverse, as indicated by a decreasing betweenness centrality, and attained a higher global efﬁciency (deﬁned as the averaged inverse of the shortest paths between all neurons in the network) than small-world networks with- out homeostasis. Our ﬁndings may account for experimental data on the topology of developing dissociated cell cultures (Downes et al., 2012). Interesting similarities were also found between the model and early human brain development (Gao et al., 2011) with respect to an increasing number of long- range connections and an increasing global efﬁciency during development. Reviewed by: Although random overlap of axons and dendrites may explain emerging connectivity, it does not account for the actual driv- ing forces underlying neurite outgrowth. There is ample evidence that electrical activity shapes neuronal morphology (Dalva et al., 1994; Wong and Ghosh, 2002; Uesaka et al., 2005; Butz et al., 2009b) and network formation (Ko et al., 2013). Electrical activ- ity inﬂuences neurite outgrowth and retraction (McKinney et al., 1999a; Konur and Ghosh, 2005; Lohmann and Wong, 2005; Tailby et al., 2005; Hutchins and Kalil, 2008), as well as the formation and deletion of axonal boutons and dendritic spines (McKinney et al., 1999b; Groc et al., 2002; Jourdain et al., 2003; Kirov et al., 2004; Hofer et al., 2009). Experimental ﬁndings further suggest that neuronal morphogenesis is driven by the need of neurons to establish and maintain a homeostatic equilibrium of their aver- age electrical activity (Kirov et al., 2004; Keck et al., 2008, 2013). Restoration of neuronal ﬁring rate after a change in neuronal input has been found experimentally after, for example, focal reti- nal lesions (Hengen et al., 2013). Based on these experimental ﬁndings, we postulated that whenever during development (van Ooyen and van Pelt, 1994; Van Ooyen et al., 1995; Tetzlaff et al., 2010) or in the mature brain (Butz et al., 2008; Butz and van Ooyen, 2013) a neuron senses a deviation of its electrical activ- ity from a homeostatic set-point, it will initiate changes in its morphology that increase the chance of synapse formation or break existing connections so that its ﬁring rate may be restored. Here we investigate what the impact is of neurons regulating their electrical activities homeostatically on network formation and emerging network topologies. Reviewed by: This observation triggered the question of this study: how does network topology develop during ontogeny when synapse The synaptic wiring of cortical networks is key to the function- ality of the brain and a precondition for all cognitive behavior (Park and Friston, 2013). How are synaptic connections set up between the brain’s billions of neurons so that cost efﬁciency (Latora and Marchiori, 2001), for example in terms of wiring length or energy consumption associated with information trans- mission, is maximized? Due to the presence of long-range con- nectivity, the brain can be regarded as a highly efﬁcient graph, in the sense that information has to pass only a few intermedi- ate neurons (“nodes”) to travel across the whole brain (Kaiser and Hilgetag, 2006). At the same time, brain connectivity is local and clustered (Hilgetag and Kaiser, 2004), making net- works less vulnerable because of the many alternative routes that exist between two nodes. Network topology that combines these two properties, long-range connectivity and high cluster- ing, is called small-world (Watts and Strogatz, 1998). Small-world networks have the advantages of local connectivity combined with a high efﬁciency brought about by a small number of April 2014 \| Volume 6 \| Article 7 \| 1 www.frontiersin.org www.frontiersin.org www.frontiersin.org Homeostatic small-world networks Butz et al. 1986, 1988; Cromme and Dammasch, 1989; Butz and Teuchert- Noodt, 2006; Butz et al., 2006) and the activity-dependent neurite outgrowth model by van Ooyen (van Ooyen and van Pelt, 1994; Van Ooyen et al., 1995). The latter model, which studied the reciprocal interactions between neuronal activity and network formation, successfully accounted for experimental data on devel- oping cell cultures (van Ooyen and van Pelt, 1994; van Oss and van Ooyen, 1997; Abbott and Rohrkemper, 2007; Tetzlaff et al., 2010). However, both models are not suited for studying topol- ogy development. The compensation model lacks topology at all, whereas the representation of neuritic ﬁelds by circles in the model by van Ooyen imposes too strong constraints on net- work topology. In van Ooyen’s model, neurons always connect to their direct neighbors before connecting to more distant neurons. Therefore, we developed our MSP (Butz and van Ooyen, 2013), in which we replaced the circle representation by discrete synaptic elements whose numbers change in an activity-dependent man- ner. Synapses are formed in a random and distance-dependent way by combining synaptic elements from different neurons. Frontiers in Synaptic Neuroscience www.frontiersin.org April 2014 \| Volume 6 \| Article 7 \| 2 April 2014 \| Volume 6 \| Article 7 \| 2 2.3. HOMEOSTATIC GROWTH RULES It is well documented that neurons change their morphology in an activity-dependent fashion during development (Butz et al., 2009b; van Ooyen, 2011). The way in which neurons change their morphology suggests that they try to maintain homeostasis of electrical activity (van Ooyen and van Pelt, 1994; Van Ooyen et al., 1995; Butz et al., 2009a; van Ooyen, 2011; Butz and van Ooyen, 2013). That is, neurons in which the average activity is too low start forming new neuritic structures, whereas neurite growth is halted or neurites are pruned when activity is higher than a desired level (homeostatic set-point). In our MSP, we abstracted away from describing detailed neuronal morphology and assumed that changes in morphology effectively result in changing numbers of axonal and dendritic elements—the con- tact sites for synaptic connections. Homeostatic adaptation of the number of axonal and dendritic elements was modeled by the fol- lowing growth rule (Figure 1), in which dz represents the change in the number of synaptic elements, with z being A, Dex or Din: The intracellular calcium concentration of a neuron is used as a low-passed ﬁltered average of its ﬁring frequency (Butz and van Ooyen, 2013). Every time a neuron ﬁres, the calcium concentra- tion is increased by a ﬁxed amount; otherwise the concentration decreases exponentially to zero: d Ca2+ i dt = ⎧ ⎪⎨ ⎪⎩ −[Ca2+]i τCa + β if v ≥30 mV −[Ca2+]i τCa otherwise (3) dz dt = ν 2 1 + e([Ca2+]−ϵ)/0.1 −1 (4) (4) where β = 0.001 ms−1 and τCa = 10,000 ms. We deﬁned a set- point ϵ = 0.7 in calcium concentration, corresponding to an intermediate level of average electrical activity. Every time the neuron’s calcium concentration deviates from ϵ, it will induce structural changes in connectivity to restore the desired average level of activity, as described below. where ϵ is the set-point of the intracellular calcium concentra- tion, corresponding to a desired average ﬁring rate of the neuron, and ν is the growth rate of synaptic elements. We chose ν = 10−4 ms−1, which is slow enough that electrical dynamics and structural dynamics do not interfere, yet fast enough not to slow down the simulations unnecessarily. For reasons of simplicity and because of a lack of detailed experimental data, we applied iden- tical sigmoid growth rules to all types of synaptic elements. 2. MATERIALS AND METHODS 2.1. NEURON MODEL The model network consisted of n = 400 neurons, of which 80% were excitatory and 20% inhibitory. Excitatory neurons were placed with some jitter on a 20 × 16 grid with a spatial dis- tance between two grid points of 150 μm. Inhibitory neurons were placed evenly between the excitatory neurons. The same net- work layout was used as in Butz and van Ooyen (2013). We used Izhikevich’s model (Izhikevich, 2003) to simulate neuronal elec- trical activity. This model has two differential equations, one for the membrane potential v (in mV) and one for a recovery variable u (in mVms−1), enabling re-polarization after an action potential: In order to study the impact of electrical activity on emerg- ing network topology, we used our recent Model of Structural Plasticity (MSP) (Butz and van Ooyen, 2013). There are impor- tant earlier models of homeostatic structural plasticity, such as the compensation model by Dammasch et al. (Dammasch et al., dv dt = k1v2 + k2v + k3 −u + I du dt = a(bv −u) (1) dv dt = k1v2 + k2v + k3 −u + I (1) du dt = a(bv −u) April 2014 \| Volume 6 \| Article 7 \| 2 www.frontiersin.org www.frontiersin.org Homeostatic small-world networks Butz et al. where k1 = 0.04 mV−1ms−1, k2 = 5 ms−1, k3 = 140 mVms−1, and t is in ms. Every time a neuron ﬁres (v ≥30 mV), v and u are reset: postsynaptic counterparts, i.e., postsynaptic receptor plates on dendritic spines or the dendritic shaft. Axonal and dendritic ele- ments are either excitatory or inhibitory. Any neuron i can form Ai axonal elements, which are excitatory if the neuron is excita- tory, or inhibitory if the neuron is inhibitory. At the same time, a neuron, irrespective of its type, can express Dex i excitatory and Din i inhibitory dendritic elements. Complementary elements can merge to form a synapse (excitatory axonal with excitatory den- dritic elements, and inhibitory axonal with inhibitory dendritic elements). Synaptic elements form and delete independently from a synaptic contact partner. In case a neuron deletes a synaptic element that is bound in a synapse, the complementary synap- tic element on the other neuron remains and becomes vacant and available again for synapse formation with a new target. Therefore MSP allows for synaptic rewiring. 2. MATERIALS AND METHODS 2.1. NEURON MODEL if v ≥30 mV, then v ←c u ←u + d (2) (2) As in Izhikevich (2003), the following parameter values were used: a = 0.1 ms−1, b = 0.2 ms−1, c = −65 mV, d = 2 mVms−1. We used the same dynamics for excitatory and inhibitory neurons. Each neuron receives input I = Isyn + Iext, consisting of synap- tic input Isyn from within the network and external input Iext. Neurons interchange electrical signals on a millisecond timescale without synaptic delay. Synaptic input consists of the incoming action potentials from the presynaptic neurons low-pass ﬁltered by an exponential ﬁlter function h(t) = exp −t μ with decay constant μ = 5 ms. Network connectivity Wi,j is deﬁned as the number of synapses from neuron j to i. If a synapse exists, it has a ﬁxed strength of 1 mVms−1. Neurons are either excitatory or inhibitory. Indices refer to excitatory neurons if i or j ∈{Ex} and to inhibitory neurons if i or j ∈{In}. As in Izhikevich (2003) and Butz and van Ooyen (2013), Iext is delivered as white noise with mean 5 mVms−1 and standard deviation 1 mVms−1. In some cases lower input values were required and speciﬁed where relevant. 2.3. HOMEOSTATIC GROWTH RULES In a random and distance-dependent recombination process, newly formed synaptic elements were distributed to matching synaptic elements, thereby forming synapses and creating the pattern of connectivity in the network. Frontiers in Synaptic Neuroscience 2.2. SYNAPSE MODEL FOR STRUCTURAL PLASTICITY During development, neurons show a pronounced formation and pruning of synapses. To simulate reorganization of synaptic connectivity, standard models of synaptic plasticity, in which con- nectivity is considered ﬁxed with plasticity merely arising from changes in the strength of existing synapses (modeled as weight factors), are not suitable. For this study on the development of neuronal networks, we therefore used our Model of Structural Plasticity (MSP) (Butz and van Ooyen, 2013). The characteristic feature of this model is that it represents synapses as consisting of two recombinable synaptic elements, namely an axonal element and a dendritic element. Axonal elements represent countable presynaptic specializations for transmitter release such as axonal terminals or boutons, while dendritic elements represent the 2.4. KERNEL FUNCTION FOR SYNAPSE FORMATION As in our previous work on MSP (Butz and van Ooyen, 2013), we assumed that synapse formation is more likely between adja- cent neurons than between distant ones. We applied a two- dimensional Gaussian-shaped kernel function centered at the x, y-coordinates of neuron i, with Ki,j the distance-dependent April 2014 \| Volume 6 \| Article 7 \| 3 www.frontiersin.org www.frontiersin.org www.frontiersin.org Homeostatic small-world networks Butz et al. FIGURE 1 \| Identical sigmoidal growth rules were used for all types of synaptic elements to determine the change dz/dt in the number of synaptic elements in dependence on the intracellular calcium concentration Ca2+ . z needs to be replaced by the respective type of element A, Dex or Din. The homeostatic set-point ϵ is the value of the calcium concentration where dz/dt = 0. of the simulation in milliseconds is not required to sum up to weeks. 2.5.1. Synapse deletion The same holds true for outgoing inhibitory synapses with j ∈{In}. (5) where posxi is the x-coordinate and posyi is the y-coordinate of postsynaptic neuron i, and posxj and posyj are the coordinates of presynaptic neuron j. The probability for autapse connections (i.e., a neuron connecting to itself) was set to zero (Ki,j = 0 for i = j). For these simulations we chose σ = 1 × 150 μm where 150 μm is the distance between two grid points. Sequentially, outgoing and incoming excitatory and inhibitory synapses were selected for deletion. For every type of synapse, the accumulated sum of Pdel i,j [see description of Equation (6) for the range of i and j] gave a probability distribution from which we drew the required number of synapses to be deleted. The selected synapse was deleted by reducing the respective entry Wi,j in the connectivity matrix by one. It can happen that more then one synapse is selected for deletion from the same connection j to i. In this case, the implementation of the algorithm makes sure that the number of synapses to be deleted did not exceed Wi,j. Whenever a neuron deletes a synaptic element that is bound in a synapse, the complementary synaptic element on the other neuron remains and becomes vacant again. In order to investigate the impact of this distance-dependency on emerging network topologies, we additionally grew networks with a ﬂat kernel, i.e., with Ki,j, i ̸= j = 1 and Ki,j = 0 for i = j. 2.5. SYNAPSE FORMATION AND DELETION The MSP algorithm (Butz and van Ooyen, 2013) proceeded in three steps to update network connectivity in an activity- dependent fashion. First, electrical activity of all neurons was computed continuously over time. Secondly, depending on the average electrical activity and according to the homeostatic growth rule (Equation 4), the number of elements changed con- tinuously, too. Thirdly, at discrete time points, network connec- tivity W was updated by synapse formation and deletion. Because of the low growth rate ν = 10−4 ms−1, changes in connectivity are very slow compared to changes in activity, so it was not nec- essary to update connectivity at every time step but only at every 100 ms. The timescale of network formation in the model corre- sponds in principle to a timescale of days or weeks. However, as described above under homeostatic growth rules, the timescale of structural changes was chosen so that the simulations were not unnecessarily slowed down, which means that the total duration Frontiers in Synaptic Neuroscience 2.5.1. Synapse deletion Since network connectivity is updated at discrete time steps but synaptic elements change continuously over time due to the activity-dependent growth rules, it can happen that a neuron has more outgoing synapses than axonal elements or more incoming synapses than dendritic elements at the time of the next update in network connectivity. In this case, the neuron has to delete the surplus of synapses and to update connectivity. To update connectivity, the algorithm needs to select which synapses are to be removed. All synapses have an equal chance of being deleted. Note, however, that multiple synapses can co-exist from neuron j to i and that the more synapses there are, the higher the chance that a synapse between neuron j and i will be deleted. The probability Pdel i,j for synapse deletion between neuron j and i is computed by the following master equation that captures four different cases: FIGURE 1 \| Identical sigmoidal growth rules were used for all types of synaptic elements to determine the change dz/dt in the number of synaptic elements in dependence on the intracellular calcium concentration Ca2+ . z needs to be replaced by the respective type of element A, Dex or Din. The homeostatic set-point ϵ is the value of the calcium concentration where dz/dt = 0. Pdel i,j = Wi,j Wk,l (6) (6) For deletion of incoming synapses, we need to distinguish between excitatory and inhibitory synapses in Equation (6). For deleting incoming excitatory synapses of neuron i ∈{In ∪Ex}, we sum up Wk,l over all l ∈{Ex}. For deleting incoming inhibitory synapses of neuron i ∈{In ∪Ex}, we sum up Wk,l over all l ∈{In}. For deletion of outgoing excitatory synapses of excitatory presy- naptic neuron j with j ∈{Ex}, in Equation (6) all synapses are considered to any postsynaptic neuron k with k ∈{In ∪Ex}. Thus, we sum up Wk,l over all k ∈{In ∪Ex}. The same holds true for outgoing inhibitory synapses with j ∈{In}. likelihood for synapse formation between neuron j and i: Ki,j, i ̸= j = e − posxj −posxi 2 + posyj −posyi 2 σ 2 For deletion of outgoing excitatory synapses of excitatory presy- naptic neuron j with j ∈{Ex}, in Equation (6) all synapses are considered to any postsynaptic neuron k with k ∈{In ∪Ex}. Thus, we sum up Wk,l over all k ∈{In ∪Ex}. 2.6. DEVELOPMENT OF NON-HOMEOSTATIC NETWORKS To investigate the impact of homeostasis in electrical activity on developing network topology, we created for every home- ostatic network a corresponding non-homeostatic network. At every update in connectivity, we took the number of synapses from the homeostatic network and distributed them in the non- homeostatic network with the same kernel function as used in the homeostatic network. Hence, the placement of synapses in the non-homeostatic network was purely dependent on the kernel function but did not meet the homeostasis criterion. The algo- rithmic implementation for placing synapses was the same for homeostatic and non-homeostatic networks, with Pform i,j = Ki,j. Instead of distributing MPotSyn synapses (Equation 8), we simply distributed the total number of synapses from the homeostatic network. Since synapse formation was not limited by numbers of vacant elements, Equation (9) was not applicable. Pform i,j = ⎧ ⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎩ j ∈{Ex} : Avac j Dex,vac i ι∈{Ex} Avac ι κ∈{Ex∪In} Dex,vac κ Kij j ∈{In} : Avac j Din,vac i ι∈{In} Avac ι κ∈{Ex∪In} Din,vac κ Kij ⎫ ⎪⎪⎪⎪⎪⎪⎬ ⎪⎪⎪⎪⎪⎪⎭ with i ∈{Ex ∪In}. (7) (7) The minor number of vacant excitatory and inhibitory axonal or dendritic elements determined how many new excitatory and inhibitory synapses, respectively, could at most be formed (so-called potential synapses) in every update of connectivity. Thus, the number of excitatory and inhibitory potential synapses equaled The minor number of vacant excitatory and inhibitory axonal or dendritic elements determined how many new excitatory and inhibitory synapses, respectively, could at most be formed (so-called potential synapses) in every update of connectivity. Thus, the number of excitatory and inhibitory potential synapses equaled MPotSyn,ex = min ι∈{Ex} Avac ι , κ∈{Ex∪In} Dex,vac κ MPotSyn,in = min ι∈{In}, Avac ι , κ∈{Ex∪In} Din,vac κ (8) (9) In every update, this condition was checked and synapse for- mation infringing this condition was rejected. Alternatively, the update of connectivity can also be implemented in a purely local fashion (Butz and van Ooyen, 2013). For small networks, the current implementation is more efﬁcient than the original MSP algorithm (Butz and van Ooyen, 2013) because run time is not dependent on the growing numbers of vacant synaptic elements but proportional to the square of the number of neurons in the network. However, for large networks with n >> 1000, n2 will quickly out-range the number of vacant synaptic elements, in which case looping over the number of synaptic elements would be faster. Particularly in Matlab, the current description of MSP allows for an elegant vectorized implementation providing additional speed up. In every update, this condition was checked and synapse for- mation infringing this condition was rejected. Alternatively, the update of connectivity can also be implemented in a purely local fashion (Butz and van Ooyen, 2013). For small networks, the current implementation is more efﬁcient than the original MSP algorithm (Butz and van Ooyen, 2013) because run time is not dependent on the growing numbers of vacant synaptic elements but proportional to the square of the number of neurons in the network. However, for large networks with n >> 1000, n2 will quickly out-range the number of vacant synaptic elements, in which case looping over the number of synaptic elements would be faster. Particularly in Matlab, the current description of MSP allows for an elegant vectorized implementation providing additional speed up. 2.7. TOPOLOGY MEASUREMENTS A neuronal network can be seen as a graph with the neu- rons being the nodes and the synapses being the edges or links between two nodes. Since the presynaptic neuron always activates the postsynaptic neuron (and never the other way around), we regard the graph as directed. At every update in connectivity, we assessed those graph theoretical measures that are indicative of small-worldness and network efﬁciency. In addition, betweenness centrality was measured to determine the importance of nodes in the network. To reduce the complexity of the assessment, we considered only the topology of excitatory synaptic connections Wex,ex between the nex excitatory neurons. For the graph theo- retical assessments, the brain connectivity toolbox by Olaf Sporns et al. (Rubinov and Sporns, 2010) was used. for every update in connectivity. From this distribution, the algorithm chose at maximum MPotSyn,ex excitatory and MPotSyn,in inhibitory connections at which new synapses were created. The respective entries Wi,j in the connectivity matrix were then increased by one. A connection was chosen by drawing a random number from a uniform dis- tribution and comparing it to the accumulated probabilities Pform i,j for all excitatory connections and all inhibitory connections of the entire network. That connection was chosen that had the high- est accumulated probability that the random number just did not exceed. If, for this try, the random number exceeded all accumu- lated probabilities, no synapse was formed. Hence, not necessarily all of the potential synapses were formed. for all excitatory connections and all inhibitory connections of the entire network. That connection was chosen that had the high- est accumulated probability that the random number just did not exceed. If, for this try, the random number exceeded all accumu- lated probabilities, no synapse was formed. Hence, not necessarily all of the potential synapses were formed. 2.7.1. Weighted characteristic path length The characteristic path length L is the average shortest path from one node to any other node in the network. Path length is deﬁned as the number of links that need to be traveled to go from one node (possibly via intermediate nodes) to any other node. On top of this deﬁnition, a direct link between two nodes in a weighted network is considered “shorter” the stronger the weight of the link is. For our network, we take the number of synapses Wex,ex i,j between two directly linked neurons j and i, with i, j ∈{Ex}, as the weight of the connection and the inverse 1/Wex,ex i,j as the length li,j of the connection. The shortest path di,j is then the smallest sum of connection lengths that lead from neuron j to i via any interme- diate neurons. Our calculation of the weighted characteristic path length was based on an implementation of Dijkstra’s algorithm for computing the shortest path in weighted directed networks by Rubinov and Sporns (2010). Additionally, synapse formation needed to fulﬁl the condition that the number W+ i,j of newly-formed synapses from neuron j to i did not exceed the number of vacant synaptic elements that neuron j and i offered: W+ i,j ≤ ⎧ ⎪⎨ ⎪⎩ j ∈{Ex} : min(Avac j , Dex,vac i ) j ∈{In} : min(Avac j , Din,vac i ) ⎫ ⎪⎬ ⎪⎭ with i ∈{Ex ∪In}. (9) 2.5.2. Synapse formation For synapse formation, the algorithm checked whether a neuron gained vacant synaptic elements, i.e., whether the total number of synaptic elements exceeded the number of bound synaptic ele- ments of this type. Matching vacant synaptic elements (vacant excitatory axonal elements Aex,vac j with vacant excitatory dendritic elements Dex,vac i , and inhibitory axonal with inhibitory dendritic elements Din,vac) were randomly distributed among each other with probability density function Pform. The probability Pform i,j for forming new synapses between neuron j and i depended on April 2014 \| Volume 6 \| Article 7 \| 4 www.frontiersin.org www.frontiersin.org www.frontiersin.org Homeostatic small-world networks Butz et al. the number of vacant synaptic elements they offered and on the Euclidean distance between neuron j and i: Frontiers in Synaptic Neuroscience 2.7.2. Weighted clustering coefﬁcient The clustering coefﬁcient is an indication of how strongly nodes in a network are interconnected. It can be measured by the num- ber of triangles, ˜tD i , one node forms with any other two nodes in the network divided by the total number of possible triangles, TD i . For weighted and directed graphs, one needs to realize that the adjacency matrix (in our case, the connectivity matrix Wex,ex of the excitatory neurons) is not symmetric and that the entries Frontiers in Synaptic Neuroscience April 2014 \| Volume 6 \| Article 7 \| 5 www.frontiersin.org www.frontiersin.org Homeostatic small-world networks Butz et al. of the adjacency matrix are not one but can have any weight. According to Fagiolo (2007), the clustering coefﬁcient of a single node in a weighted directed network ˜CD i is computed as of the adjacency matrix are not one but can have any weight. According to Fagiolo (2007), the clustering coefﬁcient of a single node in a weighted directed network ˜CD i is computed as Consequently, betweenness centrality provides a measure for how well networks are interconnected. A high local or global betweenness centrality means that individual nodes or the entire network, respectively, is badly interconnected, because all infor- mation has to travel through the same nodes in the absence of alternative routes or by-passes. Clinical data shows that after brain lesions, betweenness centrality of directly and indi- rectly affected brain areas changes (Wang et al., 2010). Note that as for the measurements above, the shortest paths are based on weighted excitatory connections Wex,ex i,j . Therefore, global betweenness centrality was computed by the formalism for weighted directed networks by Brandes (2001) as imple- mented in the brain connectivity toolbox (Rubinov and Sporns, 2010). ˜CD i Wex,ex = ˜tD i TD i = (Wex,ex)[1/3] + (Wex,ex)T[1/3]3 i,i 2 dtot i dtot i −1 −2d↔ i (10) (10) where (Wex,ex)[1/k] = w1/k i,j , the kth root of the entries of the matrix for i, j ∈{Ex}, and (Wex,ex)T is the transposed Wex,ex matrix. where nex is the number of excitatory neurons. where nex is the number of excitatory neurons. 2.7.6. Euclidean distance Although not a topology measure in a strict graph theoretical sense, the average Euclidean distance between nodes that are con- nected by synapses was measured in order to help interpret the development of characteristic path length and clustering coefﬁ- cient. To obtain the average Euclidean distance ED, we multiplied the Euclidean distances between all pairs of excitatory neurons in the network with the number of synapses between these neurons. We then summed up all of the so-weighted distances and divided the sum by the number of excitatoy synapses: s = C/Crand L/Lrand (11) (11) We replaced the clustering coefﬁcient C and the characteristic path length L by the corresponding versions for weighted directed graphs as described above. Crand and Lrand were taken from an Erd˝os-Rényi random graph generated with the same number of synapses as in the developing networks at every update in connectivity. ED = ⎛ ⎝ nex i,j posxj −posxi 2 + posyj −posyi 2 Wex,ex i,j ⎞ ⎠/ nex i,j Wex,ex i,j (14) with i, j ∈{Ex} 2.7.2. Weighted clustering coefﬁcient The variable dtot i denotes the total degree of node i (the degree counts the number of either incoming or outgo- ing edges per node, and the total degree is the sum over both the incoming and outgoing edges), and d↔ i stands for the num- ber of bilateral edges of node i (the number of nodes node i projects to and receives edges from, excluding self-interactions of node i). 2.7.3. Small-world parameter To estimate small-worldness of the developing networks, we applied the formalism by Humphries and Gurney (2008): 2.7.4. Betweenness centrality The local betweenness centrality is a measure for the impor- tance of a node in a network. A high betweenness central- ity of a node i means that many shortest paths between any two nodes k and l pass through node i. Thus, the local betweenness centrality counts the number of times, σkl(i), that node i is on a shortest path between two nodes k and l. The local betweenness centrality is normalized by the num- ber σkl of alternative shortest paths between k and l that do not pass through node i. Global betweenness centrality is the sum of all local betweenness centrality values of the individual nodes: (14) with i, j ∈{Ex} with i, j ∈{Ex} Frontiers in Synaptic Neuroscience 2.7.5. Efﬁciency Global efﬁciency is related to the inverse characteristic path length with the advantage over characteristic path length that it can be meaningfully computed also of disconnected graphs (Latora and Marchiori, 2001; Achard and Bullmore, 2007). While path lengths between disconnected cells are inﬁnite, efﬁciency becomes zero and, thus, adds neutrally to global efﬁciency. The overall clustering coefﬁcient of the network is thus ˜CD = (nex)−1 N i = 1 ˜CD i . Note that for this assessment we only con- sidered nex excitatory nodes and excitatory connections Wex,ex. For a more detailed description of Equation (10), see Fagiolo (2007). We computed the clustering coefﬁcient of the develop- ing neuronal networks at every update in connectivity by using the implementation by Mika Rubinov from the brain connectivity toolbox (Rubinov and Sporns, 2010). Eglobal = 1 nex (nex −1) i ̸= j 1 di,j (13) (13) (13) 3. RESULTS Therefore, we will call networks that resulted from distance-dependent synapse formation small-world networks and those that resulted from synapse formation without distance-dependency random networks. In networks that favor long-range over short-range connec- tions (small-world networks) (Figure 4A), s constantly increased FIGURE 2 \| Development of intracellular calcium concentration Ca2+ over time. (A) Development of calcium concentration in small-world networks arising from synapse formation that favors short-range over long-range connections. Mean calcium concentrations averaged over ﬁve simulations reach the set-point ϵ = 0.7 when the number of synaptic elements is regulated homeostatically (yellow), whereas calcium concentrations remain much lower when there is no homeostatic regulation (gray). (B) Development of calcium concentration in random networks without any distance-dependency in synapse formation. The random network with homeostatic regulation of synaptic elements (blue) also reaches the homeostatic set-point ϵ, whereas the network without homeostasis (gray) and the same number of synapses as the homeostatic network has much lower values in calcium. Shadow around the curves (hardly visible since so small) indicates the standard deviation. FIGURE 2 \| Development of intracellular calcium concentration Ca2+ 3. RESULTS We started each network simulation with zero connectivity and zero synaptic elements. Due to the homeostatic formation of axonal and dendritic elements, model neurons are able to form synapses and to adapt the number of synapses so as to reach a homeostatic equilibrium in electrical activity (Figure 2). For a wide range of set-points ϵ, model neurons adapt their aver- age ﬁring rate so that, at the end of each simulation (here after 15,000 updates in connectivity), their calcium concentra- tion [Ca2+] has converged to ϵ, with a corresponding ﬁring rate y (in Hz) that follows the linear relation y = 100 × [Ca2+] BCglobal = nex i k ̸= i ̸= l σkl(i) σkl (12) (12) Frontiers in Synaptic Neuroscience April 2014 \| Volume 6 \| Article 7 \| 6 Frontiers in Synaptic Neuroscience www.frontiersin.org www.frontiersin.org Homeostatic small-world networks Butz et al. and reached a maximum markedly greater than 10 at around 7000 updates in connectivity. Small-world networks that were set-up by an additional rule for homeostasis in electrical activity reached a plateau of about s = 10 very early but began to decrease again around the time that neuronal activities reached the homeo- static set-point ϵ. Nevertheless, small-world networks with home- ostasis maintained their small-world property throughout the whole course of the simulation (s > 5 at T = 15,000). In net- works without distance-dependency in synapse formation (ran- dom networks) (Figure 4B), s equaled 1 from the very beginning of network development. Random networks with and with- out homeostasis did not differ in the course of s (Figure 4B). Hence, homeostasis did not seem to have an impact on the development of topology in random networks. Knowing that homeostasis inﬂuenced the development of small-worldness, we further analyzed how homeostasis exerted its inﬂuence during network formation. For some simulations, s could not be com- puted for the ﬁrst few updates in connectivity because of division by zero. (Figure 3). We investigated how the topology of these self- organizing networks developed over time when neurons favor short-range connections over long-range connections, or, alter- natively, when all connections are equally likely. In the ﬁrst case, as we will show in detail later on, networks developed a distinct small-world property, with small-world parameter s markedly greater than 1, whereas in the second case, s reaches 1. 3.1. HOMEOSTASIS INFLUENCES THE CLUSTERING COEFFICIENT AND CHARACTERISTIC PATH LENGTH OF DEVELOPING SMALL-WORLD NETWORKS The mean or characteristic path length in small-world net- works without homeostasis showed, after an initial sharp rise, a steady decrease and converged toward values of around 3 (Figure 5A). Homeostatic small-world networks also started with a sharp rise and a subsequent decrease in characteris- tic path length. The decrease was even steeper than in the non-homeostatic case, and the characteristic path length con- verged toward slightly lower values than in non-homeostatic networks. In addition, networks with homeostasis showed a second but minor decrease in characteristic path length when activities reached the homeostatic set-point ϵ. The ﬁnal values of the characteristic path length after 15,000 updates in con- nectivity were stable in both homeostatic and non-homeostatic small-world networks. The initial sharp increase is caused by the limited number of synapses in the network at the begin- ning of the simulation. Characteristic path lengths in developing random networks showed an identical course with and without homeostasis (Figure 5B). The ﬁnal values in random networks FIGURE 2 \| Development of intracellular calcium concentration Ca2+ FIGURE 2 \| Development of intracellular calcium concentration Ca2+ over time. (A) Development of calcium concentration in small-world networks arising from synapse formation that favors short-range over long-range connections. Mean calcium concentrations averaged over ﬁve simulations reach the set-point ϵ = 0.7 when the number of synaptic elements is regulated homeostatically (yellow), whereas calcium concentrations remain much lower when there is no homeostatic regulation (gray). (B) Development of calcium concentration in random networks without any distance-dependency in synapse formation. The random network with homeostatic regulation of synaptic elements (blue) also reaches the homeostatic set-point ϵ, whereas the network without homeostasis (gray) and the same number of synapses as the homeostatic network has much lower values in calcium. Shadow around the curves (hardly visible since so small) indicates the standard deviation. FIGURE 3 \| Neurons develop their connectivity in order to reach a homeostatic set-point ϵ of intracellular calcium. The ﬁgure shows the ﬁring rates attained for different set-point values of calcium. For each calcium concentration, the ﬁring rates of all neurons were pooled from four different simulations recorded over the last 20,000 ms. The central mark of each box is the median ﬁring rate; boxes represent the 25th and 75th percentiles; the whiskers extend to the most extreme data points not considered “outliers”; and “outliers” are plotted individually (which show up here as thick bold lines). 3.1. HOMEOSTASIS INFLUENCES THE CLUSTERING COEFFICIENT AND CHARACTERISTIC PATH LENGTH OF DEVELOPING SMALL-WORLD NETWORKS An “outlier” is a value that is more than 1.5 times the interquartile range away from the top or bottom of the box. The ﬁring rate is proportional to ϵ by a factor of 100. For this set of simulations lower external inputs Iext with mean 2 mVms−1 were used. FIGURE 4 \| Small-worldness of developing networks. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. FIGURE 3 \| Neurons develop their connectivity in order to reach a homeostatic set-point ϵ of intracellular calcium. The ﬁgure shows the FIGURE 3 \| Neurons develop their connectivity in order to reach a homeostatic set-point ϵ of intracellular calcium. The ﬁgure shows the ﬁring rates attained for different set-point values of calcium. For each calcium concentration, the ﬁring rates of all neurons were pooled from four different simulations recorded over the last 20,000 ms. The central mark of each box is the median ﬁring rate; boxes represent the 25th and 75th percentiles; the whiskers extend to the most extreme data points not considered “outliers”; and “outliers” are plotted individually (which show up here as thick bold lines). An “outlier” is a value that is more than 1.5 times the interquartile range away from the top or bottom of the box. The ﬁring rate is proportional to ϵ by a factor of 100. For this set of simulations lower external inputs Iext with mean 2 mVms−1 were used. homeostatic set point ϵ of intracellular calcium. The ﬁgure shows the ﬁring rates attained for different set-point values of calcium. For each calcium concentration, the ﬁring rates of all neurons were pooled from four different simulations recorded over the last 20,000 ms. The central mark of each box is the median ﬁring rate; boxes represent the 25th and 75th percentiles; the whiskers extend to the most extreme data points not considered “outliers”; and “outliers” are plotted individually (which show up here as thick bold lines). An “outlier” is a value that is more than 1.5 times the interquartile range away from the top or bottom of the box. The ﬁring rate is proportional to ϵ by a factor of 100. For this set of simulations lower external inputs Iext with mean 2 mVms−1 were used. 3.1. HOMEOSTASIS INFLUENCES THE CLUSTERING COEFFICIENT AND CHARACTERISTIC PATH LENGTH OF DEVELOPING SMALL-WORLD NETWORKS FIGURE 4 \| Small-worldness of developing networks. (A) Small-world \| p g networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. April 2014 \| Volume 6 \| Article 7 \| 7 Frontiers in Synaptic Neuroscience www.frontiersin.org Homeostatic small-world networks Butz et al. in networks favoring short-range connections over long-range connections. We therefore further studied how homeostasis inﬂu- enced clustering in small-world networks. We tested the hypothe- sis that homeostasis produced more long-range connections than expected from the kernel function K (Equation 5). Computing the average Euclidean distance between the pre- and postsy- naptic neuron for every synapse indeed revealed longer average Euclidean distances in homeostatic than in non-homeostatic networks (Figure 7A). In non-homeostatic networks, the aver- age Euclidean distance was constant, because it is directly derived from the kernel function (in Equation 5: σ = 150 μm). In homeostatic networks, however, we observed two different phases. First, during initial network development ([Ca2+] << ϵ), the average Euclidean distance converged quickly toward a stable value of around 2, which was only slightly higher than in non- homeostatic networks. Secondly, when calcium concentrations approached the homeostatic set-point ϵ, the average Euclidean distances ramped up and reached values greater than 4. The con- siderable increase in the average Euclidean distance of synaptic connections coincided with a drop in clustering coefﬁcient. As the initial high clustering of the network is due to the kernel function for synapse formation favoring short- over long-range connections, we may conclude that increasing Euclidean lengths of synaptic connections give rise to the decrease in clustering right at the time neurons approach the homeostatic set-point in electrical activity. The effect was also noticeable in the course of the characteristic path length but much less pronounced. We did not observe a comparable effect of homeostasis on Euclidean dis- tances of synaptic connections in random networks (Figure 7B). were marginally lower than those in homeostatic small-world networks. The clustering coefﬁcient in developing homeostatic small- world networks took a considerably different course from the coefﬁcient in small-world networks without homeostasis (Figure 6A). 3.1. HOMEOSTASIS INFLUENCES THE CLUSTERING COEFFICIENT AND CHARACTERISTIC PATH LENGTH OF DEVELOPING SMALL-WORLD NETWORKS Whereas the clustering coefﬁcient in networks with- out homeostasis converged, with a small overshoot, toward high levels of over 1.6, networks with homeostasis generated a con- siderable overshoot and ended up at much lower values as com- pared with non-homeostatic networks. After a ramp-up phase, homeostatic networks reached a maximum clustering coefﬁ- cient of about one; thereafter, the clustering coefﬁcient decreased again with a decreasing negative slope. The maximum cluster- ing coefﬁcient was reached when the average electrical activities approached the homeostatic set-point ϵ. By contrast, we did not see an overshoot in clustering coefﬁcient in homeostatic and non-homeostatic random networks (Figure 6B). Therefore, the homeostasis in electrical activity had no effect on clustering in random networks. 3.2. HOMEOSTASIS FAVORS LONG-RANGE CONNECTIONS IN SMALL-WORLD NETWORKS Particularly the development of the clustering coefﬁcient, with its pronounced overshoot, determines the emerging small-worldness FIGURE 5 \| Characteristic path length in developing networks. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. The inset in (A) is a close-up of the time interval from 5000 to 10,000 updates in connectivity that clearly shows the decay in characteristic path length in homeostatic networks compared to non-homeostatic networks. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. FIGURE 6 \| Clustering coefﬁcient in developing networks. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. Are the increasing average Euclidean distances of synaptic con- nections caused by the fact that average neuronal activities are reaching a homeostatic equilibrium, or is this just some net- work effect that merely coincides with neurons equilibrating their activities? To answer this question, we assessed the number of all types of vacant elements (i.e., excitatory or inhibitory axonal elements, excitatory or inhibitory dendritic elements) and the spatial position of their hosting neurons at every time step of the simulation. We ﬁrst checked whether there was any bias in the spatial position of synaptic elements that could generate more FIGURE 5 \| Characteristic path length in developing networks. (A) \| Small-world networks with (orange) and without (gray) homeostasis in electrical activity. The inset in (A) is a close-up of the time interval from 5000 to 10,000 updates in connectivity that clearly shows the decay in characteristic path length in homeostatic networks compared to non-homeostatic networks. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. FIGURE 7 \| Average Euclidean distance between pre- and postsynaptic neurons for every synapse during network development. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. Inset in (A) depicts the change in Euclidean distances for homeostatic random (blue) and homeostatic small-world networks (orange) with torus boundary conditions. 3.3. HOMEOSTASIS DECREASES THE BETWEENNESS CENTRALITY IN SMALL-WORLD NETWORKS Betweenness centrality (or inbetweenness) is a measure for the importance of nodes in a network. Compared with random net- works, small-world networks without homeostatic synapse for- mation had a relatively high betweenness centrality (Figure 10A). Small-world networks with homeostasis, by contrast, revealed a pronounced decrease in betweenness centrality over develop- mental time. Values peaked in the beginning of development when neurons ﬁrst connected to their nearest neighbors only. However, right after the peak, homeostatic synapse formation generated topologies with values for betweenness centrality that were lower than in non-homeostatic networks. Betweenness centrality is high in non-homeostatic small-world networks because synapse formation is only determined by the kernel func- tion (Equation 5), which often caused that the same cell pairs were connected repeatedly. The consequence is a limited num- ber of shortest paths between any pairs of nodes in the network. FIGURE 8 \| Spatial position of vacant synaptic elements in small-world networks. The purpose of this ﬁgure is to rule out that the position of FIGURE 8 \| Spatial position of vacant synaptic elements in small-world networks. The purpose of this ﬁgure is to rule out that the position of vacant synaptic elements alone gave rise to longer-ranged synaptic connections. The left column shows vacant synaptic elements accumulated over the ﬁrst 5000 updates in connectivity, whereas the right column shows the accumulation of vacant synaptic elements over the following 5000 updates. Although some tendency of vacant synaptic elements being located at the borders of the network is visible, this effect is gone after 5000 updates in connectivity. So there is no bias in the distribution of vacant synaptic elements when activity reaches the homeostatic set-point, and therefore the position of vacant synaptic elements alone cannot account for the formation of longer-ranged synaptic connections for T > 5000. (A) Vacant axonal elements. (B) Vacant dendritic elements. Color scale indicates number of vacant synaptic elements per neuron. FIGURE 8 \| Spatial position of vacant synaptic elements in small-world networks. The purpose of this ﬁgure is to rule out that the position of vacant synaptic elements alone gave rise to longer-ranged synaptic connections. The left column shows vacant synaptic elements accumulated over the ﬁrst 5000 updates in connectivity, whereas the right column shows the accumulation of vacant synaptic elements over the following 5000 updates. 3.2. HOMEOSTASIS FAVORS LONG-RANGE CONNECTIONS IN SMALL-WORLD NETWORKS FIGURE 6 \| Clustering coefﬁcient in developing networks. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. FIGURE 7 \| Average Euclidean distance between pre- and postsynaptic neurons for every synapse during network development. (A) FIGURE 7 \| Average Euclidean distance between pre- and postsynaptic neurons for every synapse during network development. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. Inset in (A) depicts the change in Euclidean distances for homeostatic random (blue) and homeostatic small-world networks (orange) with torus boundary conditions. Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. Inset in (A) depicts the change in Euclidean distances for homeostatic random (blue) and homeostatic small-world networks (orange) with torus boundary conditions. FIGURE 6 \| Clustering coefﬁcient in developing networks. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. Frontiers in Synaptic Neuroscience April 2014 \| Volume 6 \| Article 7 \| 8 www.frontiersin.org Homeostatic small-world networks Butz et al. number of synaptic elements and therefore also the distribu- tion of vacant synaptic elements are activity-dependent, the most likely synaptic connection to be formed is a direct consequence of the neurons’ electrical activities. It turned out that in the begin- ning of development, when all neurons offer vacant synaptic elements, the most likely synaptic connections are those between adjacent neurons (Figures 9A,B). In other words, when neuronal activities were much lower than the homeostatic set-point ϵ, the kernel function had a large impact on the Euclidean length of synaptic connections. Therefore, at this stage of development, homeostatic and non-homeostatic networks did not differ much in connection lengths. 3.2. HOMEOSTASIS FAVORS LONG-RANGE CONNECTIONS IN SMALL-WORLD NETWORKS However, when the activity of all individ- ual neurons approached the set-point ϵ, vacant synaptic elements became rare and matching synaptic elements were available, if at all, only between more distant neurons. Expected distances for most likely synaptic connections therefore became much larger. Moreover, the distribution of expected Euclidean lengths of new synaptic connections did not follow the Gaussian-shaped kernel any longer but became much wider and ﬂatter. At the same time, it took much longer for a synapse to form because, although longer-range synaptic connections were the most likely ones, their probability was still very low due to the kernel function. Due to a lack of shorter-range alternatives, these longer-range synaptic connections were nonetheless formed at some point in time, because the kernel function is non-zero for all distances. Taken together, the Euclidean length of synaptic connections in small-world networks is inﬂuenced by the homeostatic forma- tion of synaptic elements. As expected from the topology data, no activity-dependent effect on the Euclidean distance of synaptic connections was observed in random networks. distant connections, e.g., a placement of synaptic elements at the boundaries of the network. We accumulated the number of vacant axonal (Figure 8A) and dendritic elements (Figure 8B) for each neuron for the ﬁrst 5000 updates in connectivity with [Ca2+] < ϵ as well as for the next 5000 time steps with [Ca2+] ≈ϵ. In the beginning of the simulation, we indeed found a little more vacant synaptic elements at the network boundaries, which can be explained by the fact that neurons at boundaries have less neigh- bors than neurons in the center and compensate for this by getting a higher number of vacant dendritic elements. However, by the time that the homeostatic equilibrium is reached, vacant synaptic elements were equally distributed over the network, and therefore the placement of vacant synaptic elements cannot account for the increasing distances of synaptic connections once the network has reached the homeostatic set-point. We found a comparable course of Euclidean distances in homeostatic small-world networks with torus boundary conditions (see inset of Figure 7A). Since we could exclude a spatial bias in the distribution of vacant synaptic elements, we tested whether the increasing dis- tances of synaptic connections were a direct consequence of the activity-dependent growth rules. 3.2. HOMEOSTASIS FAVORS LONG-RANGE CONNECTIONS IN SMALL-WORLD NETWORKS On the basis of the number of vacant elements per neuron and the Euclidean distance between the host neurons, we determined the most likely synaptic connec- tion every time a new synapses was formed, which is equivalent to the maximum of Pform i,j (Equation 7). Since the change in the FIGURE 8 \| Spatial position of vacant synaptic elements in small-world networks. The purpose of this ﬁgure is to rule out that the position of vacant synaptic elements alone gave rise to longer-ranged synaptic connections. The left column shows vacant synaptic elements accumulated over the ﬁrst 5000 updates in connectivity, whereas the right column shows the accumulation of vacant synaptic elements over the following 5000 updates. Although some tendency of vacant synaptic elements being located at the borders of the network is visible, this effect is gone after 5000 updates in connectivity. So there is no bias in the distribution of vacant synaptic elements when activity reaches the homeostatic set-point, and therefore the position of vacant synaptic elements alone cannot account for the formation of longer-ranged synaptic connections for T > 5000. (A) Vacant axonal elements. (B) Vacant dendritic elements. Color scale indicates number of vacant synaptic elements per neuron. Additionally, synapse deletion could in principle also inﬂu- ence the Euclidean length of synaptic connections if, for example, with increasing neuronal activities preferentially short connec- tions would be deleted. Therefore, we further tested whether the expected Euclidean length of synaptic connections that were most likely to be deleted correlated with the current average activity in the network. However, an activity-dependent effect on synapse deletion was not observed (Figures 9C,D). 3.3. HOMEOSTASIS DECREASES THE BETWEENNESS CENTRALITY IN SMALL-WORLD NETWORKS In (A,B), each dot represents the Euclidean distances between those neurons that are most likely to form a synaptic connection with each other at this update in connectivity. For this, we took at every update in connectivity in which vacant synaptic elements were available the Euclidean distance of the connection from neuron j to i for which Pform i,j (Equation 7) was maximal. In (C,D), each dot represents the length of that connection (again in terms of the Euclidean distance between the connected neurons) for which synapse deletion was most likely, i.e., Pdel i,j (Equation 6) was maximal for every update in connectivity in which synapses had to be deleted. In (A,C), we plotted the Euclidean distances for synapse formation and deletion FIGURE 9 \| The spatial distribution of newly formed synapses change in dependence on the calcium concentration. In (A,B), each dot represents the FIGURE 9 \| The spatial distribution of newly formed synapses change in dependence on the calcium concentration. In (A,B), each dot represents the Euclidean distances between those neurons that are most likely to form a synaptic connection with each other at this update in connectivity. For this, we took at every update in connectivity in which vacant synaptic elements were available the Euclidean distance of the connection from neuron j to i for which Pform i,j (Equation 7) was maximal. In (C,D), each dot represents the length of that connection (again in terms of the Euclidean distance between the connected neurons) for which synapse deletion was most likely, i.e., Pdel i,j (Equation 6) was maximal for every update in connectivity in which synapses had to be deleted. In (A,C), we plotted the Euclidean distances for synapse formation and deletion over time. The black curve (right y-axis) indicates the course of the calcium concentration Ca2+ . In (B,D), we plotted synapse formation and deletion in dependence on Ca2+ . The color code in all panels indicates the density of the dots in the diagrams, with blue and red representing low and high densities of dots, respectively. The ﬁgure essentially shows that before calcium reaches the homeostatic set-point ϵ, the distribution for synapse formation is rather Gaussian, following the Kernel function K (Equation 5). The distribution becomes random and scattered, with increased Euclidean distances, when calcium is at the set-point. 3.3. HOMEOSTASIS DECREASES THE BETWEENNESS CENTRALITY IN SMALL-WORLD NETWORKS The stripes in the distribution arise from the fact that not all Euclidean distances are possible due to the grid layout of the network. There is no change in the distribution for synapse deletion. FIGURE 10 \| Changing betweenness centrality over time. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. Because the variety of shortest paths in non-homeostatic net- works is limited, betweenness centrality reached a stable plateau in these networks. Interestingly, the betweenness centrality in homeostatic networks initially also converged toward a quasi- stable level. However, as soon as activities approached the home- ostatic set-point ϵ, betweenness centrality strongly decreased. Over time, the rate of decrease slowed down. The decrease in betweenness centrality precisely coincided with the increase in Euclidean lengths of synaptic connections. Because the kernel function favors short- over long-range connections and therefore initially creates networks with high betweenness centrality, we may conclude that the formation of longer-range connections (in an Euclidean sense) lead to a decreasing betweenness centrality. Any new long-range connection not present in the network before creates new shortest paths, which in turn decreases betweenness centrality. By contrast, homeostasis did not affect the course of betweenness centrality in random networks (Figure 10B). FIGURE 10 \| Changing betweenness centrality over time. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. By contrast, in homeostatic networks, synapse formation depends on the availability of synaptic elements, which can force synapses to be formed that are less likely according to the kernel. This may increase the number of alternative paths between two neu- rons and therefore also increase the number of multiple shortest paths through the network. Hence, betweenness centrality quickly decreased over time. 3.3. HOMEOSTASIS DECREASES THE BETWEENNESS CENTRALITY IN SMALL-WORLD NETWORKS Although some tendency of vacant synaptic elements being located at the borders of the network is visible, this effect is gone after 5000 updates in connectivity. So there is no bias in the distribution of vacant synaptic elements when activity reaches the homeostatic set-point, and therefore the position of vacant synaptic elements alone cannot account for the formation of longer-ranged synaptic connections for T > 5000. (A) Vacant axonal elements. (B) Vacant dendritic elements. Color scale indicates number of vacant synaptic elements per neuron. April 2014 \| Volume 6 \| Article 7 \| 9 Frontiers in Synaptic Neuroscience www.frontiersin.org Butz et al. Homeostatic small-world networks FIGURE 9 \| The spatial distribution of newly formed synapses change in dependence on the calcium concentration. In (A,B), each dot represents the Euclidean distances between those neurons that are most likely to form a synaptic connection with each other at this update in connectivity. For this, we took at every update in connectivity in which vacant synaptic elements were available the Euclidean distance of the connection from neuron j to i for which Pform i,j (Equation 7) was maximal. In (C,D), each dot represents the length of that connection (again in terms of the Euclidean distance between the connected neurons) for which synapse deletion was most likely, i.e., Pdel i,j (Equation 6) was maximal for every update in connectivity in which synapses had to be deleted. In (A C) we plotted the Euclidean distances for synapse formation and deletion over time. The black curve (right y-axis) indicates the course of the calcium concentration Ca2+ . In (B,D), we plotted synapse formation and deletion in dependence on Ca2+ . The color code in all panels indicates the density of the dots in the diagrams, with blue and red representing low and high densities of dots, respectively. The ﬁgure essentially shows that before calcium reaches the homeostatic set-point ϵ, the distribution for synapse formation is rather Gaussian, following the Kernel function K (Equation 5). The distribution becomes random and scattered, with increased Euclidean distances, when calcium is at the set-point. The stripes in the distribution arise from the fact that not all Euclidean distances are possible due to the grid layout of the network. There is no change in the distribution for synapse deletion FIGURE 9 \| The spatial distribution of newly formed synapses change in dependence on the calcium concentration. 3.4. SMALL-WORLD NETWORKS BECOME MORE EFFICIENT BY HOMEOSTASIS Efﬁcient information transmission is probably the most-desired property in computational networks. Small-world networks are April 2014 \| Volume 6 \| Article 7 \| 10 Frontiers in Synaptic Neuroscience www.frontiersin.org Butz et al. Homeostatic small-world networks With additional long-range connections betweenness central- ity decreases. Networks with low betweenness centrality are more robust against lesions because all nodes are equally important. By contrast, networks with high betweenness centrality are very vulnerable to lesions. If neurons that are part of many short- est paths are lost, the characteristic path length will immediately increase, with a signiﬁcant impact on information transmission. It is remarkable that a self-organizing process that forms net- works by striving toward homeostasis in electrical activity as a side effect produces topologies with lower betweenness centrality that contribute to a higher robustness against lesions. very efﬁcient because they combine a high clustering coefﬁcient with a short characteristic path length. Nevertheless, their efﬁ- ciency is still markedly lower than that of random networks. Homeostatic small-world networks, however, generated efﬁciency levels that during the whole course of development exceeded the levels in non-homeostatic small-world networks (Figure 11A). Remarkably, at the time when average activities reached the homeostatic set-point ϵ, efﬁciency levels of homeostatic net- works further increased and almost reached the levels in random networks (Figure 11B). Consequently, favoring more distant con- nections in combination with homeostasis in electrical activity led to a more efﬁcient network topology than achieved without homeostasis. g g Key to the homeostasis-driven change in topology is the increasing Euclidean length of connections. Since we did not observe an increase in connection length in networks with- out homeostasis, we concluded that the increase in connection lengths was caused by homeostasis in electrical activity. To create networks without homeostasis, we took the number of synapses from the homeostatic network at every update in connectiv- ity and distributed them randomly under the same kernel in the non-homeostatic network. There are, in fact, other ways to create networks without homeostasis. We additionally built non- homeostatic networks by initially giving all model neurons a ﬁxed number of vacant synaptic elements and then running updates in connectivity until no more synapses could be formed. Also in this scenario we did not see an increase in connection length. In a third scenario, we added a few vacant synaptic elements to all neurons before every update in connectivity. 3.4. SMALL-WORLD NETWORKS BECOME MORE EFFICIENT BY HOMEOSTASIS No matter how few vacant elements we added and how long we ran the network, we did not obtain more long-range connections than expected from the kernel. We only observed more long-range connections when we slowly added vacant elements at a few randomly selected neu- rons at a time (i.e., spatial sparseness in the formation of synaptic elements). From these observations we concluded that the contri- bution of homeostasis is not only to limit the number of available vacant synaptic elements but also to generate a certain sparseness in the formation of vacant elements. In fact, as long as the neurons were far away from the homeostatic set-point, synaptic element formation was not sparse at all as all neurons added elements roughly at the same time at equal rates. Only when the neurons approached the homeostatic set-point did sparseness in synap- tic element formation arise. Homeostasis creates this sparseness because balancing the activity of one neuron immediately affects the activity in other neurons, which in turn may be driven fur- ther away from the homeostatic set-point and then start forming new vacant elements. The presence of inhibitory neurons would further reinforce this process. 4. DISCUSSION We have shown that network formation favoring short-range over long-range connections produced networks with a pronounced small-world structure. Networks with homeostasis in electrical activity developed a weaker small-world structure in favor of more efﬁcient wiring of connections. Global efﬁciency particu- larly increased when network activity reached the homeostatic set-point. Increased global efﬁciency was caused by the fact that homeostasis favored longer-ranged connections, which affected clustering as well as characteristic path length. Thus, network topology continued to change even after the network had reached a homeostatic equilibrium in electrical activity. Adding more long-range connections to a small-world net- work makes the network more efﬁcient but also more ran- dom. This is apparent in our simulations, too, by a decreas- ing clustering coefﬁcient and a decreasing characteristic path length. Nevertheless, the small-world property of the net- works was preserved throughout the whole course of devel- opment, and the decrease in clustering coefﬁcient was slow- ing down over time. However, networks would most likely turn into random networks if rewiring continued indeﬁnitely. Consequently, there seems to be a trade-off in network devel- opment between high clustering and strong small-worldness on the one hand and more randomness and higher efﬁciency on the other hand. The latter particularly arises when networks con- tinue to rewire their circuitry when they are in a homeostatic equilibrium. FIGURE 11 \| Global efﬁciency changes over time due to network development. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. Homeostasis in electrical activity is one way by which a local process can give rise to a change in global topology. Another com- parable mechanisms was provided by Kaiser et al. (2009). In their model study, they showed that a simple axonal growth process can generate the experimentally observed exponential decrease in number of connections with increasing connection length. As a result of the growth process, most connections become short- range, although long-range connections also arise, but in lower numbers. The idea of their model is that axons grow out until they hit a postsynaptic target. The capacity of model neurons to receive connections is limited, and if nearby target neurons are FIGURE 11 \| Global efﬁciency changes over time due to network development. 4. DISCUSSION (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. FIGURE 11 \| Global efﬁciency changes over time due to network development. (A) Small-world networks with (orange) and without (gray) homeostasis in electrical activity. (B) Random networks with (blue) and without (gray) homeostasis in electrical activity. Means over ﬁve simulations per scenario. Shadings of the curves indicate standard deviations. E 11 \| Global efﬁciency changes over time due to network April 2014 \| Volume 6 \| Article 7 \| 11 Frontiers in Synaptic Neuroscience www.frontiersin.org www.frontiersin.org Homeostatic small-world networks Butz et al. 1991; Abbott and Nelson, 2000) and a variety of plasticity mech- anisms can act homeostatically. Scaling of synaptic strengths (Turrigiano, 1999), for example, has been reported as a mech- anism acting at existing synapses to stabilize postsynaptic ﬁring in cortical, hippocampal and spinal cord networks (Lissin et al., 1998; O’Brien et al., 1998; Turrigiano and Nelson, 1998). Even in the mature brain, not only the strength of synapses but also the formation of new synapses can contribute to the stabilization of neuronal activity, for example after focal retinal lesions (Butz and van Ooyen, 2013). In developing dissociated cell cultures, we showed that homeostasis in electrical activity may be a precon- dition for the emergence of self-organized criticality in neuronal ﬁring (Tetzlaff et al., 2010). In another study, we showed that homeostasis in electrical activity can regulate the synaptic embed- ding of newly formed neurons in the mature hippocampal dentate gyrus (adult neurogenesis) and can account for the experimen- tally observed counter-intuitive inverse relationship between cell proliferation rate and synaptogenesis (Butz et al., 2008). completely occupied by incoming connections, axons continue to grow out until they hit a vacant target. Hence, Euclidean connec- tion length increases over developmental time. In this model, the spatial growth process in combination with a hard boundary on the number of incoming synapses per model neuron generates the increase in connection lengths. There are striking similarities between the topology of our model networks and that of developing dissociated cell cultures. It is well known that cultured neuronal networks can form small- world topology (Bettencourt et al., 2007; Yu et al., 2008; Gerhard et al., 2011). Downes et al. 4. DISCUSSION (2012) reported an increase in clus- tering coefﬁcient of dissociated cell cultures between 14 days in vitro (DIV) and 28 DIV and a subsequent drop until 35 DIV, a course of development that is comparable to the course of devel- opment in our model networks with homeostasis. Between 14 and 35 DIV, the mean shortest path length did not change signif- icantly, only showing a slight drop around 28 DIV. Consequently, small-worldness reached its maximum around 28 DIV. Moreover, the experimental data indicated the presence of longer synaptic connections from 28 DIV onwards that had not been not present at 21 DIV. From our previous studies we know that dissociated cell cultures reach homeostasis around this time (Tetzlaff et al., 2010). Therefore, we may hypothesize that the increase in con- nection length in dissociated cell cultures may be due to neurons reaching a homeostatic equilibrium in electrical activity. Other synaptic plasticity mechanisms not currently incorporated in our model may of course also have contributed to network formation in developing cell cultures. In summary, we conclude that homeostatic regulation of elec- trical activity together with simple distance-dependent forma- tion of connections is capable of creating, in a self-organizing manner, neuronal networks that are more robust and more efﬁcient than networks grown without homeostatic regulation. Strikingly, the growth process revealed features of developing topologies that are also observed in dissociated cell cultures and infant human brains. The formation of network topology by a self-organizing, local growth process may also be relevant for automatically generating the connectivity structure of large-scale neuronal networks (Potjans and Diesmann, 2014) that are cur- rently studied in enterprizes such as the Human Brain Project (www.humanbrainproject.eu). On a macroscopic scale, functional imaging data reveal a devel- opment of small-world topology that also has interesting similar- ities with the self-organizing network formation in our model. The infant human brain has small-world properties already at the third post-natal week (Fransson et al., 2011). During the follow- ing 2 years, network topology undergoes a signiﬁcant reﬁnement: brain networks increase their small-worldness, global efﬁciency and number of long-distance connections (Gao et al., 2011). Although our network model shows only an initial transient increase in small-worldness, it may offer a simple explanation for the sudden increase in number of long-range connections and the associated increase in global efﬁciency. Frontiers in Synaptic Neuroscience FUNDING This work was partly funded by the Helmholtz Association through the Helmholtz Portfolio Theme “Supercomputing and Modeling for the Human Brain.” Markus Butz and Ines D. Steenbuck were also partly supported by the NETFORM project (grant number 635.100.017, awarded to Arjen van Ooyen) of the Computational Life Sciences program of the Netherlands Organization for Scientiﬁc Research (NWO) and by a starting grant awarded to Markus Butz from the collaborative research center (SFB 874) funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). 4. DISCUSSION Could it be that even in the human brain, neurons establishing a homeostatic equilib- rium in electrical activity produce—as an emergent property of the homeostatic growth process—more long-range connections? Remarkably, the increase in number of long-range connections occurs not during a genetically-encoded formation of an initial embryonic layout of projections but during the post-natal critical period (Gao et al., 2011), during which neurons are highly sensi- tive to afferent input. 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D., Bijma, F., et al. (2014). Independently outgrowing neurons and geometry-based synapse formation produce networks with realistic synaptic connectivity. PLoS ONE 9:e85858. doi: 10.1371/journal.pone.0085858 This article was submitted to the journal Frontiers in Synaptic Neuroscience. This article was submitted to the journal Frontiers in Synaptic Neuroscience. Copyright © 2014 Butz, Steenbuck and van Ooyen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. van Ooyen, A., and van Pelt, J. (1994). Activity-dependent neurite outgrowth and neural network development. Prog. 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https://openalex.org/W4387158776	https://jurnal.stisipwidyapuri-smi.ac.id/index.php/mimbar/article/download/86/86	Indonesian	null	EFEKTIVITAS PROGRAM KELUARGA BERENCANA (KB) DALAM PENGGUNAAN ALAT KONTRASEPSI DI UPTD DINAS PENGENDALIAN PENDUDUK DAN KELUARGA BERENCANA (DPPKB) KECAMATAN CARINGIN KABUPATEN SUKABUMI	Mimbar Administrasi Mandiri	2,023	cc-by	2,640	Volume 19 Nomor 2 September 2023 DOI : doi.org/10.37949/mimbar19286 Volume 19 Nomor 2 September 2023 DOI : doi.org/10.37949/mimbar19286 Mimbar Administrasi Mandiri e-ISSN : 2721-2459 p-ISSN : 1907-0683 DOI : doi.org/10.37949/mimbar Mimbar Administrasi Mandiri e-ISSN : 2721-2459 p-ISSN : 1907-0683 DOI : doi.org/10.37949/mimbar EFEKTIVITAS PROGRAM KELUARGA BERENCANA (KB) DALAM PENGGUNAAN ALAT KONTRASEPSI DI UPTD DINAS PENGENDALIAN PENDUDUK DAN KELUARGA BERENCANA (DPPKB) KECAMATAN CARINGIN KABUPATEN SUKABUMI Rena Fadilah Maliki Email: renafadilahm@stitfatahillah.ac.id STIT Fatahillah words: Effectiveness, Family Planning Program, Contraception (KB). Keywords: Effectiveness, Family Planning Program, Contraception (KB). ABSTRACT This study aims to assess the effectiveness of the Family Planning Program (KB) in the use of contraceptives in the UPTD of the Population Control and Family Planning Office of Caringin Sub- district, Sukabumi District. The KB program has an important role in regulating births, marriage age, and increasing family resilience and welfare. This activity involves married couples and requires the support of husbands. Indonesia's high population growth and geographical issues pose challenges to family planning implementation. The interview results show that by 2023, around 79% of people in Caringin Sub-district will have used contraceptives. This shows an increase in community awareness of the program. However, there are still 21% of the community who have not joined the program. The UPTD of the Population Control and Family Planning Office has the responsibility to provide explanations to those who have not joined the program. In this case, it requires maximum understanding and efficient use of the budget. The family planning program in the use of contraceptives in the UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana Kecamatan Caringin Kabupaten Sukabumi has been running quite well. Public awareness of the program has increased, and the services provided are effective. Coordination with related agencies and directed procedures also play an important role in the success of this program. ABSTRAK Penelitian ini bertujuan untuk mengkaji efektivitas Program Keluarga Berencana (KB) dalam penggunaan alat kontrasepsi di UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana Kecamatan Caringin Kabupaten Sukabumi. Program KB memiliki peran penting dalam mengatur kelahiran, usia perkawinan, serta meningkatkan ketahanan dan kesejahteraan keluarga. Kegiatan ini melibatkan pasangan suami istri dan memerlukan dukungan suami. Masih tingginya pertumbuhan penduduk dan masalah geografis di Indonesia menimbulkan tantangan dalam implementasi KB. g g g p Hasil wawancara menunjukkan bahwa pada tahun 2023, sekitar 79% masyarakat di Kecamatan Caringin telah menggunakan alat kontrasepsi. Ini menunjukkan peningkatan 216 kesadaran masyarakat terhadap program ini. Namun, masih ada 21% masyarakat yang belum mengikuti program ini. UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana memiliki tanggung jawab untuk memberikan penjelasan kepada mereka yang belum bergabung dalam program. Dalam hal ini, diperlukan pemahaman yang maksimal dan penggunaan anggaran yang efisien. kesadaran masyarakat terhadap program ini. Namun, masih ada 21% masyarakat yang belum mengikuti program ini. UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana memiliki tanggung jawab untuk memberikan penjelasan kepada mereka yang belum bergabung dalam program. Dalam hal ini, diperlukan pemahaman yang maksimal dan penggunaan anggaran yang efisien. Program KB dalam penggunaan alat kontrasepsi di UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana Kecamatan Caringin Kabupaten Sukabumi telah berjalan dengan cukup baik. Kesadaran masyarakat terhadap program ini meningkat, dan pelayanan yang diberikan efektif. Koordinasi dengan dinas terkait dan prosedur yang terarah juga berperan penting dalam keberhasilan program ini. g p g Efektivitas, Program KeluargaiBerencana, Alat Kontrasepsi (KB). Kata Kunci : Efektivitas, Program KeluargaiBerencana, Alat Kontrasepsi (KB). \| \| Kata Kunci : Efektivitas, Program KeluargaiBerencana, Alat Kontrasepsi (KB). Submitted: 23-09-2023\| Accepted: 23-09-2023\| Published: 30-09-2023 Submitted: 23-09-2023\| Accepted: 23-09-2023\| Published: 30-09-2023 2. Kajian Pustaka Mott dalam Hoy dan Miskel (1992:341), menyebutkan bahwa efektivitas merupakan upaya mengintegrasikan kuantitas dan kualitas, produk, efisiensi, adaptasi, dan fleksibilitas dalam mencapai tujuan. Kriteria - kriteria pengukuran efektivitas organisasi di atas terdapat beberapa pendekatan yang diantaranya berbeda satu sama lain. Hal ini dikarenakan banyaknya bentuk atau orientasi organisasi yang berbeda pula baik itu kriteria pengukuran efektivitas yang kompleks maupun yang sederhana yang dalam pelaksanaan penilainnya di sesuaikan dengan keadaan dari organisasi tersebut. Program ini bertujuan untuk mengatur program kelahiran, pendewasaan dalam usia perkawinan, serta peningkatan dalam hal ketahanan dan kesejahteraan keluarga. Pengaturan kelahiran merupakan cara yang diambil. Alat yang dapat digunakan dengan tujuan untuk mencegah terjadinya kehamilan disebut sebagai alat kontrasepsi. Penggunaan alat kontrasepsi diharapkan dapat mencegah penggabungan sel sperma dan sel telur menjadi sebuah kehamilan tersebut. 1. PENDAHULUANi Tindakan yang membantu pasangan suami istri dalam hal mengatur jarak kelahiran, dan menentukan jumlah anak dalam keluarga merupakan hal yang disebut sebagai kegiatan Keluarga Berencana (KB). Penggunaan kontrasepsi merupakan tanggung jawab bersama antara pasangan. Pengalaman istri dalam penggunaan kontrasepsi yang dipilih akan dijadikan acuan untuk mengikuti program keluarga berencana. Dukungan suami juga mempengaruhi penggunaan kontrasepsi, karena istri yang mendapat dukungan dari suami akan menggunakan kontrasepsi secara terus menerus sedangkan yang tidak mendapatkan dukungan akan sedikit yang menggunakan kontrasepsi. Masih tingginya tingkat pertumbuhan penduduk dengan angka kelahiran lebih cepat dan masih tingginya tingkat kematian menjadikan permasalahan di dalam hal kependudukan dan Program Keluarga Berencana di Indonesia. Masalah lain selain tingkat pertumbuhan yang tinggi adalah penyebaran penduduk yang juga kurang merata karena disebabkan oleh keadaan geografis yang berbeda-beda di setiap daerah. Pertumbuhan penduduk yang tidak disertai dengan pertumbuhan yang cukup dalam produksi nasional dapat juga menimbulkan berbagai masalah yang berkaitan dengan kurangnya fasilitas pendidikan, kurangnya penyediaan makanan bergizi, pelayanan kesehatan, kesempatan kerja dan lain sebagainya. Salah satu program pembangunan yang terus dilaksanakan secara berkelanjutan oleh Pemerintah adalah program KeluargaiBerencana. Adapun implementasi dari peraturan tersebut tertuang dalam Peraturan Pemerintah Nomor 87 Tahun 2014 tentang perkembangan kependudukan, Keluarga Berencana dan sistem informasi keluarga. Sukses dan tidaknya suatu program, tergantung dari peran aktif masyarakat yang terlibat di dalamnya. Permasalahan yang penulis dapatkan berdasar hasil prasurvey diantaranya adalah : 217 1) Masyarakat kurang memahami permaksudan dan tujuan dari diadakannya Program Keluarga Berencana Untuk Menggunakan alat kontrasepsi agar mewujudkan kesejahteraan pada masing- masing keluarga yang mengikuti Program Keluarga Berencana; 2) Masih kurangnya sosialisai Petugas lapangan terkait dengan Program Keluarga Berencana yang diberikan oleh petugas lapangan (KB) Sehingga tingkat kesadaran masyarakat terhadap Program Keluarga Berencana masih rendah; 3) Masih kurangnya prosedur pelaksanaan program Keluarga Berencana. memasuki lapangan, selama di lapangan, dan setelah selasai di lapangan. memasuki lapangan, selama di lapangan, dan setelah selasai di lapangan. 4. Hasil Dan Pembahasan Berdasar informasi yang didapatkan setelah melakukan wawancara dengan para informan diketahui bahwa pada tahun 2023 jumlah peserta yang menggunakan alat kontrasepsi cukup banyak sekitar 79%. Berdasar hal ini, maka terlihat kepedulian dan kemauan masyarakat di Kecamatan Caringin untuk ikut serta dalam program alat kontrasepsi sudah meningkat. Jika dihitung, maka jumlah KK yang ada masih ada 21% lagi masyarakat yang belum mengikuti program alat kontrasepsi. Berdasar hal tesebut, maka UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana sebagai pelaksana program alat kontrasepsi menjadi memiliki sebuah keharusan serta loyalitas untuk memberikan penjelasan kepada yang belum mengikuti program. Dampak yang mungkin terjadi adalah kemungkinan dalam hal melaksanakan sosialisasi serta penyuluhan dimugkinkan akan terbentur dengan pembiayaan dan tenaga penyuluh itu sendiri. Maksimalisasi pemahaman dan penjelasan kepada masyarakat diharapkan dapat dilakukan UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana dalam banyak hal terutama pemaksimalan dalam memanfaatkan anggaran. 3. Metodeipenelitian Pendekatan kualitatif dilakukan dalam penelitian ini. Metode yang dipakai adalah metode deskriptif. Metode ini merupakan cara kerja penelitian untuk menggambarkan, melukiskan, memaparkan keadaan suatu objek pada saat penelitian itu dilakukan. Dalam penelitian ini, unit analisisnya adalah pimpinan sebanyak 1 orang, petugas pelayanan alat kontrasepso (Bidan/Nakes) sebanyak 2 orang, dan PUS/Akseptor sebanyak 2 orang, sehingga total sebanyak 5 orang. Kemudian settingiiinforman yang digunakan adalah purposive sampling dan snowbal sampling. Setelah itu, dilakukan pengumpulan data, validasi data dan analisis data yang dilakukan sejak sebelum 218 4.1. Analisis TerhadapiOutput Pernyataan hasil pada tingkat pencapaian jangka pendek yang secara langsung dapat diperoleh hasil dari kegiatan yang dilakukan serta secara keseluruhan di dalam sebuah kendali manajemen organisasi disebut sebagai output. Spesifikasi sebuah misi biasanya dimiliki setiap organisasi, dalam hal pencapaian tujuan serta keberhasilan didirikannya organisasi tersebut. Penentuan sebuah tujuan harus memiliki lingkup yang lebih luas dan berorientasi kemasa depan serta mampu menggerakkan segenap energi dan sumber daya organisasi untuk mewujudkan tujuan organisasi itu sendiri, tidak hanya tidak bisa dibuat untuk memenuhi hal-hal yang bersifat sementara saja. Pelaksanaan program alat kontrasepsi dapat dikatakan efektif dilihat dari Output cara pelayanan yang diberikan kepada masyarakat sehingga tujuan dari program ini dapat tercapai. Cara pelayanan yang diberikan dapat mempengaruhi pencapaian target yang diharapkan dari program ini. Berdasarkan hasil wawancara output dalam pelaksanaan program ala 219 kontrasepsi di UPTD DPPKB Kecamatan Caringin Kabupaten Sukabumi cukup baik. Hal ini berdasarkan pelaksanaan yang diberikan oleh pihak UPTD sudah dilakukan serta pelayanan yang diberikan kepada masyarakat bahwa program KB ini bertujuan untuk peningkatan dalam hal terjamin serta terkendalinya pertumbuhan penduduk dan mewujudkan sasaran dari Norma Keluarga Kecil Bahagia Sejahtera (NKKBS) yang menjadi dasar terwujudnya pengendalian kelahiran agar terwujudnya masyarakat yang lebih sejahtera. Hal ini didukung pula denga dengan fakta bahwa masyarakat sudah banyak yang mengikuti program KB hingga 79% dan adanya pelayanan program alat kontrsepsi yang tersedia di bidan ataupun puskesmas. 4.2. Analisis Terhadap Kebijakan Serangkaian tindakan yang mesti diikuti dan dilakukan para pelaksananya untuk pencapaian mempunyai tujuan tertentu dalam hal memecahkan suatu masalah disebut sebagai kebijakan. Dalam kaitannya kebijakan merupakan ukuran suatu organisasi yang saling berkaitan dalam hal pencapaian tujuan yang layak dan dapat dicapai serta saling berkaitan. Hal ini dimaksudkan agar program dapat berjalan secara lebih efektif dan efisien. Efektivitasi Program Keluarga Berencana (KB) Dalam Penggunaan Alat Kontrasepsi di UPTD DinasPengendalian Penduduk dan Keluarga Berencana Kecamatan Caringin Kabupaten Sukabumi. Tentunya untuk mencapai tujuan program yang diharapkan diawal, karena itu diketahui bersama bahwa sebuah program akan tercapai apabila dalam kebijakannya jelas dan terarah. Hal tersebut sesuai dengan wawancara yang telah di laksanakan. Agar pelaksanaan program berjalan dengan efektif pada tujuan yang telah ditetapkan, maka perlu kebijakan yang terarah terlebih dahulu. Kebijakan terkait Porgram Keluarga Berencana dalam penggunaan alat kontrasepsi di UPTD Dinas pengendalilan Penduduk dan Keluarga Berencana (DPPKB) Kecamatan Caringin Kabupaten Sukabumi dilakukan dengan cara berkoordinasi dengan dinas terkait agar kebijakan Program Keluarga Berencana terlaksana dengan baik karena Program Keluarga Berencana tidak bisa bekerja sendiri seperti bekerja sama dengan dinas kesehatan yaitupuskesmas dan kecamatan sebagai pemangku kebijakan di wilayah tersebut. Kesimpulan hasil wawancara adalah kebijakan dalam Pelaksanaan Keluarga 220 Berencana cukup baik dilihat dari proses dan sosialisasi yang diberikan kepada masyarakat. Masyarakat menjadi lebih paham dengan dilaksanakannya Program Keluarga Berencana (KB) dalam penggunaan alat kontrasepsi dengan demikian masyarakat bisa melaksanakan Program alat kontrasepsi dengan mandiri atas kesadaran sendiri untuk mencapai keluarga kecil bahagia dan sejahtera. Serta didukung dengan fakta bahwa pada proses mengikuti program alat kontrasepsi, masyarakat selalu memdapatkan pembinaan dan pengarahan supaya masyarakat mudah dan tidak mendapatkan kesulitan dalam mengikuti program KB serta dengan adanya sosialisasi yang terus diberikan kepada masyarakat disetiap acara kegiatan kemasyarakat maupun dari kegiatan KB itu sendiri. 4.3.Analisis Terhadap Prosedur Prosedur merupakan faktor yang penting didalam suatu peleksanaan program dimana prosedur ini merupakan suatu rangkaian dari tata kerja yang salingberhubungan satu dengan yang lain dimana terlihat adanya suatu urutan tahap demi tahap. Dalam pelaksanaan program prosedur merupakan aksi yang detail atau disebut tindakan yang harus di jalankan dengan cara yang sama sesuai dengan yangtertera pada teks prosedur supaya mendapatkan hal yang sama, agar suatu program berjalan dengan efektif maka harus dilakukan sesuai dengan prosedur yang ada. Setiap oranisasi mempunyai aturan. Tujuan dibuatnya peraturan ini tentunya untuk menjelaskan secara rinci bagaimana seluruh sumber daya manusia yang ada di dalam organisasi bertindak sesuai dengan standar yang ada, sehingga akan menjadikan arus kerja yang teratur dan efektif dan tentunya dengan adanya Prosedur mampu memudahkan pekerjaan seluruh pegawai. Dari hasil wawancara dapat disimpulkan bahwa terkait Prosedur Program Keluarga Berencana (KB) Dalam Penggunaan Alat Kontrasepsi di UPTD DPPKB Kecamatan Caringin Kabupaten Sukabumi sudah berjalan dengan cukup baik yaitu dilihat dari arahan dan komunikasi yang diberikan pimpinan yaitu Kepala UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana (DPPKB) Kecamatan Caringin Kabupaten Sukabumi kepada bawahannya yaitu Petugas Lapangan Keluarga Berencana (PLKB) agar melaksanakan tugas sesuai dengan prosedur yang sudah ditetapkan, sehingga realisasi program memperoleh hasil yang diinginkan, serta didukung dengan fakta bahwa dalam perealisasian program alat kontrasepsi yang rata-rata dari masyarakat itu sudah mengikuti program penggunaan alat kontrasepsi dandengan dibentuknya group 221 WhatsApp supaya memudahkan masyakarat mendapatkan informasi dan berkomunikasi antara satu sama lain. DAFTAR PUSTAKA Gustina,Eni. 2020. Pedoman Pelayanan Kontrasepsi Dan Keluarga Berencana. Menteri Kesehatan Republik Indonesia p Handoko, Hani. 2014. Manajemen. Yogyakarta: BPFE-Yogyakarta. Indrawijaya, Ibrahim. 2014. Teori, Perilaku Dan Organisasi. Bandung: PT. Refika Aditama. Listyawardani, Dwi. 2017. Aman Dan Sehat Menggunakan Kontrasepsi. BKKBN J awa barat Listyawardani, Dwi. 2017. Aman Dan Sehat Menggunakan Kontrasepsi. BKKBN J awa barat Makmur. 2015. Efektivitas Kebijakan Kelembagaan Pengawasan. Bandung: PT. Refika Aditama Moleong, Lexy J. 2011. Metode Penelitian Kualitatif. Bandung: PT. RemajaRosda Karya. Nashar. 2020. Kualitas Pelayanan Akan Meningkatkan Kepercayaan Masyarakat. pamekasan: Duta Media Publishing Sulistiyawati, Ari. 2011. Pelayanan Keluarga Berencana. Jakarta: Salemba Medika. Tangkilisan, Hessel.2005. Manajemen Publik. Jakarta: PT Grasindo Ulum, Ihyaul. 2012. Audit Sektor Publik. Jakarta: Bumi Aksara. W dh i Hiti 2016 P i K li K h t R d k i BKKB P t Duta Media Publishing Sulistiyawati, Ari. 2011. Pelayanan Keluarga Berencana. Jakarta: Salemba M Tangkilisan, Hessel.2005. Manajemen Publik. Jakarta: PT Grasindo Ulum, y , y g J Tangkilisan, Hessel.2005. Manajemen Publik. Jakarta: PT Grasindo Ulum, g j Ihyaul. 2012. Audit Sektor Publik. Jakarta: Bumi Aksara. Wardhani, Hitima. 2016. Promosi Konseling Kesehatan Reproduksi.BKKB Pusat Wardhani, Hitima. 2016. Promosi Konseling Kesehatan Rep 5. KESIMPULAN Berdasar atas hasil Penelitian beserta pembahasan mengenai Efektivitas Program Keluarga Berencana Dalam Penggunaan Alat Kontrasepsi di UPTD dinas Pengendalian Penduduk dan Keluarga Berencana (DPPKB) Kecamatan Caringin Kabupaten Sukabumi sebagai berikut: 1. Output Program Keluarga Berencana Dalam Penggunaan Alat Kontrasepsi di UPTD DinasiPengendalian Penduduk dan KeluargaiBerencana (DPPKB) Kecamatan Caringin Kabupaten Sukabumi CukupiBaik. Hal ini dapat diketahui dari pelaksanaan program sudah dilakukan sesuai dengan ketentuan yang sudah ditetapkan. Pelayanan yang diberikan kepada masyarakat bahwa Program Keluarga Berencana bertujuan untuk membentuk keluarga kecil sesuai dengan sosial ekonomi suatu keluarga, dengan cara pengaturan kelahiran anak agar diperoleh suatu keluarga bahagia dan sejahtera yang dapat memenuhi kebutuhan hidupnya. Serta didukung dengan fakta dalam pelayanan sudah dibentuknya Sub-sub KB yaitu puskesmas, bidan ataupun kader di setiap desanya supaya memudahkan masyarakat dalam mengikuti pelaksanaan program Penggunaan Alat Kontrasepsi sesuai dengan arahan dan binaan yang petugas berikan serta didukung dengan fakta bahwa sudah banyaknya masyarakat yang mengikuti program Alat Kontrasepsi ini hingga 87%. 2. Kebijakan dalam Pelaksanaan Keluarga Berencana cukup baik dilihat dari proses dan Sosialisasi yang telah diberikan oleh UPTD DPPKB Kecamatan Caringin Kabupaten Sukabumi kepada masyarakat agar masyarakat lebih paham dengan dilaksanakannya Program Penggunaan Alat Kontrasepsi. dan didukung dengan fakta bahwa dalam proses pelaksanaan program Penggunaan Alat Kontrasepsi selalu membantu dan mengarahkan masyarakat dalam proses mengikuti program Penggunaan Alat Kontrasepsi sehingga bisa memudahkan masyarakat. 2. 3. Prosedur pelaksanaan program di dalam menggunakan alat kontrasepsi di UPTD DinasiiPengendalian Penduduk dan KeluargaiiBerencana (DPPKB) Kecamatan Caringin Kabupaten Sukabumi sudah berjalan dengan cukup baik yaitu dilihat 3. Prosedur pelaksanaan program di dalam menggunakan alat kontrasepsi di UPTD DinasiiPengendalian Penduduk dan KeluargaiiBerencana (DPPKB) Kecamatan Caringin Kabupaten Sukabumi sudah berjalan dengan cukup baik yaitu dilihat 222 dari arahan dan komunikasi yang diberikan Kepala UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana (DPPKB) Kecamatan Caringin Kabupaten Sukabumi kepada bawahannya yaitu Petugas Lapangan Keluarga Berencana (PLKB), dan Bidan agar melaksanakan tugas sesuai dengan prosedur yang sudah ditetapkan untuk mencapai target pada sasaran yang tidak menggunaan Alat Kontrasepsi. Serta didukung dengan fakta bahwa dalam komunikasi yang baik satu sama lain dalam pelaksanaan program Penggunaan Alat Kontrasepsi di UPTD Dinas Pengendalian Penduduk dan Keluarga Berencana dapat terealisasinya. Sumber Hukum Undang-Undang Nomer 10 Tahun 1992 Tentang Perkembangan Kependudukan dan Pembangunan Keluarga Sejahtera. Undang-Undang Nomer 10 Tahun 1992 Tentang Perkembangan Kependudukan dan Pembangunan Keluarga Sejahtera. Undang-Undang Nomer 52 tahun 2009 tentang Perkembangan kependudukan dan pembangunan keluarga. Peraturan Pemerintah Nomer 87 tahun 2014 tentang kesehatan Reproduksi, menetapakan bahwa untuk menjamin pemenuhan hak-hak reproduksi setiap orang diperoleh melalui pelayanan kesehatan yang bermutu, aman dan dapat dipertanggung jawabkan. Peraturan Pemerintah Nomer 87 tahun 2014 tentang kesehatan Reproduksi, menetapakan bahwa untuk menjamin pemenuhan hak-hak reproduksi setiap orang diperoleh melalui pelayanan kesehatan yang bermutu, aman dan dapat dipertanggung jawabkan. 223 224
https://openalex.org/W2758091987	http://real.mtak.hu/166115/1/1701.06940.pdf	English	null	Search for new phenomena with multiple charged leptons in proton–proton collisions at $$\sqrt{s}= 13$$ s = 13 $$\,\text {TeV}$$ TeV	European physical journal. C, Particles and fields	2,017	cc-by	24,251	UROPEAN ORGANIZATION FOR NUCLEAR RESEARCH (CERN CMS-SUS-16-003 Search for new phenomena with multiple charged leptons in proton-proton collisions at √s = 13 TeV v:1701.06940v2 [hep-ex] 25 Sep 2017 The CMS Collaboration∗ arXiv:1701.06940v2 [hep-ex] 25 Sep Abstract arXiv:1701.06940v2 [hep-ex] Results are reported from a search for physics beyond the standard model in ﬁnal states with at least three charged leptons, in any combination of electrons or muons. The data sample corresponds to an integrated luminosity of 2.3 fb−1 of proton-proton collisions at √s = 13 TeV, recorded by the CMS experiment at the LHC in 2015. Two jets are required in each event, providing good sensitivity to strong production of gluinos and squarks. The search regions, sensitive to a range of different new physics scenarios, are deﬁned using the number of jets tagged as originating from bottom quarks, the sum of the magnitudes of the transverse momenta of the jets, the im- balance in the overall transverse momentum in the event, and the invariant mass of opposite-sign, same-ﬂavor lepton pairs. The event yields observed in data are con- sistent with the expected background contributions from standard model processes. These results are used to derive limits in terms of R-parity conserving simpliﬁed mod- els of supersymmetry that describe strong production of gluinos and squarks. Model- independent limits are presented to facilitate the reinterpretation of the results in a broad range of scenarios for physics beyond the standard model. lished in the European Physical Journal C as doi:10.1140/epjc/s10052-017-5182-1 c⃝2017 CERN for the beneﬁt of the CMS Collaboration. CC-BY-3.0 license 1 Introduction Many types of beyond-the-standard-model (BSM) theories can produce multilepton events (three or more leptons) with a wide array of unique signatures [1–5], including a number of supersymmetric (SUSY) models [6–15]. In these models, multilepton ﬁnal states can arise from the decay of multiple vector bosons, e.g., in tt production with t →cH followed by H →WW∗ or H →ZZ∗, or in strong production of pairs of squarks or gluinos, which often initiate com- plex decay chains that can result in multiple W and/or Z bosons. The standard model (SM) processes that produce this ﬁnal state are also characterized by multiple bosons and are well- understood both theoretically [16–30] and experimentally [31–35]. This paper describes a search for new physics in ﬁnal states with three or more leptons, elec- trons or muons, produced at the CERN LHC, in proton-proton (pp) collisions at a center-of- mass energy of 13 TeV, with the CMS detector. The data correspond to an integrated luminosity of 2.3 fb−1 collected in 2015. The expected irreducible backgrounds come from diboson pro- duction (WZ and ZZ) or other SM processes, including ttW, ttZ, and ttH. These backgrounds are modeled using Monte Carlo (MC) simulations that have appropriate corrections applied to match the behavior of reconstructed objects in data. Reducible backgrounds are processes that produce one or more misidentiﬁed or nonprompt leptons, i.e. those that arise from jets or meson decays, that pass all reconstruction, identiﬁcation, and isolation criteria. Estimates of the probabilities of observing misidentiﬁed or nonprompt leptons based on control samples in data are used. As an example of the type of BSM models for which this search has sensitivity, we interpret the results of this analysis in the context of SUSY models that feature strong production of pairs of squarks (eq) or gluinos (eg). In addition to multiple leptons, these models predict that events can contain multiple jets, b-tagged jets, and missing transverse momentum. Searches probing similar models have been carried out by the ATLAS and CMS Collaborations using pp collisions at 8 TeV [36–44], and at 13 TeV [45–52]. Previous searches exclude models with gluino mass less than approximately 1500 GeV, for a neutralino mass of 50 GeV, and models with bottom squark mass less than 830 GeV. The result of the search, which is consistent with SM expectation, can also be used to constrain other BSM models not explicitly considered in this paper. 1 Introduction To this end, we also provide upper limits on possible BSM contributions in the kinematic tail of the search variables in terms of the product of cross section, detector acceptance, and selection efﬁciency. c⃝2017 CERN for the beneﬁt of the CMS Collaboration. CC-BY-3.0 license ∗See Appendix A for the list of collaboration members 1 3 Event selection and Monte Carlo simulation Events used in this analysis are selected by the triggers that collect dilepton and multilepton events for later study, using variables constructed by the HLT. One set of triggers requires two leptons satisfying loose isolation criteria and transverse momentum pT > 17 GeV for the leading lepton and pT > 12 (8) GeV for the subleading lepton in the case of electrons (muons). The second set of triggers places no requirements on the isolation, has a lower pT threshold for the two leptons, pT > 8 GeV, and also requires that the scalar sum of jets with pT > 40 GeV reconstructed in the HLT be greater than 300 GeV. Electron candidates are reconstructed using tracking and electromagnetic calorimeter informa- tion by combining Gaussian sum ﬁlter tracks and ECAL energy deposits [54]. The electron identiﬁcation is performed using a multivariate discriminant built with shower shape, track cluster matching, and track quality variables. The working point for the selection is chosen to maintain approximately 90% efﬁciency for accepting electrons produced in the decays of W and Z bosons and also to efﬁciently reject candidates originating from jets. To reject elec- trons originating from photon conversions, electrons are required to have hits in all possible inner layers of the tracker and to be incompatible with any secondary vertices containing only another electron. The selected electron candidates must have \|η\| < 2.5. Muon candidates are reconstructed in a global ﬁt to the combined information from both the silicon tracker and the muon spectrometer [55]. An identiﬁcation is performed using the qual- ity of the geometrical matching between measurements in the tracker and the muon system. To ensure the candidates are within the ﬁducial volume of the detector, we require that the candidate pseudorapidities satisfy \|η\| < 2.4. The reconstructed vertex with the largest value of summed physics-object p2 T is taken to be the primary pp interaction vertex. The physics objects are the objects returned by a jet ﬁnding algo- rithm [56, 57] applied to all charged tracks associated with the vertex, plus the corresponding associated missing transverse momentum. Both electron and muon candidates are required to have a transverse (longitudinal) impact parameter of less than 0.5 (1.0) mm from the primary vertex. In addition, a requirement on the three-dimensional impact parameter signiﬁcance is applied. 2 The CMS detector The CMS detector features a superconducting solenoid of 6 m internal diameter that creates a magnetic ﬁeld of 3.8 T. Inside the magnet volume are a silicon pixel and strip tracker, an electromagnetic calorimeter (ECAL) made of lead tungstate crystals, and a hadron calorimeter (HCAL) made of brass and scintillator, each composed of a barrel and two endcap sections. Forward calorimeters provide additional pseudorapidity (η) coverage for the HCAL. Muons are detected in gas-ionization chambers embedded in the steel ﬂux-return yoke outside the solenoid. The ﬁrst level of the CMS trigger system, composed of specialized hardware proces- sors, uses information from the calorimeters and muon detectors to select the most interesting events in a ﬁxed time interval of less than 4 µs. The high-level trigger (HLT) processor farm further decreases the event rate from approximately 100 kHz to less than 1 kHz, before data storage. A more detailed description of the CMS detector, together with a deﬁnition of the coordinate system used and the relevant kinematic variables, can be found in Ref. [53]. 3 Event selection and Monte Carlo simulation 2 3 Event selection and Monte Carlo simulation This variable is the value of the impact parameter divided by its uncertainty and is required to be less than 4 for both electrons and muons. The rejection of nonprompt leptons is more efﬁcient using the impact parameter signiﬁcance than the value of impact parameter for similar prompt-lepton acceptance. Lepton isolation is constructed using three different variables. The mini isolation, Imini, is the ratio of the amount of measured energy in a cone to the transverse momentum of the lepton. The radius is pT-dependent: Riso = 10 GeV/min(max(pT(ℓ), 50 GeV), 200 GeV), resulting in radii between 0.05 and 0.2. Requiring Imini to be below a given threshold ensures that the lepton is locally isolated, even in Lorentz-boosted topologies. The second variable is the ratio of the lepton pT and the pT of the jet matched to the lepton: pratio T = pT(ℓ)/pT(jet). This jet must be separated by no more than 0.4 in ∆R from the lepton it is matched to, where ∆R = √ ∆φ2 + ∆η2. In most cases, this is the jet containing the lepton. If no jet is found within ∆R < 0.4, then pratio T = 1. The use of pratio T is a simple way to identify nonprompt low-pT leptons originating from low-pT b-quarks that decay with larger opening angles than the one used in the mini isolation. The last variable is prel T , which is calculated by subtracting the lepton momentum from the momentum vector of the geometrically matched jet described above and then ﬁnding the com- ponent of the lepton momentum that is transverse to this new vector. If there is no matched jet, 3 prel T = 0. This variable allows us to recover leptons from accidental overlap with jets in events where some of the ﬁnal state particles are close together in Lorentz-boosted topologies. prel T = 0. This variable allows us to recover leptons from accidental overlap with jets in events where some of the ﬁnal state particles are close together in Lorentz-boosted topologies. Using the three variables above, a lepton is considered isolated if Imini < I1 and that either pratio T > I2 or prel T > I3. The Ii values depend on the ﬂavor of the lepton. The probability to misidentify a jet is higher for electrons, so tighter isolation values are used. 3 Event selection and Monte Carlo simulation The logic behind this isolation is that a lepton should be locally isolated (Imini) and should carry the major part of the energy of the corresponding jet (pratio T ) unless its overlap with the jet is accidental (prel T ). For electrons (muons), the tight selection requirements are I1 = 0.12 (0.16), I2 = 0.76 (0.69), and I3 = 7.2 (6.0) GeV. The loose lepton isolation is relaxed to Imini < 0.4, and the other re- quirements are dropped. The loose leptons are used for background estimates. These selection requirements were optimized using MC simulations. The ofﬂine selection requires at least three well-identiﬁed leptons in the event and any pair of opposite sign and same ﬂavor (OSSF) leptons having an invariant mass greater than 12 GeV to reject low mass Drell–Yan and quarkonium processes. The leptons must pass ofﬂine pT thresholds of 20, 15, and 10 GeV for the ﬁrst, second, and third lepton, respectively, when pT- ordered. For this ofﬂine selection, the trigger efﬁciency is above 99%. Jets are reconstructed from particle-ﬂow candidates [58] clustered using the anti-kT algorithm [56] with a distance parameter of 0.4 as implemented in the FASTJET package [57]. Only jets with pT > 30 GeV and within the tracker acceptance \|η\| < 2.4 are considered. Additional criteria are applied to reject events containing noise and mismeasured jets [59–61]. To avoid double count- ing, the closest matching jets to leptons are not considered if they are separated from the lepton by less than 0.4 in ∆R. From those selected jets, the quantity HT is deﬁned by HT = ∑jets \|⃗pT\|, for all jets that satisfy the above-mentioned criteria. Jet energies are corrected for a shift in the energy scale, contributions from additional, simultaneous pp collisions (pileup), and residual nonuniformity and nonlinearity differences between data and simulation [60]. The combined secondary vertex algorithm [62, 63] is used to assess the likelihood that a jet orig- inates from a bottom quark (“b jet”). Jets in this analysis are considered to be b tagged if they pass the algorithm’s medium working point, which has a tagging efﬁciency of approximately 70% and a mistag rate of approximately 1% for light quarks and gluons. The missing transverse momentum ⃗pmiss T is deﬁned as the negative vector sum of transverse momenta of all particle-ﬂow candidates reconstructed in the event. Its magnitude is referred to as pmiss T . 3 Event selection and Monte Carlo simulation Jet energy corrections are propagated to the pmiss T following the procedure described in Ref. [64]. To estimate the contribution of SM processes to the signal regions (described in Section 4) and to calculate the efﬁciency for new physics models, MC simulations are used. All the SM sam- ples are generated using the MADGRAPH5 aMC@NLO 2.2.2 [65–67] program at leading order (LO) or next-to-leading order (NLO) in perturbative QCD, with the exception of the diboson production samples (WZ and ZZ) that are generated using POWHEG v2 [68–72] at NLO pre- cision. The NNPDF3.0 [73] LO (NLO) parton distribution function (PDF) set is used in MC simulations generated at LO (NLO). Parton showering and hadronization are simulated using PYTHIA 8.205 [74] with the underlying event tune CUETP8M1 [75]. The CMS detector response is determined using a GEANT4-based model [76]. Events corresponding to the production of SUSY processes are generated with MADGRAPH5 aMC@NLO at LO precision, allowing up to two additional partons in the matrix element calculations. The SUSY particle decays, parton showering, and hadronization are simulated with PYTHIA. The detector response for signal events is simulated using a CMS fast-simulation package [77] that 4 Search strategy 4 P1 P2 ˜g ˜g ¯t t eχ0 1 eχ0 1 ¯t t P1 P2 ˜g ˜g eχ0 2 eχ± q ¯q′ W±(∗) eχ0 1 eχ0 1 Z(∗) ¯q q P1 P2 eb1 eb1 eχ+ 1 eχ− 1 ¯t W+ eχ0 1 eχ0 1 W− t Figure 1: Diagrams for gluino and bottom squark pair production leading to multilepton events for simpliﬁed models of supersymmetry: (left) T1tttt, (middle) T5qqqqWZ, and (right) T6ttWW. P1 P2 ˜g ˜g eχ0 2 eχ± q ¯q′ W±(∗) eχ0 1 eχ0 1 Z(∗) ¯q q q ¯q′ q q Figure 1: Diagrams for gluino and bottom squark pair production leading to multilepton events for simpliﬁed models of supersymmetry: (left) T1tttt, (middle) T5qqqqWZ, and (right) T6ttWW. is validated against the GEANT4-based model. Cross sections for SUSY signal processes, cal- culated at NLO with next-to-leading-log (NLL) gluon resummation, are taken from the LHC SUSY Cross Section Working Group [78–83]. All simulated events are processed with the same reconstruction procedure as data. They include the effects of additional interactions, which can occur in the same or adjacent beam crossings (pileup). The distribution of additional interac- tions is matched to that observed in data. 4 Search strategy The goal of this analysis is to search for possible excesses over the expected yields from SM processes in different categories of events with three or more leptons. With the 2.3 fb−1 data sample at √s = 13 TeV, the search is focused on strongly produced SUSY particles, which beneﬁt most from the increase of the production cross section with respect to 8 TeV. A few examples of diagrams of simpliﬁed models of SUSY processes [84, 85] that can give rise to multilepton ﬁnal states are shown in Fig. 1. In these models, SUSY particles that are not directly included in the diagrams are assumed to be too heavy to be accessible at the LHC. Therefore, the free parameters in these models are usually the mass of the produced particles: gluinos and squarks, as well as the mass of the lightest supersymmetric particle (LSP). Typical SUSY processes relevant for this work include T1tttt, which corresponds to gluino pair production where each gluino decays to a tt pair and the LSP (Fig. 1-left). Another model, referred to as T5qqqqWZ, involves gluino pair production, where each gluino decays to a pair of light quarks (u, d, s, and c) and a neutralino (eχ0 2) or chargino (eχ± 1 ), followed by decay of the neutralino or the chargino to a W or Z boson, respectively, and the LSP (Fig. 1-middle). The probability for the decay to proceed via eχ+ 1 , eχ− 1 , or eχ0 2 is 1/3 for each case, leading to the probabilities of having WW, ZZ or WZ bosons in the ﬁnal state to be about 44.5%, 11.1%, and 44.5%, respectively. Only the ﬁnal state with WZ bosons contributes signiﬁcantly to the acceptance of this search. Final states with WW bosons do not contribute, and the contribution from ZZ ﬁnal states decaying to four leptons is negligibly small. In this scenario the neutralino and chargino are assumed to be mass-degenerate. A model called T6ttWW, features bottom squark pair production with their subsequent cascade decays via top quarks and W bosons (Fig. 1-right). The LSP is a neutralino in all of these models. For the deﬁnition of the signal regions (SRs) we use several event variables: the number of b-tagged jets (Nb), HT, pmiss T , and a classiﬁcation depending on whether the event contains any OSSF dilepton pairs with an invariant mass between 76 and 106 GeV, i.e. 3 Event selection and Monte Carlo simulation The pileup interactions are simulated by overlaying the primary interaction with additional minimum bias events, which are generated with the same PYTHIA conﬁguration as described above. 4 Search strategy consistent with the Z boson (called “on-Z” if so and “off-Z” otherwise in the following). Events that do not contain 5 Table 1: Deﬁnition of multilepton signal regions. These regions are the same for the on-Z and off-Z regions. Table 1: Deﬁnition of multilepton signal regions. These regions are the same for the on-Z and off-Z regions. off-Z regions. Nj Nb pmiss T (GeV) 60 ≤HT < 400 GeV 400 ≤HT < 600 GeV HT ≥600 GeV ≥2 0 50–150 SR 1 SR 3 SR 14 150–300 SR 2 SR 4 1 50–150 SR 5 SR 7 150–300 SR 6 SR 8 2 50–150 SR 9 SR 11 150–300 SR 10 SR 12 ≥3 50–300 SR 13 ≥0 ≥300 SR 15 any OSSF pairs are included in the off-Z sample. any OSSF pairs are included in the off-Z sample. ny OSSF pairs are included in the off-Z sample. The separation in b-tagged jet multiplicities maximizes signal-to-background ratios for dif- ferent signal models. For example, the T1tttt model features several b jets, which would be categorized into SRs which are almost free of WZ background owing to the b-tagged jet re- quirement. Including the zero b-tagged SRs keeps the analysis sensitive to signatures such as the T5qqqqWZ model. Additionally, a categorization in HT and pmiss T is useful to distinguish between compressed and noncompressed SUSY spectra, i.e. models with small or large mass differences between the SUSY particles in the decay chain. A baseline selection is applied to the data set to select events of interest: three or more electrons or muons satisfying the requirements pT ≥20, 15, and 10 GeV; mℓℓ≥12 GeV; at least two jets; HT ≥60 GeV; and pmiss T ≥50 GeV. Events containing additional leptons with pT > 10 GeV are included in the event selection. Table 1 shows the deﬁnition of the subdivision of the baseline selection into two sets of SRs for events that contain on-Z and off-Z dilepton pairs. There are 15 SRs for each of the two groups, hence in total 30 SRs. A set of four SRs with low or medium HT and low or medium pmiss T are deﬁned for each of the b-tagged jet multiplicities 0, 1, and 2. 4 Search strategy Motivated by the low expected yield of events with Nb ≥3, SR 13 is deﬁned for high b-tagged jet multiplicities and also has pmiss T < 300 GeV and HT < 600 GeV. Two additional SRs with large HT (SR 14) and large pmiss T (SR 15), respectively, have been deﬁned as nearly background-free SRs, since noncompressed SUSY models can yield events with very large values of pmiss T or HT. Both of these SRs are inclusive in the number of b-tagged jets, and every selected event with pmiss T ≥300 GeV is categorized in SR 15, while SR 14 is populated with events with pmiss T < 300 GeV and HT ≥600 GeV. 5 Background estimation Backgrounds in the multilepton ﬁnal states can be divided in three categories: 1. Nonprompt or misidentiﬁed leptons are those arising from heavy-ﬂavor decays, misiden- tiﬁed hadrons, electrons from unidentiﬁed photon conversions, or muons from light- meson decays in ﬂight. For this analysis, tt events can enter the SRs if nonprompt leptons are present in addition to the prompt leptons from the W boson decays. These non- prompt leptons typically originate from semileptonic decays of hadrons containing a b quark, which, in this case, is not reconstructed as a jet. Therefore, tt events typically have low HT and pmiss T and predominately populate SR 1 and SR 5, with 0 and 1 b-tagged jets, respectively. 5 Background estimation 6 In addition to tt, Drell–Yan events can enter the baseline selection, although they are largely suppressed by the pmiss T > 50 GeV requirement. Processes that yield only one prompt lepton, e.g. W+jets and single top quark production, are effectively suppressed by the three-lepton requirement because of the low probability that the two nonprompt leptons pass the tight identiﬁcation and isolation requirements. In addition to tt, Drell–Yan events can enter the baseline selection, although they are largely suppressed by the pmiss T > 50 GeV requirement. Processes that yield only one prompt lepton, e.g. W+jets and single top quark production, are effectively suppressed by the three-lepton requirement because of the low probability that the two nonprompt leptons pass the tight identiﬁcation and isolation requirements. In addition to tt, Drell–Yan events can enter the baseline selection, although they are largely suppressed by the pmiss T > 50 GeV requirement. Processes that yield only one prompt lepton, e.g. W+jets and single top quark production, are effectively suppressed by the three-lepton requirement because of the low probability that the two nonprompt leptons pass the tight identiﬁcation and isolation requirements. 2. Diboson production could yield multilepton ﬁnal states with up to three prompt leptons in WZ production and up to four prompt leptons in ZZ production. Especially in signal regions without b-tagged jets, WZ production has a sizable contribution. The normal- ization of this background is obtained from a dedicated control region enriched in WZ events. 3. Other SM processes that can yield three or more leptons are ttW, ttZ, and triboson pro- duction VVV where V stands for a W or Z boson. 5 Background estimation We also include the contribution from the SM Higgs boson produced in association with a vector boson or a pair of top quarks in this category of backgrounds. Processes that produce additional leptons from internal conversions, which are events that contain a virtual photon that decays to leptons, are also included here as X+γ, where X is predominantly tt or Z. Those backgrounds are obtained from simulation and appropriate systematic uncertainties are assigned. The background contribution from nonprompt and misidentiﬁed leptons is estimated using the “tight-to-loose ratio” method [52]. The tight-to-loose ratio f is the probability for a nonprompt lepton that satisﬁes the loose requirements to also satisfy the full set of requirements. The nonprompt background contribution is obtained from the number of events in an application region containing events with at least one of the leptons failing the full set of tight identiﬁca- tion and isolation requirements, but passing the loose requirements, weighted by f /(1 −f ). This ratio is measured in a control sample of QCD multijet events that is enriched in non- prompt leptons (measurement region), by requiring exactly one lepton passing the loose ob- ject selection and one recoiling jet with ∆R(jet, ℓ) > 1.0. To suppress events with leptons from W and Z boson decays, pmiss T < 20 GeV and MT < 20 GeV are also required, where MT = √ 2pmiss T pT(ℓ)(1 −cos ∆φ) and ∆φ is the difference in azimuthal angle between the lepton and ⃗pmiss T . The remaining contribution from these electroweak processes within the measure- ment region is subtracted using estimates from MC simulations. The background contribution from nonprompt and misidentiﬁed leptons is estimated using the “tight-to-loose ratio” method [52]. The tight-to-loose ratio f is the probability for a nonprompt lepton that satisﬁes the loose requirements to also satisfy the full set of requirements. The nonprompt background contribution is obtained from the number of events in an application region containing events with at least one of the leptons failing the full set of tight identiﬁca- tion and isolation requirements, but passing the loose requirements, weighted by f /(1 −f ). This ratio is measured in a control sample of QCD multijet events that is enriched in non- prompt leptons (measurement region), by requiring exactly one lepton passing the loose ob- ject selection and one recoiling jet with ∆R(jet, ℓ) > 1.0. 5 Background estimation To suppress events with leptons from W and Z boson decays, pmiss T < 20 GeV and MT < 20 GeV are also required, where MT = √ 2pmiss T pT(ℓ)(1 −cos ∆φ) and ∆φ is the difference in azimuthal angle between the lepton and ⃗pmiss T . The remaining contribution from these electroweak processes within the measure- ment region is subtracted using estimates from MC simulations. The dependence of the tight-to-loose ratio on the ﬂavor of the jet from which the nonprompt lepton originates is reduced by parameterizing the ratio as a function of a variable that is more strongly correlated with the parent parton pT than with lepton pT. This variable is calculated by correcting the lepton pT as a function of the energy in the isolation cone around it. This def- inition leaves the pT of the leptons passing the isolation requirement unchanged and modiﬁes the pT of those failing the requirement, so that it is a better proxy for the parent parton pT and results in a ﬂatter tight-to-loose ratio as a function of the parent parton pT. The cone correction signiﬁcantly improves the results of the method when applying it in simulation. The ﬂavor de- pendence, which is much more important for the case of electrons, is also reduced by adjusting the loose object selection to obtain similar ratios for nonprompt electrons that originate from both light- and heavy-ﬂavor jets. To avoid experimental biases, the tight-to-loose ratio is also measured as a function of η. The tight-to-loose ratio method of estimating the nonprompt background is validated in a con- trol region exclusive to our baseline selection with minimal signal contamination. This region is deﬁned by having three tight leptons, one or two jets, 20 < pmiss T < 50 GeV, and an off-Z dilepton pair. We ﬁnd agreement of the order of 20% between the predicted and observed 7 yields in this control region in data, which validates the predictions and uncertainties of this method. The WZ process is one of the main backgrounds in the regions with zero b-tagged jets. 6 Systematic uncertainties Systematic uncertainties are characterized as either experimental, theoretical, or arising from the limited size of simulated event samples. These sources of uncertainties and their magni- tudes are described below, and are summarized in Table 2. The table also provides the effect of varying the uncertainties by ±1 standard deviation (s.d.) on the signal and background yields. The jet energy scale uncertainty and the uncertainty in the b tagging efﬁciency are the only ones that can cause simulated events to migrate between signal regions. The major experimental source of uncertainty is the knowledge of the jet energy scale (JES), which accounts for differences between kinematical variables from data and simulation and affects signal and background events that are taken from simulation samples [60, 61]. For the data set used in this analysis, the uncertainties on the JES vary from 2 to 8%, depending on the pT and η of the jet. The impact of these uncertainties is assessed by shifting the jet energy correction factors for each jet up and down by ±1 s.d. and recalculating all of the kinematic quantities. The JES uncertainties are propagated to the missing transverse momentum and all variables derived from jets (numbers of jets and b-tagged jets, and HT) used in this analysis; this propagation results in 1–20% variation in the MC background estimation in the regions with higher data yields. A similar approach is used for the uncertainties associated with the corrections for the b tagging efﬁciencies for light-, charm-, and bottom-ﬂavour jets, which are parametrized as a function of pT and η [62, 63]. The variation of the scale factor correcting for the differences between data and simulation is at maximum 5–10%, and leads to an effect of 1–20% on the yields, depending on the SR and on the topology of the events under study. If one considers only highly populated SRs to get an overview of the main effects on the background yields, the bulk of the ttW yield varies by ∼10% and the WZ yield by ∼13%. Lepton identiﬁcation scale factors have been measured by comparing efﬁciencies in data and simulation using the “tag-and-probe” method [54, 55] and are applied as a function of lepton pT and η. The corresponding uncertainties on the scale factors have been evaluated and are approximately 2% for both electrons and muons. Trigger efﬁciency scale factors have been found to be very close to unity. 5 Background estimation The relative contribution of this process in various SRs is estimated from the MC simulation at NLO, but the normalization is taken from a control region that is highly enriched for this process: three leptons pass nominal identiﬁcation and isolation requirements, two leptons form an OSSF pair with \|mℓℓ−mZ\| < 15 GeV, the number of jets is zero or one, the number of b-tagged jets is zero, 30 < pmiss T < 100 GeV, and the MT of the third lepton (not in the pair forming the Z) is required to be at least 50 GeV to suppress contamination from Drell–Yan processes. The expected WZ purity in the selected sample is 84%. Using this control region, we ﬁnd that the WZ background predictions from simulation are consistent with data. The ratio between the prediction and data obtained with 2.3 fb−1 of data is 1.13 ± 0.17. The uncertainty on the normalization of the WZ background includes the statistical uncertainty related to the event yield in the CR and a systematic component related to a small contamination of the CR due to other processes. large number of samples. large number of samples. All these uncertainties related to corrections of the simulation (JES corrections, b tagging ef- ﬁciency scale factors, lepton identiﬁcation and trigger scale factors) have been estimated also for the fast simulation used for the signal samples. We propagate them to the expected signal yields following the same procedure. The uncertainties in the renormalization (µR) and factorization scales (µF) and the PDF are considered for some of the rare processes, namely ttW, ttZ, and ttH. Both the changes in the acceptance and cross sections due to those effects are taken into account. For the study of the renormalization and factorization scale uncertainties, variations up and down by a factor of two with respect to the nominal values of µR and µF are considered. The maximum difference in the yields with respect to the nominal case is observed when both scales are varied simultaneously up and down. The effect on the overall cross section is found to be about 13% for ttW and about 11% for ttZ. An additional uncertainty in the acceptance corresponding to different signal regions is included. This is found to be between 3 and 18% depending on the SR and process. The uncertainty related to the PDFs is estimated from the 100 NNPDF 3.0 replicas by computing the deviation with respect to the nominal yields for each of them, and for each signal region (the cross section and acceptance effects are considered together) [86]. The root mean square of the variations is taken as the value of the systematic uncertainty. Since no signiﬁcant variations among the different signal regions are seen, a ﬂat uncertainty of 3(2)% is applied to the ttW (ttZ) background. This value also includes the deviation resulting from varying the strong coupling strength αS(MZ), which is added in quadrature, and whose magnitude is similar to or smaller than that of the PDF set uncertainty. For the ttH process, the same uncertainties as estimated for ttZ are applied. A theoretical uncertainty of 50% is assigned to the remaining rare processes. In signal samples, the uncertainty due to initial-state radiation is computed as a function of the pT of the gluino pair using the methods described in Ref. [87]. For values below 400 GeV, no uncertainty is applied. For values between 400 and 600 GeV, a 15% uncertainty is assigned, and above 600 GeV this uncertainty is increased to 30%. 6 Systematic uncertainties An uncertainty of 3% in the scale factors has, however, been assigned to cover the difference between trigger efﬁciencies measured in simulation over a 6 Systematic uncertainties 8 large number of samples. The limited size of the generated MC samples represents an additional source of uncertainty. The uncertainty in signal processes and backgrounds such as ttW, ttZ, and ttH, is calculated from the number of MC events entering each of the signal regions. For the nonprompt and misidentiﬁed lepton background, we assign several systematic uncer- tainties. The statistical uncertainty resulting from the limited number of events in the applica- tion region used to estimate this background contribution varies from 1 to 100%. The regions where these uncertainties are large are generally regions where the overall contribution of this background is small. When no events are observed in the application region, the upper limit of the background expectation is set to 0.35, which is found by applying the most probable tight-to-loose ratio as if the application region contained an event count equal to the variance of a Poisson distribution with a mean of zero. The systematic uncertainties related to the extrapolation from the control regions to the SRs for the nonprompt lepton background are estimated to be 30%. This magnitude has been extracted from the level of closure achieved in a test that was performed with MC samples yielding nonprompt leptons to validate background predictions based on control samples in data, as described in Section 5. 9 Table 2: Summary of the sources of uncertainties and their magnitudes. The third column provides the changes in yields of signal and background induced by one s.d. changes in the magnitude of uncertainties. Source Magnitude (%) Effect on yield (%) Integrated luminosity [88] 2.7 2.7 ∗ Limited MC sample sizes 1–100 1–100 ∗ Jet energy scale 2–8 1–20 ∗ b tagging efﬁciency 5–10 1–20 ∗ Pileup 5 3 ∗ Renormalization and factorization scales −50 / +100 11–13 (cross-section) / 3–18 (acceptance) (ttW,ttZ,ttH) PDF — 2–3 (ttW,ttZ,ttH) Other backgrounds 50 50 (rare processes, tribosons, etc.) Lepton efﬁciencies 2 6 ∗ Trigger efﬁciencies 3 3 ∗ FastSim lepton efﬁciencies 3–10 3–10 FastSim signals FastSim trigger efﬁciencies 5 5 FastSim signals Tight-to-loose ratio control region statistical uncertainty 1–100 1–100 (nonprompt bkg. only) Tight-to-loose ratio systematic uncertainty 30 30 (nonprompt bkg. only) EW subtraction in tight-to-loose ratio 100 (ewk. SF) 1–5 (nonprompt bkg. only) WZ control region normalization 15 15 (WZ only) WZ extrapolation 10–30 2–30 (WZ only) ∗Applied to both signal and background simulation samples. large number of samples. The uncertainty associated with the electroweak (EW) background subtraction in the tight- to-loose ratio computation is propagated through the full analysis process by replacing the nominal tight-to-loose ratio with another value obtained when the scale factor applied to the electroweak processes in the measurement region is varied by 100% of its difference from unity. The overall effect on the nonprompt background yield lies between 1 and 5% depending on the SR considered. The estimate of the WZ background is assigned a 15% normalization uncertainty using the measurement in a dedicated control region. This uncertainty is compatible with the one quoted for the experimental measurement of this process in Ref. [33]. Additional uncertainties for the extrapolation from the control region to the signal regions of 10 – 30% are taken into account depending on the SR. These uncertainties are dominated by the JES and b tagging uncertainties described earlier. Finally the uncertainty on the integrated luminosity is 2.7% [88]. 7 Results and interpretations Expected event yields are compared to the observation in Tables 3 and 4. Comparisons of dis- tributions of HT, pmiss T , Nj, Nb, leading lepton pT, subleading lepton pT, and trailing lepton pT measured in data with those predicted by the background estimation methods are shown in Fig. 2 (Fig. 3), using all the events satisfying the off-Z (on-Z) SR selection criteria. The non- prompt lepton background comes from the technique described in Section 5. The hatched band represents the total background uncertainty in each bin. A graphical summary of predicted backgrounds and observed event yields in individual SRs is also shown. In these ﬁgures, the “rare” component is the sum over several SM processes, such as triboson production, associ- ated Higgs production, tttt, and other lower cross section processes. The number of events observed in data is found to be consistent with predicted SM background yields. 7 Results and interpretations The results are used to calculate upper limits on the production cross section of gluinos 10 7 Results and interpretations 100 200 300 400 500 600 Events / 50 GeV 0 2 4 6 8 10 12 14 16 18 20 22 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T H 100 200 300 400 500 600 Data/pred 0 1 2 3 50 100 150 200 250 300 350 400 450 Events / 50 GeV 0 10 20 30 40 50 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) miss T p 100 200 300 400 Data/pred 0 1 2 3 2 3 4 5 6 7 Events 0 10 20 30 40 50 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS jets N 2 4 6 Data/pred 0 1 2 3 0 1 2 3 4 Events 0 5 10 15 20 25 30 35 40 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS bjets N 0 1 2 3 4 Data/pred 0 2 4 6 50 100 150 200 Events / 10 GeV 0 2 4 6 8 10 12 14 16 18 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T Leading lepton p 50 100 150 200 Data/pred 0 1 2 3 20 40 60 80 100 Events / 10 GeV 0 2 4 6 8 10 12 14 16 18 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T Subleading lepton p 20 40 60 80 100 Data/pred 0 1 2 3 20 40 60 80 100 Events / 10 GeV 0 5 10 15 20 25 30 35 40 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T Trailing lepton p 20 40 60 80 100 Data/pred 0 1 2 3 2 4 6 8 10 12 14 Events 0 5 10 15 20 25 30 35 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS Signal region 2 4 6 8 10 12 14 Data/pred 0 1 2 3 Off-Z samples: from left to right, top to bottom, distributions of HT, pmiss T , Nj, s for the predicted backgrounds and for the data in the off-Z baseline selection plots the rightmost bin contains the overﬂow from counts outside the range of th ottom-right corner the total predicted background and the number of events ob off-Z SRs is shown. 7 Results and interpretations Events / 50 GeV Events / 50 GeV Data/pred Events / 10 GeV Data/pred Data/pred Data/pred Figure 2: Off-Z samples: from left to right, top to bottom, distributions of HT, pmiss T , Nj, Nb, pT of leptons for the predicted backgrounds and for the data in the off-Z baseline selection region, in these plots the rightmost bin contains the overﬂow from counts outside the range of the plot. On the bottom-right corner the total predicted background and the number of events observed in the 15 off-Z SRs is shown. 7 Results and interpretations Events / 50 GeV 00 γ + ata GeV) 00 50 100 150 200 250 300 350 400 450 Events / 50 GeV 0 10 20 30 40 50 60 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (GeV) miss T p 100 200 300 400 Data/pred 0 1 2 3 7 γ + ata TeV) jets N 0 1 2 3 4 Events 0 10 20 30 40 50 60 70 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS bjets N 0 1 2 3 4 Data/pred 0 1 2 3 200 γ + ata TeV) GeV) 200 20 40 60 80 100 Events / 10 GeV 0 2 4 6 8 10 12 14 16 18 20 22 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T Subleading lepton p 20 40 60 80 100 Data/pred 0 1 2 3 100 γ + ata TeV) GeV) 100 2 4 6 8 10 12 14 Events 0 10 20 30 40 50 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS Signal region 2 4 6 8 10 12 14 Data/pred 0 1 2 3 op to bottom, distributions of HT, pmiss T , Nj, Nb, pT d for the data in the on-Z baseline selection region, overﬂow from counts outside the range of the plot. ed background and the number of events observed Events Data/pred Events / 10 GeV Data/pred 20 40 60 80 100 Events / 10 G 0 5 10 15 20 25 30 WZ Rare Uncertainties Data (GeV) T Trailing lepton p 20 40 60 80 100 Data/pred 0 1 2 3 2 4 6 8 10 12 14 Ev 0 10 20 30 40 50 WZ Rare Uncertainties Data Signal region 2 4 6 8 10 12 14 Data/pred 0 1 2 3 Data/pred Figure 3: On-Z samples: from left to right, top to bottom, distributions of HT, pmiss T , Nj, Nb, pT of leptons for the predicted backgrounds and for the data in the on-Z baseline selection region, in these plots the rightmost bin contains the overﬂow from counts outside the range of the plot. 7 Results and interpretations 11 100 200 300 400 500 600 Events / 50 GeV 0 2 4 6 8 10 12 14 16 18 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T H 100 200 300 400 500 600 Data/pred 0 1 2 3 50 100 150 200 250 300 350 400 450 Events / 50 GeV 0 10 20 30 40 50 60 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) miss T p 100 200 300 400 Data/pred 0 1 2 3 2 3 4 5 6 7 Events 0 10 20 30 40 50 60 70 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS jets N 2 4 6 Data/pred 0 1 2 3 0 1 2 3 4 Events 0 10 20 30 40 50 60 70 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS bjets N 0 1 2 3 4 Data/pred 0 1 2 3 50 100 150 200 Events / 10 GeV 0 2 4 6 8 10 12 14 16 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T Leading lepton p 50 100 150 200 Data/pred 0 1 2 3 20 40 60 80 100 Events / 10 GeV 0 2 4 6 8 10 12 14 16 18 20 22 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T Subleading lepton p 20 40 60 80 100 Data/pred 0 1 2 3 20 40 60 80 100 Events / 10 GeV 0 5 10 15 20 25 30 35 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS (GeV) T Trailing lepton p 20 40 60 80 100 Data/pred 0 1 2 3 2 4 6 8 10 12 14 Events 0 10 20 30 40 50 Nonprompt W tt Z/H tt γ X+ WZ Rare Uncertainties Data (13 TeV) -1 2.3 fb CMS Signal region 2 4 6 8 10 12 14 Data/pred 0 1 2 3 On-Z samples: from left to right, top to bottom, distributions of HT, pmiss T , N for the predicted backgrounds and for the data in the on-Z baseline selection ots the rightmost bin contains the overﬂow from counts outside the range of ttom-right corner the total predicted background and the number of events o n-Z SRs is shown. 7 Results and interpretations On the bottom-right corner the total predicted background and the number of events observed in the 15 on-Z SRs is shown. 7 Results and interpretations 12 Table 3: Off-Z SRs: Comparison of observed event yields in data with predicted background yields. Table 3: Off-Z SRs: Comparison of observed event yields in data with predicted background yields. able 3: Off-Z SRs: Comparison of observed event yields in data with predicted backg elds. Nb HT (GeV) pmiss T (GeV) Predicted Observed SR (off-Z) 0 b-tags 60-400 50-150 19.26+4.81 −4.80 18 SR 1 150-300 1.16+0.31 −0.20 4 SR 2 400-600 50-150 1.20+0.47 −0.40 3 SR 3 150-300 0.29+0.44 −0.09 0 SR 4 1 b-tags 60-400 50-150 16.57 ± 4.52 24 SR 5 150-300 2.32+0.80 −0.76 1 SR 6 400-600 50-150 0.67+0.45 −0.09 2 SR 7 150-300 0.48+0.29 −0.07 0 SR 8 2 b-tags 60-400 50-150 4.49+1.81 −1.79 4 SR 9 150-300 0.31+0.44 −0.09 1 SR 10 400–600 50–150 0.40+0.27 −0.26 0 SR 11 150–300 0.08+0.43 −0.08 0 SR 12 ≥3 b-tags 60–600 50-300 0.13+0.43 −0.09 0 SR 13 ≥0 b-tags >600 50-300 1.84+0.44 −0.37 3 SR 14 ≥0 b-tags ≥0 ≥300 1.62+1.22 −1.19 0 SR 15 or squarks for the various models discussed in Section 4, as a function of the gluino or squark, and the chargino or neutralino masses. For each mass hypothesis, the observation, background predictions, and expected signal yields from all on-Z and off-Z SRs are combined to extract an upper limit on the cross section, at 95% conﬁdence level (CL) using the asymptotic formulation of the LHC-style CLs method [89–92]. Log-normal nuisance parameters are used to describe the systematic uncertainties listed in Section 6. or squarks for the various models discussed in Section 4, as a function of the gluino or squark, and the chargino or neutralino masses. For each mass hypothesis, the observation, background predictions, and expected signal yields from all on-Z and off-Z SRs are combined to extract an upper limit on the cross section, at 95% conﬁdence level (CL) using the asymptotic formulation of the LHC-style CLs method [89–92]. Log-normal nuisance parameters are used to describe the systematic uncertainties listed in Section 6. or squarks for the various models discussed in Section 4, as a function of the gluino or squark, and the chargino or neutralino masses. or squarks for the various models discussed in Section 4, as a function of the gluino or squark, and the chargino or neutralino masses. For each mass hypothesis, the observation, background predictions, and expected signal yields from all on-Z and off-Z SRs are combined to extract an upper limit on the cross section, at 95% conﬁdence level (CL) using the asymptotic formulation of the LHC-style CLs method [89–92]. Log-normal nuisance parameters are used to describe the systematic uncertainties listed in Section 6. 7 Results and interpretations upper limit on cross section (pb) (GeV) b~ m 300 400 500 600 700 800 900 (GeV) 1 ± χ∼ m 200 400 600 800 1000 -2 10 -1 10 1 10 1 ± χ∼ = m b~ m (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼ tW → 1 b~ , 1 b~ 1 b~ → pp = 50 GeV 1 0 χ∼ m 1 s.d. (theory) ± Observed 1 s.d. (experiment) ± Expected 95% C.L. upper limit on cross section (pb) (GeV) g~ m 600 700 800 900 1000 1100 1200 1300 (GeV) 0 1 χ∼ m 0 200 400 600 800 1000 1200 1400 -2 10 -1 10 1 10 1 0 χ∼ = m g~ m (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼ 'W/Z q q → g~ , g~ g~ → pp ) 1 0 χ∼ + m g~ = 0.5 (m 1 ± χ∼ m 1 s.d. (theory) ± Observed 1 s.d. (experiment) ± Expected 95% C.L. upper limit on cross section (pb) Figure 4: Exclusion contours as a function of meg or meb, and meχ0 or meχ±, for the simpliﬁed SUSY models (top-left) T1tttt, (top-right) T6ttWW, and (bottom) T5qqqqWZ. The color scale indicates the 95% CL observed upper limits on the cross section. The observed (expected) exclusion curves are indicated by the solid (dashed) lines using NLO+NLL production cross sections, along with the corresponding ±1 s.d. theoretical (experimental) uncertainties. (GeV) g~ m 800 1000 1200 1400 1600 (GeV) 0 1 χ∼ m 0 200 400 600 800 1000 1200 1400 1600 -2 10 -1 10 1 10 ) b + m W = 2 (m 1 0 χ∼ - m g~ m (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼t t → g~ , g~ g~ → pp 1 s.d. (theory) ± Observed 1 s.d. (experiment) ± Expected 95% C.L. upper limit on cross section (pb) (GeV) b~ m 300 400 500 600 700 800 900 (GeV) 1 ± χ∼ m 200 400 600 800 1000 -2 10 -1 10 1 10 1 ± χ∼ = m b~ m (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼ tW → 1 b~ , 1 b~ 1 b~ → pp = 50 GeV 1 0 χ∼ m 1 s.d. (theory) ± Observed 1 s.d. (experiment) ± Expected 95% C.L. 7 Results and interpretations For each mass hypothesis, the observation, background predictions, and expected signal yields from all on-Z and off-Z SRs are combined to extract an upper limit on the cross section, at 95% conﬁdence level (CL) using the asymptotic formulation of the LHC-style CLs method [89–92]. Log-normal nuisance parameters are used to describe the systematic uncertainties listed in Section 6. These upper limits are used to calculate exclusion contours on the concerned sparticles mass plane, shown in Fig. 4 for the simpliﬁed models under consideration. In these ﬁgures, the thick black lines delineate the observed exclusion region, which is at the lower masses side. The uncertainty in the observed limit, represented by the thinner black lines, is the propagation of the NLO+NLL cross section uncertainties for the relevant signal process [78–81]. The red dashed lines represent the expected limits with the uncertainties reﬂecting those discussed in Section 6. The yields and background predictions can be used to test additional BSM physics scenarios. To facilitate such reinterpretations, we provide limits on the number of multilepton events as a function of the pmiss T threshold in the kinematic tails of this search. These limits are ob- tained based on the tails of our SRs, in particular we consider events with HT > 400 GeV, both with and without an on-Z lepton pair, employing the LHC-style CLs method carried out with pseudo-experiments [89–91]. They are shown in Fig. 5 in terms of the product of cross section (σ), detector acceptance (A), and selection efﬁciency (ϵ). As we increase the pmiss T threshold, the observed and expected limits converge to 1.3 fb. 13 (GeV) g~ m 800 1000 1200 1400 1600 (GeV) 0 1 χ∼ m 0 200 400 600 800 1000 1200 1400 1600 -2 10 -1 10 1 10 ) b + m W = 2 (m 1 0 χ∼ - m g~ m (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼t t → g~ , g~ g~ → pp 1 s.d. (theory) ± Observed 1 s.d. (experiment) ± Expected 95% C.L. 7 Results and interpretations Nb HT (GeV) pmiss T (GeV) Predicted Observed SR (on-Z) 0 b-tags 60–400 50–150 38.01 ± 5.92 39 SR 1 150–300 4.48+0.84 −0.75 3 SR 2 400–600 50–150 4.88+1.49 −1.47 4 SR 3 150–300 1.88+0.47 −0.39 3 SR 4 1 b-tags 60–400 50–150 11.84+2.28 −2.26 14 SR 5 150–300 1.53+0.42 −0.34 1 SR 6 400–600 50–150 1.18+0.49 −0.23 1 SR 7 150–300 0.42+0.44 −0.10 3 SR 8 2 b-tags 60–400 50–150 2.55+0.67 −0.51 2 SR 9 150–300 0.72+0.76 −0.28 0 SR 10 400–600 50–150 0.55+0.45 −0.13 0 SR 11 150–300 0.31+0.51 −0.17 0 SR 12 ≥3 b-tags 60–600 50–300 0.21+0.44 −0.13 0 SR 13 ≥0 b-tags >600 50–300 4.22+0.68 −0.63 5 SR 14 ≥0 b-tags ≥0 ≥300 1.41+0.50 −0.25 1 SR 15 threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, on-Z T H (13 TeV) -1 2.3 fb CMS threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, off-Z T H (13 TeV) -1 2.3 fb CMS Figure 5: Limits on the product of cross section, detector acceptance, and selection efﬁciency, σAϵ, for the production of multilepton events with (left) or without (right) an on-Z lepton pair as a function of the pmiss T threshold. Table 4: On-Z SRs: Comparison of observed event yields in data with predicted background yields. Table 4: On-Z SRs: Comparison of observed event yields in data with predicted background yields. 7 Results and interpretations Nb HT (GeV) pmiss T (GeV) Predicted Observed SR (on-Z) 0 b-tags 60–400 50–150 38.01 ± 5.92 39 SR 1 150–300 4.48+0.84 −0.75 3 SR 2 400–600 50–150 4.88+1.49 −1.47 4 SR 3 150–300 1.88+0.47 −0.39 3 SR 4 1 b-tags 60–400 50–150 11.84+2.28 −2.26 14 SR 5 150–300 1.53+0.42 −0.34 1 SR 6 400–600 50–150 1.18+0.49 −0.23 1 SR 7 150–300 0.42+0.44 −0.10 3 SR 8 2 b-tags 60–400 50–150 2.55+0.67 −0.51 2 SR 9 150–300 0.72+0.76 −0.28 0 SR 10 400–600 50–150 0.55+0.45 −0.13 0 SR 11 150–300 0.31+0.51 −0.17 0 SR 12 ≥3 b-tags 60–600 50–300 0.21+0.44 −0.13 0 SR 13 ≥0 b-tags >600 50–300 4.22+0.68 −0.63 5 SR 14 ≥0 b-tags ≥0 ≥300 1.41+0.50 −0.25 1 SR 15 threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, on-Z T H (13 TeV) -1 2.3 fb CMS threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, off-Z T H (13 TeV) -1 2.3 fb CMS Figure 5: Limits on the product of cross section, detector acceptance, and selection efﬁcien σAϵ, for the production of multilepton events with (left) or without (right) an on-Z lepton p as a function of the pmiss T threshold. Table 4: On-Z SRs: Comparison of observed event yields in data with predicted background yields. 7 Results and interpretations upper limit on cross section (pb) (GeV) g~ m g b (GeV) g~ m 600 700 800 900 1000 1100 1200 1300 (GeV) 0 1 χ∼ m 0 200 400 600 800 1000 1200 1400 -2 10 -1 10 1 10 1 0 χ∼ = m g~ m (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼ 'W/Z q q → g~ , g~ g~ → pp ) 1 0 χ∼ + m g~ = 0.5 (m 1 ± χ∼ m 1 s.d. (theory) ± Observed 1 s.d. (experiment) ± Expected 95% C.L. upper limit on cross section (pb) g (GeV) g~ m 600 700 800 900 1000 1100 1200 1300 (GeV) 0 1 χ∼ m 0 200 400 600 800 1000 1200 1400 -2 10 -1 10 1 10 1 0 χ∼ = m g~ m (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼ 'W/Z q q → g~ , g~ g~ → pp ) 1 0 χ∼ + m g~ = 0.5 (m 1 ± χ∼ m 1 s.d. (theory) ± Observed 1 s.d. (experiment) ± Expected 95% C.L. upper limit on cross section (pb) Figure 4: Exclusion contours as a function of meg or meb, and meχ0 or meχ±, for the simpliﬁed SUSY models (top-left) T1tttt, (top-right) T6ttWW, and (bottom) T5qqqqWZ. The color scale indicates the 95% CL observed upper limits on the cross section. The observed (expected) exclusion curves are indicated by the solid (dashed) lines using NLO+NLL production cross sections, along with the corresponding ±1 s.d. theoretical (experimental) uncertainties. 7 Results and interpretations 14 14 7 Results and interpretatio Table 4: On-Z SRs: Comparison of observed event yields in data with predicted backgrou yields. 7 Results and interpretations Nb HT (GeV) pmiss T (GeV) Predicted Observed SR (on-Z) 0 b-tags 60–400 50–150 38.01 ± 5.92 39 SR 1 150–300 4.48+0.84 −0.75 3 SR 2 400–600 50–150 4.88+1.49 −1.47 4 SR 3 150–300 1.88+0.47 −0.39 3 SR 4 1 b-tags 60–400 50–150 11.84+2.28 −2.26 14 SR 5 150–300 1.53+0.42 −0.34 1 SR 6 400–600 50–150 1.18+0.49 −0.23 1 SR 7 150–300 0.42+0.44 −0.10 3 SR 8 2 b-tags 60–400 50–150 2.55+0.67 −0.51 2 SR 9 150–300 0.72+0.76 −0.28 0 SR 10 400–600 50–150 0.55+0.45 −0.13 0 SR 11 150–300 0.31+0.51 −0.17 0 SR 12 ≥3 b-tags 60–600 50–300 0.21+0.44 −0.13 0 SR 13 ≥0 b-tags >600 50–300 4.22+0.68 −0.63 5 SR 14 ≥0 b-tags ≥0 ≥300 1.41+0.50 −0.25 1 SR 15 threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, on-Z T H (13 TeV) -1 2.3 fb CMS threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, off-Z T H (13 TeV) -1 2.3 fb CMS Figure 5: Limits on the product of cross section, detector acceptance, and selection efﬁciency, σAϵ, for the production of multilepton events with (left) or without (right) an on-Z lepton pair as a function of the pmiss T threshold. threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, on-Z T H (13 TeV) -1 2.3 fb CMS threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. 8 Summary We have presented the search for beyond-the-standard-model physics in ﬁnal states with at least 3 leptons, electrons or muons, using proton-proton data collected with the CMS detector at √s = 13 TeV, corresponding to an integrated luminosity of 2.3 fb−1. The analysis makes use of techniques based on control samples in data to estimate reducible backgrounds and to validate the simulation for use in estimating irreducible backgrounds. To maximize sensitivity to a broad range of possible signal models, we investigate 30 exclusive signal regions. The event yields observed in data are in agreement with the standard model background predictions. This search is designed to be sensitive to multiple BSM models. As an example, we interpret the result in the context of a gluino-pair production model that features cascade decays producing four top quarks in the ﬁnal state. In this simpliﬁed model, we exclude gluinos with a mass of up to 1175 GeV in the case of a massless lightest supersymmteric particle (LSP). For gluino masses up to approximately 1150 GeV, neutralino masses below 650 GeV are excluded. These are the ﬁrst CMS results reported in this ﬁnal state at √s = 13 TeV. In a bottom squark pair production model with cascade decays that contain two top quarks and two additional W± bosons, we also set limits on the masses of the bottom squark and the chargino. We exclude bottom squarks with a mass of up to 450 GeV in the case of a chargino with a mass of 200 GeV. For bottom squark masses up to approximately 450 GeV, neutralino masses below 300 GeV are excluded. In a similar search at √s = 8 TeV [42], the bottom squark mass limit was slightly larger and the chargino mass limit was approximately the same. An additional interpretation is presented in a gluino pair production model with four light quarks and two vector bosons in the ﬁnal state. For the case of one W and one Z boson in the ﬁnal state, we exclude gluino masses up to 825 GeV when the LSP mass is 100 GeV, and LSP masses up to 500 GeV for 700 GeV gluinos. Finally, limits on the number of multilepton events with HT > 400 GeV as a function of pmiss T threshold are also presented in terms of the product of cross section, detector acceptance, and selection efﬁciency. 8 Summary For a pmiss T threshold greater than 500 GeV, the observed and expected limits are 1.3 fb. 7 Results and interpretations ± Expected exclusion limit ε A σ Model independent > 400 GeV, off-Z T H (13 TeV) -1 2.3 fb CMS Figure 5: Limits on the product of cross section, detector acceptance, and selection efﬁcienc σAϵ, for the production of multilepton events with (left) or without (right) an on-Z lepton pa as a function of the pmiss T threshold. threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, off-Z T H (13 TeV) -1 2.3 fb CMS threshold (GeV) miss T p 300 400 500 600 700 800 limit at 95% CL (fb) ε A σ 0 1 2 3 4 5 Observed 2 s.d. ± 1 s.d. ± Expected exclusion limit ε A σ Model independent > 400 GeV, on-Z T H (13 TeV) -1 2.3 fb CMS limit at 95% CL (fb) ε A σ limit at 95% CL (fb) ε A σ Figure 5: Limits on the product of cross section, detector acceptance, and selection efﬁciency, σAϵ, for the production of multilepton events with (left) or without (right) an on-Z lepton pair as a function of the pmiss T threshold. 15 Acknowledgments Fi- nally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the following funding agencies: the Austrian Federal Min- istry of Science, Research and Economy and the Austrian Science Fund; the Belgian Fonds de la Recherche Scientiﬁque, and Fonds voor Wetenschappelijk Onderzoek; the Brazilian Fund- ing Agencies (CNPq, CAPES, FAPERJ, and FAPESP); the Bulgarian Ministry of Education and Science; CERN; the Chinese Academy of Sciences, Ministry of Science and Technology, and Na- tional Natural Science Foundation of China; the Colombian Funding Agency (COLCIENCIAS); the Croatian Ministry of Science, Education and Sport, and the Croatian Science Foundation; the Research Promotion Foundation, Cyprus; the Secretariat for Higher Education, Science, Technology and Innovation, Ecuador; the Ministry of Education and Research, Estonian Re- 16 References search Council via IUT23-4 and IUT23-6 and European Regional Development Fund, Estonia; the Academy of Finland, Finnish Ministry of Education and Culture, and Helsinki Institute of Physics; the Institut National de Physique Nucl´eaire et de Physique des Particules / CNRS, and Commissariat `a l’´Energie Atomique et aux ´Energies Alternatives / CEA, France; the Bundes- ministerium f¨ur Bildung und Forschung, Deutsche Forschungsgemeinschaft, and Helmholtz- Gemeinschaft Deutscher Forschungszentren, Germany; the General Secretariat for Research and Technology, Greece; the National Scientiﬁc Research Foundation, and National Innova- tion Ofﬁce, Hungary; the Department of Atomic Energy and the Department of Science and Technology, India; the Institute for Studies in Theoretical Physics and Mathematics, Iran; the Science Foundation, Ireland; the Istituto Nazionale di Fisica Nucleare, Italy; the Ministry of Science, ICT and Future Planning, and National Research Foundation (NRF), Republic of Ko- rea; the Lithuanian Academy of Sciences; the Ministry of Education, and University of Malaya (Malaysia); the Mexican Funding Agencies (BUAP, CINVESTAV, CONACYT, LNS, SEP, and UASLP-FAI); the Ministry of Business, Innovation and Employment, New Zealand; the Pak- istan Atomic Energy Commission; the Ministry of Science and Higher Education and the Na- tional Science Centre, Poland; the Fundac¸˜ao para a Ciˆencia e a Tecnologia, Portugal; JINR, Dubna; the Ministry of Education and Science of the Russian Federation, the Federal Agency of Atomic Energy of the Russian Federation, Russian Academy of Sciences, and the Russian Foundation for Basic Research; the Ministry of Education, Science and Technological Devel- opment of Serbia; the Secretar´ıa de Estado de Investigaci´on, Desarrollo e Innovaci´on and Pro- grama Consolider-Ingenio 2010, Spain; the Swiss Funding Agencies (ETH Board, ETH Zurich, PSI, SNF, UniZH, Canton Zurich, and SER); the Ministry of Science and Technology, Taipei; the Thailand Center of Excellence in Physics, the Institute for the Promotion of Teaching Science and Technology of Thailand, Special Task Force for Activating Research and the National Sci- ence and Technology Development Agency of Thailand; the Scientiﬁc and Technical Research Council of Turkey, and Turkish Atomic Energy Authority; the National Academy of Sciences of Ukraine, and State Fund for Fundamental Researches, Ukraine; the Science and Technology Facilities Council, UK; the US Department of Energy, and the US National Science Foundation. Acknowledgments Individuals have received support from the Marie-Curie programme and the European Re- search Council and EPLANET (European Union); the Leventis Foundation; the A. P. Sloan Foundation; the Alexander von Humboldt Foundation; the Belgian Federal Science Policy Of- ﬁce; the Fonds pour la Formation `a la Recherche dans l’Industrie et dans l’Agriculture (FRIA- Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT-Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Sci- ence and Industrial Research, India; the HOMING PLUS programme of the Foundation for Polish Science, coﬁnanced from European Union, Regional Development Fund, the Mobil- ity Plus programme of the Ministry of Science and Higher Education, the National Science Center (Poland), contracts Harmonia 2014/14/M/ST2/00428, Opus 2013/11/B/ST2/04202, 2014/13/B/ST2/02543 and 2014/15/B/ST2/03998, Sonata-bis 2012/07/E/ST2/01406; the Thalis and Aristeia programmes coﬁnanced by EU-ESF and the Greek NSRF; the National Pri- orities Research Program by Qatar National Research Fund; the Programa Clar´ın-COFUND del Principado de Asturias; the Rachadapisek Sompot Fund for Postdoctoral Fellowship, Chu- lalongkorn University and the Chulalongkorn Academic into Its 2nd Century Project Advance- ment Project (Thailand); and the Welch Foundation, contract C-1845. 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Carrera Jarrin Academy of Scientiﬁc Research and Technology of the Arab Republic of Egypt, Egyptian Network of High Energy Physics, Cairo, Egypt Y. Assran9,10, T. Elkafrawy11, S. Khalil12 National Institute of Chemical Physics and Biophysics, Tallinn, Estonia B. Calpas, M. Kadastik, M. Murumaa, L. Perrini, M. Raidal, A. Tiko, C. Veelken Department of Physics, University of Helsinki, Helsinki, Finland P. Eerola, J. Pekkanen, M. Voutilainen Helsinki Institute of Physics, Helsinki, Finland J. H¨ark¨onen, T. J¨arvinen, V. Karim¨aki, R. Kinnunen, T. Lamp´en, K. Lassila-Perini, S. Lehti, T. Lind´en, P. Luukka, J. Tuominiemi, E. Tuovinen, L. Wendland 27 Lappeenranta University of Technology, Lappeenranta, Finland J. Talvitie, T. Tuuva IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France M. Besancon, F. Couderc, M. Dejardin, D. Denegri, B. Fabbro, J.L. Faure, C. Favaro, F. Ferri, S. Ganjour, S. Ghosh, A. Givernaud, P. Gras, G. Hamel de Monchenault, P. Jarry, I. Kucher, E. Locci, M. Machet, J. Malcles, J. Rander, A. Rosowsky, M. Titov, A. Zghiche IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France y M. Besancon, F. Couderc, M. Dejardin, D. Denegri, B. Fabbro, J.L. Faure, C. Favaro, F. Ferri, S. Ganjour, S. Ghosh, A. Givernaud, P. Gras, G. Hamel de Monchenault, P. Jarry, I. Kucher, E. Locci, M. Machet, J. Malcles, J. Rander, A. Rosowsky, M. Titov, A. Zghiche Laboratoire Leprince-Ringuet, Ecole Polytechnique, IN2P3-CNRS, Palaiseau, France A. Abdulsalam, I. Antropov, S. Bafﬁoni, F. Beaudette, P. Busson, L. Cadamuro, E. Chapon, C. Charlot, O. Davignon, R. Granier de Cassagnac, M. Jo, S. Lisniak, P. Min´e, M. Nguyen, C. Ochando, G. Ortona, P. Paganini, P. Pigard, S. Regnard, R. Salerno, Y. Sirois, T. Strebler, Y. Yilmaz, A. Zabi Institut Pluridisciplinaire Hubert Curien (IPHC), Universit´e de Strasbourg, CNRS-IN2P3 J.-L. Agram13, J. Andrea, A. Aubin, D. Bloch, J.-M. Brom, M. Buttignol, E.C. Chabert, N. Chanon, C. Collard, E. Conte13, X. Coubez, J.-C. Fontaine13, D. Gel´e, U. Goerlach, A.-C. Le Bihan, K. Skovpen, P. Van Hove Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France S. Gadrat Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France S. Gadrat Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France S. Beauceron, C. Bernet, G. Boudoul, E. Bouvier, C.A. Carrillo Montoya, R. Chierici, D. Contardo, B. Courbon, P. Depasse, H. El Mamouni, J. Fan, J. Fay, S. Gascon, M. Gouzevitch, Universite de Lyon, Universite Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France S. Beauceron, C. Bernet, G. Boudoul, E. Bouvier, C.A. Carrillo Montoya, R. Chierici, D. Contardo, B. Courbon, P. Depasse, H. El Mamouni, J. Fan, J. Fay, S. Gascon, M. Gouzevitch, G. Grenier, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, S. Perries, A. Popov14, D. Sabes, V. Sordini, M. Vander Donckt, P. Verdier, S. Viret S. Beauceron, C. Bernet, G. Boudoul, E. Bouvier, C.A. Carrillo Montoya, R. Chierici, D. Contardo, B. Courbon, P. Depasse, H. El Mamouni, J. Fan, J. Fay, S. Gascon, M. Gouzevitch, G. Grenier, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, S. Perries, A. Popov14, D. Sabes, V. Sordini, M. Vander Donckt, P. Verdier, S. Viret Georgian Technical University, Tbilisi, Georgia A. Khvedelidze8 University of Hamburg, Hamburg, Germany University of Hamburg, Hamburg, Germany V. Blobel, M. Centis Vignali, A.R. Draeger, T. Dreyer, E. Garutti, D. Gonzalez, J. Haller, M. Hoffmann, A. Junkes, R. Klanner, R. Kogler, N. Kovalchuk, T. Lapsien, T. Lenz, I. Marchesini, D. Marconi, M. Meyer, M. Niedziela, D. Nowatschin, F. Pantaleo15, T. Peiffer, A. Perieanu, J. Poehlsen, C. Sander, C. Scharf, P. Schleper, A. Schmidt, S. Schumann, J. Schwandt, H. Stadie, G. Steinbr¨uck, F.M. Stober, M. St¨over, H. Tholen, D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald Institut f¨ur Experimentelle Kernphysik, Karlsruhe, Germany p p y , , y M. Akbiyik, C. Barth, S. Baur, C. Baus, J. Berger, E. Butz, R. Caspart, T. Chwalek, F. Colombo, W. De Boer, A. Dierlamm, S. Fink, B. Freund, R. Friese, M. Giffels, A. Gilbert, P. Goldenzweig, D. Haitz, F. Hartmann15, S.M. Heindl, U. Husemann, I. Katkov14, S. Kudella, P. Lobelle Pardo, H. Mildner, M.U. Mozer, Th. M¨uller, M. Plagge, G. Quast, K. Rabbertz, S. R¨ocker, F. Roscher, M. Schr¨oder, I. Shvetsov, G. Sieber, H.J. Simonis, R. Ulrich, J. Wagner-Kuhr, S. Wayand, M. Weber, T. Weiler, S. Williamson, C. W¨ohrmann, R. Wolf Institute of Nuclear and Particle Physics (INPP), NCSR Demokritos, Aghia Paraskevi, Greece G. Anagnostou, G. Daskalakis, T. Geralis, V.A. Giakoumopoulou, A. Kyriakis, D. Loukas, I. Topsis-Giotis National and Kapodistrian University of Athens, Athens, Greece S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi Tbilisi State University, Tbilisi, Georgia Z. Tsamalaidze8 RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, S. Beranek, L. Feld, A. Heister, M.K. Kiesel, K. Klein, M. Lipinsk M Preuten F Raupach S Schael C Schomakers J Schulz T Verlage H Web RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, S. Beranek, L. Feld, A. Heister, M.K. Kiesel, K. Klein, M. Lipinski, A. Ostapchuk, M. Preuten, F. Raupach, S. Schael, C. Schomakers, J. Schulz, T. Verlage, H. Weber, V. Zhukov14 RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany A. Albert, M. Brodski, E. Dietz-Laursonn, D. Duchardt, M. Endres, M. Erdmann, S. Erdweg, T. Esch, R. Fischer, A. G¨uth, M. Hamer, T. Hebbeker, C. Heidemann, K. Hoepfner, S. Knutzen, M. Merschmeyer, A. Meyer, P. Millet, S. Mukherjee, M. Olschewski, K. Padeken, T. Pook, M. Radziej, H. Reithler, M. Rieger, F. Scheuch, L. Sonnenschein, D. Teyssier, S. Th¨uer RWTH Aachen University, III. Physikalisches Institut B, Aachen, Germany V. Cherepanov, G. Fl¨ugge, F. Hoehle, B. Kargoll, T. Kress, A. K¨unsken, J. Lingemann, T. M¨uller, A. Nehrkorn, A. Nowack, I.M. Nugent, C. Pistone, O. Pooth, A. Stahl15 Deutsches Elektronen Synchrotron, Hamburg, Germany M. Aldaya Martin, T. Arndt, C. Asawatangtrakuldee, K. Beernaert, O. Behnke, U. Behrens, A.A. Bin Anuar, K. Borras16, A. Campbell, P. Connor, C. Contreras-Campana, F. Costanza, C. Diez Pardos, G. Dolinska, G. Eckerlin, D. Eckstein, T. Eichhorn, E. Eren, E. Gallo17, J. Garay Garcia, A. Geiser, A. Gizhko, J.M. Grados Luyando, P. Gunnellini, A. Harb, A The CMS Collaboration 28 J. Hauk, M. Hempel18, H. Jung, A. Kalogeropoulos, O. Karacheban18, M. Kasemann, J. Keaveney, C. Kleinwort, I. Korol, D. Kr¨ucker, W. Lange, A. Lelek, J. Leonard, K. Lipka, A. Lobanov, W. Lohmann18, R. Mankel, I.-A. Melzer-Pellmann, A.B. Meyer, G. Mittag, J. Mnich, A. Mussgiller, E. Ntomari, D. Pitzl, R. Placakyte, A. Raspereza, B. Roland, M. ¨O. Sahin, P. Saxena, T. Schoerner-Sadenius, C. Seitz, S. Spannagel, N. Stefaniuk, G.P. Van Onsem, R. Walsh, C. Wissing A. Mussgiller, E. Ntomari, D. Pitzl, R. Placakyte, A. Raspereza, B. Roland, M.O. Sahin, P. Saxena, T. Schoerner-Sadenius, C. Seitz, S. Spannagel, N. Stefaniuk, G.P. Van Onsem, R. Walsh, C. Wissing Indian Institute of Technology Madras, Madras, India P.K. Behera Bhabha Atomic Research Centre, Mumbai, India R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty15, P.K. Netrakanti, L.M. Pant, P. Shukla, A. Topkar R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty15, P.K. Netrakanti, L.M. Pant, P. Shukla, A. Topkar Tata Institute of Fundamental Research-A, Mumbai, India T. Aziz, S. Dugad, G. Kole, B. Mahakud, S. Mitra, G.B. Mohanty, B. Parida, N. Sur, B. Sutar Tata Institute of Fundamental Research B, Mumbai, India S. Banerjee, S. Bhowmik24, R.K. Dewanjee, S. Ganguly, M. Guchait, Sa. Jain, S. Kumar, M. Maity24, G. Majumder, K. Mazumdar, T. Sarkar24, N. Wickramage25 Indian Institute of Science Education and Research (IISER), Pune, India S. Chauhan, S. Dube, V. Hegde, A. Kapoor, K. Kothekar, S. Pandey, A. Rane, S. Sharma Institute for Research in Fundamental Sciences (IPM), Tehran, Iran Institute for Research in Fundamental Sciences (IPM), Tehran, Iran H. Behnamian, S. Chenarani26, E. Eskandari Tadavani, S.M. Etesami26, A. Fahim27, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi28, F. Rezaei Hosseinabadi, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi28, F. Rezaei Hosseinabadi, B. Safarzadeh29, M. Zeinali University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald University of Io´annina, Io´annina, Greece Sharan, S. Thakur University of Io´annina, Io´annina, Greece University of Io´annina, Io´annina, Greece I. Evangelou, G. Flouris, C. Foudas, P. Kokkas, N. Loukas, N. Manthos, I. Papadopoulos, E. Paradas MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University, Budapest, Hungary N. Filipovic Wigner Research Centre for Physics, Budapest, Hungary G. Bencze, C. Hajdu, P. Hidas, D. Horvath19, F. Sikler, V. Veszpremi, G. Vesztergombi20, A.J. Zsigmond Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi21, A. Makovec, J. Molnar, Z. Szillasi Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi21, A. Makovec, J. Molnar, Z. Szillasi Institute of Physics, University of Debrecen M. Bart´ok20, P. Raics, Z.L. Trocsanyi, B. Ujvari Institute of Physics, University of Debrecen M. Bart´ok20, P. Raics, Z.L. Trocsanyi, B. Ujvari National Institute of Science Education and Research, Bhubaneswar, India S. Bahinipati, S. Choudhury22, P. Mal, K. Mandal, A. Nayak23, D.K. Sahoo, N. Sahoo, S.K. Swain Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, National Institute of Science Education and Research, Bhubaneswar, India S. Bahinipati, S. Choudhury22, P. Mal, K. Mandal, A. Nayak23, D.K. Sahoo, N. Sahoo, S.K. Swain Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, P. Kumari, A. Mehta, M. Mittal, J.B. Singh, G. Walia Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, P. Kumari, A. Mehta, M. Mittal, J.B. Singh, G. Walia 29 University of Delhi, Delhi, India Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, S. Keshri, S. Malhotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma University of Delhi, Delhi, India Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, S. Keshri, S. Malhotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma Saha Institute of Nuclear Physics, Kolkata, India R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh, N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur Saha Institute of Nuclear Physics, Kolkata, India R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh, N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, D. Roy, S. Roy Chowdhury, S. Sarkar, M. University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Tabarelli de Fatisa,b INFN Sezione di Napoli a, Universit`a di Napoli ’Federico II’ b, Napoli, Italy, Universit`a della Basilicata c, Potenza, Italy, Universit`a G. Marconi d, Roma, Italy S. Buontempoa, N. Cavalloa,c, G. De Nardo, S. Di Guidaa,d,15, M. Espositoa,b, F. Fabozzia,c, F. Fiengaa,b, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,15, P. Paoluccia,15, C. Sciaccaa,b, F. Thyssen INFN Sezione di Padova a, Universit`a di Padova b, Padova, Italy, Universit`a di Trento c, Trento, Italy P A ia 15 N B h tt a L B t a b D Bi ll a b A B l ttia b R C li a b A C lh INFN Sezione di Padova a, Universit`a di Padova b, Padova, Italy, Universit`a di Trento c, Trento, Italy P. Azzia,15, N. Bacchettaa, L. Benatoa,b, D. Biselloa,b, A. Bolettia,b, R. Carlina,b, A. Carvalho Antunes De Oliveiraa,b, P. Checchiaa, M. Dall’Ossoa,b, P. De Castro Manzanoa, T. Dorigoa, U. Dossellia, F. Gasparinia,b, U. Gasparinia,b, A. Gozzelinoa, S. Lacapraraa, M. Margonia,b, A.T. Meneguzzoa,b, J. Pazzinia,b, N. Pozzobona,b, P. Ronchesea,b, F. Simonettoa,b, E. Torassaa, M. Zanetti, P. Zottoa,b, G. Zumerlea,b P. Azzia,15, N. Bacchettaa, L. Benatoa,b, D. Biselloa,b, A. Bolettia,b, R. Carlina,b, A. Carvalho Antunes De Oliveiraa,b, P. Checchiaa, M. Dall’Ossoa,b, P. De Castro Manzanoa, T. Dorigoa, U. Dossellia, F. Gasparinia,b, U. Gasparinia,b, A. Gozzelinoa, S. Lacapraraa, M. Margonia,b, A.T. Meneguzzoa,b, J. Pazzinia,b, N. Pozzobona,b, P. Ronchesea,b, F. Simonettoa,b, E. Torassaa, M. Zanetti, P. Zottoa,b, G. Zumerlea,b M. Zanetti, P. Zottoa,b, G. Zumerlea,b INFN Sezione di Pavia a, Universit`a di Pavia b, Pavia, Italy A. Braghieria, A. Magnania,b, P. Montagnaa,b, S.P. Rattia,b, V. Rea, C. Riccardia,b, P. Salvinia, I. Vaia,b, P. Vituloa,b INFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy L Al i S l i ia b G M Bil ia D Ci i ia b L F ` a b P L INFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy L. Alunni Solestizia,b, G.M. Bileia, D. Ciangottinia,b, L. Fan`oa,b, P. Laricciaa,b, R. Leonardia,b, G. Mantovania,b, M. Menichellia, A. Sahaa, A. Santocchiaa,b INFN Sezione di Pisa a, Universit`a di Pisa b, Scuola Normale Superiore di Pisa c, Pisa, Italy K. Androsova,30, P. Azzurria,15, G. Bagliesia, J. Bernardinia, T. Boccalia, R. Castaldia, M.A. Cioccia,30, R. Dell’Orsoa, S. Donatoa,c, G. Fedi, A. Giassia, M.T. Grippoa,30, F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy-Navarroa,31, P. Spagnoloa, R. Tenchinia, G. University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald INFN Sezione di Bari a, Universit`a di Bari b, Politecnico di Bari c, Bari, Italy M. Abbresciaa,b, C. Calabriaa,b, C. Caputoa,b, A. Colaleoa, D. Creanzaa,c, L. Cristellaa,b, N. De Filippisa,c, M. De Palmaa,b, L. Fiorea, G. Iasellia,c, G. Maggia,c, M. Maggia, G. Minielloa,b, S. Mya,b, S. Nuzzoa,b, A. Pompilia,b, G. Pugliesea,c, R. Radognaa,b, A. Ranieria, G. Selvaggia,b, L. Silvestrisa,15, R. Vendittia,b, P. Verwilligena INFN Sezione di Bologna a, Universit`a di Bologna b, Bologna, Italy G. Abbiendia, C. Battilana, D. Bonacorsia,b, S. Braibant-Giacomellia,b, L. Brigliadoria,b, R. Campaninia,b, P. Capiluppia,b, A. Castroa,b, F.R. Cavalloa, S.S. Chhibraa,b, G. Codispotia,b, M. Cufﬁania,b, G.M. Dallavallea, F. Fabbria, A. Fanfania,b, D. Fasanellaa,b, P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria, F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,15 C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria, F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,15 INFN Sezione di Catania a, Universit`a di Catania b, Catania, Italy S. Albergoa,b, M. Chiorbolia,b, S. Costaa,b, A. Di Mattiaa, F. Giordanoa,b, R. Potenzaa,b, A. Tricomia,b, C. Tuvea,b INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy , , , y G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, V. Goria,b, P. Lenzia,b, M. Meschinia, S. Paolettia, G. Sguazzonia, L. Viliania,b,15 g , , , , , , , M. Meschinia, S. Paolettia, G. Sguazzonia, L. Viliania,b,15 M. Meschinia, S. Paolettia, G. Sguazzonia, L. Viliania,b,15 30 A The CMS Collaboration INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera15 INFN Sezione di Genova a, Universit`a di Genova b, Genova, Italy V. Calvellia,b, F. Ferroa, M. Lo Veterea,b, M.R. Mongea,b, E. Robuttia, S. Tosia,b INFN Sezione di Milano-Bicocca a, Universit`a di Milano-Bicocca b, Milano, Italy L. Brianza15, M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, A. Ghezzia,b, P. Govonia,b, M. Malberti, INFN Sezione di Milano-Bicocca a, Universit`a di Milano-Bicocca b, Milano, Italy L. Brianza15, M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, A. Ghezzia,b, P. Govonia,b, M. Malberti, S. Malvezzia, R.A. Manzonia,b,15, D. Menascea, L. Moronia, M. Paganonia,b, D. Pedrinia, S. Pigazzini, S. Ragazzia,b, T. Tabarelli de Fatisa,b S. Malvezzia, R.A. Manzonia,b,15, D. Menascea, L. Moronia, M. Paganonia,b, D. Pedrinia, S. Pigazzini, S. Ragazzia,b, T. University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Tonellia,b, A. Venturia, P.G. Verdinia INFN Sezione di Roma a, Universit`a di Roma b, Roma, Italy L. Baronea,b, F. Cavallaria, M. Cipriania,b, D. Del Rea,b,15, M. Diemoza, S. Gellia,b, E. Longoa,b, F Margarolia,b B Marzocchia,b P Meridiania G Organtinia,b R Paramattia F Preiatoa,b F. Margarolia,b, B. Marzocchia,b, P. Meridiania, G. Organtinia,b, R. Paramattia, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b INFN Sezione di Torino a, Universit`a di Torino b, Torino, Italy, Universit`a del Piemonte Orientale c, Novara, Italy N A a b R A idi a c 15 S A i a b M A d a c N B t ika R B ll a b C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, b b b b b oa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, oa,b, B. Kiania,b, C. Mariottia, S. Masellia, E. Migliorea,b, V. Monacoa,b, E. Monteila,b, g M.M. Obertinoa,b, L. Pachera,b, N. Pastronea, M. Pelliccionia, G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa,b, M. Ruspaa,c, R. Sacchia,b, K. Shchelinaa,b, V. Solaa, A. Solanoa,b, A. Staianoa, P. Traczyka,b 31 INFN Sezione di Trieste a, Universit`a di Trieste b, Trieste, Italy S. Belfortea, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, A. Zanettia Kyungpook National University, Daegu, Korea D.H. Kim, G.N. Kim, M.S. Kim, S. Lee, S.W. Lee, Y.D. Oh, S. Sekmen, D.C. Son, Y.C. Yang Chonbuk National University, Jeonju, Korea A. Lee INFN Sezione di Trieste a, Universit`a di Trieste b, Trieste, Italy S. Belfortea, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, A. Zanettia Chonnam National University, Institute for Universe and Elementary Particles, Kwangju, Korea H. Kim Hanyang University, Seoul, Korea J.A. Brochero Cifuentes, T.J. Kim Hanyang University, Seoul, Korea Korea University, Seoul, Korea S. Cho, S. Choi, Y. Go, D. Gyun, S. Ha, B. Hong, Y. Jo, Y. Kim, B. Lee, K. Lee, K.S. Lee, S. Lee, J. Lim, S.K. Park, Y. Roh Seoul National University, Seoul, Korea J. Almond, J. Kim, H. Lee, S.B. Oh, B.C. Radburn-Smith, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu University of Seoul, Seoul, Korea M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu University of Seoul, Seoul, Korea M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu Sungkyunkwan University, Suwon, Korea Y. Choi, J. Goh, C. Hwang, J. Lee, I. Yu Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia I. Ahmed, Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali32, F. Mohamad Idris33, W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia I. Ahmed, Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali32, F. Mohamad Idris33, W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada, R. Lopez-Fernandez, R. Maga˜na Villalba, J. Mejia Guisao, A. Sanchez-Hernandez Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada, R. Lopez-Fernandez, R. Maga˜na Villalba, J. Mejia Guisao, A. Sanchez-Hernandez R. Lopez-Fernandez, R. Maga˜na Villalba, J. Mejia Guisao, A. Sanchez-Hernandez Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, C. Oropeza Barrera, F. Vazquez V Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, C. Oropeza Barrera, F. Vazquez Valencia Benemerita Universidad Autonoma de Puebla, Puebla, Mexico S. Carpinteyro, I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Benemerita Universidad Autonoma de Puebla, Puebla, Mexico S. Carpinteyro, I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Benemerita Universidad Autonoma de Puebla, Puebla, Mexico S. Carpinteyro, I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico A. Morelos Pineda Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico A. Morelos Pineda University of Auckland, Auckland, New Zealand D. Krofcheck University of Canterbury, Christchurch, New Zealand P.H. Butler National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, A. Saddique, M.A. Shah, M. National Centre for Nuclear Research, Swierk, Poland National Centre for Nuclear Research, Swierk, Poland National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland K. Bunkowski, A. Byszuk35, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Misiura, M. Olszewski, M. Walczak Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho, M G lli J H ll N L d L Ll t I l i M V N ll di J R d i Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho, M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Rodrigues Antunes J Seixas O Toldaiev D Vadruccio J Varela P Vischia ¸ p , , g P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho, M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Rodrigues Antunes, J. Seixas, O. Toldaiev, D. Vadruccio, J. Varela, P. Vischia M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Rodrigues Antunes, J. Seixas, O. Toldaiev, D. Vadruccio, J. Varela, P. Vischia Joint Institute for Nuclear Research, Dubna, Russia Joint Institute for Nuclear Research, Dubna, Russia V. Alexakhin, I. Golutvin, I. Gorbunov, A. Kamenev, V. Karjavin, G. Kozlov, A. Lanev, A. Malakhov, V. Matveev36,37, V. Palichik, V. Perelygin, M. Savina, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, N. Voytishin, A. Zarubin Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia L. Chtchipounov, V. Golovtsov, Y. Ivanov, V. Kim38, E. Kuznetsova39, V. Murzin, V. Oreshkin, V. Sulimov, A. Vorobyev Institute for Nuclear Research, Moscow, Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov, N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin Institute for Theoretical and Experimental Physics, Moscow, Russia Institute for Theoretical and Experimental Physics, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, V. Popov, I. Pozdnyakov, G. Safronov, A. Spiridonov, M. Toms, E. Vlasov, A. Zhokin Moscow Institute of Physics and Technology, Moscow, Russia A. Hanyang University, Seoul, Korea Shoaib, M. Waqas National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, A. Saddique, M.A. Shah, M. Shoaib, M. Waqas A The CMS Collaboration 32 Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain Mad d, Spa J. Alcaraz Maestre, M. Barrio Luna, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Colino, B. De La Cruz, A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, M.S. Soares J. Alcaraz Maestre, M. Barrio Luna, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Colino, B. De La Cruz, A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, M.S. Soares Universidad Aut´onoma de Madrid, Madrid, Spain J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad de Oviedo, Oviedo, Spain Universidad de Oviedo, Oviedo, Spain J. Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, E. Palencia Cortezon, S. Sanchez Cruz, I. Su´arez Andr´es, J.M. Vizan Garcia , , p J. Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, E. Palencia Cortezon, S. Sanchez Cruz, I. Su´arez Andr´es, J.M. Vizan Garcia Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain I.J. Cabrillo, A. Calderon, J.R. Casti˜neiras De Saa, E. Curras, M. Fernandez, J. Garcia-Ferrero, G. Gomez, A. Lopez Virto, J. Marco, C. Martinez Rivero, F. Matorras, J. Piedra Gomez, T. Rodrigo, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain I.J. Cabrillo, A. Calderon, J.R. Casti˜neiras De Saa, E. Curras, M. Fernandez, J. Garcia-Ferrero, G. Gomez, A. Lopez Virto, J. Marco, C. Martinez Rivero, F. Matorras, J. Piedra Gomez, T. Rodrigo, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte CERN, European Organization for Nuclear Research, Geneva, Switzerland D. Abbaneo, E. Auffray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Barney, P. Bloch, A. Bocci, A. Bonato, C. Botta, T. Camporesi, R. Castello, M. Cepeda, G. Cerminara, M. D’Alfonso, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola, A. De Roeck, E. Di Marco44, M. Dobson, B. Dorney, T. du Pree, D. Duggan, M. D¨unser, N. Dupont, A. Elliott- Peisert, S. National Centre for Nuclear Research, Swierk, Poland Soares Universidad Aut´onoma de Madrid, Madrid, Spain J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad de Oviedo, Oviedo, Spain J. Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, E. Palencia Cortezon, S. Sanchez Cruz, I. Su´arez Andr´es, J.M. Vizan Garcia National Centre for Nuclear Research, Swierk, Poland Bylinkin37 Moscow Institute of Physics and Technology, Moscow, Russia A. Bylinkin37 National Research Nuclear University ’Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia M. Chadeeva40, O. Markin, E. Tarkovskii P.N. Lebedev Physical Institute, Moscow, Russia y V. Andreev, M. Azarkin37, I. Dremin37, M. Kirakosyan, A. Leonidov37, S.V. Rusakov, A. Terkulov Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 41 A. Baskakov, A. Belyaev, E. Boos, M. Dubinin41, L. Dudko, A. Ershov, A. Gri A. Baskakov, A. Belyaev, E. Boos, M. Dubinin , L. Dudko, A. Ershov, A. Gribushin, V. Klyukhin, O. Kodolova, I. Lokhtin, I. Miagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev Novosibirsk State University (NSU), Novosibirsk, Russia V. Blinov42, Y.Skovpen42, D. Shtol42 Novosibirsk State University (NSU), Novosibirsk, Russia V. Blinov42, Y.Skovpen42, D. Shtol42 State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, D. Elumakhov, V. Kachanov, A. Kalinin, D. Konstantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov 33 University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P. Adzic43, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain J. Alcaraz Maestre, M. Barrio Luna, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Colino, B. De La Cruz, A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, M.S. Soares University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P. Adzic43, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain J. Alcaraz Maestre, M. Barrio Luna, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Colino, B. De La Cruz, A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, M.S. University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P Ad i 43 P Ci k i D D t k M D d i J Mil i V R k i P. Adzic43, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain J. Alcaraz Maestre, M. Barrio Luna, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Colino, B. De La Cruz, A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I Redondo L Romero M S Soares Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain Fartoukh, G. Franzoni, J. Fulcher, W. Funk, D. Gigi, K. Gill, M. Girone, F. Glege, D. Gulhan, S. Gundacker, M. Guthoff, J. Hammer, P. Harris, J. Hegeman, V. Innocente, P. Janot, J. Kieseler, H. Kirschenmann, V. Kn¨unz, A. Kornmayer15, M.J. Kortelainen, K. Kousouris, M. Krammer1, C. Lange, P. Lecoq, C. Lourenc¸o, M.T. Lucchini, L. Malgeri, M. Mannelli, A. Martelli, F. Meijers, J.A. Merlin, S. Mersi, E. Meschi, P. Milenovic45, F. Moortgat, S. Morovic, M. Mulders, H. Neugebauer, S. Orfanelli, L. Orsini, L. Pape, E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiffer, M. Pierini, A. Racz, T. Reis, G. Rolandi46, M. Rovere, M. Ruan, H. Sakulin, J.B. Sauvan, C. Sch¨afer, C. Schwick, M. Seidel, A. Sharma, P. Silva, P. Sphicas47, J. Steggemann, M. Stoye, Y. Takahashi, M. Tosi, D. Treille, A. Triossi, A. Tsirou, V. Veckalns48, G.I. Veres20, N. Wardle, H.K. W¨ohri, A. Zagozdzinska35, W.D. Zeuner Paul Scherrer Institut, Villigen, Switzerland W. Bertl, K. Deiters, W. Erdmann, R. Horisberger, Q. Ingram, H.C. Kaestli, D. Kotlinski, U. Langenegger, T. Rohe Ukraine National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine L. Levchuk, P. Sorokin Universit¨at Z¨urich, Zurich, Switzerland Universit¨at Z¨urich, Zurich, Switzerland T.K. Aarrestad, C. Amsler50, L. Caminada, M.F. Canelli, A. De Cosa, C. Galloni, A. Hinzmann, T. Hreus, B. Kilminster, J. Ngadiuba, D. Pinna, G. Rauco, P. Robmann, D. Salerno, Y. Yang, A. Zucchetta T.K. Aarrestad, C. Amsler50, L. Caminada, M.F. Canelli, A. De Cosa, C. Galloni, A. Hinzmann, T. Hreus, B. Kilminster, J. Ngadiuba, D. Pinna, G. Rauco, P. Robmann, D. Salerno, Y. Yang, A. Zucchetta National Central University, Chung-Li, Taiwan National Central University, Chung-Li, Taiwan y g V. Candelise, T.H. Doan, Sh. Jain, R. Khurana, M. Konyushikhin, C.M. Kuo, W. Lin, Y.J. Lu, A. Pozdnyakov, S.S. Yu National Taiwan University (NTU), Taipei, Taiwan National Taiwan University (NTU), Taipei, Taiwan Arun Kumar, P. Chang, Y.H. Chang, Y.W. Chang, Y. Chao, K.F. Chen, P.H. Chen, C. Dietz, F. Fiori, W.-S. Hou, Y. Hsiung, Y.F. Liu, R.-S. Lu, M. Mi˜nano Moya, E. Paganis, A. Psallidas, J.f. Tsai, Y.M. Tzeng J.f. Tsai, Y.M. Tzeng Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand B. Asavapibhop, G. Singh, N. Srimanobhas, N. Suwonjandee Cukurova University - Physics Department, Science and Art Faculty A Adiguzel S Cerci51 S Damarseckin Z S Demiroglu C Dozen I Dumanoglu S Cukurova University - Physics Department, Science and Art Faculty A. Adiguzel, S. Cerci51, S. Damarseckin, Z.S. Demiroglu, C. Dozen, I. Dumanoglu, S G. Gokbulut, Y. Guler, I. Hos, E.E. Kangal52, O. Kara, A. Kayis Topaksu, U. Kiminsu, M. O Cukurova University - Physics Department, Science and Art Faculty 51 Cukurova University - Physics Department, Science and Art Faculty y y p y A. Adiguzel, S. Cerci51, S. Damarseckin, Z.S. Demiroglu, C. Dozen, I. Dumanoglu, S. Girgis, G. Gokbulut, Y. Guler, I. Hos, E.E. Kangal52, O. Kara, A. Kayis Topaksu, U. Kiminsu, M. Oglakci, G. Onengut53, K. Ozdemir54, D. Sunar Cerci51, H. Topakli55, S. Turkcapar, I.S. Zorbakir, C. Zorbilmez Middle East Technical University, Physics Department, Ankara, Turkey B. Bilin, S. Bilmis, B. Isildak56, G. Karapinar57, M. Yalvac, M. Zeyrek Bogazici University, Istanbul, Turkey E. G¨ulmez, M. Kaya58, O. Kaya59, E.A. Yetkin60, T. Yetkin61 Istanbul Technical University, Istanbul, Turkey A. Cakir, K. Cankocak, S. Sen62 Istanbul Technical University, Istanbul, Turkey A. Cakir, K. Cankocak, S. Sen62 Institute for Scintillation Materials of National Academy of Science of Ukraine, Kharkov, Ukraine B. Grynyov Institute for Particle Physics, ETH Zurich, Zurich, Switzerland Institute for Particle Physics, ETH Zurich, Zurich, Switzerland Institute for Particle Physics, ETH Zurich, Zurich, Switzerland F. Bachmair, L. B¨ani, L. Bianchini, B. Casal, G. Dissertori, M. Dittmar, M. Doneg`a, C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, P. Lecomte†, W. Lustermann, B. Mangano, M. Marionneau, P. Martinez Ruiz del Arbol, M. Masciovecchio, M.T. Meinhard, D. Meister, F. Micheli, P. Musella, F. Nessi-Tedaldi, F. Pandolﬁ, J. Pata, F. Pauss, G. Perrin, L. Perrozzi, M. Quittnat, M. Rossini, M. Sch¨onenberger, A. Starodumov49, V.R. Tavolaro, K. Theoﬁlatos, R. Wallny 34 A The CMS Collaboration Universit¨at Z¨urich, Zurich, Switzerland National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine L. Levchuk, P. Sorokin University of Bristol, Bristol, United Kingdom R. Aggleton, F. Ball, L. Beck, J.J. Brooke, D. Burns, E. Clement, D. Cussans, H. Flacher, J. Goldstein, M. Grimes, G.P. Heath, H.F. Heath, J. Jacob, L. Kreczko, C. Lucas, D.M. Newbold63, S. Paramesvaran, A. Poll, T. Sakuma, S. Seif El Nasr-storey, D. Smith, V.J. Smith Rutherford Appleton Laboratory, Didcot, United Kingdom K.W. Bell, A. Belyaev64, C. Brew, R.M. Brown, L. Calligaris, D. Cieri, D.J.A. Cockerill, J.A. Coughlan, K. Harder, S. Harper, E. Olaiya, D. Petyt, C.H. Shepherd-Themistocleous, A. Thea, I.R. Tomalin, T. Williams Imperial College, London, United Kingdom M. Baber, R. Bainbridge, O. Buchmuller, A. Bundock, D. Burton, S. Casasso, M. Citron, D. Colling, L. Corpe, P. Dauncey, G. Davies, A. De Wit, M. Della Negra, R. Di Maria, P. Dunne, A. Elwood, D. Futyan, Y. Haddad, G. Hall, G. Iles, T. James, R. Lane, C. Laner, R. Lucas63, L. Lyons, A.-M. Magnan, S. Malik, L. Mastrolorenzo, J. Nash, A. Nikitenko49, J. Pela, B. Penning, 35 M. Pesaresi, D.M. Raymond, A. Richards, A. Rose, C. Seez, S. Summers, A. Tapper, K. Uchida, M. Vazquez Acosta65, T. Virdee15, J. Wright, S.C. Zenz M. Pesaresi, D.M. Raymond, A. Richards, A. Rose, C. Seez, S. Summers, A. Tapper, K. Uchida, M. Vazquez Acosta65, T. Virdee15, J. Wright, S.C. Zenz Brunel University, Uxbridge, United Kingdom J.E. Cole, P.R. Hobson, A. Khan, P. Kyberd, D. Leslie, I.D. Reid, P. Symonds, L. Teodorescu, M. Turner Baylor University, Waco, USA Baylor University, Waco, USA A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika y y, , A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika The University of Alabama, Tuscaloosa, USA O. Charaf, S.I. Cooper, C. Henderson, P. Rumerio, C. West The University of Alabama, Tuscaloosa, USA O. Charaf, S.I. Cooper, C. Henderson, P. Rumerio, C. West Boston University, Boston, USA D. Arcaro, A. Avetisyan, T. Bose, D. Gastler, D. Rankin, C. Richardson, J. Rohlf, L. Sulak, D. Zou Brown University, Providence, USA G. Benelli, E. Berry, D. Cutts, A. Garabedian, J. Hakala, U. Heintz, J.M. Hogan, O. Jesus, K.H.M. Kwok, E. Laird, G. Landsberg, Z. Mao, M. Narain, S. Piperov, S. Sagir, E. Spencer, R. Syarif University of California, Davis, Davis, USA University of California, Davis, Davis, USA R. Breedon, G. Breto, D. Burns, M. Calderon De La Barca Sanchez, S. National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine L. Levchuk, P. Sorokin Chauhan, M. Chertok, J. Conway, R. Conway, P.T. Cox, R. Erbacher, C. Flores, G. Funk, M. Gardner, W. Ko, R. Lander, C. Mclean, M. Mulhearn, D. Pellett, J. Pilot, S. Shalhout, J. Smith, M. Squires, D. Stolp, M. Tripathi, S. Wilbur, R. Yohay University of California, Los Angeles, USA C. Bravo, R. Cousins, A. Dasgupta, P. Everaerts, A. Florent, J. Hauser, M. Ignatenko, N. Mccoll, D. Saltzberg, C. Schnaible, E. Takasugi, V. Valuev, M. Weber University of California, Riverside, Riverside, USA K. Burt, R. Clare, J. Ellison, J.W. Gary, S.M.A. Ghiasi Shirazi, G. Hanson, J. Heilman, P. Jandir, E. Kennedy, F. Lacroix, O.R. Long, M. Olmedo Negrete, M.I. Paneva, A. Shrinivas, W. Si, H. Wei, S. Wimpenny, B. R. Yates S. Wimpenny, B. R. Yates University of California, San Diego, La Jolla, USA Cornell University, Ithaca, USA Cornell University, Ithaca, USA J. Alexander, J. Chaves, J. Chu, S. Dittmer, K. Mcdermott, N. Mirman, G. Nicolas Kaufman, J.R. Patterson, A. Rinkevicius, A. Ryd, L. Skinnari, L. Sofﬁ, S.M. Tan, Z. Tao, J. Thom, J. Tucker, P. Wittich, M. Zientek University of California, San Diego, La Jolla, USA J.G. Branson, G.B. Cerati, S. Cittolin, M. Derdzinski, R. Gerosa, A. Holzner, D. Klein, V. Krutelyov, J. Letts, I. Macneill, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tadel, A. Vartak, S. Wasserbaech66, C. Welke, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della Porta V. Krutelyov, J. Letts, I. Macneill, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tadel, A. Vartak, S. Wasserbaech66, C. Welke, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della Porta y M. Tadel, A. Vartak, S. Wasserbaech66, C. Welke, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della Porta University of California, Santa Barbara - Department of Physics, Santa Barbara, USA N Amin R Bhandari J Bradmiller-Feld C Campagnari A Dishaw V Dutta K F University of California, Santa Barbara - Department of Physics, Santa Barbara, USA N. Amin, R. Bhandari, J. Bradmiller-Feld, C. Campagnari, A. Dishaw, V. Dutta, K. Flowers, M. Franco Sevilla, P. Geffert, C. George, F. Golf, L. Gouskos, J. Gran, R. Heller, J. Incandela, S.D. Mullin, A. Ovcharova, J. Richman, D. Stuart, I. Suarez, J. Yoo California Institute of Technology, Pasadena, USA D. Anderson, A. Apresyan, J. Bendavid, A. Bornheim, J. Bunn, Y. Chen, J. Duarte, J.M. Lawhorn, A. Mott, H.B. Newman, C. Pena, M. Spiropulu, J.R. Vlimant, S. Xie, R.Y. Zhu Carnegie Mellon University, Pittsburgh, USA M.B. Andrews, V. Azzolini, T. Ferguson, M. Paulini, J. Russ, M. Sun, H. Vogel, I. Vorobiev, M. Weinberg 36 A The CMS Collaboration University of Colorado Boulder, Boulder, USA Florida Institute of Technology, Melbourne, USA Florida Institute of Technology, Melbourne, USA University of Illinois at Chicago (UIC), Chicago, USA M.R. Adams, L. Apanasevich, D. Berry, R.R. Betts, I. Bucinskaite, R. Cavanaugh, O. Evdokimov, L. Gauthier, C.E. Gerber, D.J. Hofman, K. Jung, P. Kurt, C. O’Brien, I.D. Sandoval Gonzalez, P. Turner, N. Varelas, H. Wang, Z. Wu, M. Zakaria, J. Zhang The University of Iowa, Iowa City, USA B. Bilki68, W. Clarida, K. Dilsiz, S. Durgut, R.P. Gandrajula, M. Haytmyradov, V. Khristenko, J.-P. Merlo, H. Mermerkaya69, A. Mestvirishvili, A. Moeller, J. Nachtman, H. Ogul, Y. Onel, F. Ozok70, A. Penzo, C. Snyder, E. Tiras, J. Wetzel, K. Yi Johns Hopkins University, Baltimore, USA University of Colorado Boulder, Boulder, USA University of Colorado Boulder, Boulder, USA J.P. Cumalat, W.T. Ford, F. Jensen, A. Johnson, M. Krohn, T. Mulholland, K. Stenson, S.R. Wagner J.P. Cumalat, W.T. Ford, F. Jensen, A. Johnson, M. Krohn, T. Mulholland, K. Stenson, S.R. Wagner University of Florida, Gainesville, USA y D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Brinkerhoff, A. Carnes, M. Carver, D. Curry, S. Das, R.D. Field, I.K. Furic, J. Konigsberg, A. Korytov, J.F. Low, P. Ma, K. Matchev, H. Mei, G. Mitselmakher, D. Rank, L. Shchutska, D. Sperka, L. Thomas, J. Wang, S. Wang, J. Yelton Florida International University, Miami, USA S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez Florida State University, Tallahassee, USA A. Ackert, J.R. Adams, T. Adams, A. Askew, S. Bein, B. Diamond, S. Hagopian, V. Hagopian, K.F. Johnson, A. Khatiwada, H. Prosper, A. Santra Florida Institute of Technology, Melbourne, USA M.M. Baarmand, V. Bhopatkar, S. Colafranceschi67, M. Hohlmann, D. Noonan, T. Roy, F. Yumiceva Fairﬁeld University, Fairﬁeld, USA D. Winn Fermi National Accelerator Laboratory, Batavia, USA y, , S. Abdullin, M. Albrow, G. Apollinari, S. Banerjee, L.A.T. Bauerdick, A. Beretvas, J. Berryhill, P.C. Bhat, G. Bolla, K. Burkett, J.N. Butler, H.W.K. Cheung, F. Chlebana, S. Cihangir†, M. Cremonesi, V.D. Elvira, I. Fisk, J. Freeman, E. Gottschalk, L. Gray, D. Green, S. Gr¨unendahl, O. Gutsche, D. Hare, R.M. Harris, S. Hasegawa, J. Hirschauer, Z. Hu, B. Jayatilaka, S. Jindariani, M. Johnson, U. Joshi, B. Klima, B. Kreis, S. Lammel, J. Linacre, D. Lincoln, R. Lipton, M. Liu, T. Liu, R. Lopes De S´a, J. Lykken, K. Maeshima, N. Magini, J.M. Marrafﬁno, S. Maruyama, D. Mason, P. McBride, P. Merkel, S. Mrenna, S. Nahn, C. Newman-Holmes†, V. O’Dell, K. Pedro, O. Prokofyev, G. Rakness, L. Ristori, E. Sexton-Kennedy, A. Soha, W.J. Spalding, L. Spiegel, S. Stoynev, J. Strait, N. Strobbe, L. Taylor, S. Tkaczyk, N.V. Tran, L. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal, M. Wang, H.A. Weber, A. Whitbeck, Y. Wu University of Florida, Gainesville, USA Kansas State University, Manhattan, USA A. Ivanov, K. Kaadze, Y. Maravin, A. Mohammadi, L.K. Saini, N. Skhirtladze, S. Toda A. Ivanov, K. Kaadze, Y. Maravin, A. Mohammadi, L.K. Sain Lawrence Livermore National Laboratory, Livermore, USA F. Rebassoo, D. Wright Massachusetts Institute of Technology, Cambridge, USA gy g D. Abercrombie, B. Allen, A. Apyan, R. Barbieri, A. Baty, R. Bi, K. Bierwagen, S. Brandt, W. Busza, I.A. Cali, Z. Demiragli, L. Di Matteo, G. Gomez Ceballos, M. Goncharov, D. Hsu, Y. Iiyama, G.M. Innocenti, M. Klute, D. Kovalskyi, K. Krajczar, Y.S. Lai, Y.-J. Lee, A. Levin, P.D. Luckey, B. Maier, A.C. Marini, C. Mcginn, C. Mironov, S. Narayanan, X. Niu, C. Paus, C. Roland, G. Roland, J. Salfeld-Nebgen, G.S.F. Stephans, K. Sumorok, K. Tatar, M. Varma, D. Velicanu, J. Veverka, J. Wang, T.W. Wang, B. Wyslouch, M. Yang, V. Zhukova University of Minnesota, Minneapolis, USA A.C. Benvenuti, R.M. Chatterjee, A. Evans, A. Finkel, A. Gude, P. Hansen, S. Kalafut, S.C. Kao, Y. Kubota, Z. Lesko, J. Mans, S. Nourbakhsh, N. Ruckstuhl, R. Rusack, N. Tambe, J. Turkewitz i i f i i i i f d Lawrence Livermore National Laboratory, Livermore, USA F. Rebassoo, D. Wright University of Maryland, College Park, USA C. Anelli, A. Baden, O. Baron, A. Belloni, B. Calvert, S.C. Eno, C. Ferraioli, J.A. Gomez, N.J. Hadley, S. Jabeen, R.G. Kellogg, T. Kolberg, J. Kunkle, Y. Lu, A.C. Mignerey, F. Ricci-Tam, Y.H. Shin, A. Skuja, M.B. Tonjes, S.C. Tonwar Johns Hopkins University, Baltimore, USA J p y, , I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsan, P. Maksimovic, C. Martin, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. G g g P. Maksimovic, C. Martin, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You P. Maksimovic, C. Martin, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin 37 The University of Kansas, Lawrence, USA A. Al-bataineh, P. Baringer, A. Bean, S. Boren, J. Bowen, C. Bruner, J. Castle, L. Forthomme, R.P. Kenny III, S. Khalil, A. Kropivnitskaya, D. Majumder, W. Mcbrayer, M. Murray, S. Sanders, R. Stringer, J.D. Tapia Takaki, Q. Wang The University of Kansas, Lawrence, USA Kansas State University, Manhattan, USA University of Mississippi, Oxford, USA J.G. Acosta, S. Oliveros University of Nebraska-Lincoln, Lincoln, USA E. Avdeeva, R. Bartek, K. Bloom, D.R. Claes, A. Dominguez71, C. Fangmeier, R. Gonzalez Suarez, R. Kamalieddin, I. Kravchenko, A. Malta Rodrigues, F. Meier, J. Monroy, J.E. Siado, G.R. Snow, B. Stieger State University of New York at Buffalo, Buffalo, USA State University of New York at Buffalo, Buffalo, USA State University of New York at Buffalo, Buffalo, USA State University of New York at Buffalo, Buffalo, USA M. Alyari, J. Dolen, J. George, A. Godshalk, C. Harrington, I. Iashvili, J. Kaisen, A. Kharchilava, State University of New York at Buffalo, Buffalo, USA M. Alyari, J. Dolen, J. George, A. Godshalk, C. Harrington, I. Iashvili, J. Kaisen, A. Kharchilava, A. Kumar, A. Parker, S. Rappoccio, B. Roozbahani Northeastern University, Boston, USA G. Alverson, E. Barberis, A. Hortiangtham, A. Massironi, D.M. Morse, D. Nash, T. Orimoto, R. Teixeira De Lima, D. Trocino, R.-J. Wang, D. Wood Northeastern University, Boston, USA Northwestern University, Evanston, USA S. Bhattacharya, K.A. Hahn, A. Kubik, A. Kumar, N. Mucia, N. Odell, B. Pollack, M.H. Schmitt, K. Sung, M. Trovato, M. Velasco University of Notre Dame, Notre Dame, USA N. Dev, M. Hildreth, K. Hurtado Anampa, C. Jessop, D.J. Karmgard, N. Kellams, K. Lannon, N. Marinelli, F. Meng, C. Mueller, Y. Musienko36, M. Planer, A. Reinsvold, R. Ruchti, G. Smith, S. Taroni, M. Wayne, M. Wolf, A. Woodard 38 A The CMS Collaboration The Ohio State University, Columbus, USA J. Alimena, L. Antonelli, J. Brinson, B. Bylsma, L.S. Durkin, S. Flowers, B. Francis, A. Hart, C. Hill, R. Hughes, W. Ji, B. Liu, W. Luo, D. Puigh, B.L. Winer, H.W. Wulsin Princeton University, Princeton, USA S. Cooperstein, O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, D. Lange, J. Luo, D. Marlow, J. Mc Donald, T. Medvedeva, K. Mei, M. Mooney, J. Olsen, C. Palmer, P. Pirou´e, D. Stickland, A. Svyatkovskiy, C. Tully, A. Zuranski Princeton University, Princeton, USA S. Cooperstein, O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, D. Lange, J. Luo, D. M p , g , , J , , g , J , , J. Mc Donald, T. Medvedeva, K. Mei, M. Mooney, J. Olsen, C. Palmer, P. Pirou´e, D. Stickland, A. Svyatkovskiy, C. Tully, A. Zuranski Purdue University, West Lafayette, USA y, y , A. Barker, V.E. Barnes, S. Folgueras, L. Gutay, M.K. Jha, M. Jones, A.W. Jung, D.H. Miller, N. Neumeister, J.F. Schulte, X. Shi, J. Sun, F. Wang, W. Xie, L. Xu A. Barker, V.E. Barnes, S. Folgueras, L. Gutay, M.K. Jha, M. Jones, A.W. Jung, D.H N. Neumeister, J.F. Schulte, X. Shi, J. Sun, F. Wang, W. Xie, L. Xu N. Neumeister, J.F. Schulte, X. Shi, J. Sun, F. Wang, W. Xie, L. X Purdue University Calumet, Hammond, USA N. Parashar, J. Stupak University of Rochester, Rochester, USA University of Rochester, Rochester, USA B. Betchart, A. Bodek, P. de Barbaro, R. Demina, Y.t. Duh, T. Ferbel, M. Galanti, A. Garcia- Bellido, J. Han, O. Hindrichs, A. Khukhunaishvili, K.H. Lo, P. Tan, M. Verzetti y B. Betchart, A. Bodek, P. de Barbaro, R. Demina, Y.t. Duh, T. Ferbel, M. Galanti, A. Garcia- Bellido, J. Han, O. Hindrichs, A. Khukhunaishvili, K.H. Lo, P. Tan, M. Verzetti Rutgers, The State University of New Jersey, Piscataway, USA Rutgers, The State University of New Jersey, Piscataway, USA A. Agapitos, J.P. Chou, E. Contreras-Campana, Y. Gershtein, T.A. G´omez Espinosa, E. Halkiadakis, M. Heindl, D. Hidas, E. Hughes, S. Kaplan, R. Kunnawalkam Elayavalli, S. Kyriacou, A. Lath, K. Nash, H. Saka, S. Salur, S. Schnetzer, D. Shefﬁeld, S. Somalwar, R. Stone, S. Thomas, P. Thomassen, M. Walker , , , g , p , y , S. Kyriacou, A. Lath, K. Nash, H. Saka, S. Salur, S. Schnetzer, D. Shefﬁeld, S. Somalwar, R. Stone, S. Thomas, P. Thomassen, M. Walker University of Tennessee, Knoxville, USA A.G. Delannoy, M. Foerster, J. Heideman, G. Riley, K. Rose, S. Spanier, K. Thapa University of Tennessee, Knoxville, USA A.G. Delannoy, M. Foerster, J. Heideman, G. Riley, K. Rose, S. Spanier, K. Thapa Texas A&M University, College Station, USA O. Bouhali72, A. Celik, M. Dalchenko, M. De Mattia, A. Delgado, S. Dildick, R. Eusebi, J. Gilmore, T. Huang, E. Juska, T. Kamon73, R. Mueller, Y. Pakhotin, R. Patel, A. Perloff, L. Perni`e, D. Rathjens, A. Rose, A. Safonov, A. Tatarinov, K.A. Ulmer Rice University, Houston, USA Rice University, Houston, USA y A. Adair, B. Akgun, Z. Chen, K.M. Ecklund, F.J.M. Geurts, M. Guilbaud, W. Li, B. Michlin, M. Northup, B.P. Padley, R. Redjimi, J. Roberts, J. Rorie, Z. Tu, J. Zabel Moscow, Russia 15: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland p g 16: Also at RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany 16: Also at RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germa 17: Also at University of Hamburg, Hamburg, Germany 17: Also at University of Hamburg, Hamburg, Germany 18: Also at Brandenburg University of Technology, Cottbus, Germany 18: Also at Brandenburg University of Technology, Cottbus, Germany 19: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 19: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary g y 20: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University Budapest Hungary g y 20: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os 20: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University Budapest Hungary Texas Tech University, Lubbock, USA Texas Tech University, Lubbock, USA N. Akchurin, C. Cowden, J. Damgov, F. De Guio, C. Dragoiu, P.R. Dudero, J. Faulkner, E. Gurpinar, S. Kunori, K. Lamichhane, S.W. Lee, T. Libeiro, T. Peltola, S. Undleeb, I. Volobouev, Z. Wang Vanderbilt University, Nashville, USA S. Greene, A. Gurrola, R. Janjam, W. Johns, C. Maguire, A. Melo, H. Ni, P. Sheldon, S. Tuo, J. Velkovska, Q. Xu J. Velkovska, Q. Xu University of Virginia, Charlottesville, USA M.W. Arenton, P. Barria, B. Cox, J. Goodell, R. Hirosky, A. Ledovskoy, H. Li, C. Neu, T. Sinthuprasith, X. Sun, Y. Wang, E. Wolfe, F. Xia Wayne State University, Detroit, USA C. Clarke, R. Harr, P.E. Karchin, J. Sturdy Wayne State University, Detroit, USA C. Clarke, R. Harr, P.E. Karchin, J. Sturdy 39 University of Wisconsin - Madison, Madison, WI, USA D.A. Belknap, C. Caillol, S. Dasu, L. Dodd, S. Duric, B. Gomber, M. Grothe, M. Herndon, A. Herv´e, P. Klabbers, A. Lanaro, A. Levine, K. Long, R. Loveless, I. Ojalvo, T. Perry, G.A. Pierro, G. Polese, T. Ruggles, A. Savin, N. Smith, W.H. Smith, D. Taylor, N. 28: Also at Yazd University, Yazd, Iran 29: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran 29: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran University, Budapest, Hungary 21: Also at Institute of Physics, University of Debrecen, Debrecen, Hungary 21: Also at Institute of Physics, University of Debrecen, Debrecen, Hungary 22: Also at Indian Institute of Science Education and Research, Bhopal, India 22: Also at Indian Institute of Science Education and Research, Bhopal, Indi 23: Also at Institute of Physics, Bhubaneswar, India 23: Also at Institute of Physics, Bhubaneswar, India 24: Also at University of Visva-Bharati, Santiniketan, India 24: Also at University of Visva-Bharati, Santiniketan, India 25: Also at University of Ruhuna, Matara, Sri Lanka 25: Also at University of Ruhuna, Matara, Sri Lanka 26: Also at Isfahan University of Technology, Isfahan, Iran 26: Also at Isfahan University of Technology, Isfahan, Iran 27: Also at University of Tehran, Department of Engineering Science, Tehran, Iran 27: Also at University of Tehran, Department of Engineering S 28: Also at Yazd University, Yazd, Iran Texas Tech University, Lubbock, USA Woods 1: Also at Vienna University of Technology, Vienna, Austria 1: Also at Vienna University of Technology, Vienna, Austria 2: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China 2: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China 3: Also at Institut Pluridisciplinaire Hubert Curien (IPHC), Universit´e de Strasbourg, CNRS/IN2P3, Strasbourg, France 3: Also at Institut Pluridisciplinaire Hubert Curien (IPHC), Universit´e de Strasbourg, CNRS/IN2P3, Strasbourg, France 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 5: Also at Universidade Federal de Pelotas, Pelotas, Brazil 5: Also at Universidade Federal de Pelotas, Pelotas, Brazil 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 9: Also at Suez University, Suez, Egypt 10: Now at British University in Egypt, Cairo, Egypt 10: Now at British University in Egypt, Cairo, Egypt 11: Also at Ain Shams University, Cairo, Egypt y gyp 12: Also at Zewail City of Science and Technology, Zewail, Egypt y gy 13: Also at Universit´e de Haute Alsace, Mulhouse, France 13: Also at Universit´e de Haute Alsace, Mulhouse, France 14: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 14: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 14: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia University, Tehran, Iran Lebedev Physical Institute, Moscow, Russia 41: Also at California Institute of Technology, Pasadena, USA 41: Also at California Institute of Technology, Pasadena, USA gy 42: Also at Budker Institute of Nuclear Physics, Novosibirsk, Russia 42: Also at Budker Institute of Nuclear Physics, Novosibirsk, Russia y 43: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 43: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia y y y g g 44: Also at INFN Sezione di Roma; Universit`a di Roma, Roma, Italy 44: Also at INFN Sezione di Roma; Universit`a di Roma, Roma, Italy y 45: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia 45: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia g 46: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy g 46: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy y 47: Also at National and Kapodistrian University of Athens, Athens, Greece 47: Also at National and Kapodistrian University of Athens, Athens, Greec 48: Also at Riga Technical University, Riga, Latvia 48: Also at Riga Technical University, Riga, Latvia 49: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 49: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 50: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 50: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 51: Also at Adiyaman University, Adiyaman, Turkey 51: Also at Adiyaman University, Adiyaman, Turkey 52: Also at Mersin University, Mersin, Turkey 52: Also at Mersin University, Mersin, Turkey 53: Also at Cag University, Mersin, Turkey University, Tehran, Iran 30: Also at Universit`a degli Studi di Siena, Siena, Italy 30: Also at Universit`a degli Studi di Siena, Siena, Italy 31: Also at Purdue University, West Lafayette, USA 31: Also at Purdue University, West Lafayette, USA y y 2: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 33: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 33: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 34: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexic 4: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 35: Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Poland 35: Also at Warsaw University of Technology, Institute of Electronic System 36: Also at Institute for Nuclear Research, Moscow, Russia 37: Now at National Research Nuclear University ’Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia 37: Now at National Research Nuclear University ’Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia ( ) 38: Also at St. Petersburg State Polytechnical University, St. Petersburg, Russia 38: Also at St. Petersburg State Polytechnical University, St. Petersburg, Russia A The CMS Collaboration 40 39: Also at University of Florida, Gainesville, USA y 40: Also at P.N. Lebedev Physical Institute, Moscow, Russia 40: Also at P.N. 54: Also at Piri Reis University, Istanbul, Turkey 54: Also at Piri Reis University, Istanbul, Turkey 54: Also at Piri Reis University, Istanbul, Turkey 55: Also at Gaziosmanpasa University, Tokat, Turkey 55: Also at Gaziosmanpasa University, Tokat, Turkey 56: Also at Ozyegin University, Istanbul, Turkey 56: Also at Ozyegin University, Istanbul, Turkey 57: Also at Izmir Institute of Technology, Izmir, Turkey 57: Also at Izmir Institute of Technology, Izmir, Turkey 58: Also at Marmara University, Istanbul, Turkey 58: Also at Marmara University, Istanbul, Turkey 59: Also at Kafkas University, Kars, Turkey 60: Also at Istanbul Bilgi University, Istanbul, Turkey 60: Also at Istanbul Bilgi University, Istanbul, Turkey 61: Also at Yildiz Technical University, Istanbul, Turkey 61: Also at Yildiz Technical University, Istanbul, Turkey 62: Also at Hacettepe University, Ankara, Turkey 62: Also at Hacettepe University, Ankara, Turkey 63: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 63: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 64: Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdom United Kingdom 65: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain Also at Utah Valley University, Orem, USA 67: Also at Facolt`a Ingegneria, Universit`a di Roma, Roma, Ita 69: Also at Erzincan University, Erzincan, Turkey 69: Also at Erzincan University, Erzincan, Turkey y y 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey y y 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 71: Now at The Catholic University of America, Washington, USA 71: Now at The Catholic University of America, Washington, USA 72: Also at Texas A&M University at Qatar, Doha, Qatar 72: Also at Texas A&M University at Qatar, Doha, Qatar 73: Also at Kyungpook National University, Daegu, Korea 73: Also at Kyungpook National University, Daegu, Korea
https://openalex.org/W4394880213	https://revistes.ub.edu/index.php/matter/article/download/46480/41623	English	null	The Archive as a World-Making Apparatus in the Anthropocene	Matter	2,024	cc-by	8,880	MATTER journal of new materialist research Universitat de Barcelona ARTICLE ISSUE 9 The Archive as a World-Making Apparatus in the Anthropocene El archivo como aparato creador de mundos en el Antropoceno L'arxiu com a aparell que fa el món a l'antropocè Gabriela Galati (0000-0002-6126-9455) Nuova Accademia di Belle Arti, Milan, Italy Date of submission: September 2022 Accepted in: January 2024 Published in: April 2024 DOI: https://doi.org/10.1344/jnmr.v9.46480 Recommended citation: Galati, Gabriela (2024). The Archive as a World-Making Apparatus in the Anthropocene. Matter: Journal of New Materialist Research, 9, 1-14. University of Barcelona. https://doi.org/10.1344/jnmr.v9.46480 The texts published in this journal are – unless otherwise indicated – covered by the Creat Commons Spain Attribution 4.0 International licence. The full text of the licence can be consu here:http://creativecommons.org/licenses/by/4.0/ Date of submission: September 2022 Accepted in: January 2024 Published in: April 2024 DOI: https://doi.org/10.1344/jnmr.v9.46480 Recommended citation: Galati, Gabriela (2024). The Archive as a World-Making Apparatus in the Anthropocene. Matter: Journal of New Materialist Research, 9, 1-14. University of Barcelona. htt //d i /10 1344/j 9 46480 The texts published in this journal are – unless otherwise indicated – covered by the Creative Commons Spain Attribution 4.0 International licence. The full text of the licence can be consulted here:http://creativecommons.org/licenses/by/4.0/ 1 2024, Gabriela Galati 1 2024, Gabriela Galati 1 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9 (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) Resumen Este texto tiene como objetivo explorar el archivo como una fuerza poderosa que da forma a la vida. En consecuencia, busca desarrollar un marco ético para el archivo desde una perspectiva feminista. La de-extinción puede vincularse al archivo como un aparato estabilizador y un escenario de responsabilidad (Wolfe, 2018) que implica nuestro compromiso ético de reconocer la radical pasividad de aquellos que ya no existen en este mundo, tanto como individuos como especies. Este escenario de responsabilidad está entrelazado con la agencia del archivo. En otras palabras, el archivo actúa como un aparato creador de vida al dar forma a las condiciones para la lectura, el futuro y, en cierta medida, la realidad misma. Al establecer los fundamentos para una ética del archivo como una práctica que crea vida, este texto tiene como objetivo reformular el discurso en torno a la extinción y la de-extinción. Palabras clave Archivo-des-extinción; Antropoceno; precaridad; contraapocalipsis. Keywords Archive-de-extinction; Anthropocene; precartity; counterapocalypse. Abstract This text aims to explore the archive as a powerful force that shapes life. Consequently, it seeks to develop an ethical framework for the archive from a feminist perspective. De-extinction can be linked to the archive as a stabilizing apparatus and a scene of responsibility (Wolfe 2018) that entails our ethical commitment to acknowledge the radical passivity of those who no longer exist in this world, both as individuals and species. This scene of responsibility is intertwined with the agency of the archive. In other words, the archive acts as a life-creating apparatus by shaping the conditions for reading, the future, and, to some extent, reality itself. By establishing the foundations for an ethics of the archive as a practice that creates life, this text aims to reframe the discourse surrounding extinction and de-extinction. Keywords Introduction In fact, the most tragic and characteristic event of the Anthropocene is to be in the midst, or at the beginning, depending on one’s point of view, of the Sixth Mass Extinction. What could make the difference, then, is the way one intends to cope with it and, eventually, find, if not solutions, ways of doing less harm to other species as well as our own. Focusing on a series of topics which range from ‘the animal question’ and mal d’archive (Derrida, 1996) to more recent discussions on extinction and de-extinction, the present work proposes that the archive be considered as a ‘a life-shaping force’ (Zylinska, 2017, p. 2), advancing an ethics of the archive from a feminist perspective. The central question to this work is: if we consider the archive as an apparatus that creates life—at least in part—can it then be a tool for understanding and changing our actions towards the living? In other words, would it be possible to advance an ethics of the archive? Focusing on a series of topics which range from ‘the animal question’ and mal d’archive (Derrida, 1996) to more recent discussions on extinction and de-extinction, the present work proposes that the archive be considered as a ‘a life-shaping force’ (Zylinska, 2017, p. 2), advancing an ethics of the archive from a feminist perspective. The central question to this work is: if we consider the archive as an apparatus that creates life—at least in part—can it then be a tool for understanding and changing our actions towards the living? In other words, would it be possible to advance an ethics of the archive? As Stefano Mancuso and others have pointed out, our time as a species on the planet is quite limited, just 300,000 years, compared to the average life-span of all living species on the planet that ranges from 3 to 5 million years (Mancuso, 2018, p. 29). Thus, to start considering that the planet has existed long before us and will continue to exist afterwards is a good way to try to move away from an anthropocentric view, to begin thinking in geological, non-human time as a first step toward perceiving human existence as just a tiny moment in the planet’s long history (Parikka, 2015; Zylinska, 2017). 1 For example, as Joseph Masco notes, ‘Donna Haraway has recently critiqued the Anthropocene, suggesting that it naturalizes a specific historical–political formation—capitalism—as the only human mode. She suggests, along with Jason Moore, that instead of Anthropocene, it should be Capitalocene—to mark the specifically destructive qualities of a petrochemical-based capitalist system, or perhaps the Chthulucene’ (Masco, 2018, p. 77). 2 According to Joanna Zylinska, it could be located in the eighteenth century, with the second Industrial Revolution, or by the mid-twentieth century (Zylinska, 2018, p. 4); or, according to Stefano Mancuso, the impact of human action on the environment began with the development of agriculture (Mancuso, 2019). Resum Aquest text té com a objectiu explorar l'arxiu com una força poderosa que dóna forma a la vida. En conseqüència, busca desenvolupar un marc ètic per a l'arxiu des d'una perspectiva feminista. La de-extinció pot vincular-se a l'arxiu com un aparell estabilitzador i un escenari de responsabilitat (Wolfe, 2018) que implica el nostre compromís ètic de reconèixer la radical passivitat d'aquells que ja no existeixen en aquest món, tant com a individus com a espècies. Aquest escenari de responsabilitat està entremesclat amb l'agència de l'arxiu. En altres paraules, l'arxiu actua com un aparell creador de vida en donar forma a les condicions per a la lectura, el futur i, en certa mesura, la realitat mateixa. En establir els fonaments per a una ètica de l'arxiu com una pràctica que crea vida, aquest text té com a objectiu reformular el discurs entorn de l'extinció i la de-extinció. Paraules clau Arxiu-des-extinció; Antropocè; precaritat; contraapocalipsi. Arxiu-des-extinció; Antropocè; precaritat; contraapocalipsi. 2 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) In fact, the most tragic and characteristic event of the Anthropocene is to be in the midst, or at the beginning, depending on one’s point of view, of the Sixth Mass Extinction. What could make the difference, then, is the way one intends to cope with it and, eventually, find, if not solutions, ways of doing less harm to other species as well as our own. Introduction It is precisely this type of anthropocentric vision of other species—in which the idea of the human species as the superior one is evidenced by the fact that the proof we would leave of our exceptionality is 3 2024, Gabriela Galati 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) passivity are what all sentient beings share and constitute the ground for rethinking humanity’s relationships with other animals and the foundational basis for a new ethics in this regard (Derrida 2006). Precarity, or passivity, introduces a common ground between all living beings on the basis of which a non-anthropocentric ethics, one of responsibility towards others, can be developed and shared. This common ground is the ‘feminist counterapocalypse’ that ‘promises liberation from the form of subjectivity pinned to a competitive, overachieving, and overreaching masculinity. It also prompts us all to ask: If unbridled progress is no longer an option, what kinds of coexistences and collaborations do we want to create in its aftermath?’ (Zylinska 2018, p. 59). not addressed to all future species, but only to the ‘dominant’ ones—which has brought us to the present situation in the first place. This is the kind of apocalyptic, masculinist posture when confronting the Anthropocene that Zylinska criticises in her short book, The End of Man. A Feminist Counterapocalypse (2018). Drawing on the work of Donna Haraway and Karen Barad, Zylinska proposes considering the concept of relationality as a more solid and compelling way to understand subjectivity. What relationality considers is a ‘prior existence of relations between clusters of matter and energy that temporarily stabilize for us humans into entities—on a molecular, cellular, and social level’ (p. 53). This approach avoids the typical masculinist view of the subject that ‘disinterestedly looks at the world as its possession and playground’ (p. 53). And, it can be added, to continue with Haraway’s ideas, it is a view from above, as a disembodied drone (the bird’s-eye view) which controls its property without physically mingling with it. Introduction In fact, Haraway’s idea of situatedness implies that, from a feminist stance, all knowledge and all vision are embodied and situated: ‘I would like a doctrine of embodied objectivity that accommodates paradoxical and critical feminist science projects: Feminist objectivity means quite simply situated knowledges.’ (Haraway, 1988, p. 581) Introduction This question acquires particular relevance in relation to recent and pressing discussions on the Anthropocene, the current geological era characterised by the irreversible effects of human action on a geological scale. There is no universal consensus in the academic community about the term Anthropocene (Parikka, 2015, p. 17), and several alternatives have been suggested1, nor is there consensus about when exactly to locate its inception2. However, there is scholarly agreement regarding the fact that ‘the human’s impact upon the geomorphological and biological setup of planet Earth has become both momentous and irreversible, via processes such as excavation, deforestation, urbanization, and globalization. It is also a period that is experiencing a mass extinction of various species as a result of anthropogenic factors’ (Zylinska, 2017, p. 93). This does not mean in any way adopting empty and aestheticising attitudes like that of MoMA curator Paola Antonelli who has repeatedly stated that ‘humans will inevitably become extinct due to environmental breakdown, but we have the power to design ourselves a “beautiful ending”’ (Pownall, 2019), or, in another version, ‘we can design a more elegant extinction in order to make sure that the next dominant species will remember us with respect’. It is hard to think of a more superficial way of saying it: talking about elegance or beauty in relation to the death and suffering of millions of species, including humans, seems like a lack of respect and, above all, a lack of intellectual and critical depth. Furthermore, when it comes to being ‘remembered with respect’ by future ‘dominant species’, Antonelli's example compares the human species with the dinosaur: ‘we talk about how small their brains were, so we talk about them with fear but not with respect’ (BBC, 2020). Extinction Christopher Preston, in The Synthetic Age, defines it is as follows: finitude we experience in our subjection to a radically ahuman technicity or mechanicity of language, a technicity that has profound consequences, of course, for what we too hastily think of as “our” concepts, which are therefore in an important sense not “ours” at all’ (2009, p. 88). Located at an extreme end of the interventionist spectrum, deextinctionists—or extinction reversalists—embrace the possibility of not just reorganizing ecosystems by moving species around but recreating extinct species so that lost biodiversity can be regained. It turns out that the same techniques now available in synthetic biology for building genomes can be put to use reconstructing the DNA of extinct animals. Extinction, these biologists propose, need not be forever after all. (2018, p. 94) So, Wolfe asks, ‘when a being, human or nonhuman, dies, what goes out of the world? When an entire species becomes extinct, what world leaves the world, the world we are left with? To begin to answer these questions is to realize that extinction, whatever else it may be, is never a generic event’ (2009, p. 88). These questions have twofold interest: Firstly, they make evident human responsibility in extinguishing a world that leaves this world—namely the world of the extinct species. Secondly, if de-extinction is going to be considered, conversely asking which worlds should be brought back has also an ethical valence. And, equally important: ‘What do beasts and men have in common?’ (Wolfe, 2018, p. 108). Derrida’s first answer to this question is very well known: they share the passivity that is made evident through the repositioning of Jeremy Bentham’s question ‘Can they suffer?’, namely being sentient beings, and sharing the same finitude of death (Derrida, 2006). But there is a further level of this passivity which Derrida explains in the seminar published as The Beast and the Sovereign I and II (2009; 2011) and that is briefly analysed in Wolfe’s article: Death is something that is not given to us to know—it is always something that happens to the others3. Derrida points out that to be dead means ‘to be delivered over’ to someone who will decide what to do of my remains. This is the scene of responsibility of the ones who remain: The responsibility towards the radical passivity of the dead (Derrida, 2011, p. 113). Ursula K. 3 As the epitaph Marcel Duchamp wrote for himself, inscribed on his gravestone in Rouen, says: 'D'ailleurs, c'est toujours les autres qui meurent’ [Anyway, it is always the others who die]. Extinction Discussions of extinction inevitably lead to contemporary discussions of de-extinction and its relation to the archive. When asking what kind of event extinction is, Cary Wolfe asserts that it is both the ‘most natural thing in the world’ yet, [and] at the same time, it can never be natural (Wolfe, 2018, p. 107): if it is true that 99.9% of the species that have lived on the planet have become extinct, there is also hardly one thing we can still call ‘nature’. And this is due not only to the fact that ‘nature’ is already a cultural, an often human-centred idea, but also because the machinic apparatus through which ‘nature’ is conceptualised, namely language, is always already technological, at the same time that it is inherently ‘natural’ to humankind: this is the second kind of ‘passivity’ which, together with finitude, puts every living being on the same ontological plane: there is no nature/culture divide. (Derrida, 2006; Wolfe, 2009, p. 88). In Wolfe’s words: ‘The first type (physical vulnerability, embodiment, and eventually mortality) is paradoxically made unavailable, inappropriable, to us by the very thing that makes it available—namely, a second type of “passivity” or “not being able,” which is the p. 581) In opposition to the ‘masculinist posture’ explained above, Zylinska proposes a ‘counterapocalypse’ which poses an alternative and feminist approach to extinction, in particular, to move away from anthropocentric exceptionalism—the sort of view, like Antonelli’s, that considers humanity’s ability to go extinct ‘beautifully’ as an aesthetic choice that other living beings would not have. An alternative concept to thinking of a counterapocalypse could be Anna Lowenhaupt Tsing’s idea of precarity as ‘the condition of being vulnerable to others’, a ‘life without the promise of stability’ (2015, p. 20). I think precarity shares an evident point of contact with Jacques Derrida’s ideas of finitude and passivity. Both finitude and 4 4 2024, Gabriela Galati 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) So, what is de-extinction? Extinction Heise analyses three very different yet significative fiction projects in light of these de-extinction processes, including Steven Spielberg's Jurassic Park (1993), based on the novel by Michael Crichton (1990), and its sequel, The Lost World (1997). The author sees de-extinction, and the fantasies regarding it conveyed in these projects, as sophisticated ‘techno-fixes’ which, however, do not imply thinking critically about the factors that led to these circumstances in the first place—the most obvious being anthropocentrism (Heise, 2003, pp. 59–73). In fact, other authors, such as Wolfe, have related de-extinction to the archive (2018, p. 118) and, as I will argue below, de-extinction also seems to be closely related to mal d’archive, ‘archive fever’, full of destructive drives without which the archive would not exist, but which simultaneously threaten the archive constantly from within (Derrida, 1995). In 'Biocapitalism and De-extinction’, Ashley Dawson addresses the question: ‘What is to be done in response to the Sixth Extinction?’ (2018, p. 174) and, perhaps more interestingly, what is not to be done. Analysing science journalist Elisabeth Kolbert’s conclusions on the Sixth Extinction, Dawson deeply criticises Kolbert’s universalist view regarding responsibility for the Anthropocene. Kolbert’s account seems to assume that ‘the world’s flora and fauna cannot adapt to the De-Extinction 5 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) but also the engineering of new forms of life—which, as US lawyers have already begun to anticipate, ‘should be eligible for patenting’ (Dawson, 2018, p. 179). Thus, ‘de-extinction provides a mouthwatering opportunity for a new round of capital accumulation based on generating, and acquiring intellectual property rights over, living organisms’ (Dawson, 2018, p. 179). but also the engineering of new forms of life—which, as US lawyers have already begun to anticipate, ‘should be eligible for patenting’ (Dawson, 2018, p. 179). Thus, ‘de-extinction provides a mouthwatering opportunity for a new round of capital accumulation based on generating, and acquiring intellectual property rights over, living organisms’ (Dawson, 2018, p. 179). accelerated rate of change human beings are imposing on the world’ (Dawson, 2018, p. 174). De-extinction and the archive In this context, extinction emerges as a ‘new opportunity to capital for a new round of accumulation’ at the same time that it is ‘the leading edge of contemporary capitalism’s contradictions’: I would like to propose a complementary vision to Dawson’s on de-extinction by considering the archive’s agency or, in other words, the archive as a life-making practice. The questions that need to be asked, then, are: what kind of archive(s) do we want or do we need? What kind of archive(s) are we creating? If capital must expand at an ever-increasing rate or go into crisis, 'development’ is now consuming entire ecosystems and thereby threatening the planetary environment as a whole. The catastrophic rate of extinction today and the broader decline of biodiversity thus represent a direct threat to the reproduction of capital. Indeed, there is no clearer example of the tendency of capital accumulation to destroy its own conditions of reproduction than the sixth extinction. (Dawson, 2018, p. 176) To better analyse this, I will refer to the conception of the archive proposed by Derrida in two brief but dense articles which are instrumental for thinking about this topic: ‘Freud and the Scene of Writing’ in Writing and Difference (1967) and, more specifically, Archive Fever: A Freudian Impression (1995). To summarise Derrida’s conception on the archive, I will only point out to two central ideas from the aforementioned texts. There are two contradictory tendencies regarding the archive in Freudian theory. The first considers the archive as a prosthetic, technological and external memory. In this sense, there is a metaphysical return to the origin or original, which would be kept in this external prosthetic memory. This is exactly what Derrida intends to avoid. The second tendency has its root in the concept of ‘original repetition’, which turns the archive into ‘the origin exposed to the outside’ (Vergani, 2000, p. 109); it is thus ‘the non-origin that is original’ (Derrida, 1967, p. 303). This last conception indicates that the question of the archive is not only a question regarding memory and the past but it is more In other words, when consuming and destroying complete ecosystems in the name of ‘progress’, capitalism is also destroying the resources necessary for its own reproducibility. Extinction, then, becomes another possibility for capital accumulation through de-extinction or, as Dawson prefers to call it, re-genesis (2018, p. 177). Extinction Thus, granting responsibility for this event to all humanity at the same level: ‘Humanity is represented as unified and undifferentiated, as if we are all equally culpable for the current wave of extinction’ (p. 174). The result of this kind of reasoning is that, if everyone is guilty, no one is actually accountable. According to Dawson, this universal responsibility is at the base of the scapegoating, for instance, of indigenous people in Amazonia who are attributed the same level of responsibility for the climate crises as, say, oil extraction companies. On top of this, an obvious issue is how problematic it is to re-insert extinct species back into the same ecosystems in which they became extinct in the first place. De-extinction and the archive Derrida dedicates the first half of the conference lecture to conceptualise the characteristics of the archive in detail. In the first place, he establishes that the only meaning of the word ‘archive’ has to do with its ‘domiciliation’: As is the case for the Latin archivum or archium (a word that is used in the singular, as was the French archive, formerly employed as a masculine singular: un archive), the meaning of 'archive,' its only meaning, comes to it from the Greek arkheion: initially a house, a domicile, an address, the residence of the superior magistrates, the archons, those who commanded. (1995, p. 2) (1995, p. 2) So, in this sense, the archive takes place in a clear location, in a home, in a certain address. This permanent address is what signs the passage from private to public: the possibility of finding the archive, of acceding to it, of knowing that it is in that place and not in another, of its becoming public, or shared. Almost thirty years later, in Archive Fever: A Freudian Impression, Derrida offers a slightly more literal reflection on the topic of the archive. The publication is based on a conference lecture that he gave at the Freud Museum in London in 1994, and the issue that Derrida actually addresses in Archive Fever is the implication of Freudian theory for the conceptualisation of a new archive, namely, the one enabled by the computer and digital technologies—and also of Freud’s Museum as an archive—, of the unconscious as archive, and archive fever (mal d’archive) itself. In the second place, Derrida stresses what he calls the ‘power of consignation’, not in the sense of depositing or consigning something, but in the sense of ‘gathering together signs’: Consignation aims to coordinate a single corpus, in a system or a synchrony in which all the elements articulate the unity of an ideal configuration. In an archive, there should not be any absolute dissociation, any heterogeneity or secret which could separate (secernere), or partition, in an absolute manner. The archontic principle of the archive is also a principle of consignation, that is, of gathering together. (1995, p. 3) Mal d’archive is described, then, as the (unconscious) double tendency, guided by the death drive inhabiting any subject to a greater or lesser measure, to save, register, remember, keep everything—every trauma— in order to repeat it. De-extinction and the archive The new developments in biotechnologies are, in fact, increasing humanity’s godlike capacities, as Freud anticipated long ago (1929). At the same time, they are adapting life to the ‘dictates of corporate profit’ (Dawson, 2018, p. 178)—because these new capacities not only include the re-genesis of extinguished species 6 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) the archive from within as the same impulse to conserve is ultimately the drive that will try to knock down everything from within. What permits the archive to exist, its very conditions of possibility, are the seeds of its own destruction: the archive always works, a priori, against itself (Derrida, 1995, p. 14). the archive from within as the same impulse to conserve is ultimately the drive that will try to knock down everything from within. What permits the archive to exist, its very conditions of possibility, are the seeds of its own destruction: the archive always works, a priori, against itself (Derrida, 1995, p. 14). importantly about the future. The archive links past experiences and mourning with the possibilities of what is yet to come (p. 110). Mourning here is intended in the sense that what is kept in the archive of the unconscious—which the subject would not be able to access if not by metonymic traces, through psychoanalysis or in the form of trauma—is the repressed Oedipus Complex, and thus it is the mourning of the acceptance of castration, of the impossibility for the subject to blend with her object of desire, the father or the mother (Laplanche-Pontalis, 1967). This intense love is the non-origin of a first time that will repeat in different, more or less neurotic forms throughout the subject’s entire life, but that is not the real first time, it is already a trace, an absence, a repetition. Past experiences, sometimes traumatic, will create future ones, which is the reason for the recursivity of the archive/unconscious. In this sense, the archive is alive, it is neither fixed nor determined and allows for creation and unpredictability. Its repetitions are not controllable because they are traces, they are pure différance. De-extinction and the archive Yet, somehow, hidden in the desire to hold onto things lies a second tendency towards erasing, losing, forgetting and destroying everything that was supposed to be kept safe. Thus, mal d’archive menaces 7 7 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) meaning’ is also structured, modified and determined by the archive’s logic, characteristics and structures (p. 18). meaning’ is also structured, modified and determined by the archive’s logic, characteristics and structures (p. 18). Interestingly enough, this aspect of the archive implies that an archive should have a certain coherence, follow a certain taxonomy. Yet this suggested guideline to order and read the archive, is nonetheless not a call to complete it, as it should not dissociate (the user?) ‘in an absolute manner’. The relatively thematic looseness of the archive must also leave room for a great deal of creativity in both its creation and its actualisation. In a certain way, and of course with a very different vocabulary, Derrida already foresees what is going to be theorised as the advent of the posthuman, that is to say, the emergence of a different subjectivity—different from the ‘self-regulating subject of liberal humanism’ (Hayles, 1999, p. 86)—that has co-emerged in her interaction with digital technologies: Derrida questions the limit of the archive’s exteriorisation: if the archive, beginning with the print, is an externalisation of memory—a prosthetic memory in Freud’s terms—where does it begin? The archive is never completely external, even if its exteriorisation is determinant: ‘There is no archive without a place of consignation, without a technique of repetition, and without a certain exteriority. No archive without outside’ (Derrida, 1995, p. 11). Neither of these hypotheses can be reduced to the other. Because if the upheavals in progress affected the very structures of the psychic apparatus, for example in their spatial architecture and in their economy of speed, in their processing of spacing and of temporalization, it would be a question no longer of simple continuous progress in representation, in the representative value of the model, but rather of an entirely different logic. (Derrida, 1995, p. De-extinction and the archive But if it is examined more closely it will be found that its construction shows a remarkable agreement with my hypothetical structure of our perceptual apparatus and that it can in fact provide both an ever-ready receptive surface and permanent traces of the notes that have been made upon it. The Mystic Pad is a slab of dark brown resin or wax with a paper edging; over the slab is laid a thin transparent sheet, the top end of which is firmly secured to the slab while its bottom end rests upon it without being fixed to it. This transparent sheet is the more interesting part of the little device. It itself consists of two layers, which can be detached from each other except at their two ends. The upper layer is a transparent piece of celluloid; the lower layer is made of thin translucent waxed paper. When the apparatus is not in use, the lower surface of the waxed paper adheres lightly to the upper surface of the wax slab. If one wishes to destroy what has been written, all that is necessary is to raise the double covering-sheet from the wax slab by a light pull, starting from the free lower end. […]. The Mystic Pad is now clear of writing and ready to receive fresh notes. […] If we lift the entire covering-sheet–both the celluloid and the waxed paper–off the wax slab, the writing vanishes and, as I have already remarked, does not re-appear again. But it is easy to discover that the permanent trace of what was written is retained upon the wax slab itself and is legible in suitable lights. Thus the Pad provides not only a receptive surface that can be used over and over again, like a slate, but also permanent traces of what has been written, like an ordinary paper pad: it solves the problem of combining the two functions by dividing them between two separate but interrelated component parts or systems. But this is precisely the way in which, according to the hypothesis which I mentioned just now, our mental apparatus performs its perceptual function’ (Freud, 1925, p. 209–210). This is the power of the archive as a life-shaping force, thus its ethical valence becomes apparent, as it is further explained below. De-extinction and the archive 22) the screen, the letters remaining as if suspended and floating yet at the surface of a liquid element, I pushed a certain key to ‘save' a text undamaged, in a hard and lasting way, to protect marks from being erased, so as thus to ensure salvation and indemnity, to stock, to accumulate, and, in what is at once the same thing and something else, to make the sentence thus available for printing and for reprinting, for reproduction? (Derrida, 1995, p. 22) ‘Mystic Writing Pad’)4 (Derrida, 1967; 1995) seems also valid in this case as, even when it is 'erased' on the surface, traces are left on deeper layers: I asked myself what is the moment proper to the archive, if there is such a thing, the instant of archivization strictly speaking, which is not, […], so-called live or spontaneous memory (mneme or anamnesis), but rather a certain hypomnesic and prosthetic experience of the technical substrate. Was it not at this very instant that, having written something or other on In this sense, the archive can only exist as an event, as a constant actualisation and modification, as a block of notes on which one can comment, contribute, alter and consult, but which is continuously modifying one’s experience of it, and of its contents, as Derrida says, not only of its contents of past events, but also of the future. This is partly a risk, but also the main interest of an archive as event, of an archive that is, somehow, alive. 4 In the very short article ‘A Note upon the “Mystic Writing Pad”’ (1925), Freud compares the relationship between memory, perception, and the psychic apparatus with the Mystic Writing Pad, an ancestor of the more contemporary Magic Slate. In the article Freud states that prosthetic memory devices such as sheets of paper or slates either are emptied too soon, or are not permanent. This is not the way human memory, perception and the unconscious work, because they work in a more similar way to the Wunderblock (in German), or Mystic Writing Pad: ‘It claims to be nothing more than a writing-tablet from which notes can be erased by an easy movement of the hand. De-extinction and the archive 16) Most importantly, Derrida asks if the structure of the psychic apparatus, of the mind, of the unconscious as well as the conscious, and its relationship with memory and the perceived events or things, such as Freud had studied it, is different, better or worse represented, or influenced by the current techno-sciences of storage and reproduction (Derrida, 1995, p. 15). Even more interestingly to Derrida, the archive has a hypomnesic sense; it is not just memory, an external and auxiliary memory, but it is creative: it implies reflection, comments in the margins and constant possibilities of modification—it works, in fact, as a notebook. Moreover, Freud’s famous Wunderblock (the In part, the answer is yes; not in the sense of a better or worse influence, but in the sense of a definitive change in what the archive produces. As a prosthesis of memory, the archive is not only the place of its storage of the past, but it is also a projection to the future, there is no doubt that the archive gives shape to its object of storage, with its different structures, its different techniques and technologies: ‘The archivization produces as much as it records the event. This is also our political experience of the so-called news media’ (Derrida, 1995, p. 17). Derrida remarks that it is not so much that the archive determines what is conserved, ‘but rather the very institution of the archivable event’ (p. 18). Here again, it is possible to think about the archive as a construction of the future: one lives a present event according to how it is archived, and its meaning, its ‘archivable 8 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) the screen, the letters remaining as if suspended and floating yet at the surface of a liquid element, I pushed a certain key to ‘save' a text undamaged, in a hard and lasting way, to protect marks from being erased, so as thus to ensure salvation and indemnity, to stock, to accumulate, and, in what is at once the same thing and something else, to make the sentence thus available for printing and for reprinting, for reproduction? (Derrida, 1995, p. 5 See: Trout Fishing in America and Other stories (n. d.). snæbjörnsdóttir / wilson. https://snaebjornsdottirwilson.com/projects/trout-fishing-i n-america/trout-fishing-in-america-and-other-stories/ [accessed November 2023]. 5 See: Trout Fishing in America and Other stories (n. d.). snæbjörnsdóttir / wilson. https://snaebjornsdottirwilson.com/projects/trout-fishing-i n-america/trout-fishing-in-america-and-other-stories/ [accessed November 2023]. De-extinction and the archive Art, Extinction and the Construction of Counter-Apocalypses Wolfe has recently addressed the topic of de-extinction and the archive—albeit with a different focus. He analyses an artistic project by Bryndís Snæbjörnsdóttir and Mark Wilson called Trout Fishing in America and Other Stories (2015), in which the bodies of California condors—a species that has been de-extinct and reintroduced to its original habitat—are the protagonists of the work5, and are part of an archive. His focus in this analysis is on the archive as a ‘stabilizing apparatus’, as Derrida called it, on its legal 9 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) commodities, to feed on? Why do these deserve to be tortured, anonymised, forgotten? And the ‘extraordinary’ ones do not? aspects which are inevitably linked to questions of power: aspects which are inevitably linked to questions of power: to What better way to mark this fact [that the archive is linked to questions of power], in these images, than the strange cohabitation, within the same frame, the same 'place', of these singular dead animal bodies, subject to the laws of chemistry, decay, rigor mortis, and the like—'ultra-natural' objects, in that sense, whose decay we try to control through technological means—and what Derrida has called the machinalité of any semiotic code whose epitome is, of course, mathematics, here represented by the 'anonymous' numbers that mark each bird’s wing tag but only to become, in time, a kind of emotionally charged “proper name” for this particular creature—all of which redoubles and accumulates in the seriality of the photographic series itself. (Wolfe, 2018, p. 118) [The] animals who are deemed 'killable but not murderable'—the animals that sustain these carrion feeders—and those who, like the condor, are ‘rare,' ‘threatened,’ and ‘protected,' with the full backing of scientific and political apparatuses. The archive, in other words, may record the 'official story’ of body weight, reproductive rate, legal status, and so on, but it also actualizes something more, and in that other space, that other scene, we discover that the world is not given but made. We thus discover, in short, a scene of responsibility. (Wolfe, 2018, p. 120) Thus, this ‘scene of responsibility’ is twofold. De-extinction and the archive This period concluded with the occurrence of What is our responsibility regarding the animals we breed on an industrial scale, as 10 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) The Archive as a World-Making Apparatus in the Anthropoce Matter: Journal of New Materialist Research, 9th Issue (202 www.revistes.ub.edu/matter / ISSN: 2604-7551(1) Despite focusing on nature, the museum fails to acknowledge the presence of living beings within its premises, some of which may contradict its mission. the largest mass extinction event ever recorded. Notably, up to 96% of marine species and 70% of terrestrial vertebrate species faced extinction. Additionally, this event stands as the sole documented mass extinction of insects. Owing to this historical association, the local Regional Museum of Natural History predominantly showcases Permian fossils. the largest mass extinction event ever recorded. Notably, up to 96% of marine species and 70% of terrestrial vertebrate species faced extinction. Additionally, this event stands as the sole documented mass extinction of insects. Owing to this historical association, the local Regional Museum of Natural History predominantly showcases Permian fossils. As Yulia Glazryna, Head of the Natural History Department of the Perm Regional Museum, put it in her text for the book that accompanies the exhibition: I believe that this could also establish a global precedent: creating a new collection of insects without leaving the museum’s building. Axel’s idea of assembling the ‘victims’ of the museum’s pest extermination program within the walls of the museum itself may seem somewhat odd and even questionable: is it really acceptable for ‘surplus’ insects to reside in the collection? But the idea also presents the museum with very important questions. For example, are we really aware of our ‘neighbors’ and, in a more general sense, is it possible for anything on this planet to be ‘absolutely sterile’ (with the exception of the Large Hadron Collider, of course)? Where does the museum set the boundary between life and death, and what does it value: insects as display items or insects as living things? De-extinction and the archive And what does it mean to organize today’s world within the conditions of an urban ecosystem? The Permian Projects consists of two research projects: The Permian Collection and The Dioramas from the Perm Regional Museum that are the results of a residency program at the Perm Regional Museum in Russia, in which the artist was invited to participate in 2019. Taking the Museum’s natural history collection as a point of departure—a collection that ranges from paleontology, geology, botany, zoology, and entomology—Straschnoy’s intervention reflects, and invites the viewer to do so too, on the Permian Mass Extinction and, more broadly, on the complex yet topical issue of the Anthropocene. The Dioramas from the Perm Regional Museum consists in a series of lenticular, three-dimensional photographs showcasing some of the taxidermised animals of the Museum's collection, now in storage. Dioramas usually represent the animals in their natural habitats, most of which no longer exist because of extinction. As the dioramas were dismantled, different storage rooms and offices have now become the animals' natural biome. We are eternally grateful to Axel for challenging us to consider these issues, and in doing so to conceive of new doorways and paths for the future. (Glazryna, 2020, p. 12) We are eternally grateful to Axel for challenging us to consider these issues, and in doing so to conceive of new doorways and paths for the future. (Glazryna, 2020, p. 12) The second project, The Permian Collection, was inspired by the extermination of live insects that the Perm Museum conducts twice a year to protect the already dead insects of its entomological collection. At Straschnoy’s request, the Museum has now started collecting the insects it kills to protect its entomological collection and has created a new collection called the Perm Regional Museum's Insect Collection. These insects have been archived and catalogued like the other collections in the museum. Straschnoy has taken portraits of each of the insects producing a series of prints, and a book. De-extinction and the archive In the first place, there is the one that Wolfe proposes, namely, our responsibility related to the memory we are preserving of those who are no longer with us, of the killable animals, of the exceptional ones, like the California Condor, and of the living at large. In the second place, I would like to propose that the scene of responsibility is also related to the archive’s agency, that is to say, to the archive as a life-creating apparatus because the archive produces its conditions of possibility, of reading, of the future, as explained above; in simpler words, it shapes (a part of) reality. So there is a responsibility also regarding which kinds of archives we are creating: what kind of future will they construct, at least partially? This question is fundamental to the issue of de-extinction: what kind of future will the seed banks, or the DNA of extinct species help form? And again, for whom? And then to the question of adestination: for whom is the archive? For whom are we preserving the traces of the ones we are responsible for? (Wolfe, 2018, p. 120). This is our scene of responsibility, our ethical commitment to the radical passivity (that we share) of those who are not here anymore, namely, what we decide to do with their memory and remains. Derrida also notes how the biblical commandment ‘thou shalt not kill’ seems not to apply to nonhuman animals (2011, p. 104-105), and ‘does killing necessarily mean putting to death? Isn’t it also “letting die”?’ (Derrida, 2003, p. 108). Thus here, another fundamental question emerges, as Wolfe suggests: how do we decide which are the ‘extraordinary animals’ who deserve to be part of the archive, to be preserved, to be de-extinct, to have and create a future, and which do not? Which are the animals that we will ‘let die’, and which are the ones worth de-extinguishing? I consider that an artistic project that analyses most of what was exposed above (with an ironic twist) is Axel Straschnoy’s The Permian Projects, 2020. The Permian period, which derives its name from the Russian city of Perm, spanned approximately 300 to 250 million years ago. Conclusions It is in a work like Straschnoy’s that we can also clearly see the double logic of the mal d’archive, for which the very drive that spurred the museum’s desire to maintain its impressive entomological collection is the same one that leads them to kill all other living insects in the museum. 11 2024, Gabriela Galati 2024, Gabriela Galati The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) whom. Moreover, part of this responsibility implies being aware of the instability and impermanence of memory in an archive that is somehow alive, an archive with a logic of its own. In a broader sense, and regarding de-extinction, would it be far-fetched to advance the idea that the same logic that caused the destruction of entire ecosystems (the one cited above, as explained by Dawson, in terms of capitalist extractionism) and which has caused the extinction of millions of species, is the same one that now wants to de-extinguish them all using archives (of seeds, of animal DNA and the like.)? And then, there is still the ‘radical finitude’ shared by all animals (Derrida, 2006), which at the beginning of this article was also called ‘precarity’ (Tsing, 2015; Zylinska, 2018)—a trait inevitably linked to the archive and mal d’archive, and one that goes beyond the simple forgetfulness of repression because it is based on the inevitability of death shared by all living beings: ‘there would indeed be no archive desire without radical finitude, without the possibility of a forgetfulness which does not limit itself to a repression’ (Derrida, 1995, p. 19). To conclude, I want to propose that one possible way of assuming the responsibility that this double ‘scene of responsibility’ implies is to consider the ethical valence of the trace: for Derrida, ‘life protects itself by repetition, trace, différance (deferral)’; moreover, ’life must be thought of as trace before Being may be determined as presence’ (Derrida, 1967, p. 254–255). In order to start building an ethics of the archive, it is necessary to recognise that what mobilises our actions and the archive’s agency is an absence, différance, a recursive inscription whose sense is in constant revision and creation. As in the archive of the unconscious, what gives life and sense to the archive is something that is not there and has never been, pure différance. Conclusions And, at the same time, this sense is actualised retroactively, recursively, it is in constant creation, undergoing revision, parts are erased (though not completely) and rewritten, others remain, however partially, exactly as in the Wunderblock. Therefore, the trace, an absence, points to a shared responsibility in front of the ones who are no longer here: to the ways of preserving their memory, and for Because the desire to archive, to preserve is not only linked to the phenomenon of oblivion caused by repression, but is ultimately linked to vulnerability, to the precariousness of life which represents the awareness of the inevitability of death in which precisely oblivion will be total. It is therefore also in this sense that I propose to rethink extinction and de-extinction through the framework of an ethics of the archive, as briefly put forward here, with the aim of contributing to relevant previous and ongoing efforts to build counter-apocalypses; namely, to be engaged in a critical work that, even though it is incapable of avoiding a situation of extinction, is at least able to take responsibility for the archives that will survive it. Bibliography Dawson, Ashley (2018). Bio-capitalism and de-extinction. In Richard Grusin (Ed.), After extinction (pp. 173–200), University of Minnesota Press. Derrida, Jacques (1995). Archive fever: A Freudian impression. Trans. Eric Prenowitz, Chicago University Press. 12 The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) Derrida, Jacques (2003). Autoinmunity: Real and symbolic suicides. A dialogue with Jacques Derrida. In Borradori, Giovanna, Habermas, Jürgen, & Derrida, Jacques, Philosophy in a time of terror, University of Chicago Press. Derrida, Jacques (2006). L’animal que donc je suis. Galilée. Derrida, Jacques (2009; 2011). The Beast and the Sovereign. Trans. Geoffrey Bennington, University of Chicago Press. Freud, Sigmund (1950 [1925]). A note upon the ‘Mystic Writing Pad’. In The Standard Edition of the Complete Psychological Works of Sigmund Freud, trans. James Strachey & Anna Freud (pp. 227–232). Hogarth. Freud, Sigmund (1961 [1929]). Civilization and its discontents. Trans. James Strachey, W.W. Norton & Company Inc. Glazryna, Yulia (2020). Welcome to the Anthropocene! In Axel Straschnoy, The Permian Collection (pp. 4–9). Bombus. Haraway, Donna (1988). Situated knowledges: The science question in feminism and the privilege of partial perspective. In Feminist Studies, Vol. 14, No. 3 (Autumn), 575–599. Hayles, N. Katherine (1999). How we became posthuman: Virtual bodies in cybernetics, literature, and informatics. The Chicago University Press. Heise, Ursula K. (2003). From extinction to electronics: Dead frogs, live dinosaurs, and electric sheep. In Wolfe, C. (Ed.), Zoontologies. The question of the animal (pp. 59–82), University of Minnesota Press. Labarre, Suzanne (2019, August 1). MoMA curator: “[Humanity] will become extinct. We need to design an elegant ending”. Fast Company. https://www.fastcompany.com/90280777/moma-curator-we-will-become-extinct-we-need-to-design-an g g g p y https://www.fastcompany.com/90280777/moma-curator-we-will-become-extinct-we-need-to-design-an -elegant-ending Lowenhaupt Tsing, Anna (2015). The mushroom at the end of the world: On the possibility of life in capitalist ruins. Princeton University Press. Mancuso, Stefano (2019). La nazione delle piante. Laterza. Parikka, Jussi (2015). A geology of media. University of Minnesota Press. Pownall, Augusta (2019, February 22). "We don’t have the power to stop our extinction” says Paola Antonelli. dezzeen. https://www.dezeen.com/2019/02/22/paola-antonelli-extinction-milan-triennale-broken-nature-exhibitio n/ Serres, Michel (1997). Le parasite. Hachette. Snæbjörnsdóttir, Bryndís, Wilson, Mark (n.d.). Trout fishing in America and other stories. https://snaebjornsdottirwilson.com/projects/trout-fishing-in-america/trout-fishing-in-america-and-other- stories/ Why humanity should embrace its ‘inevitable’ extinction, (2020, January 20). BBC. https://www.bbc.com/reel/video/p080w0dg/why-humanity-should-embrace-its-inevitable-extinction Wolfe, Cary (Ed.). (2003). Zoontologies. The question of the animal. University of Minnesota Press. Wolfe, Cary (2010). What is posthumanism?. University of Minnesota Press. Wolfe, Cary (2018). The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) Zylinska, Joanna (2017). Nonhuman photography. The MIT Press. Zylinska, Joanna (2018). The end of men. A feminist counterapocalypse. University of Minnesota Press. The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) Zylinska, Joanna (2017). Nonhuman photography. The MIT Press. Zylinska, Joanna (2018). The end of men. A feminist counterapocalypse. University of Minnesota Press. The Archive as a World-Making Apparatus in the Anthropocene Matter: Journal of New Materialist Research, 9th Issue (2024) www.revistes.ub.edu/matter / ISSN: 2604-7551(1) Zylinska, Joanna (2017). Nonhuman photography. The MIT Press. Zylinska, Joanna (2018). The end of men. A feminist counterapocalypse. University of Minnesota Press. Author information Gabriela Galati (0000-0002-6126-9455) Bibliography Condors at the end of the world. In R. Grusin (Ed.), After extinction, University of Minnesota Press. Wolfe, Cary (2018). Condors at the end of the world. In R. Grusin (Ed.), After extinction, University of Minnesota Press. 13 2024, Gabriela Galati 13 2024, Gabriela Galati 13 2024, Gabriela Galati 13 13 2024, Gabriela Galati e-mail: gabriela.galati@icloud.com Dr. Gabriela Galati is professor of Theory and Methodology of Mass Media and Media Art Theory at NABA, Nuova Accademia di Belle Arti Milano and at IED, Turin, and of Aesthetics of New Media, Phenomenology of Contemporary Arts II and of Digital Cultures also in NABA. In the past she taught Research Methodology at the University of Buenos Aires, the University of the Argentine Social Museum and Media Art Theory at Domus Academy, Milan. She has published Duchamp Meets Turing: Art, modernism, posthuman (Postmedia Books, 2017), and (as Editor) the volume Complex ecologies: Thinking art beyond the human (Meltemi 2021). She writes reviews on art, philosophy, science and technology for Leonardo Reviews / Leonardo Journal (MIT Press), and regularly collaborates with AdVersus, Leonardo Journal, Acoustic Space Journal and Mimesis Scenari with scientific publications. She edited for Mimesis Scenari the issues # 10 and # 11 dedicated to art and posthuman theory, and the first Italian translation of the book The End of Man. A feminist counter-apocalypse by media theorist Joanna Zylinska (Rogas edizioni 2021). Galati is co-founder and director of the contemporary art gallery IPERCUBO. 14 2024, Gabriela Galati 2 14 2024, Gabriela Galati
https://openalex.org/W4309857876	https://rusjbpc.ru/en/storage/download/101800	Russian	null	SYMMETRY BREAKING IS THE PHYSICAL BASIS FOR THE PERFORMANCE OF "USEFUL WORK" BY BIOLOGICAL MOLECULAR MACHINES	Aktualʹnye voprosy biologičeskoj fiziki i himii	2,022	cc-by	4,557	Актуальные вопросы биологической физики и химии, 2022, том 7, № 4, с. 552-556 НАРУШЕНИЕ СИММЕТРИИ – ФИЗИЧЕСКАЯ ОСНОВА СОВЕРШЕНИЯ «ПОЛЕЗНОЙ РАБОТЫ» БИОЛОГИЧЕСКИМИ МОЛЕКУЛЯРНЫМИ МАШИНАМИ Твердислов В.А. Живое можно охарактеризовать как системную совокупность физических принципов, реализуемых химическим инструментарием и проявляющуюся в единстве биологических структур и функций, способных к устойчивой эволюции земных форм жизни. Основным биологическим признаком живого на Земле является клетка, основным физическим признаком – молекулярная машина. Принципы живого во Вселенной, по- видимому, универсальны, формы живого на Земле – уникальны. Живое можно охарактеризовать как системную совокупность физических принципов, реализуемых химическим инструментарием и проявляющуюся в единстве биологических структур и функций, способных к устойчивой эволюции земных форм жизни. Основным биологическим признаком живого на Земле является клетка, основным физическим признаком – молекулярная машина. Принципы живого во Вселенной, по- видимому, универсальны, формы живого на Земле – уникальны. Наряду с преобразованием форм энергии, присущим синергетическим процессам самоорганизации, существенную роль в живых системах выполняют симметрийные факторы – явления нарушения симметрии при эволюционном развитии систем. Живая природа базируется на двух фундаментальных асимметриях: клеточной – ионной и молекулярной – хиральной. Клеточным системам свойственна ионная асимметрия между внутриклеточной и внеклеточной средой, обеспечивающая термодинамическую неравновесность клеток, электрические явления и возбудимость. Для молекулярного уровня живого свойственна базовая гомохиральность и череда нарушений симметрии – смена знака хиральности при формировании внутримолекулярных и надмолекулярных клеточных структур. Наряду с преобразованием форм энергии, присущим синергетическим процессам самоорганизации, существенную роль в живых системах выполняют симметрийные факторы – явления нарушения симметрии при эволюционном развитии систем. Живая природа базируется на двух фундаментальных асимметриях: клеточной – ионной и молекулярной – хиральной. Клеточным системам свойственна ионная асимметрия между внутриклеточной и внеклеточной средой, обеспечивающая термодинамическую неравновесность клеток, электрические явления и возбудимость. Для молекулярного уровня живого свойственна базовая гомохиральность и череда нарушений симметрии – смена знака хиральности при формировании внутримолекулярных и надмолекулярных клеточных структур. О природе и сущности макроскопических и молекулярных машин. В неживой природе машин не существует, имеются лишь естественные преобразователи энергии и вещества, которые не могут совершать «полезной работы». Полезная работа – как раз и есть биологическая функция машин-ферментов, насосов, сократительных систем, рецепторов и пр. Следует отметить, что одними из первых разработок проблемы молекулярных биологических машин были работы наших соотечественников Ю.И. Хургина, С.Э. Шноля, Л.А. О природе и сущности макроскопических и молекулярных машин. В неживой природе машин не существует, имеются лишь естественные преобразователи энергии и вещества, которые не могут совершать «полезной работы». Полезная работа – как раз и есть биологическая функция машин-ферментов, насосов, сократительных систем, рецепторов и пр. Следует отметить, что одними из первых разработок проблемы молекулярных биологических машин были работы наших соотечественников Ю.И. Хургина, С.Э. Шноля, Л.А. НАРУШЕНИЕ СИММЕТРИИ – ФИЗИЧЕСКАЯ ОСНОВА СОВЕРШЕНИЯ «ПОЛЕЗНОЙ РАБОТЫ» БИОЛОГИЧЕСКИМИ МОЛЕКУЛЯРНЫМИ МАШИНАМИ Твердислов В.А. рд Московский государственный университет имени М.В. Ломоносова, ул. Ленинские Горы, 1, стр. 2, г. Москва, 119234, РФ; e-mail: tverdislov@mail.ru Поступила в редакцию 09.08.2022. DOI: 10.29039/rusjbpc.2022.0559 Аннотация. Физической основой функционирования живых систем являются молекулярные машины. Выполнение «полезной работы» составляет существо их биологических функций. Молекулярные машины являются хиральными иерархически организованными устройствами (конструкциями). Они циклически осуществляют преобразование формы энергии за счет смены или переключений симметрий в её хиральных структурных элементах, которые как раз и реализуют в них выделенные «квазимеханические» степени свободы. Феномен хиральности позволяет формировать дискретные хирально знакопеременные иерархии структур в макромолекулярных машинах в процессе фолдинга: начиная с уровня асимметричного углерода в дезоксирибозе и аминокислотах. Ранее нами была выявлена и проанализирована тенденция чередования знака хиральности внутримолекулярных структурных уровней D-L-D-L для ДНК и L-D-L-D для белков. Проявлениями хиральности выступают также спиральность и суперспиральность внутримолекулярных и надмолекулярных структур. Также, в рамках развиваемых представлений, хиральное расщепление свойств элементов структур обеспечивает однонаправленное движение машин по энергетическому циклу за счет нелинейных вентильных свойств спиральных структур. Спиральные структуры могут служить несимметричными, нелинейными, механическими, в том числе переключающими, элементами конструкций молекулярных машин (подобно устройству «храповик-собачка») по вращательным степеням свободы. лючевые слова: молекулярные машины, белки, термодинамика, симметрии, хиральность, моорганизация, нелинейность, фолдинг, спирали, суперспирали, вентильность. Со времён утверждения классической физики в биологии складывалось мнение о том, что в основе развития живых систем, включая биологическую эволюцию в целом, лежит, в первую очередь, энергетический фактор, поддерживающий процессы самоорганизации [1]. По-видимому, это не вся истина. Сами процессы самоорганизации, включающие упорядочивание в системе, с необходимостью включают обратные связи, пространственно канализирующие процессы диссипации энергии. Потоки и преобразования форм энергии неразрывно связаны с другим фундаментальным физическим фактором, геометрически характеризуемым особым качеством материи – симметрией. В последнее время в научном сообществе всё более укрепляется мнение, ранее утвердившееся в современной физике, о том, что понятие симметрии является значимым не столько в её проявлениях в структурах неживой и живой природы, сколько в фундаментальных представлениях о формировании и эволюции неживой и живой материи. Как известно, максимальной симметрией обладает изотропное бесконечное пространство, не способное выполнять функции машины, поскольку, в принципе, не обладает свойствами создавать «конструкции» [2]. Понижение энтропии в развивающихся сложных системах в ходе эволюции сопровождается понижением ранга симметрии специализированных, эволюционно отбираемых и фиксируемых элементов. Фактор симметрий в конечном счете определяет движение системы по «координате реакции», особенно на уровне супрамолекулярных конструкций, выполняющих функции молекулярных машин в живых системах. ОБЩАЯ БИОФИЗИКА ОБЩАЯ БИОФИЗИКА 552 GENERAL BIOPHYSICS 553 Блюменфельда, Д.С. Чернавского и других [2-4]. Для современного состояния общих представлений о биологических молекулярных машинах характерно разрозненное описание их структурных, термодинамических и кинетических характеристик [5-7]. Вместе с тем, рассмотрение симметрийных особенностей системы позволяет связать воедино означенные аспекты. Самые общие представления о молекулярных биологических машинах можно сформулировать следующим образом. Во-первых, это принципиально не тепловые, а изотермические электромеханохимические машины, характеризующиеся на уровне своих моторов КПД до 80% и выше, что по понятным причинам недостижимо для биополимерных устройств, будь они тепловыми машинами (разность температур между холодильником и нагревателем должна бы была составить многие сотни градусов). Во-вторых, имея молекулярные размеры, биологические машины находятся в условиях теплового шума, что принципиально затрудняет прецезирование (отслеживание) позиционного состояния деталей их конструкций в ходе движения по координате механохимической реакции в ходе машинного цикла [2,8]. По этой причине молекулярные машины работают, в отличие от макроскопических машин, в релаксационном режиме, фиксируя начальную фазу цикла по «правильному» присоединению к ним всех участников процесса и конечное состояние системы по завершении цикла. Траектория перехода между крайними состояниями определяется конструкцией устройства. Перманентное прецезирование системы в условиях тепловых флуктуаций в молекулярном устройстве потребовало бы подключения внешнего резервуара свободной энергии, существенно превышающего масштабы собственных преобразований энергии молекулярной машиной. Макроскопические же машины контролируют прохождение машиной рабочего цикла или непрерывно, или по многим позициям, что возможно в макромашинах, поскольку процессы контроля и управления в них по энергетической стоимости существенно ниже, чем реализуемые ими преобразования энергии, вещества и информации. р у р р р ф р Молекулярные моторы – энергетические преобразователи, двигатели машин – являются ключевым элементом конструкции молекулярных машин. В молекулярных машинах, в отличие от макроскопических, «рабочее тело», передающее и преобразующее формы энергии в соответствии с конструкцией устройства, идентично их конструктивным элементам [9]. Более того, в них не локализованы, а структурно сопряжены управляющий (регулирующий) и исполнительный уровни функционирования. По-видимому, из-за того, что в молекулярных машинах на микроуровне в условиях теплового шума, как мы говорили, энергетически не реализуем режим прецезирования, то система запуска цикла функционирует в «ждущем режиме», а сам цикл осуществляется как релаксационный процесс [2,7]. Запуск, например, ферментативного цикла, осуществляется при присоединении полного числа участников процесса – субстрата и эффекторов. Так, для запуска транспортного цикла мембранного Na-насоса (Na,K-АТФазы) к ферменту должны «правильно» и в нужном порядке присоединиться MgАТФ, 3 иона Na, 2 иона K, только после чего насос-машина проведет весь белковый комплекс по «координате реакции» – сложной релаксационной последовательности структурных (конформационных) перестроек и переходов [10]. НАРУШЕНИЕ СИММЕТРИИ – ФИЗИЧЕСКАЯ ОСНОВА СОВЕРШЕНИЯ «ПОЛЕЗНОЙ РАБОТЫ» БИОЛОГИЧЕСКИМИ МОЛЕКУЛЯРНЫМИ МАШИНАМИ Твердислов В.А. Актуальные вопросы биологической физики и химии, 2022, том 7, № 4, с. 552-556 Russian Journal of Biological Physics and Chemistry, 2022, vol. 7, No. 4, pp. 552-556 GENERAL BIOPHYSICS детерминированная, которую задаёт симметрийность. Случайные блуждания характерны для неупорядоченных элементов пептидной цепи, а макроскопические структурированные элементы в своих движениях детерминированы, поскольку, в наших представлениях, есть конструктивно заданное релаксационное движение при фолдинге и при работе машины. В одном случае – к нативной квазистационарной структуре при самоукладке – фолдинге, а во втором случае - от «возбужденного» состояния из неравновесных конформаций – к тому же уровню, но уже с другой стороны, по координате химической реакции. Это уже не химия, а механохимия. Ранее мы высказывали соображение о том, что формирование α-спиралей и суперспиралей, образованных их взаимным скручиванием, с физической точки зрения можно рассматривать как процесс автоволновой самоорганизации в активных средах [11,15,16]. Распределенный ресурс энергии по энтропийной компоненте создаётся исходной гомохиральностью первичной аминокислотной структуры – пептидной цепи, созданной с затратой энергии (АТФ) т-РНК при отборе левых аминокислот из присутствующей в клеточной среде смеси левых и правых аминокислот. Понижая свободную энергию системы и повышая энтропию, левая аминокислотная цепочка скручивается в правую α-спираль. Водородные же связи стабилизируют правую спиральную структуру (энтальпийный компонент). Далее процесс скручивания продолжается, уже на уровне суперспиралей, уже с правым знаком, что ещё более понижает уровень энергии и повышает энтропию. В этом примере наглядно видно, как явления смены/нарушения типа симметрии напрямую связаны с преобразованием энергии. Важно отметить, что формирование регулярных структур в белковых цепях существенным образом связано с их движением в вязкой среде в процессе фолдинга при выходе из рибосомного туннеля [17]. В принципе, подобный подход может быть использован и для описания функционирования нуклеиновых кислот, иерархически также хирально знакопеременных [11], но в данной работе мы не предполагаем обсуждать данный аспект их фолдинга и функционирования. Сейчас речь шла о фолдинге. Но ещё более впечатляющим представляется соображение о важности симметрийного фактора уже в работе белковых молекул в качестве молекулярных машин. Чуть более 100 лет назад в 1918 году Эмми Нётер доказала систему теорем о том, что законам сохранения соответствуют различного рода симметрии (см. [18]). На сегодняшний день это стало целым направлением теоретической физики. Вместе с тем, нам не известны случаи применения этих соображений к работе машин вообще, начиная с тепловых, и вплоть до молекулярных. Другое дело, что, к примеру, наш великий соотечественник физик Н.А. Умов, обсуждая работу паровоза, отмечал, что машина «умеет» понижать энтропию в системе за счет сторонних сил (энергии). Молекулы пара движутся хаотично, а конструкция цилиндров и поршней переводит движение в направленное. Теперь в теории машин можно перейти к формулировкам Нётер – машины понижают ранг симметрии. Актуальные вопросы биологической физики и химии, 2022, том 7, № 4, с. 552-556 GENERAL BIOPHYSICS Цикличность как принципиальная характеристика машины с необходимостью предполагает обязательное наличие симметрий в конструкции, а для движения по циклу в «правильном» направлении – несимметричного элемента, обладающего свойством «вентиля», то есть конструкции типа «храповик – собачка» [8]. Спиральные конструкции, по нашим представлениям, способны быть нелинейными вентильными устройствами по вращательным степеням свободы. Простейшее механическое соображение: плотно завитая пружинка легко раскручивается в одну сторону, но резко тормозит закручивание в другую. В макромолекулах они должны представлять собой макроскопические конструкции, поскольку должны характеризоваться долгоживущими состояниями по сравнению с тепловыми. Молекулярная машины являются иерархическими динамическими устройствами, циклически сопрягающими преобразование формы энергии, необходимое для совершения полезной работы, и череду преобразований симметрии в конструктивных элементах («рабочем теле»), составляющих «выделенные (квази)механическими степенями свободы» при самосборке и переключающих их при функционировании. Ранее нами была сформулирована и обоснована концепция, согласно которой, физической основой молекулярной биологии и молекулярных машин является периодическая система связанных симметрическими соотношениями знакопеременных хиральных структур во внутри- и надмолекулярных иерархиях структур важнейших классов биомакромолекул. Выявлена общая закономерность: начиная с уровня асимметричного углерода в дезоксирибозе и аминокислотах, прослежена тенденция чередования знака хиральности внутримолекулярных структурных уровней D-L-D-L для ДНК и L-D-L-D для белков [11-13]. Проявлениями хиральности выступают также спиральность и суперспиральность структур. Составленное этими иерархиями ядро молекулярной биологии интегрально составляет уже ахиральный инвариант, а сами эти структуры непосредственно реализуют выделенные механические степени свободы молекулярных машин. Кстати, напомним, что описанное явление чередования хиральных уровней при структурообразовании в гомохиральных системах есть общее их свойство, характерное и для искусственных полимерных систем, а не только биополимерных [11-13]. Левинталь перешел от двумерной потенциальной ямы, принятой как образ в представлениях квантовой механики, к трехмерной модели [11-14]. Но, по существу, потенциальная ловушка Левинталя – многомерное понятие, т.к. многие физические факторы определяют её характеристики. Один из них –хиральность, дополненная электрическими несимметриями. В физической трактовке ловушки Левинталя имеется заведомая двоякость: есть стохастическая компонента движения по воронке за счет флуктуаций и блужданий, а есть Russian Journal of Biological Physics and Chemistry, 2022, vol. 7, No. 4, pp. 552-556 ОБЩАЯ БИОФИЗИКА 554 детерминированная, которую задаёт симметрийность. Случайные блуждания характерны для неупорядоченных элементов пептидной цепи, а макроскопические структурированные элементы в своих движениях детерминированы, поскольку, в наших представлениях, есть конструктивно заданное релаксационное движение при фолдинге и при работе машины. В одном случае – к нативной квазистационарной структуре при самоукладке – фолдинге, а во втором случае - от «возбужденного» состояния из неравновесных конформаций – к тому же уровню, но уже с другой стороны, по координате химической реакции. Это уже не химия, а механохимия. GENERAL BIOPHYSICS Хаотическое движение молекул пара – высшая симметрия, а движение паровоза есть вектор. В целом же о машинах, в частности о молекулярных машинах, которые для производства любой полезной работы обязательно преобразуют форму энергии, можно сказать, что они с необходимостью делают это сопряженно с последовательным нарушением типа симметрии. Применительно к молекулярным машинам мы можем привязать последовательное преобразование форм энергии к цепочке нарушений симметрии посредством смены типа симметрии и знака хиральности при движении по машинному циклу. Сразу подчеркнём, что любая машина – хиральный объект, поскольку цикл должен быть однонаправленным. Феномен хиральности позволяет формировать дискретные хирально знакопеременные иерархии структур в макромолекулярных машинах в процессе фолдинга, а также, в рамках развиваемых представлений, обеспечивает однонаправленное движение машин по энергетическому циклу за счет нелинейных вентильных свойств спиральных внутри- и надмолекулярных структур. Нелинейность вентиля-α-спирали не только в том, что несимметричны правые и левые вращения относительно хиральной спирали, но разнятся и физические причины нарушения симметрии: переключается механизм: водородные связи держат в одном направлении, а «механика» – в обратном. На данный момент актуален теоретический анализ вентильных свойств спиральных и суперспиральных структур белковых макромолекул. А сами белки-машины в силу их структурной неоднородности и иерархичности как механические системы следует отнести к неголономным системам, в которых, кроме геометрических, накладываются и кинематические связи, которые нельзя свести к геометрическим. Как известно, математическое описание динамики подобных систем вызывает значительные сложности, даже в относительно простых случаях. Наподобие того как скелет млекопитающих имеет подвижные нежесткие связи между костями, что в целом обеспечивает сложно организованное движение организма в пространстве, так и жесткими деталями конструкций молекулярных машин (α-спиралями, суперспиралями и β-структурами), соединёнными нерегулярными фрагментами пептидной цепи, обеспечивается скоординированная в пространстве и времени работа молекулярных машин. В этой связи представляются реальными представления о коллективном поведении квазикристаллической белковой глобулы или фибриллы в ходе фолдинга или машинного цикла («апериодический кристалл» в представлениях Э. Шредингера [19]). Аминокислотная гомохиральность первичной структуры создаёт распределённую в пространстве термодинамическую неравновесность за счет нарушения симметрии (энтропийный компонент запаса свободной энергии), а связывание лиганда, субстрата или лекарства с макромолекулой создаёт «энтальпийное возмущение» в ней и запускает релаксационный механизм машинного цикла. Рецепторы-мишени лекарственных препаратов также можно отнести к классу макромолекул-машин. Среди используемых в настоящее время лекарственных средств больше половины составляют хиральные препараты, а, в свою очередь, большая часть этих хиральных лекарств представляет собой рацемат. Более половины Актуальные вопросы биологической физики и химии, 2022, том 7, № 4, с. 552-556 GENERAL BIOPHYSICS 555 разрабатываемых за последние годы лекарств также состоят из хиральных молекул. Список литературы / References: Блюменфельд Л.А. Молекулярные машины живой клетки. Природа, 1981, № 6, с. 66-73. [Blumenfeld L.A. Molecular machines of the living cell. Nature, 1981, no. 6, pp. 66-73. (In Russ.)] p p y p p 7. Блюменфельд Л.А. Молекулярные машины живой клетки. Природа, 1981, № 6, с. 66-73. [Blumenfeld L.A. Molecular machines of the living cell. Nature, 1981, no. 6, pp. 66-73. (In Russ.)] g pp ( )] 8. Фейнман Р., Лейтон Р., Сэндс М. Фейнмановские лекции по физике. Либроком, 2017, т. 4, 264 с. [Feynman R., Layton R., Sands M. Feynman Lectures on Physics. Librocom, 2017, vol. 4, 264 p. (In Russ.)] g pp ( )] 8. Фейнман Р., Лейтон Р., Сэндс М. Фейнмановские лекции по физике. Либроком, 2017, т. 4, 264 с. [Feynman R., Layton R., Sands M. Feynman Lectures on Physics. Librocom, 2017, vol. 4, 264 p. (In Russ.)] , y , y y , , , p ( )] 9. Аветисов В.А. Молекулярные машины, и как их делать. [Avetisov V.A. Molecular machines and how to make them. https://www.youtube.com/watch?v=NWP1eaR9Zj4 (In Russ.)] 9. Аветисов В.А. Молекулярные машины, и как их делать. [Avetisov V.A. Molecular machines and how to make them. https://www.youtube.com/watch?v=NWP1eaR9Zj4 (In Russ.)] p y j ( )] 10. Твердислов В.А., Тихонов А.Н., Яковенко Л.В. Физические механизмы функционирования биологических мембран. Изд. МГУ, М.: 1987, 189 с. [Tverdislov V.A., Tikhonov A.N., Yakovenko L.V. Physical mechanisms of functioning of biological membranes. Ed. Moscow State University, Moscow: 1987, 189 p. (In Russ.)] 10. Твердислов В.А., Тихонов А.Н., Яковенко Л.В. Физические механизмы функционирования биологических мембран. Изд. МГУ, М.: 1987, 189 с. [Tverdislov V.A., Tikhonov A.N., Yakovenko L.V. Physical mechanisms of functioning of biological membranes. Ed. Moscow State University, Moscow: 1987, 189 p. (In Russ.)] f f g f g y p ( )] 11. Твердислов В.А., Малышко Е.В. О закономерностях спонтанного формирования структурных иерархий в хиральных системах неживой и живой природы. Успехи физических наук, 2019, т. 189, № 4, с. 375-385. [Tverdislov V.A., Malyshko E.V. On the patterns of spontaneous formation of structural hierarchies in chiral systems of inanimate and living nature. Uspekhi fizicheskikh nauk, 2019, vol. 189, no. 4, pp. 375-385. (In Russ.)] 11. Твердислов В.А., Малышко Е.В. О закономерностях спонтанного формирования структурных иерархий в хиральных системах неживой и живой природы. Успехи физических наук, 2019, т. 189, № 4, с. 375-385. [Tverdislov V.A., Malyshko E.V. On the patterns of spontaneous formation of structural hierarchies in chiral systems of inanimate and living nature. Russian Journal of Biological Physics and Chemistry, 2022, vol. 7, No. 4, pp. 552-556 Список литературы / References: Список литературы / References: 1. Пригожин И., Кондепуди Д. Современная термодинамика. От тепловых двигателей до диссипативных структур. М.: УРСС, 2002, 461 с. [Prigogine I., Kondepudi D. Modern thermodynamics. From heat engines to dissipative structures. M.: URSS, 2002, 461 p. (In Russ.)] Списо литературы / efe ences: 1. Пригожин И., Кондепуди Д. Современная термодинамика. От тепловых двигателей до диссипативных структур. М.: УРСС, 2002, 461 с. [Prigogine I., Kondepudi D. Modern thermodynamics. From heat engines to dissipative structures. M.: URSS, 2002, 461 p. (In Russ.)] 2. Блюменфельд Л.А. Проблемы биологической физики. Изд. 2-е. М.: Гл. ред. физ.-мат. лит.изд-ва «Наука», 1977, 336 с. [Blumenfeld L.A. Problems of biological physics. Ed. 2nd. M.: Ch. ed. Phys.-Math. lit.izd-va "Nauka", 1977, 336 p. (In Russ.)] 2. Блюменфельд Л.А. Проблемы биологической физики. Изд. 2-е. М.: Гл. ред. физ.-мат. лит.изд-ва «Наука», 1977, 336 с. [Blumenfeld L.A. Problems of biological physics. Ed. 2nd. M.: Ch. ed. Phys.-Math. lit.izd-va "Nauka", 1977, 336 p. (In Russ.)] 3. Шноль С.Э., Чернавский, Д.С., Хургин Ю.И. Молекула белка-фермента как механическая система. В сб. Колебательные процессы в биол. и хим. системах. М.: Наука, 1967, с. 42-50. [Shnol S.E., Chernavsky D.S., Khurgin Yu.I. The protein-enzyme molecule as a mechanical system. In comp. Oscillatory processes in biol. and chem. systems. M.: Nauka, 1967, pp. 42-50. (In Russ.)] , , pp ( )] 4. Чернавский Д.С., Чернавская Н.М. Белок - машина. Биологические макромолекулярные конструкции. М.: Янус-К, 1999, 256 с. [Chernavsky D.S., Chernavskaya N.M. Protein is a machine. Biological macromolecular structures. Moscow: Janus-K, 1999, 256 p. (In Russ.)] pp ( )] 4. Чернавский Д.С., Чернавская Н.М. Белок - машина. Биологические макромолекулярные конструкции. М.: Янус-К, 1999, 256 с. [Chernavsky D.S., Chernavskaya N.M. Protein is a machine. Biological macromolecular structures. Moscow: Janus-K, 1999, 256 p. (In Russ.)] 5. Drexler K. Eric Engines of Creation: The Coming Era of Nanotechnology. Anchor Books, New York, 1986. 6. Kolomeisky A.B. Motor Proteins and Molecular. Motors CRC Press Taylor & Francis Group 2015 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business, 195 p. 5. Drexler K. Eric Engines of Creation: The Coming Era of Nanotechnology. Anchor Books, New York, 1986. 6. Kolomeisky A.B. Motor Proteins and Molecular. Motors CRC Press Taylor & Francis Group 2015 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business, 195 p. p, p y p, , p 7. GENERAL BIOPHYSICS Хиральные препараты используют в лечении широкого спектра заболеваний, в том числе сердечно-сосудистых и желудочно-кишечных. Получение оптически чистых форм вещества является сложной и дорогостоящей задачей, однако их использование во многих случаях могло бы уменьшить дозировку и количество побочных действий препарата. Стереоспецифичность взаимодействия лекарства и молекулы-мишени важно учитывать при создании лекарственных препаратов, так как одна хиральная форма лекарственного препарата может обладать терапевтическим эффектом, а другая не усваиваться, быть менее активной или даже вызывать серьезные осложнения, являться токсичной. Стереоспецифичность взаимодействия лекарства и молекулы-мишени важно учитывать при создании лекарственных препаратов, так как одна хиральная форма лекарственного препарата может обладать терапевтическим эффектом, а другая не усваиваться, быть менее активной или даже вызывать серьезные осложнения, являться токсичной. Заключение. До сих пор остаётся без внятного общепринятого ответа давний вопрос, зачем природа сделала белки-ферменты и другие белки большими, тогда как биохимиками и молекулярными биологами принято считать достаточными комплементарные взаимодействия в активных центрах. Однако, ответ на этот вопрос имеется, причем ответ вразумительный и четкий. Универсальный принцип молекулярной биологии – комплементарность взаимодействий, обеспечивающая специфичность функционирования биомакромолекул, реально обеспечивает стадию распознавания («ключ – замок»), а функциональные стадии, к примеру, ферментативного цикла реализуются всей белковой макромолекулой – квазимеханическими внутри- и межмолекулярными конструкциями молекулярных машин, о которых шла речь в настоящей работе. В качестве одного из обоснований существования внутримолекулярных дальних взаимодействий в белковой глобуле имеет смысл вспомнить об аллостерической регуляции в ферментах [20] и о важности её взаимодействия с водным и ионным окружением [21]. ру [ ] С учетом высказанных выше соображений приведём следующее определение молекулярной машины. Молекулярная машина есть иерархическое устройство, циклически сопрягающее преобразование формы энергии, необходимое для совершения полезной работы, и череду преобразований или переключений симметрии в её регулярных структурных хиральных элементах, реализующих выделенные «квазимеханические» степени свободы и контролирующих движение системы по заданному направлению цикла. С учетом высказанных выше соображений приведём следующее определение молекулярной машины. Молекулярная машина есть иерархическое устройство, циклически сопрягающее преобразование формы энергии, необходимое для совершения полезной работы, и череду преобразований или переключений симметрии в её регулярных структурных хиральных элементах, реализующих выделенные «квазимеханические» степени свободы и контролирующих движение системы по заданному направлению цикла. Можно полагать, что с использованием новейших экспериментальных возможностей и новейшей вычислительной техники, новых моделей, алгоритмов и программ появляется возможность уже в скором времени изучить и понять тонкие детали внутримолекулярной динамики конкретных молекулярных машин – ферментов разных классов, мембранных насосов, сократительных систем, рецепторов-мишеней лекарств и пр. 12. Tverdislov V.A., Malyshko E.V. Chiral dualism as an instrument of hierarchical structure formation in molecular biology. Symmetry, 2020, vol. 12, no. 4, p. 587. Список литературы / References: [Schrodinger E What is life? Cambridge: University Press 1944 176 p (In Russ )] 100 years. Elements. https://elementy.ru/novosti_nauki/433257/Velikoy_teoreme_Emmi_Nyoter_100_let (In Russ.)] 19. Шрёдингер Э. Что такое жизнь? Физический аспект живой клетки, 3-е изд., Ижевск: РХД, 2002, 176 с. [Schrodinger E. What is life? Cambridge: University Press, 1944, 176 p. (In Russ.)] 19. Шрёдингер Э. Что такое жизнь? Физический аспект живой клетки, 3-е изд., Ижевск: РХД, 2002, 176 с. [Schrodinger E. What is life? Cambridge: University Press, 1944, 176 p. (In Russ.)] 20. Корниш-Боуден Э. Основы ферментативной кинетики. М.: Мир, 1979, 280 с. [Corn Fundamentals of enzymatic kinetics. M.: Mir, 1979, 280 p. (In Russ.)] 21. Уэй Т. Физические основы молекулярной биологии. Учебное пособие, Долгопрудный: Издательский дом «Интеллект», 2010, 363 с. [Way T. Physical foundations of molecular biology. Textbook, Dolgoprudny: Intellect Publishing House, 2010, 363 p. (In Russ.)] Список литературы / References: Uspekhi fizicheskikh nauk, 2019, vol. 189, no. 4, pp. 375-385. (In Russ.)] 12. Tverdislov V.A., Malyshko E.V. Chiral dualism as an instrument of hierarchical structure formation in molecular biology. Symmetry, 2020, vol. 12, no. 4, p. 587. Russian Journal of Biological Physics and Chemistry, 2022, vol. 7, No. 4, pp. 552-556 ОБЩАЯ БИОФИЗИКА 556 13. Малышко Е.В., Муртазина А.Р., Твердислов В.А. Хиральность как физическая основа иерархической периодизации структур биомакромолекул. Биофизика, 2020, т. 65, № 2, с. 213-218. [Malyshko E.V., Murtazina A.R., Tverdislov V.A. Chirality as a physical basis for hierarchical periodization of biomacromolecule structures. Biophysics, 2020, vol. 65, no. 2, pp. 213-218. (In Russ.)] 14. Финкельштейн А.В., Птицын О.Б. Физика белка: Курс лекций с цветными и стереоскопическими иллюстрациями и задачами. М.: КДУ, 2012, 524 с. [Finkelstein A.V., Ptitsyn O.B. Protein Physics: A course of lectures with color and stereoscopic illustrations and tasks M : KDU 2012 524 p (In Russ )] p p ( )] 15. Твердислов В.А., Малышко Е.В., Ильченко С.А. От автоволновых механизмов самоорганизации к молекулярным машинам. Известия Российской академии наук. Серия физическая, 2015, т. 79, № 3, с. 1728-1732. [Tverdislov V.A., Malyshko E.V., Ilchenko S.A. From autowave mechanisms of self-organization to molecular machines. News of the Russian Academy of Sciences. Physical series, 2015, vol. 79, no. 3, pp. 1728-1732. (In Russ.)] 16. Сидорова А.Э., Левашова Н.Т., Малышко Е.В., Твердислов В.А. Автоволновая самоорганизация в фолдинге белков. ВМУ, 2019, № 3, с. 3-14. [Sidorova A.E., Levashova N.T., Malyshko E.V., Tverdislov V.A. Autowave self-organization in protein folding. VMU, 2019, no. 3, pp. 3-14. (In Russ.)] g p g pp ( )] 17. Шайтан К.В. Эффекты скрытой симметрии в динамике линейных полимеров и биополимеров. Биофизика, 2022, т. 67, № 3, с. 492-515. [Shaitan K.V. Effects of hidden symmetry in the dynamics of linear polymers and biopolymers. Biophysics, 2022, vol. 67, no. 3, pp. 492-515. (In Russ.)] p y p y pp ( )] 18. Левин А. Великой теореме Эмми Нётер - 100 лет. Элементы. [Levin A. Emmy Noether's Great Theorem - 100 years. Elements. https://elementy.ru/novosti_nauki/433257/Velikoy_teoreme_Emmi_Nyoter_100_let (In Russ.)] 19 Ш ё Э Ч ? Ф й й 3 И РХД 2002 176 p y p y pp ( )] 18. Левин А. Великой теореме Эмми Нётер - 100 лет. Элементы. [Levin A. Emmy Noether's Great Theorem - 100 years. Elements. https://elementy.ru/novosti_nauki/433257/Velikoy_teoreme_Emmi_Nyoter_100_let (In Russ.)] 19. Шрёдингер Э. Что такое жизнь? Физический аспект живой клетки, 3-е изд., Ижевск: РХД, 2002, 176 с. Актуальные вопросы биологической физики и химии, 2022, том 7, № 4, с. 552-556 Key words: molecular machines, proteins, thermodynamics, symmetries, chirality, self-organizatio nonlinearity, folding, helices, supercoils, ventilarity. 13. Малышко Е.В., Муртазина А.Р., Твердислов В.А. Хиральность как физическая основа иерархической периодизации структур биомакромолекул. Биофизика, 2020, т. 65, № 2, с. 213-218. [Malyshko E.V., Murtazina A.R., Tverdislov V.A. Chirality as a physical basis for hierarchical periodization of biomacromolecule structures. Biophysics, 2020, vol. 65, no. 2, pp. 213-218. (In Russ.)] SYMMETRY BREAKING IS THE PHYSICAL BASIS FOR THE PERFORMANCE OF "USEFUL WORK" BY BIOLOGICAL MOLECULAR MACHINES Tverdislov V.A. M.V. Lomonosov Moscow State University st. Leninskie Gory, 1, building 2, Moscow, 119234, Russia; e-mail: tverdislov@mail.ru Received 09.08.2022. DOI: 10.29039/rusjbpc.2022.0559 Tverdislov V.A. M.V. Lomonosov Moscow State University st. Leninskie Gory, 1, building 2, Moscow, 119234, Russia; e-mail: tverdislov@mail.ru Received 09.08.2022. DOI: 10.29039/rusjbpc.2022.0559 Abstract. The physical basis for the functioning of living systems are molecular machines. The performance of "useful work" is the essence of their biological functions. Molecular machines are chiral hierarchically organized devices (constructions). They cyclically transform the form of energy by changing or switching symmetries in its chiral structural elements, which just realize the selected “quasi-mechanical” degrees of freedom in them. The phenomenon of chirality allows the formation of discrete chirally sign- alternating hierarchies of structures in macromolecular machines in the process of folding: starting from the level of asymmetric carbon in deoxyribose and amino acids. Previously, we have identified and analyzed the tendency of alternation of the sign of chirality of the intramolecular structural levels D-L-D-L for DNA and L-D-L-D for proteins. Helicity and superhelicity of intramolecular and supramolecular structures are also manifestations of chirality. Also, within the framework of the developed ideas, the chiral splitting of the properties of the elements of the structures ensures the unidirectional movement of machines along the energy cycle due to the nonlinear valve properties of the spiral structures. Spiral structures can serve as asymmetric, non-linear, mechanical, including switching, structural elements of molecular machines (like a ratchet-pawl device) in terms of rotational degrees of freedom. Актуальные вопросы биологической физики и химии, 2022, том 7, № 4, с. 552-556
https://openalex.org/W2804486098	https://akjournals.com/downloadpdf/journals/650/159/22/article-p870.pdf	Hungarian	null	A levoszimendán perioperatív alkalmazása a szívsebészetben. <i>Magyar ajánlás</i>	Orvosi hetilap	2,018	cc-by	5,804	EREDETI KÖZLEMÉNY EREDETI KÖZLEMÉNY A levoszimendán perioperatív alkalmazása a szívsebészetben Magyar ajánlás Szudi László dr.1 ■ Székely László dr.2 ■ Sápi Erzsébet dr.3 Prodán Zsolt dr.3 ■ Szolnoky Jenő dr.4 ■ Csomós Ákos dr.4 Nyolczas Noémi dr.4 ■ Paulovich Erzsébet dr.5 ■ Németh Endre dr.5 Hartyánszky István dr.6 ■ Zima Endre dr.6 ■ Sax Balázs dr.6 Bertalan Andrea dr.7 ■ Hejjel László dr.7 ■ Bogáts Gábor dr.8 Babik Barna dr.9 ■ Gombocz Károly dr.10 ■ Szerafin Tamás dr.11 Koszta György dr.12 ■ Molnár Andrea dr.11 Gottsegen György Országos Kardiológiai Intézet, 1Központi Aneszteziológiai és Intenzív Terápiás Osztály, 2Szívsebészeti Osztály, 3Gyermek Szívgyógyászati Központ, Budapest 4Magyar Honvédség Egészségügyi Központ, Budapest Semmelweis Egyetem, Általános Orvostudományi Kar, 5Aneszteziológiai és Intenzív Terápiás Klinika, 6Városmajori Szív- és Érgyógyászati Klinika, Budapest 7Pécsi Tudományegyetem, Általános Orvostudományi Kar, Szívgyógyászati Klinika, Pécs Szegedi Tudományegyetem, Általános Orvostudományi Kar, 8II. Belgyógyászati Klinika Szívsebészeti Osztály, 9Klinikai Központ Aneszteziológiai és Intenzív Terápiás Intézet, Szeged 10Zala Megyei Szent Rafael Kórház, Zalaegerszeg Debreceni Egyetem, Általános Orvostudományi Kar, Klinikai Központ, 11Kardiológiai és Szívsebészeti Klinika, 12Aneszteziológiai és Intenzív Terápiás Tanszék, Debrecen Gottsegen György Országos Kardiológiai Intézet, 1Központi Aneszteziológiai és Intenzív Terápiás Osztály, 2Szívsebészeti Osztály, 3Gyermek Szívgyógyászati Központ, Budapest 4Magyar Honvédség Egészségügyi Központ, Budapest Semmelweis Egyetem, Általános Orvostudományi Kar, 5Aneszteziológiai és Intenzív Terápiás Klinika, 6Városmajori Szív- és Érgyógyászati Klinika, Budapest 7Pécsi Tudományegyetem, Általános Orvostudományi Kar, Szívgyógyászati Klinika, Pécs Szegedi Tudományegyetem, Általános Orvostudományi Kar, 8II. Belgyógyászati Klinika Szívsebészeti Osztály, 9Klinikai Központ Aneszteziológiai és Intenzív Terápiás Intézet, Szeged 10Zala Megyei Szent Rafael Kórház, Zalaegerszeg Debreceni Egyetem, Általános Orvostudományi Kar, Klinikai Központ, 11Kardiológiai és Szívsebészeti Klinika, 12Aneszteziológiai és Intenzív Terápiás Tanszék, Debrecen Az alacsony perctérfogat szindróma jelentősen emeli a szívműtétek szövődményeit és a halálozást, megnyújtja az in tenzív osztályos és kórházi tartózkodási időt. A kezelésére alkalmazott katecholaminterápiának nemkívánatos sziszté más és kardiális mellékhatásai lehetnek. A levoszimendán érzékenyebbé teszi a szívizom kalciumcsatornáit kalciumra, és megnyitja az adenozin-trifoszfát (ATP)-szenzitív káliumcsatornákat (KATP) is. Ennek köszönhetően javítja a szív teljesítményét, nem növeli a szívizom oxigénigényét, valamint védőhatást fejt ki a szívre és számos egyéb szervre is. A korábbiakban megjelent irodalom és szakértői vélemények alapján 2015-ben publikálták a szakértői véleményeket tartalmazó európai dokumentumot a levoszimendán szívsebészeti perioperatív alkalmazásáról. Ennek figyelembevé telével, továbbá a hét magyar szívcentrum és a gyermekszívcentrum (szívsebész, aneszteziológus és kardiológus képviselőinek) bevonásával kidolgoztuk a magyar ajánlást, melynek két meghatározó pillére van: az irodalmi eviden ciák és a magyar centrumok képviselőinek tapasztalatai. Az áttekintett területek: koszorúérműtétek, billentyűműté tek, keringéstámogató eszközök és szívtranszplantáció, mind felnőtt, mind gyermek szívsebészeti beavatkozások vo natkozásában. Orv Hetil. 2018; 159(22): 870–877. A levoszimendán perioperatív alkalmazása a szívsebészetben Kulcsszavak: alacsony perctérfogat szindróma, szívműtét, levoszimendán, kardioprotektív hatás Idegen szavak, rövidítések tív = műtét előtti időszak; Posztoperatív = műtét utáni időszak; Pulzatilitási index = (ASV – ADV) / AMV, ahol ASV: szisztolés áramlási sebesség, ADV: diasztolés áramlási sebesség, AMV: átlagos artériás áramlási sebesség Doppler-ultrahanggal mérve; Reperfúzió = újra meginduló véráramlás; SIRS = szisztémás gyulladásos válasz szindróma, a szervezet reakciója külső ag resszióra, amely a szepszishez hasonló képet ad, de nincs kór okozó; Szívindex = a testfelszínre vonatkoztatott, egy perc alatt továbbított vér; TAPSE = (tricuspidalis annular plane systolic excursion) szisztoléban a tricuspidalis annulus elmozdulása a szívcsúcs felé; TNFα = tumornekrózis-faktor, a gyulladásos válaszreakcióban játszik szerepet; VAD = (ventricular assist device) keringéstámogató eszköz; Vasodilatator = értágító gyógyszer; Vasopressor = érösszehúzó gyógyszer Antiinflammatorikus = gyulladás kialakulását gátló; Anti-stun ning = csökkenti annak valószínűségét, hogy az oxigénhiányos állapot után egyes szívizomrostok átmenetileg ne működjenek; Antiischaemiás = véd az oxigénhiányos állapottal szemben; ATP = adenozin-trifoszfát – a sejtek energiaigényes folyamata ihoz szükséges; BNP = B-típusú natriureticus peptid; CABG = coronariaartéria-bypassgraft; Citokinek = a sejtkommunikáció ban, így az immunválaszban jelentős szerepet játszanak; ECMO = extracorporalis membránoxigenizáció; EF = ejekciós frakció, hány százalékát löki ki a szív a diasztoléban befolyt vér nek; FAC = (fractional area change) a szív egy bizonyos met szetében a kamra területének százalékos változása szisztoléban- diasztoléban; Foszfodiészterázgátló = a foszfodiészteráz bontja a ciklikus adenozin-monofoszfátot (cAMP-t), s ennek gátlása javítja a szívizom-kontrakciót; Hibernált szívizom = életképes, de átmeneti oxigénhiány miatt nem működő szívizom; Hi poperfúzió = a szövetek nem kapnak elég vért az anyagcseréjük fenntartására; IABP = intraaorticus ballonpumpa, az arteria fe moralison felvezetett eszköz, amely a vért diasztoléban a coro nariák és az agy felé tereli az alsó testfél rovására; IL6 = interle ukin-6, citokin, a gyulladásos válaszreakcióban szerepe van; Inodilatator = fokozza a szívizom-összehúzódás erejét, és tá gítja a perifériás ereket; Inotrop támogatás = olyan gyógyszer, amely a szívizom összehúzódásának erejét fokozza; Ischaemi ás-reperfúziós károsodás = az oxigénhiány után újra induló keringés oxigén-szabadgyökök és más káros anyagok felszaba dulásával jár; Kalciumérzékenyítő = fokozza a szívizom érzé kenységét kalciumra, ezáltal alacsonyabb kalciumkoncentráció mellett jobb kontrakció lesz; Kamrai remodelling = megválto zik a kamra alakja, ezzel csökken az összehúzódás hatékonysá ga; Coronaria-bypassműtét = a szívkoszorúér szűkületét áthi daló műtét vénával vagy artériával; Luzitrop = a bal kamra ellazulását elősegítő gyógyszer; Metabolit = lebomlási termék; NYHA = (New York Heart Association) New York-i Szívbeteg séggel Foglalkozó Társaság; PCI = percutan coronariainter venció – a koszorúerek katéteres úton történő megnyitása; Perctérfogat = a szív által 1 perc alatt kilökött vér mennyisége; Perioperatív időszak = közvetlenül a műtét előtti, alatti és utáni időszak; Prekondicionáló = felkészíti a szívet az oxigénhiányos állapotra; Permeabilitás = a sejthártya átjárhatósága; Preopera Ellentétben az eseménytelen műtét utáni 1% körüli halá lozással, a szívműtétek perioperatív időszakában fellépő alacsony perctérfogat szindróma miatti halálozás a 16,9%-ot is elérheti [1]. (Beérkezett: 2018. február 9.; elfogadva: 2018. március 11.) Hungarian recommendation Low output syndrome significantly increases morbidity and mortality of cardiac surgery and lengthens the durations of intensive care unit and hospital stays. Its treatment by catecholamines can lead to undesirable systemic and cardiac complications. Levosimendan is a calcium sensitiser and adenosine triphosphate (ATP)-sensitive potassium channel (IK,ATP) opener agent. Due to these effects, it improves myocardium performance, does not influence adversely the 2018 ■ 159. évfolyam, 22. szám ■ 870–877. DOI: 10.1556/650.2018.31083 ■ © Szerző(k) 870 Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC EREDETI KÖZLEMÉNY balance between O2 supply and demand, and possesses cardioprotective and organ protective properties as well. Based on the scientific literature and experts’ opinions, a European recommendation was published on the periop erative use of levosimendan in cardiac surgery in 2015. Along this line, and also taking into consideration cardiac surgeon, anaesthesiologist and cardiologist representatives of the seven Hungarian heart centres and the children heart centre, the Hungarian recommendation has been formulated that is based on two pillars: literature evidence and Hungarian expert opinions. The reviewed fields are: coronary and valvular surgery, assist device implantation, heart transplantation both in adult and pediatric cardiologic practice. Keywords: low output syndrome, cardiac surgery, levosimendan, cardioprotective effect Szudi L, Székely L, Sápi E, Prodán Zs, Szolnoky J, Csomós Á, Nyolczas N, Paulovich E, Németh E, Hartyánszky I, Zima E, Sax B, Bertalan A, Hejjel L, Bogáts G, Babik B, Gombocz K, Szerafin T, Koszta Gy, Molnár A. [Periopera tive use of levosimendan in cardiac surgery. Hungarian recommendation]. Orv Hetil. 2018; 159(22): 870–877. Szudi L, Székely L, Sápi E, Prodán Zs, Szolnoky J, Csomós Á, Nyolczas N, Paulovich E, Németh E, Hartyánszky I, Zima E, Sax B, Bertalan A, Hejjel L, Bogáts G, Babik B, Gombocz K, Szerafin T, Koszta Gy, Molnár A. [Periopera tive use of levosimendan in cardiac surgery. Hungarian recommendation]. Orv Hetil. 2018; 159(22): 870–877. Idegen szavak, rövidítések Neurohormonális és antiinflammatorikus hatás Krónikus szívelégtelenségben a levoszimendán csökkenti a BNP-szintet, és párhuzamosan javítja a szív szisztolés és diasztolés funkcióit. Ilyenkor csökken a TNFα, az IL6 és a proapoptotikus faktorok (sFas- és Fas-ligand) szint je. Szepszisben és akut szívelégtelenségben (AHF) gátol ja a reaktív oxigéngyökök felszabadulását a granulo cytákból. Ezeken kívül javítja az endothelfunkciót, és ezáltal javítja a koszorúér-keringést is [12]. A szívműtétek során történő levoszimendánalkalma zás körülményei a vérvesztés, a folyadékterek közti gyors átrendeződés és a kialakuló SIRS miatt mások, mint a kardiológiai alkalmazások kapcsán. Emiatt célszerű a le voszimendán alkalmazását a szívműtétek körülményeire adaptálni, ami az indikációkat, a beadás módját és a mel lékhatások kezelését is érinti [10]. Mivel az egyes centru mok tapasztalata némileg eltérő, ezért szükségesnek lát tuk egy közös magyar ajánlás kidolgozását az európai mellett. Anti-stunning, antiischaemiás hatás Összességében a levoszimendán kedvező hatásában az inodilatator hatás mellett komoly szerepet játszik antiinf lammatorikus és szervprotektív hatása. Szívizom-„stunning” akkor történik, amikor az akut oxi génhiányt követően a keringés újraindulása átmenetileg károsítja a szívizom összehúzódó funkcióját. Az ismételt oxigénhiányos epizódoknak összegződő hatásuk van, amely kamradiszfunkcióhoz vezet. Több tanulmány bi zonyította, hogy levoszimendán alkalmazásakor akut co ronaria szindrómás és infarktusos betegeknél csökken a PCI utáni rosszul mozgó és nem mozgó szívizomszeg mensek száma [16–18]. Kísérletes körülmények között megelőzésképpen adva csökkentette az oxigénhiányos terület kiterjedését is [19]. A gyomormucosa oxigenizációja A levoszimendán farmakológiai prekondicionáló hatásá ért a mitokondriumok ATP-szenzitív K+-csatornáinak kinyitása lehet felelős, mely az ischaemiás-reperfúziós ká rosodást is mérsékli [13]. Ezzel összhangban állatkísérle tes modellekben az infarktus kiterjedése koszorúér-elzá ródás után csökkent [14, 15]. Schwarte és mtsai [24] a levoszimendánnak, a milrinon nak és a dobutaminnak a gyomormucosa oxigenizációjá ra kifejtett hatását hasonlították össze kutyákon. A levo szimendán jobban fokozta a bélmucosa oxigenizációját azonos O2-szállítás mellett, és nem növelte az O2-fo gyasztást. A levoszimendán farmakokinetikája A levoszimendán pozitív inotrop hatása a troponin-C- hez történő kötődésével és kalciumérzékenyítő hatásával magyarázható. Értágító tulajdonsága az érfalsimaizom felszíni membránja (sarcolemma) KATP-csatornáinak nyi tásával állhat összefüggésben. A sarcolemma és a mito kontrium ATP-szenzitív K+-csatornáinak aktiválásával csökkenti a Ca++ felszaporodását, stabilizálja a mitokond rium belső membránjának permeabilitását, ezáltal csök ken a sejthalál valószínűsége és az ischaemiás-reperfúziós károsodás mértéke [11]. A levoszimendán olyan citoki nek szintjét is csökkenti [12], melyek révén a kamrai re modelling mértéke is mérsékelhető. A fenti hatásokon kívül foszfodiészterázgátló hatással is rendelkezik. A vesefunkcióra gyakorolt hatás Bragadottir és mtsai [20] a levoszimendán és a dopamin veseműködésre gyakorolt hatását hasonlították össze. A dopamin csak a vese vérátáramlását növelte, de a glome rularis filtrációt nem befolyásolta. A levoszimendán nö velte a vese vérátáramlását, csökkentette a veseerek ellen állását, és növelte a glomerularis filtrációt is. Szívműtétek adatait vizsgáló metaanalízisben szignifikánsan csökken tette a dialízisigényt, és javította a vesefunkciót [9, 20– 22]. Májfunkció Alvarez és mtsai [23] huszonöt, alacsony perctérfogat szindrómás beteget randomizáltak dobutaminra és levo szimendánra. A levoszimendán jobban emelte a szívin dexet, a portalis keringést és a pulzatilitási indexet. A dobutamin csak a v. portae keringését fokozta, a levoszi mendán mind a v. portae, mind az a. hepatica keringését javította. Idegen szavak, rövidítések Szív-tüdő motorral fenntartott mesterséges keringés, úgynevezett extracorporalis kerin gés kapcsán előfordulhat, hogy a szövetek vérellátása nem kielégítő. Emellett szinte mindig kialakul valami lyen fokú gyulladásos reakció, amely szisztémás gyulla dásos válasz szindrómát (SIRS) és többszervi elégtelen séget is okozhat. Az utóbbiak között a műtét utáni veseelégtelenség a leggyakoribb [2, 3]. A perioperatív időszakban alkalmazott inotrop támo gatás javítja a balkamra-funkciót, ugyanakkor károsíthat ja az életképes, de hibernált szívizmot, és ritmuszavarok hoz vezet. Ez akár a halálozás növekedéséhez vezethet [4]. A kalciumérzékenyítő levoszimendánnak pozitív inotrop és értágító hatásai mellett szívvédő, prekondici onáló [5], antiischaemiás és anti-stunning [6] hatásai is vannak. A levoszimendán nem befolyásolja hátrányosan a szívizom O2 ellátás-felhasználás egyensúlyát [7]. Har rison és mtsai metaanalízisében rossz balkamra-funkciójú betegeknek a műtét előtt adva szignifikánsan csökkentet te a halálozást, a műtétet követő veseelégtelenséget és a 871 2018 ■ 159. évfolyam, 22. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC EREDETI KÖZLEMÉNY szívizom-károsodást [8]. A Bayesian-hálózat metaanalí zisben [9] a levoszimendán volt az egyetlen inodilatator, mely a halálozást csökkentette. Neurohormonális és antiinflammatorikus hatás A levoszimendán farmakodinamikája A levoszimendán fél életideje 1 óra. Telítő dózis nélkül az egyensúlyi koncentrációt 4 óra alatt éri el. Az infúzió megszüntetése után a plazma koncentrációja gyorsan esik, de van egy aktív metabolitja, az OR-1896, amely nek eliminációs fél életideje 80 óra. 24 órás infúzió után szívelégtelen betegekben az OR-1896 csúcskoncentráci óját körülbelül 48 óra múlva éri el, és két hét alatt tűnik 2018 ■ 159. évfolyam, 22. szám ORVOSI HETILAP 872 Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC EREDETI KÖZLEMÉNY el a keringésből. Hemodinamikai hatása körülbelül 1 hé tig tart. Szívműtétek során az aktív metabolit szintézise késik, csúcskoncentrációját a negyedik, ötödik napon éri el [10]. Harrison és mtsai [8] (14 tanulmány, 1155 beteg) analízisében az alacsony ejekciós frakciójú (EF) csoport ban a levoszimendánnak köszönhetően a halálozás szig nifikánsan csökkent (–7,0%, 95% CI –11,0%, –3,1%; p<0,001), míg a megtartott EF csoportjában nem volt szignifikáns különbség. A levoszimendánnal kezelt cso portban szintén szignifikánsan csökkent a dialízisigény, a műtét utáni pitvarfibrilláció előfordulási gyakorisága és a szívizom-károsodás mértéke. Módszer Az irodalmi áttekintést a PubMed, az Index Medicus, az Excerpta Medica, a Reference Update és a BIOSIS adat bázisából nyertük. Az anyaggyűjtést 2017. augusztussal zártuk. A humán tanulmányok közül a prospektív, ran domizált, kettős vaktanulmányokat és a metaanalíziseket vontuk be. Az egyes szerzők adatainak összehasonlítását nehezítette, hogy a levoszimendánt különböző módo kon és körülmények között alkalmazták. A szakértői csa pat aneszteziológusokból, szívsebészekből és kardioló gusokból állt, mind a hét magyar szívcentrum és a gyermekszívcentrum képviselőit magában foglalta. A konszenzuskonferencia után a szakmai anyagot végleges formájának elnyerése érdekében e-mailen többször kör beküldtük, és a felmerülő újabb észrevételeket a doku mentumba beépítettük. Greco és mtsai [9] Bayesian-hálózat analízist végeztek (46 tanulmány, 2647 beteg), és az inodilatatoroknak a szívsebészeti műtétek túlélésére gyakorolt hatását vizs gálták. A következő gyógyszereket hasonlították össze: dobutamin, enoximon, milrinon, levoszimendán. Csak a levoszimendán csökkentette a placebóval szemben a ha lálozást. Lim és mtsai [30] (14 tanulmány, 965 beteg) vizsgála tában, amelybe csak az alacsony EF-jú betegeket válogat ták be, levoszimendán hatására szignifikánsan (9,5%-ról 4,2%-ra) csökkent a korai mortalitás. Zhou és mtsai [31] (13 tanulmány, 1345 beteg) a mű tét utáni akut veseelégtelenséget, a vesepótló kezelés szükségességét, a lélegeztetés hosszát, az intenzív osztá lyos tartózkodási időt és a halálozást vizsgálták. A levo szimendánnal kezelt csoport eredményei minden egyes esetben jobbak voltak a kontrollcsoportéinál. A halálozás vonatkozásában az OR 0,41 (95% CI, 0,27–0,62; p<0,002) volt. Metaanalízisek Pollesello és mtsai [25] 2016-ban az addig megjelent 25 metaanalízist tekintették át, melyek több mint 6000 be teg adatait foglalják össze. Ebből 10 metaanalízis készült szívsebészeti beteganyagon. Nyolc végpontja a halálozás volt [9, 10, 26–31] és mindegyik statisztikailag szignifi káns rizikócsökkenést mutatott. Két metaanalízis vég pontja a veseelégtelenség volt [21, 22,], mindkét eset ben statisztikailag szignifikáns volt a rizikóredukció. Részletezve a metaanalíziseket: Niu és mtsai [21] (5 tanulmány, 529 beteg), valamint Bove és mtsai [22] (33 tanulmány, 3879 beteg) metaana líziseikben az akut veseelégtelenség előfordulását vizs gálták szívműtét után. A levoszimendáncsoport adatai mindkét analízisben szignifikánsan kedvezőbbek voltak a kontrolléinál. Niu: OR, 0,44; 95% CI, 0,22–0,85; p = 0,02; Bove: OR, 0,52; 95% CI, 0,32–0,86; p = 0,01. , ; , , ; , , , ; p , Az utóbbi időben megjelent három tanulmányban – CHEETAH [32], LEVO-CTS [33] és LICORN [34] – nem igazoltak szignifikáns mortalitáscsökkenést, de ezekben máshogy alkalmazták a levoszimendánt, mint az előző vizsgálatokban. A CHEETAH-tanulmány ban például a szívműtét után kialakuló alacsony perctér fogat kezelésére adták oly módon, hogy a standard terá piához adták hozzá vagy a levoszimendánt, vagy a placebót. Így nem tudták kihasználni a levoszimendán nak a szívre és az egyéb szervekre kifejtett védőhatását. Vélhetően ezért nem találtak különbséget a két csoport mortalitásában. A LEVO-CTS- és a LICORN-tanul mányban a levoszimendán alkalmazását 0,1 ug/kg/min dózisban az anesztézia indukciójánál kezdték, így a szer szintén nem fejthette ki szervvédő hatását olyan mérték ben, mint a korábban megjelent tanulmányokban. A LE VO-CTS-tanulmányban a rossz balkamra-funkcióval rendelkező koszorúérműtött betegek alcsoportjában még így is szignifikánsan jobb volt a halálozás. Mindhá rom tanulmány biztonságosnak találta a levoszimendán alkalmazását. Zangrillo és mtsai [26] készítették az első tanulmányt (139 beteg, 5 tanulmány), melynek elsődleges végpontja a posztoperatív troponinfelszabadulás volt. A levoszi mendáncsoportban szignifikánsan alacsonyabb volt a troponinszint. Landoni és mtsai [27] aktualizálták Zangrillóék analí zisét (440 beteg, 10 tanulmány). Megállapításuk szerint a levoszimendán szignifikánsan csökkentette a halálozást (12,7%-ról 4,7%-ra). Maharaj és mtsai [28] (17 tanulmány, 729 beteg) munkájában koszorúérműtek során a levoszimendán 11,4%-ról 4,9%-ra csökkentette a halálozást. Szintén szignifikánsan csökkent az intenzív osztályon eltöltött idő, a pitvarfibrillációk száma, a troponinfelszabadulás, és nőtt a szívindex. Maharaj és mtsai [28] (17 tanulmány, 729 beteg) munkájában koszorúérműtek során a levoszimendán 11,4%-ról 4,9%-ra csökkentette a halálozást. Szintén szignifikánsan csökkent az intenzív osztályon eltöltött idő, a pitvarfibrillációk száma, a troponinfelszabadulás, és nőtt a szívindex. Szakértői ajánlás A levoszimendán alkalmazásával kapcsolatban coronaria- bypassműtétek esetén található a legtöbb közlemény. A legjelentősebb evidencia az alacsony EF-jú betegek szív- tüdő motoron végzett koszorúérműtéteire vonatkozik. Levin és mtsai [35] 252 koszorúérbeteget randomizál tak, akiknek EF-értéke 25%-nál kisebb volt. Műtét előtt 10 ug/kg bolus dózist adtak 1 órán át, majd 0,1 ug/kg/ min infúziót 23 órán át. Ennek hatására szignifikánsan csökkent a halálozás (12,8%-ról 3,9%-ra), az intraaorti cus ballonpumpa (IABP) használata (30,4%-ról 6,3%- ra), valamint a második inotrop szer és a vasopressor használata is. Ezt erősíti Harrison és mtsai metaanalízise is [8], amelyben szignifikáns halálozási különbség csak az alacsony EF csoportjában volt a levoszimendán javára, a megtartott balkamra-funkciójú betegek esetében nem volt szignifikáns különbség. Az irodalmi adatok, a hét magyar szívcentrum és a gyer mekszívcentrum tapasztalatai alapján a magyar szakértői ajánlás a levoszimendán alkalmazására a következőkben foglalható össze: 1. A levoszimendán alkalmazása: CABG-műtétek ese tén, ha az EF≤25%, akkor javasolt, 25%<EF≤30% EF esetén megfontolandó, 30–35%-os EF esetén meg fontolható a kísérőbetegségek (vese, máj), a NYHA-stádium, az egyidejűleg fennálló egyéb szív betegségek (billentyűbetegségek, jobbkamra-funk ció) alapján. 2. Billentyű szűkülete esetén a levoszimendán az úgy nevezett „low flow low gradiens” aortaszűkület kap csán adható, de ilyenkor az fokozott elővigyázatos ságot igényel (volumenstátusz, vasopressor) a vérnyomásesés veszélyessége miatt. Ebben az eset ben szóba jön csak a műtőben történő alkalmazása. gi g Billentyű- és kombinált műtétek esetén kisebb számú beteganyag áll rendelkezésre. Lahtinen és mtsai [36] bil lentyűműtétek és kombinált koszorúér- és billentyűmű tétek esetén alkalmazták a levoszimendánt az anesztézia indukciója után. 24 ug/kg bolus dózist adtak 30 perc alatt, majd 0,2 ug/kg/min 24 órán át. A műtét utáni szívelégtelenség szignifikánsan ritkábban fordult elő a le voszimendáncsoportban (15%), mint a placebocsoport ban (58%). A levoszimendán alkalmazásával összefüg gésben szintén szignifikánsan kevesebb volt az IABP és más inotrop szer használata, de a halálozásban nem volt különbség. Aortabillentyű-szűkület műtéti megoldása során Jörgensen és mtsai [37] a levoszimendán pozitív lu zitrop hatását mutatták ki balkamra-hipertrófia esetén. Szívultrahanggal vizsgálva a betegeket, a levoszimendán csoportban minden diasztolés funkcióra vonatkozó ult rahangos paraméter szignifikánsan jobb volt, mint a pla cebocsoportban (izovolumetriás relaxációs idő, a korai diasztolés telődés decelerációs lejtőjének időtartama, a korai és késői diasztolés telődés csúcssebessége). Ezzel párhuzamosan nőtt a szív egy összehúzódásával kilökött vér mennyisége is. A mitralis billentyű és a koszorúér kombinált műtéteinél [38] a levoszimendánkezelést az IABP-vel összehasonlítva szignifikánsan kevesebb volt az intenzív osztályos tartózkodási idő (2,5, illetve 5 nap), de nem volt szignifikáns a halálozási különbség. 3. Szakértői ajánlás Aortabillentyű-elégtelenség esetén, ha az EF<35%, a levoszimendán alkalmazása javasolt. j 4. Mitralisbillentyű-elégtelenség fennállásakor a levo szimendán alkalmazása javasolt: a) ha funkcionális elégtelenség esetén az EF<35%; b) ha organikus az elégtelenség, vagy ha jobbkamra- diszfunkció, pulmonalis hipertónia áll fenn, és az EF 35–45% vagy kevesebb. b) ha organikus az elégtelenség, vagy ha jobbkamra- diszfunkció, pulmonalis hipertónia áll fenn, és az EF 35–45% vagy kevesebb. 5. Mitralisbillentyű-szűkület: súlyos jobbkamra-disz funkció esetén egyedi megfontolás alapján a levoszi mendánkezelés alkalmazható. 5. Mitralisbillentyű-szűkület: súlyos jobbkamra-disz funkció esetén egyedi megfontolás alapján a levoszi mendánkezelés alkalmazható. 6. Jobbkamra-elégtelenség és pulmonalis hipertónia kapcsán a levoszimendánkezelés szintén felmerül. Akár elsődleges a jobbkamra-diszfunkció, akár pul monalis hipertónia vagy balszívfél-elégtelenség kö vetkezménye, javasolt a levoszimendán adása, ha a) a TAPSE ≤12 mm; b) a TAPSE ≤16 mm, a FAC≤36%, és mindehhez súlyos tricuspidalisbillentyű-elégtelenség és csök kent jobbkamra-funkció társul; c) pulmonalis hipertónia áll fenn (pulmonalis közép nyomás ≥35 Hgmm), a pulmonalis érellenállás ≥3 Wood-egység, és egyidejűleg már csökkent a jobbkamra-funkció (jobb kamra ≥40 mm és/vagy súlyos tricuspidalis regurgitatio és/vagy TAPSE ≤16 mm). ) 7. Kombinált műtétek esetén a levoszimendánkezelés megfontolható egyéni rizikófelmérés alapján, amikor az előzőekben leírt kritériumok egyike sem áll fenn, de több rizikófaktor összeadódik. Például: közepe sen csökkent bal- és/vagy jobbkamra-funkció, súlyos kísérő betegségek (tüdő, vese, máj), 120 percnél hosszabb aortalefogás stb. ) 7. Kombinált műtétek esetén a levoszimendánkezelés megfontolható egyéni rizikófelmérés alapján, amikor az előzőekben leírt kritériumok egyike sem áll fenn, de több rizikófaktor összeadódik. Például: közepe sen csökkent bal- és/vagy jobbkamra-funkció, súlyos kísérő betegségek (tüdő, vese, máj), 120 percnél hosszabb aortalefogás stb. Metaanalízisek Hernández és mtsai [29] (13 tanulmány, 654 beteg) analízisében a levoszimendán szignifikáns mértékben, 12,6%-ról 5,2%-ra csökkentette a halálozást. Hernández és mtsai [29] (13 tanulmány, 654 beteg) analízisében a levoszimendán szignifikáns mértékben, 12,6%-ról 5,2%-ra csökkentette a halálozást. 2018 ■ 159. évfolyam, 22. szám 873 ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC EREDETI KÖZLEMÉNY Megbeszélés A magyar és az európai szakértői vélemény között van némi eltérés [10]: Az európai ajánlásban 35%-nál húzzák meg a rossz balkamra-funkciót, és az alatt koszorúérmű téteknél javasolják. A magyar javaslatban 25% EF szere pel, és 25–35% között a kezelőorvosra bízza a választást. Billentyűbetegségek esetén az európai javaslatban sokan neutrális álláspontot képviseltek, és százalékos arányban jelentették meg az eltérő véleményeket. Ez annak kö szönhető, hogy sok centrumnak Európában nincs ta pasztalata a levoszimendán alkalmazásával billentyűbe tegségek esetén. A magyar javaslatban viszont egyhangú volt a vélemény a levoszimendán alkalmazása mellett a fentebb részletezett kitételekkel. A monitorozásnál van még eltérés, az európai vélemény egyértelműen minden esetben invazív artériás nyomásmérést javasol. Mind az európai, mind a magyar javaslatban megfogalmazódnak a centrumok eltérő álláspontjai. Ezek még nem letisztul tak, érvek-ellenérvek vannak, úgy gondoltuk, hogy he lyes, ha megjelenítjük a még vitatott és még egyértelmű en nem bizonyított álláspontokat. 9. Szívtranszplantáció esetén levoszimendán műtét alatti és utáni adagolása megfontolandó. 9. Szívtranszplantáció esetén levoszimendán műtét alatti és utáni adagolása megfontolandó. 10. Gyermekkori adagolás: A gyermekszívcentrumban az indikációk hasonlóak a felnőttekéihez, de jelenleg Magyarországon a levoszimendán alkalmazása gyer mekkorban „off-label” kategóriának tekintett. Java soljuk, hogy gyermek- és csecsemőellátás esetén is törzskönyvezve legyen a levoszimendán Magyaror szágon. 10. Gyermekkori adagolás: A gyermekszívcentrumban az indikációk hasonlóak a felnőttekéihez, de jelenleg Magyarországon a levoszimendán alkalmazása gyer mekkorban „off-label” kategóriának tekintett. Java soljuk, hogy gyermek- és csecsemőellátás esetén is törzskönyvezve legyen a levoszimendán Magyaror szágon. 10. Gyermekkori adagolás: A gyermekszívcentrumban az indikációk hasonlóak a felnőttekéihez, de jelenleg Magyarországon a levoszimendán alkalmazása gyer mekkorban „off-label” kategóriának tekintett. Java soljuk, hogy gyermek- és csecsemőellátás esetén is törzskönyvezve legyen a levoszimendán Magyaror szágon. A mellékhatások elkerülése, kezelése 1. A volumenstátusz rendezése. 2. A vasodilatatio ellensúlyozására norepinefrint adjunk. 3. A K+-szintet tartsuk 4 mmol/liter felett a ritmuszava rok elkerülésére. Jobbkamra-funkció A levoszimendán javította a jobb kamra összehúzódásá nak erejét, és csökkentette a pulmonalis érellenállást [39]. Továbbá a levoszimendán javította a jobbkamra- funkciót a bal kamrát helyettesítő műszív beültetésekor is [40]. Levoszimendán-előkezelés mellett a bal kamrai műszívbeültetésen átesett betegek 20 hónapos túlélése javult [41]. 8. Bal kamrai keringéstámogató eszköz beültetésekor a jobb kamra támogatására megfontolandó a levoszi mendán adása. Mechanikus keringéstámogató esz 8. Bal kamrai keringéstámogató eszköz beültetésekor a jobb kamra támogatására megfontolandó a levoszi mendán adása. Mechanikus keringéstámogató esz 2018 ■ 159. évfolyam, 22. szám 874 ORVOSI HETILAP ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC EREDETI KÖZLEMÉNY közről (ECMO-ról, VAD-ról) történő leszoktatás kor szintén javasolt. Más gyógyszerekkel való kombinációja 1. Második inotrop szernek elsőként dobutamint ad junk, de ha kell, epinefrinnel vagy milrinonnal is kom binálhatjuk. Hármas kombináció nem javasolt. Két intézet nem javasolja a dopaminnal történő kombiná ciót. 1. Második inotrop szernek elsőként dobutamint ad junk, de ha kell, epinefrinnel vagy milrinonnal is kom binálhatjuk. Hármas kombináció nem javasolt. Két intézet nem javasolja a dopaminnal történő kombiná ciót. 2. Ha nem elég a kombináció, IABP bevezetése javasolt. 3. Béta-blokkoló megtartása, ACE-gátlók leállítása java solt. A levoszimendánalkalmazás módja a mellékhatások elkerülése érdekében Műtét előtt 24 órával folyamatos infúzióban bolus nél kül. Így lehet kihasználni a szívre és más szervekre gya korolt protektív, antiinflammatorikus hatását. Ha a műtőben kezdjük el, a bolus adása megfontolha tó. A bolus nagysága 6–12 µg/kg. A fenntartó infúzió nagysága 6 centrumban 0,1 µg/kg/min, 1 centrumban 0,2 µg/kg/min. Az irodalomban publikált metaanalízisek és az utolsó három kettős kontrollált randomizált vaktanulmány eredményei között lényeges különbség áll fenn. Az el lentmondás adódhat az adagolás módjából, mivel a 3 utóbbi tanulmányban nem preventíven, a műtétet meg előző 24 órában adták a levoszimendánt, így az nem tudta szív- és veseprotektív, antiinflammatorikus hatásait kifejteni. A LEVO-CTS-tanulmány tervezésekor felme rült ez a probléma, de az Amerikai Egyesült Államokban uralkodó gyakorlat szerint a beteg a műtét napján érke zik, így nem lehet 24 órás előkezelést végezni. Viszont minden tanulmány megegyezett abban, hogy alkalmazá sa biztonságos. Az irodalom és a hazai centrumok ta pasztalatai alapján jelenleg a súlyosan csökkent bal- és/ vagy jobbkamra-funkció a levoszimendán fő indikációja szívműtétek során. A legmeggyőzőbb bizonyítékok ko szorúérműtétek kapcsán állnak rendelkezésre. A legtöbb nyereséggel akkor jár alkalmazása, ha a műtét előtt 24 órával kezdjük el infúzióban alkalmazni, kihasználva a szívre és más szervekre kifejtett protektív, antiinflamma torikus hatásait. Beadásához szubintenzíves körülmé nyek, kompetens személyzet, monitorozás szükséges. Súlyos billentyűszűkület esetén invazív vérnyomásmérés szükséges a vérnyomásesés azonnali észlelésére és kezelé sére. Ezen esetekben megfontolandó a műtét előtt köz vetlenül a műtőben elkezdeni adagolását. Többszöri alkalmazásra lehet szükség a posztoperatív szakban az aktív metabolit szintézisének késése miatt. A mellékhatások elkerülése, kezelése Anyagi támogatás: Az ajánlás elkészítését az Orion Phar ma Kft. támogatta. Szerzői munkamegosztás: Az első szerző készítette el a közlemény vázát, koordinálta a konszenzus vitáját. Anyagi támogatás: Az ajánlás elkészítését az Orion Phar ma Kft. támogatta. Irodalom [20] Bragadottir G, Redfors B, Ricksten SE. Effects of levosimendan on glomerular filtration rate, renal blood low, and renal oxygena tion after cardiac surgery with cardiopulmonary bypass: a ran domised placebo-controlled study. Crit Care Med. 2013; 41: 2328–2335. [1] Rudiger A, Businger F, Streit M, et al. Presentation and outcome of critically ill medical and cardiac-surgery patients with acute heart failure. Swiss Med Wkly 2009; 139: 110–116. [2] Hobson CE, Yavas S, Segal MS, et al. Acute kidney injury is as sociated with increased long-term mortality after cardiothoracic surgery. Circulation 2009; 119: 2444–2453. [21] Niu ZZ, Wu SM, Sun WY, et al. Perioperative levosimendan therapy is associated with a lower incidence of acute kidney in jury after cardiac surgery: a meta-analysis. 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Anaesth Intensive Care 2013; 41: 719–727. [6] Jamali IN, Kersten JR, Pagel PS, et al. Intracoronary levosi mendan enhances contractile function of stunned myocardium. Anesth Analg. 1997; 85: 23–29. [24] Schwarte LA, Picker O, Bornstein SR, et al. Levosimendan is superior to milrinone and dobutamine in selectively increasing microvascular gastric mucosal oxygenation in dogs. Crit Care Med. 2005; 33: 135–142. [7] Papp Z, Édes I, Fruhwald S, et al. Levosimendan: molecular mechanisms and clinical implications: consensus of experts on the mechanisms of action of levosimendan. Int J Cardiol. 2012; 159: 82–87. [25] Pollesello P, Parissis J, Kivikko M, et al. Levosimendan meta- analyses: Is there a pattern in the effect on mortality? Int J Car diol. 2016; 209: 77–83. Irodalom [8] Harrison RW, Hasselblad V, Mehta RH, et al. Effect of levosi mendan on survival and adverse events after cardiac surgery: a meta-analysis. J Cardiothorac Vasc Anesth. 2013; 27: 1224– 1232. [26] Zangrillo A, Biondi-Zoccai G, Mizzi A, et al. Levosimendan re duces cardiac troponin release after cardiac surgery: a meta-ana lysis of randomized controlled studies. J Cardiothorac Vasc Anesth. 2009; 23: 474–478. [9] Greco T, Calabrò MG, Covello RD, et al. A Bayesian network meta-analysis on the effect of inodilatory agents on mortality. Br J Anaesth. 2015; 114: 746–756. [27] Landoni G, Mizzi A, Biondi-Zoccai G, et al. Reducing mortality in cardiac surgery with levosimendan: a meta-analysis of ran domized controlled trials. J Cardiothorac Vasc Anesth. 2010; 24: 51–57. [10] Toller W, Heringlake M, Guarracino F, et al. Preoperative and perioperative use of levosimendan in cardiac surgery: European expert opinion. Int J Cardiol. 2015; 184: 323–336. [28] Maharaj R, Metaxa V. Levosimendan and mortality after coro nary revascularisation: a meta-analysis of randomised controlled trials. Crit Care 2011; 15: R140. [11] Papp Z, Csapó K, Pollesello P, et al. Pharmacological mecha nisms contributing to the clinical efficacy of levosimendan. Car diovasc Drug Rev. 2005; 23: 71–98. [29] Hernández A, Miranda A, Parada A. Levosimendan reduces mortality in cardiac surgery: a systematic review and meta-analy sis. Rev Esp Anestesiol Reanim. 2012; 59: 6–11. [12] Nieminen MS, Altenberger J, Ben-Gal T, et al. Repetitive use of levosimendan for treatment of chronic advanced heart failure: clinical evidence, practical considerations, and perspectives: an expert panel consensus. Int J Cardiol. 2014; 174: 360–367. [30] Lim JY, Deo SV, Rababa’h A, et al. Levosimendan reduces mor tality in adults with left ventricular dysfunction undergoing car diac surgery: a systematic review and meta-analysis. J Card Surg. 2015; 30, 547–554. [13] McCully JD, Levitsky S. Mitochondrial ATP-sensitive potassium channels in surgical cardioprotection. Arch Biochem Biophys. 2003; 420: 237–245. [31] Zhou C, Gong J, Chen D, et al. Levosimendan for prevention of acute kidney injury after cardiac surgery: a meta-analysis of ran domized controlled trials. Am J Kidney Dis. 2016; 67: 408–416. [14] du Toit EF, Genis A, Opie LH, et al. A role for the RISK pathway and KATP channels in pre- and post-conditioning induced by le vosimendan in the isolated guinea pig heart. Br J Pharmacol. 2008; 154: 41–50. [32] Landoni G, Lomivorotov VV, Alvaro G, et al. Levosimendan for hemodynamic support after cardiac surgery. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. [19] Hein M, Roehl AB, Baumert JH, et al. Anti-ischemic effects of inotropic agents in experimental right ventricular infarction. Acta Anaesthesiol Scand. 2009; 53: 941–948. Preoperatív alkalmazásának feltételei 1. Kompetens személyzet. 1. Kompetens személyzet. 2. Minimum szubintenzív elhelyezés. 3. Billentyűszűkülettel járó betegség esetén minden centrum invazív artériás nyomásmérést javasol. Anyagi támogatás: Az ajánlás elkészítését az Orion Phar ma Kft. támogatta. 4. Csak koszorúér-betegség esetén 4 centrum javasol in vazív artériás nyomásmérést, 3 centrum elegendőnek tartja a noninvazív nyomásmérést. Szerzői munkamegosztás: Az első szerző készítette el a közlemény vázát, koordinálta a konszenzus vitáját. 2018 ■ 159. évfolyam, 22. szám ORVOSI HETILAP 875 Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC EREDETI KÖZLEMÉNY A többi szerző saját intézeti tapasztalatainak leírásával és az elektronikus úton történő vitával járult hozzá a közle ményhez. [18] Husebye T, Eritsland J, Müller C, et al. Levosimendan in acute heart failure following primary percutaneous coronary interven tion-treated acute ST-elevation myocardial infarction. Results from the LEAF trial: a randomized, placebo-controlled study. Eur J Heart Fail. 2013; 15: 565–572. Irodalom N Engl J Med. 2017; 376: 2021–2031. [15] Papp JG, Pollesello P, Varró AF, et al. Effect of levosimendan and milrinone on regional myocardial ischemia/reperfusion-induced arrhythmias in dogs. J Cardiovasc Pharmacol Ther. 2006; 11: 129–135. [33] Mehta RH, Leimberger JD, van Diepen S, et al. Levosimendan in patients with left ventricular dysfunction undergoing cardiac surgery. N Engl J Med. 2017; 376: 2032–2042. [16] Sonntag S, Sundberg S, Lehtonen LA, et al. The calcium sensi tizer levosimendan improves the function of stunned myocardi um after percutaneous transluminal coronary angioplasty in acute myocardial ischemia. J Am Coll Cardiol. 2004; 43: 2177– 2182. [34] Cholley B, Caruba T, Grosjean S, et al. Effect of Levosimendan on Low Cardiac Output Syndrome in Patients With Low Ejec tion Fraction Undergoing Coronary Artery Bypass Grafting With Cardiopulmonary Bypass. The LICORN Randomized Clinical Trial. JAMA 2017; 318: 548–556. [17] Wu X, Wu J, Yan X, et al. Enhancement of myocardial function and reduction of injury with levosimendan after percutaneous coronary intervention for acute myocardial infarction: a pilot study. Cardiology 2014; 128: 202–208. [35] Levin R, Degrange M, Del Mazo C, et al. Preoperative levosi mendan decreases mortality and the development of low cardiac output in high-risk patients with severe left ventricular dysfunc 876 2018 ■ 159. évfolyam, 22. szám ORVOSI HETILAP ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC EREDETI KÖZLEMÉNY [40] Sponga S, Ivanitskaia E, Potapov E, et al. Preoperative treatment with levosimendan in candidates for mechanical circulatory sup port. ASAIO J. 2012; 58: 6–11. tion undergoing coronary artery bypass grafting with cardiopul monary bypass. Exp Clin Cardiol. 2012; 17: 125–130. y yp p [36] Lahtinen P, Pitkänen O, Pölönen P, et al. Levosimendan reduces heart failure after cardiac surgery: a prospective, randomised, placebo-controlled trial. Crit Care Med. 2011; 39: 2263–2270. [41] Theiss HD, Grabmaier U, Kreissl N, et al. Preconditioning with levosimendan before implantation of left ventricular assist devic es. Artif Organs 2014; 38: 231–234. [37] Jörgensen K, Bech-Hanssen O, Houltz E, et al. Effects of levosi mendan on left ventricular relaxation and early filling at main tained preload and afterload conditions after aortic valve replace ment for aortic stenosis. Circulation 2008; 117: 1075–1081. [38] Severi L, Lappa A, Landoni G, et al. Levosimendan versus intra- aortic balloon pump in high-risk cardiac surgery patients. J Car diothorac Vasc Anesth. 2011; 25: 632–636. (Szudi László dr., Budapest, Hun u. Irodalom 11., 1135 e-mail: lszudi@t-online.hu) [39] Nieminen MS, Fruhwald S, Heunks LM, et al. Levosimendan: current data, clinical use, and future development. Heart Lung Vessel 2013; 5: 227–245. „Ubi dolor, nos vocat.” (A fájdalom hív bennünket.) (Szudi László dr., Budapest, Hun u. 11., 1135 e-mail: lszudi@t-online.hu) A cikk a Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk nem kereskedelmi célból bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. [41] Theiss HD, Grabmaier U, Kreissl N, et al. Preconditioning with levosimendan before implantation of left ventricular assist devic es. Artif Organs 2014; 38: 231–234. Tisztelt Szerzőink, Olvasóink! Az Orvosi Hetilapban megjelenő/megjelent közlemények elérhetőségére több lehetőség kínálkozik. Rendelhető különlenyomat, melynek áráról bővebben a www.akkrt.hu honlapon (Folyóirat Szerzőknek, Különlenyomat menü pont alatt) vagy Szerkesztőségünkben tájékozódhatnak. A közlemények megvásárolhatók pdf-formátumban is, illetve igényelhető Optional Open Article (www.oopenart.com A közlemények megvásárolhatók pdf-formátumban is, illetve igényelhető Optional Open Article (www.oopenart.com). Ad díj ll éb li kö l é k bá ki á á h áfé h ők h l k ( kö l é k külö li k k k Adott díj ellenében az online közlemények bárki számára hozzáférhetők honlapunkon (a közlemények külön link így más oldalról is linkelhetővé válnak). Bővebb információ a hirdetes@akkrt.hu címen vagy különlenyomat rendelése esetén a Szerkesztőségtől kérhető. A cikk a Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk nem kereskedelmi célból bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. 2018 ■ 159. évfolyam, 22. szám 877 ORVOSI HETILAP Unauthenticated \| Downloaded 10/24/24 04:46 AM UTC
https://openalex.org/W2204770498	https://revistas.ucm.es/index.php/NOMA/article/download/51331/47618	Spanish; Castilian	null	Del sentido de la muerte en Paul Ricoeur	Nómadas	2,015	cc-by	22,203	BBeelléénn CCaasstteellllaannooss RRooddrríígguueezz IES Virgen de Covadonga (Asturias) – UNED - EMUI http://dx.doi.org/10.5209/rev_NOMA.2015.v45.n1.51331 Resumen.- Partiendo de la obra de Paul Ricoeur, el siguiente artículo presenta una reformulación no humanista de la pregunta por el sentido de la vida, con la que se trata de salir del nihilismo pero también de la idea de que el ser humano puede, felizmente, renunciar a tal interrogante y congratularse sin más, con la afirmación del caos o con la fabricación de sentidos meramente subjetivos o gregarios, provisionales o superficiales. Se muestra la necesidad de rescatar el papel de la fe, del destino y del amor sacrificial, desplazando la pregunta por el sentido de la vida a la pregunta por el sentido de la muerte que, si se logra contestataria y significante, se colocará como respuesta y acabamiento del actuar hermenéutico. Palabras clave.- Ricoeur, fe, sentido de la vida, muerte, amor, religión, Dios, hermenéutica. Abstract.- Based on the work of Paul Ricoeur, the following article presents a non- humanist reformulation of the question of the meaning of life, with which to come out of nihilism and the idea that humans can, happily, drop out this question and welcome without more, to the affirmation of chaos or manufacture of merely subjective or gregarious or provisional senses. The need to rescue the role of faith, fate and sacrificial love is show, shifting the question of the meaning of life the question to the meaning of death which, if achieved rebellious and significant, will be placed in response and finishing hermeneutic act. A Marcos Ríos, Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 DDEELL SSEENNTTIIDDO O DDEE LLAA M MUUEERRTTEE EENN PPAAUULL RRIICCO OEEUURR BBeelléénn CCaasstteellllaannooss RRooddrríígguueezz IES Virgen de Covadonga (Asturias) – UNED - EMUI Keyword.- Ricoeur, meaning of life, death, sacrificial love, religion, God, hermeneutics. puede procurar infraestructura a ningún plan. puede procurar infraestructura a ningún plan. puede procurar infraestructura a ningún plan. Así, podríamos concluir, engalanados por el pesimismo optimista del postnihilismo o por cualquier constructivismo de estos tiempos postmodernos, que debemos acabar con la rumia, que viva la imaginación, que muerte a la búsqueda de sentidos predeterminados, etc. Sin embargo, si me pregunto por qué elegí la filosofía, solo obviando o expulsando las angustias de la infancia, podría envalentonarme y acudir a esos discursos tan actuales de la independencia, de la fuerza impostada del adulto y de ese vitalismo que encubre, para disimularlo, un “refinado” academicismo, para declarar que la elegí para jugar con las perspectivas, para inventar sentidos siempre nuevos, para volver alegremente al instante cero de cualquier emprendimiento, para remontar la inocencia del inconsciente “huérfano, ateo y anarquista” (que diría Gilles Deleuze)... Sería una impostura, pues mi niñez no fue la del niño que ríe y goza por solo levantar castillos de arena para luego derrumbarlos. Pienso que sí es una niña la que eligió la filosofía, pero una niña que lloraba la muerte, que no se explicaba la crueldad del desvanecimiento de todas las cosas, que no podía tomar con levedad el triste desenlace letal de esas vidas, pequeñas vidas de pájaros, nacidas de un cúmulo incontable de circunstancias únicas en un instante de oportunidad, de tantos esfuerzos de supervivencia, de la superación de dolorosos momentos de fragilidad, de la constancia que aguanta golpes para aprender a volar; una niña, en definitiva, que lloraba la finitud. Y de la aflicción por la finitud se va a la búsqueda de la verdad. Casi podría afirmar que la Verdad, así con mayúscula, es eso que una habría alcanzado cuando sabe qué decir en un funeral. Yo no lo sé. Filosofaremos mientras no sepamos qué decir ante el drama de la finitud, pero solo a condición de que la finitud no se haya apoderado de nosotros. La Verdad es un ir respondiendo de la muerte, es decir, de la finitud con un espíritu religioso al que su religión no le baste. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Introducción. ¿Qué está ocurriendo cuando una pierde el temple para vivir? Creo poder adelantar una respuesta provisional. Si nos paramos e intentamos cifrar, fenoménicamente, qué está ocurriendo en el ocaso del brío vital, fácilmente nos daremos cuenta de que la reflexión le ha ganado la carrera al cuerpo y, en ese momento, la empecinada búsqueda de sentido da lugar a una honda sensación de sin-sentido, o, expresado en términos intrasubjetivos, a una obscura sucesión de ¿para qué? Siendo que el cuerpo debería saltar de la cama con la luz de la mañana, deseoso de un nuevo día, y la mente la que se pusiera a trabajar después, para dar sendero al impulso, las cosas, torcidas, comienzan a sucederse al contrario y es la mente la que obscura como es, cuando se ha adueñado de las pulsaciones de la existencia, despierta antes, impidiendo que el cuerpo se desperece o, agotándolo prematuramente con reflexiones y demandas de razón, intentando encontrar argumentos que justifiquen por qué debería levantarme mejor que quedarse indefinidamente bajo las mantas, termina por apercibirse de que, con razones o sin ellas, ese cuerpo ya no le responde ni © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 terriblemente nombrable: cuando cae la ilusión de conexión1. Por esa precisa razón examinaremos la legitimidad de la pregunta por el sentido e incluso de la exigencia de sentido, haciendo una apuesta religiosa por ella. 1 Téngase en cuenta que no uso el término fantasía para indicar el carácter falso, en este caso, de la sensación de conexión o de la conexión misma. Tampoco pretendo darle a dicha sensación un correlato de realidad indubitable. Es por ello que asimilo esta fantasía de conexión con la fe, no siendo la fe o la convicción, la creencia en fraudes o simulaciones ni tampoco la certeza devenida de demostraciones empíricas o racionales, es decir, fácticas o lógicas, sino ese impulso vital que se mantiene en la ascesis o aspiración a alguna verdad esencial que, como arcké o primer principio, tan solo puede ser intuido o presupuesto o, si queremos, demostrado por ese método que no es, realmente una demostración y conocemos como reducción al absurdo: aspiración hacia aquello sin lo cual todo carecería de sentido. Por esa precisa razón examinaremos la legitimidad de la pregunta por el sentido e incluso de la exigencia de sentido, haciendo una apuesta religiosa por ella. Así, propongo recoger el significado genético del término fantasía y así entenderla, al modo griego, como aparición, manifestación, imagen o, en términos generales, aquello que puede ser percibido, sentido. Si se desea enlazar con el lenguaje psicoanalítico, recordaremos que Freud se resistía a decantarse en cuento al carácter de realidad o irrealidad objetiva del motivo o argumento de la fantasía, no considerando, además que dirimir tal cuestión fuera demasiado relevante, pues el acontecimiento psíquico, en todo caso, tiene su propia verdad, su propia substancia, estructurando incluso la línea de comportamientos exteriorizados. Más aún se puede hacer este uso si pensamos en las fantasías originarias, aquellas que, impersonales y sin relación necesaria con las vivencias del sujeto, conforman la base del inconsciente a través de la herencia filogénica de los símbolos, entendiéndose entonces que más allá de lo humano, se nos interpela con signos enigmáticos que recibimos a través del alma inconsciente. Así, propongo recoger el significado genético del término fantasía y así entenderla, al modo griego, como aparición, manifestación, imagen o, en términos generales, aquello que puede ser percibido, sentido. terriblemente nombrable: cuando cae la ilusión de conexión1. terriblemente nombrable: cuando cae la ilusión de conexión1. En una biografía inquieta, abierta al erotismo, cuántas veces hemos sentido, junto a alguien, una química tan fuerte que nos hace llorar de emoción y pensar que esa atmósfera que acaba de surgir, lo ha hecho, indudablemente, de entre los dos y que nos transporta al fondo de la tierra y a lo alto de los cielos a ambos. Estamos, en esos momentos, cargados de fe (bien le digan oxitocina los biólogos o los médicos). No es tanto el desamor, sino la desconexión o la pérdida de la ilusión de conexión, la sospecha fundada o infundada o la simple no-certeza de que el otro con-vive esa redención, la que da al traste con la fe, con la expectativa del amor. No hay nada peor que empezar a pensar que la mirada del otro solo es el reflejo de la nuestra, nada peor que este tipo de solipsismo, nada peor que la irrupción de la duda cartesiana en un universo platónico. Y al punto que espero haber conseguido tramar una relación suficientemente coherente o sensualmente coherente entre la pregunta por la finitud, por la fe y por el amor, espero también haber generado el ambiente propicio para que el repaso de los escritos de Paul Ricoeur caiga en terreno propicio, es decir, venga a responder o a re-exponer mejor que yo, mejor que otros, la hondura o radicalidad de los vértigos exhibidos en esta personal introducción. 1 Téngase en cuenta que no uso el término fantasía para indicar el carácter falso, en este caso, de la sensación de conexión o de la conexión misma. Tampoco pretendo darle a dicha sensación un correlato de realidad indubitable. Es por ello que asimilo esta fantasía de conexión con la fe, no siendo la fe o la convicción, la creencia en fraudes o simulaciones ni tampoco la certeza devenida de demostraciones empíricas o racionales, es decir, fácticas o lógicas, sino ese impulso vital que se mantiene en la ascesis o aspiración a alguna verdad esencial que, como arcké o primer principio, tan solo puede ser intuido o presupuesto o, si queremos, demostrado por ese método que no es, realmente una demostración y conocemos como reducción al absurdo: aspiración hacia aquello sin lo cual todo carecería de sentido. 1 Téngase en cuenta que no uso el término fantasía para indicar el carácter falso, en este caso, de la sensación de conexión o de la conexión misma. Tampoco pretendo darle a dicha sensación un correlato de realidad indubitable. Es por ello que asimilo esta fantasía de conexión con la fe, no siendo la fe o la convicción, la creencia en fraudes o simulaciones ni tampoco la certeza devenida de demostraciones empíricas o racionales, es decir, fácticas o lógicas, sino ese impulso vital que se mantiene en la ascesis o aspiración a alguna verdad esencial que, como arcké o primer principio, tan solo puede ser intuido o presupuesto o, si queremos, demostrado por ese método que no es, realmente una demostración y conocemos como reducción al absurdo: aspiración hacia aquello sin lo cual todo carecería de sentido. Por esa precisa razón examinaremos la legitimidad de la pregunta por el sentido e incluso de la exigencia de sentido, haciendo una apuesta religiosa por ella. puede procurar infraestructura a ningún plan. Lo mismo es preguntarse por qué moriré si deseo vivir que por qué nací, si la vida ya no parece ofrecer nada más que la espera por la muerte, si ya no sé por qué dilatar el momento, si ya nada sorprendente parece acechar tras la esquina, o si ninguna conquista me queda esperar o si las conquistas ya no tienen brillo ni emoción. Todo esto constituye una definición, bien sea muy imperfecta o poco rigurosa, de la pérdida de la fe. Me atrevería a afirmar ahora, y con ello lograr cierto hilo expositivo que me dé el derecho a pasar de una cosa a la otra, que perder la fe es perder la expectativa del amor. La experiencia del amor irrumpe dando sentido y regocijo al hecho de haber nacido, incluso al haber nacido del más melancólico. Y el amor es tan extático que nos reconcilia con la muerte pues pareciera que de un trago ya tomáramos toda la eternidad en un instante. Y, ¿cuándo dejamos de creer en esta redención? Dejamos de creer en esta redención en un punto muy concreto, Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) La fantasía de conexión como dimensión religiosa. La fantasía de conexión se me antoja como la presunción esencial, la que nos es necesaria para vivir más allá de la supervivencia, para poder esperar algo de la vida y para poder agradecer algo en el momento de la muerte. Solo la fantasía de conexión permite que el laberinto de la mente no se adelante a nuestro cuerpo, angostándolo, y que nuestro cuerpo no camine sin espíritu, al margen del deseo de verdad, ya robótico entonces. Porque creo que no se puede auténticamente vivir en la nada, en el caos tedioso, al que no le siguiera ningún reordenamiento, o porque sé, al menos, que yo, como muchos, no lo podemos hacer, vale la pena interpelar, una vez más, a nuestra dimensión religiosa y demandar al mundo, a nuestro mundo, las condiciones necesarias para no tener que renunciar a ella. En otras ocasiones y a propósito de otros estudios y en compañía de otros personajes filosóficos, podría abrir y cerrar esta cuestión con un ontológico llamamiemto al Ser, a la pregunta por el Ser, por la eternidad. En cambio, me voy a arriesgar, contra la moda, a reclamar a Dios o cuanto menos, a reclamar nuestro derecho a proponer algún modo de su existencia. Sé que hablar del Ser y hablar de Dios podría tratarse de lo mismo si la religiosidad se reduce a filosofía racional o si la filosofía se instrumentara del todo al servicio de cualquier discurso teológico. Pero, como indica Ricoeur la palabra Dios añade algo que no se le presupone al Ser, que no se le pide o que incluso se le niega: el sentido: “La palabra Dios dice más que la palabra ser porque presupone todo el contexto de los relatos, las profecías, las leyes, los salmos, etc. Seguir la palabra Dios es seguir su flecha de sentido. Por flecha de sentido entiendo su poder capaz de reunir las significaciones parciales, inscriptas en los diversos discursos parciales, y abrir un horizonte que no se deja delimitar por la clausura de ningún discurso” (Ricoeur, 2008, pp. 62-63). terriblemente nombrable: cuando cae la ilusión de conexión1. Si se desea enlazar con el lenguaje psicoanalítico, recordaremos que Freud se resistía a decantarse en cuento al carácter de realidad o irrealidad objetiva del motivo o argumento de la fantasía, no considerando, además que dirimir tal cuestión fuera demasiado relevante, pues el acontecimiento psíquico, en todo caso, tiene su propia verdad, su propia substancia, estructurando incluso la línea de comportamientos exteriorizados. Más aún se puede hacer este uso si pensamos en las fantasías originarias, aquellas que, impersonales y sin relación necesaria con las vivencias del sujeto, conforman la base del inconsciente a través de la herencia filogénica de los símbolos, entendiéndose entonces que más allá de lo humano, se nos interpela con signos enigmáticos que recibimos a través del alma inconsciente. Siguiendo la tradición griega y también la psicoanalítica tomaré indistintamente la palabra fantasía o la palabra fantasma pues, lo mismo, etimológicamente significa lo que aparece, lo que se muestra, lo que brilla. Gracias al psicoanálisis y a las precisiones lacanianas, podemos también interrogarnos acerca de la actualidad o inactualidad del fantasma. Como nuestra propia intuición indica, el fantasma es actual en tanto que se nos presenta, alegrándonos, atormentándonos, o generando cualquier otro afecto. En cambio, parece provenir del submundo, del pasado. Se dice fantasma, comúnmente, de la aparición de un difunto. Así, es actual e inactual: está o es pero insistiendo más que existiendo. En definitiva, la fantasía, el fantasma, es una virtualidad. Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 2 En torno a esta temática se desarrolla mi artículo “El erotismo como fascinación ante la muerte en Georges Bataille” publicado en Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas 26 (2010.2). Traigo aquí un fragmento en el que cito la correlación entre ambos pensadores: “La sexualidad y la muerte se pertenecen la una a la otra: <<La muerte de uno es correlativa al nacimiento de otro. La vida es siempre un producto de la descomposición de la vida. Antes que nada es tributaria de la muerte, que le hace un lugar; luego, lo es de la corrupción, que sigue a la muerte y que vuelve a poner en circulación las substancias necesarias para la incesante venida al mundo de nuevos seres>> (G. Bataille: El erotismo. Ed. Tusquets. Barcelona, 2007. p. 59). De este modo, Bataille inicia un análisis que parte de la reescritura de Anaximandro. Recordemos la famosa frase de éste recogida así por Simplicio: <<El nacimiento a los seres existentes les viene de aquello en lo que convierten al perecer, “según la necesidad, pues se pagan, los unos a los otros, mutua pena y retribución por su injusticia según la disposición del tiempo”, como Anaximandro dice en términos un tanto poéticos>> (S. Kirk, J. E. Raven y M. Schofield: Los filósofos presocráticos. Ed. Gredos, Madrid, 1994. p. 177)”. La fantasía de conexión como dimensión religiosa. Consciente de que exigirle un sentido a la vida y, más aún, pretender descubrirlo, parece propio de aquel humanismo desfasado tan criticable ya y del cual yo misma he recontado y recuento mil veces sus consecuencias funestas (para el espíritu, para la historia humana, para la salud, etc.), deseo hacerme cargo de ese grave que es el sentido de la vida y pensarlo, desde otro lado que no sea el humanismo ni el nihilismo ni, tal vez, el postnihilismo, sino, simplemente, desde la necesidad. Se trata aquí y ahora de una pregunta religiosa y no humanista. Si es así es porque no estamos buscando el sentido en metas y logros ni en nada comprometido con la idea de progreso, que considero felizmente desechada por nuestra epocalidad, sino como antes manifestaba, en aquella palabra que pudiera, verdaderamente y sin mascaradas, dar consuelo al luto. Ricoeur nos dona aquí otra significación, la de Cristo, “capaz de incorporar todas las significaciones religiosas en un único símbolo, el símbolo del amor sacrificial más fuerte que la muerte” (Ricoeur, 2008, p. 63). En el imaginario bíblico, entendiendo que reúne antiguos símbolos religiosos, se nos presenta la figura de Cristo como respuesta o modo de comprender la © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) pregunta por la muerte, por la finitud y, así también por el nacimiento, las angustias, las pasiones, el espectáculo de la crueldad (nombres, al fin, de la finitud). Solo parece posible transcender la muerte por el amor, del mismo modo que el amor, como observaríamos en Georges Bataille, ofrece una experiencia de la muerte en tanto que amor y muerte implican continuidad ontológica, deshaciendo la concreción, la insuficiencia que experimentamos como entes separados, recortados, desconectados de todo lo demás. Si al nacer, nos separamos, indicaba Anaximandro, al morir volvemos a la reunión, nos reunimos, retornamos a la indeterminación de la que todas las cosas del universo brotan y a la que todas regresan2. No olvido, sin embargo que en esta ocasión deseo trazar una propuesta religiosa más allá de una ontología desnuda. Por ello, quiero no solo hablar de Dios como palabra que añade algo al Ser sino también hablar del amor como palabra que añade algo a la reunión. La fantasía de conexión como dimensión religiosa. El amor es esa disposición a dejarse interpretar y ese dejarse interpretar se hace interpretando, a su vez, al otro. Con nuestras observaciones del otro dejamos que ese otro conozca nuestras preguntas, nuestras incógnitas, nuestras curiosidades y, en definitiva, los problemas que nos constituyen. Es así como en nuestra labor interpretativa nos ofrecemos a la interpretación, tal y como en la interpretación de los textos sagrados, dejamos que ellos mismos nos digan, a su vez, quiénes somos. Hacer el amor es hacer hermenéutica. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 3 Como sabemos, Hume muestra en su obra cumbre, Investigación sobre el entendimiento humano, cómo la fe juega un papel primordial en la práctica humana. Si por razón fuera, nos quedaríamos en el impasse de la duda filosófica cuya virtud en tanto actividad intelectual, se transmutaría exceso si pretendiéramos funcionar en la urgencia de la vida o ni siquiera en el vivir cotidiano, a partir de su reinado. Si bien nada nos asegura que las cosas tal y como las conocemos seguirán ahí mañana, que el coche se detendrá ante la luz roja del semáforo, que un tornado no arrasará nuestra vivienda o que una teja no caerá funestamente rompiéndome la crisma, tal escepticismo paralizaría por completo nuestra acción y nuestras vidas, si no lo tomáramos, saludablemente, como escudo contra dogmatismos y supercherías racionalistas, sino también como guía para nuestro quehacer diario. Hume diferencia muy bien el mundo de la reflexión del mundo de la vida, en el que la fe constituye Fe, hermenéutica y amor. Antes mencionábamos el carácter estático de la experiencia amorosa en relación a esa fantasía de conexión que otorga, no tanto una despreocupación ante la muerte, sino una afirmación de la vida hasta la muerte como diría Bataille. Recién, explorábamos una nueva aproximación hacia el concepto de interpretación y de apertura hermenéutica, con el amor, la donación y la exposición como límites y condiciones suyas de posibilidad. Ambos caminos producen, entiendo, una resonancia religiosa que conduce a la fe. En la fe se reunirían tanto el aliento vital, ese que habilita al espíritu del cuerpo para encontrarse esperanzado, emocionado, dispuesto para la vida y para el Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) desafío ante una muerte que no llegaría a alcanzar nunca al éxtasis eterno del momento del amor; como la confianza radical a partir de la cual se puede vivir realmente en tanto que se arriesga la vida o, en el extremo, tal como condensa la imagen de Cristo, se sacrificaría por una todavía mayor, con la que se transciende la muerte y se conjura la finitud. Incluso resultaría extraño denominar ser finito a uno que entrega la vida por amor. Así, desde este trazado, hacer el amor es hacer hermenéutica y hacer hermenéutica es un acto posibilitado por la fe que, obviamente, escaparía al registro clausurado de toda hermenéutica. Al fin, como decíamos, la fantasía de conexión constituye esa fe a partir de la cual permitimos que alguien nos dé un nombre en el justo momento en que leemos a ese alguien. Escuchemos a Ricoeur en torno a la fe: “puede designarse a la fe como <<inquietud última>>, subyacente a todas nuestras decisiones. Podemos asimismo llamarla <<sentimiento de dependencia absoluta>> para subrayar el hecho de que nunca es más que una respuesta a un querer que nos precede. Podemos incluso llamarla <<confianza incondicionada>>, si no deseamos separarla de la esperanza que abre un camino a pesar de todos los obstáculos y que convierte toda razón de desesperar en razón de esperar” (Ricoeur, 2008, p. 64). Tener fe implica confiar en lo desconocido que transita por fuera de las redes de nuestra razón demostrativa; implica querer el misterio pues mi propio sentimiento es misterioso: “.... el misterio del sentimiento, a saber, la ligazón invisible de mi existencia con los seres y con el ser por medio del deseo y del amor” (Ricoeur, 2011. p. 107). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Fe, hermenéutica y amor. El misterio, como veremos a continuación, nombra tanto la fragilidad de un corazón inquieto como el ánimo por el que así, en su falibilidad emocionante, vive (Ricoeur, 2011. pp. 100-101). Es tanto lo que tiene que ver la fe con la existencia como con aquello que la transpasa. Y, así, la fe trabaja desde ese sin-sentido que da sentido, aun no colocándose como meta o fin ulterior al sostén de la propia vida, que necesita ser también ultravida (no solo existencia sino esencia para la existencia). Ricoeur la explica, la fe, tomando matices en diferentes ámbitos. En el proverbio oriental, la fe trata de incardinarse en el sendero de la propia existencia, permitiendo orientar y discernir. En la cultura bíblica, el proverbio profundiza en paradojas e hipérboles. (Ricoeur, 2011b. pp. 76-79). Como sea, se abren dos dimensiones: la búsqueda de signos y la apertura a lo que nunca quedará agotado o desvelado por ellos. Como sabemos, desde la sabiduría humeana3 hasta la tan distinta kierkeggardiana4, la fe procura la energía vital, Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) basada en la confianza que nos enseña a caminar entre el misterio. Asimismo, en ambos, el amor está más acá y más allá de la ética y así lo resume Ricoeur: “El mandamiento que precede a toda ley es la palabra que el amante dirige al amado: ¡Ámame! Esta distinción inesperada entre mandamiento y ley solo tiene sentido si se admite que el mandamiento de amar es el amor mismo, como si el genitivo contenido en el mandamiento fuera a la vez genitivo objetivo y genitivo subjetivo; el amor es objeto y sujeto del mandamiento; o, en otros términos, es un mandamiento que contiene las condiciones de su propia obediencia por la ternura de su reproche: ¡Ámame!” (Ricoeur, 2011b. p.38). Ciertamente, si pienso en ese impulso, en el ánimo que necesito para levantarme cada mañana, sin cavilar, sin hundirme en los para qué, me doy perfecta cuenta de que no requiero tanto de la certidumbre de lo que me depara el nuevo día y ni siquiera de la seguridad en una total ausencia de riesgos y peligros, sino de la confianza en el misterio, en que dentro de la cálida familiaridad de lo destinado, habrá lugar para la sorpresa y los desafíos. la garantía de la energía y el dinamismo que nos mueve (Hume, 2004. Sección 5). 4 Es tan basta y profunda la reflexión de S. Kierkegaard sobre la fe y cruza a tal punto el total de su obra que resultaría siempre deficiente una anotación o cifra bibliográfica. No obstante, la fe como estado superior en el que se halla el ser humano, una vez ha transitado y superado los dictados de la mera ética para adentrarse en un terreno mistérico, aceptando la inseguridad moral, por sentirse parte de una superior conciencia o ultraconciencia, ante la que las normativas del hombre quedan diminutas y, así, pausadas, pudiendo resultar en la más grande acción aquella que sería señalada como vil ante la sociedad, se muestra abundantemente a partir del pasaje abrahámico en Temor y temblor (1987). En cualquier caso, nadie como Kierkegaard nos enseña la grandeza de la incorrección política, de las causas transcendentes batalladas más allá del bien y del mal cifrado por la estrechez del aquí y del ahora de la cultura humana y el brillo de quien sabe caminar entre la obscuridad, amándola, aun en la angustia. Ver El concepto de la angustia. (2013). Y dado que no queda lejos de nuestras investigaciones presentes, sin que lo hayamos abordado, es oportuno referir este libro de Kierkegaard titulado Las obras del amor (2006)que podemos encontrar en Ed. Sígueme. Salamanca, 2006, alojado en .http://www.sigueme.es/docs/libros/las-obras- del-amor.pdf Fe, hermenéutica y amor. No son los lances eventuales los que arruinan nuestro vigor sino la falta de confianza en que seguirán apareciendo. De ahí que resulten gozosas esas señales de la experiencia vivida que, como proverbios encarnados “reorienten desorientando”5. Pienso que resulta gozosa, incluso, la sensación de tomar parte de un destino que es comprendido solo a medias. Así, la pregunta por el sentido de la vida puede librarse del marco humanist la garantía de la energía y el dinamismo que nos mueve (Hume, 2004. Sección 5). 4 Es tan basta y profunda la reflexión de S. Kierkegaard sobre la fe y cruza a tal punto el total de su obra que resultaría siempre deficiente una anotación o cifra bibliográfica. No obstante, la fe como estado superior en el que se halla el ser humano, una vez ha transitado y superado los dictados de la mera ética para adentrarse en un terreno mistérico, aceptando la inseguridad moral, por sentirse parte de una superior conciencia o ultraconciencia, ante la que las normativas del hombre quedan diminutas y, así, pausadas, pudiendo resultar en la más grande acción aquella que sería señalada como vil ante la sociedad, se muestra abundantemente a partir del pasaje abrahámico en Temor y temblor (1987). En cualquier caso, nadie como Kierkegaard nos enseña la grandeza de la incorrección política, de las causas transcendentes batalladas más allá del bien y del mal cifrado por la estrechez del aquí y del ahora de la cultura humana y el brillo de quien sabe caminar entre la obscuridad, amándola, aun en la angustia. Ver El concepto de la angustia. (2013). Y dado que no queda lejos de nuestras investigaciones presentes, sin que lo hayamos abordado, es oportuno referir este libro de Kierkegaard titulado Las obras del amor (2006)que podemos encontrar en Ed. Sígueme. Salamanca, 2006, alojado en .http://www.sigueme.es/docs/libros/las-obras- del-amor.pdf p 5 Tomo prestada la expresión de P. Ricoeur (2008. p. 77). Añado también, recogiendo su entender, que no es extraño que el proverbio y la parábola reorienten desorientando, pues su lógica es su estilo y su estilo es el propio de la metáfora. Resulta interesante quebrar la común y escolar idea de que la metáfora se reduce a una analogía, a una matemática regla de tres que no aproxima sino que da cuenta de lo aproximado, revelando una proporción. Para ello nada mejor que el planteamiento de P. Ricoeur (1988. p. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 la garantía de la energía y el dinamismo que nos mueve (Hume, 2004. Sección 5). 6 En este caso debo indicar mi gusto por el uso del término heteronomía por oponerme con él a los discursos de la independencia, que considero discursos antipolíticos (antirreligiosos: antirreligadores) y destructures de la fantasía de conexión e incluso de la conexión misma y de la esencia del amor. Sin embargo, he de hacer notar, para guardar el rigor, que Paul Ricoeur, busca, en la línea que advierte en Heidegger, un más allá de la oposición entre autonomía y heteronomía, quizás muy afortanado y digno de estudio aparte. Este más allá está emparentado con el rescate de la conciencia como dimensión no reductible a la moral (frente a Nietzsche, Freud y toda la órbita postestructuralista) y que no se impone como realidad originaria ni como autodominio sino como justa diferencia, que permite al otro ser otro y no necesariamente un otro impersonal o un apelante que nos demanda desde estructuras de poder y control desencarnadas. Ver Ricoeur, 2011b. p. 102-107. Fe, hermenéutica y amor. 25): “si la metáfora no consiste en adornar una idea con una imagen, si consiste más bien en reducir el choque entre dos ideas incompatibles , es en esta reducción de distancia, en este acercamiento, donde la semejanza desempeña un papel. En efecto, lo que está en juego en un enunciado metafórico es hacer aparecer un <<parentesco>> allí donde la visión ordinaria no percibe ninguna conveniencia mutua”. Así, la metáfora está más cerca de la paradoja que de la simetría y más cerca de la iluminación religiosa que de la razón matemática y más cerca de la desproporción mistérica que de la imagen y semajanza del antropomorfismo teológico. Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) (racionalista, individualista o incluso solipsista) para formularse desde la hermenéutica y antes que en ella, en la religación y en la celebración de la dependencia, es decir, del amor, de la heteronomía6 y de la confianza incondicional que lo funda, la fe. Asimismo, la comprensión del cristianismo puede librarse del marco personalista y, de nuevo y con más vigor, encontrar para la vida una razón más fuerte que la supervivencia y para la muerte un más allá que la use (y la burle): “Escuchar excluye fundarse. El movimiento hacia la escucha requiere por lo tanto un segundo despojamiento, el abandono de una pretensión más sutil y más tenaz que la del saber onto-teológico. Requiere el desasimiento del sí mismo humano, en su voluntad de dominio, de suficiencia y de autonomía. A tal desasimiento se aplica la expresión del Evangelio: <<Quien quiera salvar su vida, la perderá>>” (Ricoeur, 2008. pp. 94-95). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 La muerte como garantía de la verdad. El reconocimiento y la fragilidad afectiva. La niña que eligió la filosofía ansiaba tanto la verdad que muchas veces contempló la idea de dar la vida para mostrar la suya ante el cruel escepticismo paterno. Una suerte de dignidad e impotencia nos transitan tanáticamente cuando la verdad de nuestra vida es tan frágil y, al tiempo, tan profunda y segura, que no existe más demostración que la drástica entrega a la muerte. Como sabemos, vivir es tener algo por lo que morir: “Cuando la prueba de la convicción se paga con la vida, el testigo cambia de nombre: se llama mártir. Pero, ¿cambia realmente de nombre? Mártyr, en griego, es testigo (…) Que el testigo sea asimismo el acusado reclama otro análisis distinto. A saber, que la sociedad, que la opinión común, que los poderosos detestan ciertas causas, acaso las más justas. Es necesario entonces que el justo muera. Y aquí se alza un gran arquetipo histórico, el servidor sufriente, el perseguido, Sócrates, Jesús... (…) El testigo es el hombre que se identifica con la causa justa detestada por la muchedumbre y por los grandes, es quien, por esa causa arriesga su vida” (Ricoeur, 2011. p. 117) “Pero, sobre todo, se denomina testimonio a una acción, una obra, al movimiento de una vida, en tanto constituyen la señal, la prueba viviente de la convicción y la consagración de un hombre a una causa” (Ricoeur, 2011. pp. 94-95). Hoy pasaría los 40 años la persona por la que empecé a estudiar psicoanálisis. Tal vez sería más acertado, aunque trágico, decir la persona por cuya muerte me decidí a estudiarlo a fondo ¿Por qué? Porque él lo hacía, para Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) comprenderlo a él, para comprender su muerte, para comprenderme a mí y para compensar el arrepentimiento por no estar a la altura, por no haber velado, en la parte que me correspondía, por el sentido de su vida, por haber despreciado la importancia de la desdicha y la responsabilidad que todos y una tiene en la búsqueda conjunta del sentido, por no haberme dado cuenta más que tardíamente, en la recuperación de la angustia infantil, de que vivir y morir, alegrarse y sufrir, no son asunto, como quiere la autoayuda, de uno mismo sino que en juego está el reconocimiento mutuo, la escucha de la verdad que el otro y prójimo nos ofrece, en definitiva, la generosidad. La muerte como garantía de la verdad. El reconocimiento y la fragilidad afectiva. La generosidad es, en realidad, pienso ahora, anterior a la firmeza, pues no se puede exigir más firmeza a aquel al que, en su predicación, abandonamos, no recibiendo, auténticamente su palabra, no estando ahí, dejándolo con la palabra en la boca. A veces, si no siempre, se aprende muy cruelmente qué es el amor, qué es la amistad. Se aprende con la crueldad de la finitud y se aprende a partir de un arrepentimiento muy concreto: el de haberse traicionado a una misma, burlándose de la niña que fue burlada, de aquella que deseando expresar su verdad y no siendo tomada en serio, solo se le ocurría, mostrarla con el sacrificio de su vida. A veces los melancólicos nos ponemos altaneros cuando una ráfaga de levedad nos recorre y confundimos levedad con fortaleza. Pero por suerte, aunque ásperamente, la gravedad siempre vuelve para librarnos del absurdo y de una existencia vacía y descomprometida. Mi amigo, brillante, sabio, sensible y... desencantado, dio su vida para mostrar una verdad. Yo sé que esa muerte no fue vana sino terriblemente significativa. Un suicidio lleva un mensaje, al que muchos le dan la espalda pero no es esto sino cobardía pues es un mensaje religioso, es un mensaje social, político, es un mensaje de amor o para el amor. Hacer el amor es hacer hermenéutica porque hacer el amor es mantenerse a la escucha e interpretar para dejarse interpretar. Pocos meses antes de su partida, conversando, me dijo dos cosas que nunca olvidaré y que han resultado clave para mí. Hablábamos precisamente del insomnio y del martirio que nos parecía madrugar, teniendo que levantarnos a toque de despertador justo cuando el miedo a perder la conciencia había cedido y nos encontrábamos abandonados al reposo. No queríamos dormir cuando ya habíamos logrado sobrevivir al día, pero tampoco queríamos despertarnos para enfrentarnos a uno nuevo. Yo me preguntaba por qué esa especie de inercia cíclica, por qué era tan costoso levantarse por la mañana, cuando había sido tan resistido el momento de acostarse. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 7 El texto más expresivo y concentrado descriptor de tal proceso es de S. Freud: Duelo y melancolía. Podemos encontrarlo en compilaciones de textos tanto en Editorial Alianza, RBA y otras. También libre en http://www.philosophia.cl/biblioteca/freud/1917Duelo%20y%20melancol%EDa.pdf. Si perseguimos una amplitud y enorme profundidad acerca de mencionado proceso de identidad y vivencias primarias del amor, debemos acudir a los estudios de Melanie Klein: Amor, culpa y reparación, recogido en el Tomo I de las Obras Completas de la autora en RBA. Barcelona, 2006. La muerte como garantía de la verdad. El reconocimiento y la fragilidad afectiva. Todavía hoy entiendo que hay un morir en el dormir y un nacer en el despertar, que la idea de lo uno y de lo otro es altamente angustiosa, pues se trata de los hitos más dramáticos y esenciales por los que entramos en el tiempo de la sucesión, en la finitud, y porque nos vienen impuestos, hallándonos en ellos totalmente a merced del relato de cualquiera sin que tan siquiera tengamos derecho a enmienda. Pero entonces fue cuando su pregunta irrumpió, desorientando para orientarme: “¿Pero no te ocurría, cuando ibas al instituto, que esa pereza de despertar y levantarte desaparecía alguna vez, de forma muy especial cuando empezaba a gustarte un compañero de clase o un estudiante nuevo?¿No te despertabas entonces, pensando rápidamente en verlo, curiosa por saber qué pasaría ese día, si alguna nueva señal estaba al caer?”. Al instante estuve de acuerdo en que así era y de que hasta su planteo apenas me había dado cuenta. Puede Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) que desde entonces el deseo, el amor, fueran el tema filosófico de mi vida. En otra conversación, poco después, hablamos del suicidio pues él aseguraba que el suicida, se suicida por venganza. En ese caso, había sido tan cierta esa sensación en las ideaciones infantiles de suicidio contra mis padres que me causó miedo o, lo que es lo mismo, rechazo a enfrentar quiebros aparentemente superados. Sin embargo, tenía razón pero la tenía a condición de aceptar que solo nos vengamos por amor, siendo la venganza un “hacer ver”. Parece que solo la muerte podría demostrar a los que nos importan que nuestras protestas y demandas no eran frívolas. Quienes nos importan son aquellos a los que queremos importar y queremos importar a los que nos han seducido para pensar que su importar es el más importante importar. Las figuras paternas son paradigma en este caso (si bien es cierto que tanto podríamos usarlas como estructurantes de la experiencia amorosa, como estructurantes de algo bien distinto pero confundido con ella, tal como es la experiencia de poder, en cuyo caso habría que reivindicar, con Deleuze un amor por fuera del constructo edípico en el que hemos sido atrapados, pero también que un amor así podría ser un auténtico amor religioso). Sin su reconocimiento, pareciera que ni existimos. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 La muerte como garantía de la verdad. El reconocimiento y la fragilidad afectiva. Pero la fragilidad de esta existencia, en tanto que reconocida, radica en que la <<estima>> que la consagra sólo sea opinión, en que la timé sea doxa; está ahí la amenaza de una existencia especular y cuasi- fantasmática; esa posibilidad de no ser ya más que una frase del otro, esa dependencia de la frágil opinión son precisamente la ocasión para las pasiones de la gloria que injertan su vanidad en la fragilidad de la estima como opinión” (Ricoeur, 2011. pp. 137-138). Pero sabemos, con Ricoeur, que solo los seres capaces de conocimiento, son seres capaces de reconocimiento y así: “la estima de mí mismo que busco mediante el aprecio del otro no es de distinta naturaleza que la estima que yo le tengo a él” (Ricoeur, 2011. pp. 141). las relaciones interpersonales; allí es donde persigo el propósito de que se me estime, de que se me apruebe, se me reconozca. Mi existencia, para mí mismo, es tributaria de esa constitución en la opinión de los demás; la constitución de los sujetos es, por consiguiente, una constitución mutua mediante la opinión. Pero la fragilidad de esta existencia, en tanto que reconocida, radica en que la <<estima>> que la consagra sólo sea opinión, en que la timé sea doxa; está ahí la amenaza de una existencia especular y cuasi- fantasmática; esa posibilidad de no ser ya más que una frase del otro, esa dependencia de la frágil opinión son precisamente la ocasión para las pasiones de la gloria que injertan su vanidad en la fragilidad de la estima como opinión” (Ricoeur, 2011. pp. 137-138). Pero sabemos, con Ricoeur, que solo los seres capaces de conocimiento, son seres capaces de reconocimiento y así: “la estima de mí mismo que busco mediante el aprecio del otro no es de distinta naturaleza que la estima que yo le tengo a él” (Ricoeur, 2011. pp. 141). La muerte como garantía de la verdad. El reconocimiento y la fragilidad afectiva. Es complicado lograrse una identidad y una estima sin reconocimientos paternales, y es complicado sentir el reconocimiento paternal por vías claras o sin pagar con una deuda infinita. En este complejo, diremos con Freud, se aprende el amor7. La otra cara de la fe, del deseo apasionado que nos levanta de un salto, pereza conjurada, para ver si el amado nos mira, es la fragilidad afectiva. Paul Ricoeur le dedica un capítulo en Culpabilidad y finitud, acompañándose del espíritu de Inmanuel Kant y dice: “Detrás de la tercera pasión según la antropología kantiana, la pasión de <<honor>>, de gloria, aparece una demanda más originaria que aquella, la demanda de valer en la opinión del otro, la demanda de estima (…) En esta demanda de estima, late un deseo de existir, no mediante la afirmación vital de sí mismo sino gracias al reconocimiento gracioso del otro. Entre esa demanda de estima y la posición egoísta y solipsista de la vida, está toda la distancia que media entre el simple deseo y lo que la Fenomenología del espíritu, denomina el deseo del deseo”. Esta demanda de reciprocidad, de la que no puede rendir cuentas ningún querer- vivir, constituye el auténtico tránsito de la conciencia a la conciencia de sí. Sin embargo, esta demanda no se satisface ni con las relaciones interhumanas surgidas con ocasión del tener, que son relaciones de exclusión mutua, ni con las relaciones surgidas con ocasión del poder, que son relaciones asimétricas, jerarquizantes y, por consiguiente, no recíprocas. Por eso, la constitución del Sí continúa más allá de la esfera de lo económico y de lo político, en la región de Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) las relaciones interpersonales; allí es donde persigo el propósito de que se me estime, de que se me apruebe, se me reconozca. Mi existencia, para mí mismo, es tributaria de esa constitución en la opinión de los demás; la constitución de los sujetos es, por consiguiente, una constitución mutua mediante la opinión. La muerte como garantía de la verdad. El reconocimiento y la fragilidad afectiva. Comprendamos a partir de aquí cómo lo que se presenta a menudo como síntoma de enfermedad no son sino episodios de la aventura del desamor, de la desasistencia, de la falta de receptividad, de la predicación en el desierto: “porque es creído, el valor del yo se puede fingir, alegar, aparentar; también se lo puede desconocer, contestar y denunciar; y también despreciar, vilipendiar, humillar; y cuando, con razón o sin ella, se desconoce, la falta de aprecio se puede compensar sobreestimándose a sí mismo o subestimando al otro y sus valores: agresividad, represalias, resentimiento, venganza, constituyen otras tantas respuestas al desconocimiento, que sólo comprendo por el afán de reconocimiento” (Ricoeur, 2011. pp. 142). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 8 Cierto es que, comprendida la naturaleza como simbólica, como el propio Ricoeur afirm © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 8 Cierto es que, comprendida la naturaleza como simbólica, como el propio Ricoeur afirma: Desproporción entre lo sagrado simbólico y la palabra interpretativa: reivindicación del misterio. Admitiendo felizmente mi persistente romanticismo, propongo que esa serie paralela de elementos que se encuentran puntualmente, en momentos kairóticos del camino y que, tomando a Ricoeur, se darían la mano como signos y misterio, se admita y comprenda también bajo los conceptos conjugados de naturaleza y cultura. Claro que se hace absolutamente imprescindible explicar que no estoy conduciendo nuestra problemática hacia la manida dicotomía antropológica pero sí, en cambio, disfrutaría dislocándola (y deslocalizándola), de tal modo que resucitemos la physis griega degustándola hasta su agenciamiento expresionista alemán, en el que también hace presencia el temor y el temblor, dando así a la cultura, no el carácter de envés de la naturaleza (o de freno o de represión) sino el de palabra que intenta atrapar, sin inmovilizar, los fenómenos que se nos antojan, por conveniente capricho, señales de una naturaleza nouménica, que aun siéndolo, nos interpela o, no haciéndolo o haciéndolo sin querer, nos invitara a una lectura flotante y nos dijera quién y qué somos y cuál es nuestro humilde papel dentro de su eterno discurso. Puede que haya un exceso del significante respecto de la referencia pero por ser esa referencia un constructo ya recortado por la expresión verbal8. Más bien destaca el inmenso exceso de la naturaleza, pues, Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) repito, poniéndome mucho más romántica que hebrea, es en ella donde habita el inagotable misterio y así, la sacralidad: “Lo sagrado de la naturaleza se muestra diciéndose simbólicamente. El mostrar funda el decir y no a la inversa. La sacralidad es inmediata o no es” (Ricoeur, 2011. p. 71). La palabra, igualmente, puede y debe resonar en el templo en cuanto que solo ella puede dar cuenta mediática de tal inmediatez. Así, el verbo tiene su lugar en la hora del ritual de sacralización, como palabra mensajera y como palabra expropiada de sí. La Naturaleza es simbólica y enigmática; la palabra es metafórica y resuelve o indica el sentido de los enigmas: “El símbolo está ligado. El símbolo tiene raíces. El símbolo se une en la experiencia tenebrosa de la Potencia. La metáfora es sólo la superficie lingüística que debe a su bi-dimensionalidad el poder de religar lo semántico a lo pre-semántico en la profundidad de la experiencia humana” (Ricoeur, 1988. p. 28). “ninguna categorización dada da cuenta de las potencialidades semánticas tenidas en suspenso en el símbolo, pero es únicamente el trabajo del concepto el que puede testimoniar este exceso de sentido” (1988. p.29). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 testimoniar este exceso de sentido (1988. p.29). 9 Tengamos en cuenta el significado etimológico de aporía: sin camino, siendo que así se denominaba el lugar por donde no hay pasaje o en el que no puede marcarse una senda. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI EuroMed University Salento \| ISSN 1889-7231 testimoniar este exceso de sentido (1988. p.29). 9 Tengamos en cuenta el significado etimológico de aporía: sin camino, siendo que así se denominaba el lugar por donde no hay pasaje o en el que no puede marcarse una senda. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 ninguna categorización dada da cuenta de las potencialidades semánticas tenidas en suspenso en el símbolo, pero es únicamente el trabajo del concepto el que puede testimoniar este exceso de sentido” (1988. p.29). 9 Tengamos en cuenta el significado etimológico de aporía: sin camino, siendo que así se denominaba el lugar por donde no hay pasaje o en el que no puede marcarse una senda. Desproporción entre lo sagrado simbólico y la palabra interpretativa: reivindicación del misterio. Este ecologismo mío, romántico, desde el que hablo, no pretende, contra el racionalismo ilustrado, que la naturaleza deje de resultarnos temible, sino que hallemos deleite en quererla terrible (tremenda) como es. Vuelvo en este punto a los recuerdos de infancia y me pregunto cómo sería posible, sin tal deleite, la infancia misma, que se me antoja ahora como conjunto de ensoñaciones inspiradas por los cuentos. El cuento me parece, por excelencia, el género literario que aborda, esencialmente, el misterio. El cuento nos devolvía la visión fantástica de los bosques a los niños de las sociedades industrializadas y nos traía esa noticia tenue de los lobos, de los osos y de los magos, brujas y monstruos, es decir, de esos personajes fronterizos que habitan en el medio de lo humano y lo divino, entre la luz de la luna y la obscuridad de la noche. Si un humano estaba en el bosque solo podía ser que anduviera perdido y buscando, entre la emoción y el miedo, un camino de vuelta, porque si un humano vivía en el bosque, no era del todo humano y si una casita se recortaba entre la espesura, sería, sin duda, una casita encantada. Perderse en el bosque es, quizás, nuestra primera imagen del viaje iniciático. En el cuento, los niños curiosos, atraídos por los frutos y recovecos y por la abundancia de la maleza, es decir, atraídos por señales indescifradas y por el deseo de encontrar escondites secretos alejados del mundo adulto del orden y del trabajo, pues los niños son, ellos mismos, personajes también fronterizos, iniciaban un camino aporético, irreversible, un camino que no permitía trazar huellas permanentes, como no se podían tampoco trazar, decían los griegos, en el mar9. Por eso en el cuento hay un proverbio, una moraleja siempre extraña y ambivalente. El cuento es la historia de una enseñanza de la naturaleza que se manifiesta y de un aprendizaje propiciado por la subrayada irreversibilidad del camino de ida, o dicho de otra manera, porque el camino de retorno nunca podría ser una simple inversión del primero sino otro, lleno de desvíos y de desvaríos, que nos devolvería a casa con la sabiduría insólita y extranjera de un encantamiento, con unos conocimientos que aparecerán ante los adultos Nómadas. Desproporción entre lo sagrado simbólico y la palabra interpretativa: reivindicación del misterio. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) como quimeras y despropósitos de un niño que, tras los años, dudará, habituado de nuevo al interior de la caverna, de la propia realidad de aquel viaje. Así se cuenta la historia del inconsciente, de lo otro, de lo salvaje, de la fantasía (incluyamos la fantasía de conexión que hace que bajo el embrujo del amor, digamos: “estoy viviendo en un cuento de hadas”; y que enseguida nos imaginemos exiliados en un paraíso de playa y mar o de cielo y nubes...). El cuento es la intersección de la palabra y la naturaleza, una intersección en la que tratamos, con los personajes, de leer una naturaleza que no se deja textualizar del todo, ni para el escritor del cuento, que no logra eliminar la sospecha infantil de estar siendo engatusado por historias y relatos, ni para los protagonistas, cuyos “cuadernos de bitácora”, inversamente, no son tomados en serio por el mundo de los adultos. En el cuento, la fe está en juego y en el juego de orientación y desorientación está la fe. Se diría que el cuento replica el mito de múltiples formas. Por un lado, ambos tipos de narración relatan un regreso desde la vivencia del “otro lado”. Pero, además, el cuento, propio de los tiempos modernos, nos translada a eso otro que en el tiempo de los antiguos mitos preindustriales, no era todavía lo otro. Incluso podríamos ensayar la siguiente perspectiva: el cuento constituye, con respecto a los mitos, un futuro anterior, pues si en el mito, a menudo, se describe la fundación de una ciudad, en el cuento se novela la huida de la ciudad y el adentramiento en lo que pareciera, sea fábula o no, lo previo, lo que era antes del dominio humano. En todo caso, el primitivo, el niño, el loco y, así también, el religioso, por ser premodernos, no habrían olvidado aún el universo de lo sagrado. La desacralización es tan indeseable para Paul Ricoeur como lo fuera para Friedrich Nietzsche o Martin Heidegger, pero, quizás más optimista en cuanto a la posibilidad de mediación, ya entre la conservación de lo sagrado y la revelación cristiana, ya entre la conservación de lo sagrado y la política crítica, o, dicho de otro modo, los movimientos de liberación. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 10 También podríamos distinguir justicia y amor diciendo que solo se requiere justicia porque hay algo a lo que hacer justicia, algo a lo que se ama, algo en lo que hay que tener fe, o hacia lo que nos mueve la fe: “Ni las circunstancias de la justicia ni sus vías son las del amor. Todavía menos los argumentos de la justicia son los del amor. A decir verdad, el amor no argumenta...” (Ricoeur, 2011b. p. 42); “La economía del don desborda por todas partes a la ética. Todo un abanico de significaciones confiere una articulación específica a esta economía del don. En un extremo de este abanico nos encontramos con el simbolismo, él mismo muy complejo, de la creación, en el sentido más fundamental de donación originaria de la existencia...” (p. 47); “la economía del don desarrolla una lógica de sobreabundancia que, en un primer momento al menos, se opone polarmente a la lógica de equivalencia que gobierna la ética cotidiana” (p. 48-49); “El hombre entra en el mundo ético por el miedo y no por el amor” (Ricoeur, 2011. p. 193). Creo que esta distinción de Ricoeur es la que retoman o expresan también, a su modo, los filósofos de las políticas del amor y de las políticas estéticas. Pero a pesar de las simplificaciones escolares, ya en Kant se prefigura esta idea, tan propia de Bataille, del deseo como primero respecto de la razón, del don como lógica anterior a la de la justicia, y de la religión como verdadero corazón de la política. Ricoeur trata de recogerlo en su estudio sobre la síntesis práctica, con la siguiente aseveración: “El principio será solo práctico si el deber conmociona al querer” (p. 92). La vuelta moralista y la insistencia en la doblegación y fulminación del deseo se encuentra también, imposible de evadir, en otros pliegues del carácter kantiano. En ese punto Ricoeur toma distancia, pues como sabemos, en él, el saber divino se diferencia del todo de la pretensión diabólica de © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 El sentido de la muerte como respuesta a la pregunta por el sentido de la vida. La fantasía de conexión es una dimensión paradigmática de lo sagrado pues, como decíamos, solo en ella perdemos el miedo a la muerte sin dar la espalda al dramatismo de su realidad y sin evadir la pregunta por la crueldad de la finitud. La fantasía de conexión es la vivencia efectiva de la confianza incondicional que denominamos fe. La fe es moral y es política: el que carece de fe, de impulso vital, de vigor sin cuestionamientos, de compromiso con la causa antes que con cualquier otro cálculo, anda por la vida errante, lento, aturdido, anda, se dice, desmoralizado. La política solo es política a condición de perseguir e insistir en lo unitivo del lazo social, es decir, a condición de con- crear comunidad, que no mercado. A fin de cuentas, hacer política es también hacer el amor, dado que siendo la justicia la piedra angular de la política, ¿qué es el amor sino la más básica y espontánea de las justicias? El amor es la justicia que se vive en la inmediatez y no en la reparación10. Todo los cálculos 10 También podríamos distinguir justicia y amor diciendo que solo se requiere justicia porque hay algo a lo que hacer justicia, algo a lo que se ama, algo en lo que hay que tener fe, o hacia lo que nos mueve la fe: “Ni las circunstancias de la justicia ni sus vías son las del amor. Todavía menos los argumentos de la justicia son los del amor. A decir verdad, el amor no argumenta...” (Ricoeur, 2011b. p. 42); “La economía del don desborda por todas partes a la ética. Todo un abanico de significaciones confiere una articulación específica a esta economía del don. En un extremo de este abanico nos encontramos con el simbolismo, él mismo muy complejo, de la creación, en el sentido más fundamental de donación originaria de la existencia...” (p. 47); “la economía del don desarrolla una lógica de sobreabundancia que, en un primer momento al menos, se opone polarmente a la lógica de equivalencia que gobierna la ética cotidiana” (p. 48-49); “El hombre entra en el mundo ético por el miedo y no por el amor” (Ricoeur, 2011. p. 193). Creo que esta distinción de Ricoeur es la que retoman o expresan también, a su modo, los filósofos de las políticas del amor y de las políticas estéticas. Desproporción entre lo sagrado simbólico y la palabra interpretativa: reivindicación del misterio. Ricoeur nos alivia así: “La modernidad no es un hecho ni un destino, es una cuestión abierta” (Ricoeur, 2011. pp. 82); y sobre todo con este hermosísimo interrogante: “¿Se puede acaso vivir sin orientación originaria? ¿O sólo es un fenómeno residual o una protesta existencial venida de las profundidades, la que nos empuja a la búsqueda de lugares privilegiados, sea nuestra patria de nacimiento, la escena de un primer amor, o el teatro de algún gran hecho histórico -batalla, revolución, ejecución de patriotas...? Volvemos a esos lugares porque allí otra realidad distinta de la cotidiana había hecho irrupción y porque el recuerdo que vincula el acontecimiento con ese lugar nos preserva a todos de convertirnos en seres errantes ¿Pueden los actos de construir y habitar quedar enteramente desacralizados sin perder toda especie de significación? ¿Es posible abolir la simbólica del umbral de la puerta, del hogar y todo ritual de ingreso y acogida?¿es posible desacralizar enteramente el nacimiento -venida al mundo- y la muerte -ingreso al lugar del reposo?¿Es posible despojarlo de todo rito de pasaje sin degradar enteramente al hombre, convirtiéndolo en un utensilio; sin exponerlo sin resto a una manipulación que concluye con la liquidación de un desperdicio?¿Es posible abolir todos los ritos de iniciación restantes sin que los <<pasajes>> de la vida se conviertan en simples transiciones Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) insignificantes?¿Y la sexualidad no deviene totalmente insensata cuando se rompe todo vínculo entre ella y el gran juego de las uniones cósmicas?¿El tiempo de la repetición, despojado de toda ritualización, puede acaso ser otra cosa que una figura de la condenación?¿Hay acaso algo más paralizante que el eterno retorno sin regeneración?¿Es posible vivir en un tiempo sin fiestas, según un calendario absolutamente profano? Por último ¿es por casualidad que lo que en América se llama <<subcultura>> se encuentre desesperadamente en busca de la fiesta en comunicación con fuerzas de la naturaleza que de nuevo se comenzarían a admirar, sin explotarlas? Tales son las nuevas cuestiones que empezamos a plantear, más allá de la muerte de lo sagrado ¿No nos encontramos en las vísperas del renacimiento de los sagrado, si a pesar de todo el hombre está llamado a sobrevivir? (Ricoeur, 2011. pp. 82-83). El sentido de la muerte como respuesta a la pregunta por el sentido de la vida. Pero a pesar de las simplificaciones escolares, ya en Kant se prefigura esta idea, tan propia de Bataille, del deseo como primero respecto de la razón, del don como lógica anterior a la de la justicia, y de la religión como verdadero corazón de la política. Ricoeur trata de recogerlo en su estudio sobre la síntesis práctica, con la siguiente aseveración: “El principio será solo práctico si el deber conmociona al querer” (p. 92). La vuelta moralista y la insistencia en la doblegación y fulminación del deseo se encuentra también, imposible de evadir, en otros pliegues del carácter kantiano. En ese punto Ricoeur toma distancia, pues como sabemos, en él, el saber divino se diferencia del todo de la pretensión diabólica de Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) y análisis que caen fuera de la fe, en el ámbito de una pura razón, juegan su papel como elementos tácticos, más la auténtica estrategia se halla en la fe, en la convicción, en el deseo vital: “creer es afirmar y afirmar es <<hacer>>” (Ricoeur, 2011. pp. 51). Así da cuenta Ricoeur del sentido de la teología política hallando en el cristianismo la poética de la política o, lo que yo he estudiado en otras ocasiones como ontología política: “Ciertamente, la existencia humana es existencia política. Pero los textos en los cuales la existencia cristiana se comprende a sí misma sólo son políticos en la medida en que son poéticos. De modo que los modelos para una familiaridad entre Dios y su pueblo y el resto de los hombres constituyen más bien lo que denominaría una poética de la política que, para recibir una cualificación propiamente política, reclama ser articulada en análisis, saberes, intereses, organizaciones, etc. Para emplear un lenguaje weberiano, diré que esos modelos no alcanzan la política sino nutriendo una moral de convicción, imposible de reducir a la mora de responsabilidad que, no lo olvidemos, es asimismo la del uso limitado de la violencia” (Ricoeur, 2011. p. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 sentar y clasificar según lo que de antemano es el bien y el mal, y lanza su particular apuesta: “la reflexión transcendental se sitúa de entrada en el nivel de lo originario, sin tener que alcanzarlo a través de una condición depravada” (p. 96). El sentido de la muerte como respuesta a la pregunta por el sentido de la vida. 107) De este modo, podemos retomar la comprensión de la muerte como consagración de la vida, siendo la muerte la que sacraliza la vida tal y como el profeta, siendo el poeta de la política, ejemplifica en su convicción, con su fe e inmediatez y con el trabajo de dar algo para la interpretación, tanto en su vida como con su muerte, que deviene, asimismo, signo: “todo profeta, en la medida en que profetiza contra, es profeta para la vida y para la muerte” (p. 119). El profeta es poeta, ya que siendo el que resuelve enigmas activamente, genera sentido al modo de la metáfora (Ricoeur, 1988. p. 26). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Una teología política trae la naturaleza, la Potencia, al campo de lo social, a través de la acción humana con sentido, si bien con un sentido despsicologizado en tanto los actores desconocen la trama. El saber del desconocimiento marca la dimensión religiosa. La fe impulsa el hacer, el cumplir queridamente bueno de un papel, de nuestro papel, en el que tratamos de interpretar signos, ser interpretados por dicho interpretar y convertirnos, a su vez y en el mejor de los casos, en signos que troquelarán el rumbos de otros, las interpretaciones y reinterpretaciones de los demás. El otro que se nos viene en modo de afecto, en modo de textos, en modo de habla, en modo de movimientos, en modo de hábitos, nos permite situarnos, aunque semi- aleatoriamente, en ese universo sagrado que co-construimos . No es necesario, en cambio, liderar masas ni rodearse de todo el mundo en el momento periodístico, para cumplir políticamente. Es posible, de entre las maneras, el juego político en el retiro, sobre todo, es posible a tenor de los ciclos y de la consideración de los mismos: “La llamada aísla; el envío religa” (Ricoeur, 2011b. p. 87), pues no es del todo posible distinguir el papel de unos y de otros o hasta dónde llegó la acción de ellos y desde dónde comienza la mía. Por suerte, nuestros actos escapan a las simples intenciones propuestas. El sentido de la muerte como respuesta a la pregunta por el sentido de la vida. sentar y clasificar según lo que de antemano es el bien y el mal, y lanza su particular apuesta: “la reflexión transcendental se sitúa de entrada en el nivel de lo originario, sin tener que alcanzarlo a través de una condición depravada” (p. 96). Una teología política trae la naturaleza, la Potencia, al campo de lo social, a través de la acción humana con sentido, si bien con un sentido despsicologizado en tanto los actores desconocen la trama. El saber del desconocimiento marca la dimensión religiosa. La fe impulsa el hacer, el cumplir queridamente bueno de un papel, de nuestro papel, en el que tratamos de interpretar signos, ser interpretados por dicho interpretar y convertirnos, a su vez y en el mejor de los casos, en signos que troquelarán el rumbos de otros, las interpretaciones y reinterpretaciones de los demás. El otro que se nos viene en modo de afecto, en modo de textos, en modo de habla, en modo de movimientos, en modo de hábitos, nos permite situarnos, aunque semi- aleatoriamente, en ese universo sagrado que co-construimos . No es necesario, en cambio, liderar masas ni rodearse de todo el mundo en el momento periodístico, para cumplir políticamente. Es posible, de entre las maneras, el juego político en el retiro, sobre todo, es posible a tenor de los ciclos y de la consideración de los mismos: “La llamada aísla; el envío religa” (Ricoeur, 2011b. p. 87), pues no es del todo posible distinguir el papel de unos y de otros o hasta dónde llegó la acción de ellos y desde dónde comienza la mía. Por suerte, nuestros actos escapan a las simples intenciones propuestas. sentar y clasificar según lo que de antemano es el bien y el mal, y lanza su particular apuesta: “la reflexión transcendental se sitúa de entrada en el nivel de lo originario, sin tener que alcanzarlo a través de una condición depravada” (p. 96). Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) Es por ello que pasan a formar parte de los signos y de los acontecimientos: “el tiempo social no es solo algo fugaz; es también el lugar de los efectos duraderos, de pautas persistentes. Una acción deja una huella, pone su marca...” (Ricoeur, 1988. p. 66). El sentido de la muerte como respuesta a la pregunta por el sentido de la vida. La muerte es, para un ser finito, es decir, para alguien que no es un dios, la única prueba que puede dar de una verdad que lo excede, es la única comunión definitiva con lo divino, con lo otro. A menudo se critica como impostura el ritual ensalzamiento del fallecido, diciendo burlonamente: “Resulta que cuando uno muere, siempre era muy bueno, muy bueno” o “cuando uno muere, todo de él eran virtudes”, etc. En cambio, no advertimos que los rituales suelen esconder un sentido11 que, por olvidado, no deja de resultar reconfortante y es que la muerte constituye una donación, querida o no o, más bien, conscientemente querida o no, una ofrenda, un sacrificio que, como tal, constituye una prueba. El que muere es el que lleva su verdad hasta el final e, inversamente “una verdad que no captura al hombre hasta el sacrificio carece de prueba” (Ricoeur, 2011, p. 130). La muerte es una tragedia y, como tal, igual que en su acepción literaria, ennoblece:“la tragedia es una imitación de las acciones humanas que las hace parecer mejores, más elevadas...” (Ricoeur, 1988. p. 55). En este punto, nos encontramos en condiciones de contestar al por qué de la crueldad de la muerte y de calmar la búsqueda del sentido de la vida trocando, precisamente la pregunta por el sentido de la vida en búsqueda de sentido para la muerte. Pienso ahora que tal vez la vida es un preguntar, mientra que la muerte es, o debemos hacer que sea un contestar. Quizás si la muerte es contestataria, haya una esperanza para la transcendencia. Al fin y al cabo, el infinito es, para el humano, una transgresión vivida como intención de significar. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 11 “El prejuicio contra el prejuicio procede en efecto de un prejuicio profundamente arraigado contra la autoridad, que se identifica demasiado rápidamente con la donación y la violencia” (Ricoeur, 1988, p. 156). Lo infinito como transgresión: el deseo humano de significar. Siempre me llamó la atención la cuestión del pecado original y ni en mi fase más atea pretendí ni quise disolver tal interrogante de modo vulgar, condenando, por ejemplo, a las religiones por tratar de infundirnos una culpa que nos desacreditara como dueños y señores de nuestra existencia o porque nos impidiera protestar ante un destino cruel y no elegido o porque nos impeliera a la sumisión apropiada de quien tiene que pagar por un agravio del que se es autor, aun en la ignorancia. Siempre tomé la cuestión del pecado original como un enigma en el que se albergaba una verdad porque la sentía: sentía algo de eso, sentía que algo así como una pena (o dígase malestar si se prefiere el vocablo freudiano) recorría nuestra existencia: una pena o una responsabilidad originaria o algo que liga a una y la otra cosa o que las hace ser lo mismo. Pudiera ser esa pena la que se paga por la culpa de haber nacido y que se compensa con la muerte. No se indica tanto una culpa en la sentencia Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) retomada por Anaximandro, sino una deuda ontológica, la de romper con la unidad del Ser para ocupar un lugar y un tiempo. En todo caso me interesa destacar que esa deuda ontológica que se vive como un cierto dolor12, bien se presente a modo de angustia apremiante, bien se quede en una segunda pantalla, haciéndose casi imperceptible, nos permite presentir el puente vinculante entre el nacimiento y la muerte y, tal vez incluso, se ofrezca como el recuerdo de la desventura, de la ansiedad, del quebranto... del primero, y como la expectativa fúnebre de la segunda, que, al modo de cualquier susceptibilidad fóbica, nos atemoriza y atrae o nos atemoriza como lo hace siempre un fuerte deseo inobservante. El vértigo me parece la fobia por excelencia: la atracción y terror hacia el abismo. Al abismo no se llega ni, por supuesto, se asciende a él. En el abismo se cae. Su llamada no abre un camino, y ni siquiera un viaje aporético como el de los cuentos, sino que su llamada pide precipitación, un salto arrojado e irreflexivo al vacío. 12 Esa juntura precaria, esa Diferencia unitiva es lo que Heidegger denomina “dolor”. En la misma línea, Bataille, siguiendo al Freud de Más allá del principio del placer, denominará pulsión de muerte al “instinto” de reconstruir el tejido de la continuidad ontológica. Deleuze sitúa ese dolor ontológico en las grietas de las superficies que comunican el interior de lo ente con su Afuera constituyente. En cierto modo, podemos decir que en las superficies tiene lugar la diferencia ontológica. 13 Consideremos por un momento ese relato tan repetido por aquellos que sufrieron una muerte clínica fugaz ¿Por qué pensar que todos inventan esa historia acerca del túnel obscuro a cuyo término se avista una luz? No es más fácil pensar que se produce un rememoramiento de sensaciones natales? Pues, ¿no parece lógico que el bebé, que sale de la obscuridad por un conducto estrecho y obscuro, sienta por vez primera una luz casi cegadora al final, una luz que lo atrapa y trae hacia sí? © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Lo infinito como transgresión: el deseo humano de significar. Tengo el convencimiento de que así se siente el nacimiento (y en esto me apoyan muchas voces de autoridad dentro de la filosofía en incluso de la ciencia) y de que, desde esa precisa impronta imaginamos también la muerte13. Puedo que solo tengamos miedo a la muerte por lo abismático que fue el nacimiento, más sabiendo, por los cuentos, que los caminos de vuelta son todavía más retorcidos y atorados que los de ida. Continuando, por un momento, la analogía con el cuento, algo más hay seguro, más seguro si cabe: en el medio entre el emprendimiento y el retorno se produce la sensación de estar perdido. El nudo del cuento aparece justo en ese punto en el que el protagonista se da cuenta de que se ha perdido ¿Por qué entonces habría de sorprendernos el sentirnos perdidos? ¿Por qué, de hecho, lo tomamos como trastorno, siendo tal vez la esencia del estar vivo, humanamente vivo? Pues vivimos, estamos perdidos. Así el Evangelio tan reseñado por Paul Ricoeur, “el que quiera salvar la vida, la perderá”, toma, me parece, nueva luz. Caer al vacío es un sueño típico en el recién dormirse. Muchas veces nos avisó, eléctricamente, de que morimos así en la vigilia y nacemos al sueño, cambiando por ello, la orientación y estilo del argumento. Caemos en el sueño y caeremos, pensamos también entonces, en la muerte, pues se dice de la muerte “el sueño eterno”. Curiosamente también con sueño significamos ambición, anhelo, aspiración. Y, ¿no es la ambición sino ansia de eternidad, de anclar en la Memoria? ¿Cuál es, pues, tu sueño? Sin duda, debe ser el sueño Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) eterno. No sé si queremos morir, a condición de significar o queremos significar para poder enfrentarnos a la muerte, pero, consciente de que esta segunda posibilidad parece más a tono con el sentido común, quisiera hacerme cargo más bien de la primera, pues retomando las reflexiones anteriores en torno al impulso tanático que inunda nuestra impotente impaciencia por mostrar la verdad, en torno a ese recurrente “doy la vida por...”, y siempre conocedora de que la filosofía le hace la batalla al sentido común, se me antoja una línea más interesante y esclarecedora. Durante mucho tiempo creí, en cambio, que el pecado original tenía un carácter más epistemológico-ético que ontológico. Lo infinito como transgresión: el deseo humano de significar. Me subscribí a la que me parecía la incuestionable y únicamente válida interpretación del pecado original: el pecado original como metáfora de la pérdida de la inocencia. Estimo oportuno detenerme unos momentos en ella pues recién es que avisto otras perspectivas que aún no sé si sustituirán a ésta o más modestamente, la complicarán. La pérdida de la inocencia es la condición humana misma. Se trata de una pérdida inevitable, irrevocable e irrenunciable. Es por eso que aún adscrita a las filosofías postestructuralistas del deseo, en la reivindicación de la inocencia, no encontré sino la auténtica dirección para mi ecologismo (que no es poco), para mi animalismo (que no es poco), para mi religión, a saber: el respeto por el misterio, la reivindicaión de la conservación de lo sagrado y la idea de que los animales salvajes, por no ser buenos ni malos, son más buenos que nosotros. Pero, en la proximidad con los perros, que son medio animales medio humanos, encontré la fascinación por lo divino humanizado, por el mensajero de los dioses mistéricos, de los lobos, que viniendo a vivir entre nosotros, nos traían su legado para que lo entendiéramos, o mejor, para que intuyéramos la legitimidad de la fe, la razón de la fe. El perro ya no es salvaje pero procedente de lo salvaje, parece exhibir lo mejor de lo humano. Y así la figura de Cristo, tan semejante, dejó de parecerme la antropomorfización y conjura de lo divino para parecerme la figura del puro amor y de la entrega, del que da la vida para mostrar una verdad que solo con tan enorme sacrificio puede mostrarse ante la condición precaria del ser humano, ante lo que Paul Ricoeur, en su reecritura de Platón, denomina la miseria, lo “patético” de la miseria: “En efecto, es la situación misma del alma la que es miserable; en la medida en que es, por excelencia, el ser que se encuentra en el medio, no es la Idea; como mucho, es <<de la raza de las ideas>> y de lo que está <<más próximo>> a la idea; pero tampoco es una cosa perecedera: su cuerpo es lo que <<más se parece>> a lo corruptible; el alma es más bien el movimiento mismo de lo sensible hacia lo inteligible; es la anábasis, la ascensión hacia el ser; su miseria se muestra, ante todo, en estar perpleja y en buscar” (Ricoeur, 2011. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Lo infinito como transgresión: el deseo humano de significar. p. 27). El pecado original como pérdida de la inocencia o, si se quiere, como toma de conciencia, se relata en el Génesis, a mi parecer, de hecho, de modo inmejorable. La tentación diabólica del fruto del árbol del conocimiento constituye la entrada involuntaria en la moralidad. Este fruto se registró en el imaginario como manzana, al resonar con las manzanas de la mitología (las manzanas de las Hespérides, la manzana de la discordia disputada por Hera, Afrodita y Atenea...) y al repetirse en innumerables cuentos (Blancanieves, Guillermo Tell, Hansel y Gretel...) o quizás, dicen, por el parecido entre los Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) términos latinos malum (manzana) y mali (mal). A este imaginario podemos añadirle otro sedimento definitivo: el de la manzana de Newton, pues ese árbol de su relato es, sin duda, un árbol de la ciencia. Con las manzanas se prometía la inmortalidad pues, en las representaciones del mal, un diablo nos convence de que conocer es lo mismo que ser y que, conociendo los secretos del eterno Dios, sabremos cómo alcanzar su misma eternidad. Sin embargo, las manzanas prometían inmortalidad a aquellos que no sabían de su mortalidad, a aquellos que ya eran eternos o vivían como eternos, si no fuera porque en un mismo momento, la promesa de inmortalidad arruinaba y auguraba una eternidad reflexiva. Una eternidad así, de segundo grado, traída de la meditación es sospechosa, pues justamente la inmediatez define la eternidad. A cambio, se abre la dimensión religiosa, desde lo patético de la miseria y hacia la transgresión de la propia condición humana; se abre el deseo de salir de la insignificancia para significar y tener un sentido, el de un interrogante que se abre con el nacimiento y se cierra con la muerte: “Pertenece, pues, a la finitud humana no poder experimentarse a sí misma más que bajo la condición de una <<visión-sobre>> la finitud, de una mirada dominadora que ya ha empezado a transgredirla”; “Este discurso completo sobre la finitud es un discurso sobre la finitud y sobre la infinitud del hombre” (Ricoeur, 2011. p. 42). 14 Según Bataille, el ser humano no vive fuera de la animalidad sino más bien en un lugar situado entre el deseo de volver a ella y la imposibilidad de cumplirlo. La libertad humana o, mejor dicho, la libertad que el humano persigue no puede ser ya la vuelta al mundo apetitivo del animal, del mismo modo que la violencia humana no puede volverse hacia atrás y participar de la violencia inocente del animal. Tenemos que contentarnos con una animalidad rarificada, encontrada en el camino de vuelta, que no repite el de ida sino © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Lo infinito como transgresión: el deseo humano de significar. Retomaré, sin embargo, la cuestión del pecado original tomada desde aquel otro lugar, tal vez vecino, pues si nos centramos en el conocimiento para comprenderlo, no sabemos ya si hablamos de una caída o si hablamos de una ascensión, del mismo modo que queda desdibujado, borroso, si la adquisición de la moral es una caída o es una ascensión. Supongo que es una caída respecto del principio, de la inocencia del no-saber del bien y del mal, y un ascenso respecto del ya-saber del bien y del mal. Pero si la adquisición de la moral se da ahí, en el medio, es, de nuevo, un nombre del estar perdido. Ciertamente, los protagonistas de los cuentos se hallan perdidos entre el viaje de ida y el de vuelta, en el momento en el que tras una aventura de descubrimientos, comprenden que dichos descubrimientos no sirven para volver a casa más que, en todo caso, reinterpretados como signos en un metarrelato. Entre tanto, están perdidos. Puede que aquella angustia de la que hablaba, aquel dolor que en primer o en segundo plano, nos acompaña, provenga de un saber de la muerte: “la maldición no es que el hombre muera (<<pues eres barro y volverás al barro> sino que afronte la muerte con la angustia de la inminencia: la maldición consiste en la modalidad humana del morir” (Ricoeur, 2011. p. 390). Sin embargo, ya está ahí desde el nacimiento, desde antes del conocimiento y desde antes del conocimiento de la muerte. Podría ser entonces, una anticipación solo intuida de la muerte pero ya fijé, de todas maneras, que muy probablemente, la expectativa dolorosa de la muerte se produzca porque se calca de la experiencia dolorosa y ya vivida del nacimiento. Y es que el nacimiento rompió la certeza de nuestra primera fantasía de conexión, depositándonos en esa independencia que toma el humor de un desmembramiento: “la tristeza de lo finito (…) se alimenta de todas las experiencias primitivas que, por decirlo así, implican la negación: carencia, pérdida, temor, pesar, decepción, dispersión e irrevocabilidad de la duración...” (p. 157). Lo infinito como transgresión: el deseo humano de significar. Pero el humano es humano porque conoce, así que el patetismo de la miseria le es inevitable y el conocimiento es el peor de los males por radicar la distinción entre el bien y el mal, y es el mejor de los dones porque solo conociendo nos aproximamos a ese Dios que no podemos ser y porque, entonces, tenemos la oportunidad de ser buenos por determinación y no por casualidad, al distinguir entre el bien y el mal. En cualquier caso, una evidencia quedó popularmente obscurecida: en el relato bíblico no es Dios sino el diablo el que instaura el orden moral. Lo que no queda claro es si lo diabólico colabora en la trama de algún plan divino, es decir, si el hombre como ese ser distinto de los animales creados por Dios, es una producción también de Dios o del diablo o de los diabólico de la autoconciencia. No obstante, como decíamos, la pérdida de la inocencia es irrenunciable y una vez que nuestra curiosidad ha mordido el anzuelo, ya no podemos, sino por imposturas y perversas maniobras, volver al estado de inconsciencia. Podemos, tal es la invitación de Deleuze, deconstruir los códigos, pero no creamos, como solicita, que es viable hacerlo al margen de la idea de la transgresión, es decir, inocentemente. Una cosa es desestructurar los códigos y otra muy distinta y casi contraria es fingir que no los conocemos. No sé si un esquizo puede hacerlo pero una esquizofrenia no se auto-induce ni es, como el propio Deleuze indica en Lógica del sentido, una recuperación de la niñez (Deleuze, 1989. Decimotercera serie). En este aspecto concreto, mantengo con Sartre, que no darse por enterado es la más vil de las malicias. Necesitamos un proyecto ético y volver a la inocencia (a la inconsciencia) no es una opción14: “Por <<débil>>, Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) <<impotente>>, <<inconstante>> y <<ridículo>> que sea, el hombre no deja de llevar la carga de conocerse: <<Estar lleno de defectos es, sin duda, un mal; pero es un mal todavía mayor estar lleno de ellos y no quererlos reconocer, pues esto es añadir asimismo a los anteriores el de una ilusión voluntaria (fragmento 100 de los Pensamientos de Pascal)>>” (Ricoeur, 2011. p. 33). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 añadiéndole una sensación de caída, de pecado, de transgresión. La animalidad recuperada ya no es la pura animalidad: ya no tiene el sentido de la “libertad” sino el de la transgresión: “La superación de una situación no es nunca un retorno al punto de partida. En la libertad está la impotencia de la libertad” (Bataille, 2007. p. 134). 15 Debería matizar que, según Paul Ricoeur, la concepción del pecado y de la culpa pasa por varios estadios, tanto en el sucederse de las interpretaciones religiosas como en el propio paso del pecado original, más antiguo en el relato y más primario en la vivencia personal, al pecado como mal a evitar mediante la sospecha de sí, con lo que se inicia la reapropiación del proyecto y significaciones vitales. La culpa individual es más laica que religiosa pero la obra de la propia vida y la convicción que la impulsa, que es fe, perteneciendo también a la religiosidad, pues se trata de recuperar dentro de sí mismo, la mirada absoluta, constituyen el índice de una teología ético-política. En todo caso, tal programa exige de la religación, de lo que hemos denominado fantasía de conexión, que implica unidad, también en la rendición de cuentas: “No se trata de negar que la imputación personal de la culpa marca un adelanto con relación a la escandalosa responsabilidad colectiva que permite castigar a otro que al culpable. Pero hay que entender que el precio de este adelanto es la pérdida de la unidad de la especie humana, congregada <<ante Dios>> por el vínculo más que vital y más que histórico de la culpa: el pseudo-concepto de pecado original no es más que la racionalización de tercer grado, a través del mito adámico, de ese vínculo enigmático, confesado más que reconocido en el <<nosotros>> de la confesión de los pecados” (Ricoeur, 2011. p. 243). Lo infinito como transgresión: el deseo humano de significar. No es la muerte la que nos arrebata, en primera instancia, la eternidad, sino el nacimiento, pues con él resulta que no fuimos siempre, que tenemos una perspectiva concreta y no todas a la vez y que nuestra génesis está depositada en el relato de otro: “Mi nacimiento me habla, por lo demás, de mi existencia como de algo recibido; no sólo como algo que me encontré ahí, sino como algo que me dieron otros; he sido traído al mundo, desciendo de mis padres; ellos son mi ascendencia; sin embargo, ignoro lo que significa esta donación que me convierte en heredero de mi propia vida. Aquí, más que en ninguna parte, es donde me acecha el vértigo de la objetivación” (p. 81); “El añadiéndole una sensación de caída, de pecado, de transgresión. La animalidad recuperada ya no es la pura animalidad: ya no tiene el sentido de la “libertad” sino el de la transgresión: “La superación de una situación no es nunca un retorno al punto de partida. En la libertad está la impotencia de la libertad” (Bataille, 2007. p. 134). Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) punto de vista es el ineludible estrechamiento inicial de mi apertura al mundo. Pero esta necesidad no es un destino externo; no se convierte en tal sino por medio de una falsificación, cuya andadura es fácil rastrear. No distingo dicho estrechamiento de mi apertura misma sino relacionándolo, mediante una nueva regresión más allá de todos mis <<desplazamientos>> pasados, con un primer lugar que coincide con el suceso de mi nacimiento. He nacido en algún lugar: <<una vez puesto en el mundo>>, en adelante percibo ese mundo mediante una serie de cambios e innovaciones a partir de ese lugar que no he elegido y que no puedo recuperar en mi memoria. Entonces, mi punto de vista se desgaja de mí como un destino que gobierna mi vida desde fuera” (p. 41). El acontecimiento del nacer resulta, si recapitulamos, ontológicamente “injusto”, experiencialmente fracturador, cósmicamente arbitrario, físicamente doloroso y biográficamente ajeno. No es extraño que lo imaginemos acompañado de una mancha y de una condena. Podríamos llegar a decir que no nacemos con el pecado original sino que nacer es el propio pecado original. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Lo infinito como transgresión: el deseo humano de significar. Para comprenderlo bien, debo traer las clarificaciones de Ricoeur sobre el pecado: el pecado original no es individual sino que yace junto a la condición de expropiación del propio núcleo personal15; el pecado se encuentra más cerca de la pena que de la infracción, es decir, se revela indirectamente a consecuencia de la enfermedad, del sufrimiento por causalidad inversa. Pues no se esperaba como castigo, abre un interrogante acerca del sentido en medio de un sentimiento de caos, de pérdida y desorientación. “el pecado, en tanto que alienación consigo mismo, es, tal vez más que el espectáculo de la naturaleza, una experiencia sorprendente, desconcertante, escandalosa: como tal, es la fuente más rica del pensamiento interrogativo. En Los Salterios babilónicos más antiguos el creyente pregunta: <<¿Hasta cuándo, Señor?, ¿contra qué dios he pecado?, ¿qué pecado he cometido?>> El pecado me torna incomprensible a mis propios ojos; Dios está oculto; el curso de las cosas ya no tiene sentido (p. 173); “ la experiencia más conmovedora del hombre, la de estar perdido en tanto que pecador, conecta con la necesidad de comprender y suscita tomar conciencia por su carácter mismo de escándalo” (p. 174). La culpabilidad no es un “tener la culpa”. Por eso me interesa el pecad Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) original, ese en el que estamos todos por ser finitos, humanos, por tener una perspectiva limitada y querer dar cuenta, sin embargo, del infinito y querer no estar solo en nosotros mismos sino con lo demás. En esa mezcla de particularidad y apertura se abre el pecado común y la experiencia de la culpabilidad, no como culpa sino como deseo de rendir cuentas a algo más grande que nos supera. Dice Ricoeur: “La culpabilidad es la toma de conciencia de esta situación real y, si puede decirse, el <<para sí>> de esa especie de <<en sí>>” (p. 258). A veces resulta molesta o angustiosa pero, tal vez dar la espalda a esta sensación que nos recorre no es lo preferible. Puede que tratar de entender esa sensación de culpabilidad flotante, que a menudo se ha tildado demasiado prematuramente de neurótica, sea lo más sano, pues de ese modo podremos comprendernos mejor o hallar en ese dolor algo de nuestra esencia, de nuestro particular modo de habitar el mundo. Lo infinito como transgresión: el deseo humano de significar. Heidegger, por ejemplo, más allá de psicologismos, atendía al dolor ontológico como aquello que se da en la juntura, en la superficie que nos une y separa, en el abismo interno de la simultánea soledad y compañía, en lo que une y diferencia, en la religación: “El dolor en el desgarro es lo unitivo que reúne y separa. El dolor es la juntura del desgarro. Ella es el umbral. Ella lleva a término el Entre, el Medio de los dos que están separados en él. El dolor junta el desgarro de la Diferencia. El dolor es la Diferencia misma” (Heidegger, 2002. p. 20). Se trata del dolor que tiene lugar en cuanto que lo vivo se desprende del apeiron, se trata de un dolor ontológico: “... toda angustia de la desintegración no es, en verdad, más que la única semblanza (...) en la que se oculta lo “verdadero”: el dolor universalmente repartido y por siempre duradero. El dolor no es así ni repugnante ni útil. El dolor es la benevolencia de lo esencial en toda presencia” (p. 49). La religión se rompe cuando concebimos la culpabilidad, de modo laico y unidimensional, como responsabilidad individual por los actos defectuosos o malintencionados, cuando “la metáfora del tribunal invade todos los registros de la conciencia de culpabilidad; pero antes que una metáfora de la conciencia moral, el tribunal es una institución real de la ciudad; a través del canal de esta institución se rectificó la conciencia religiosa del pecado” (Ricoeur, 2011. p. 265). Y es que en nuestra dimensión religiosa, nos hallamos en la Naturaleza sagrada, en la que lo que es, es bueno y lo malo solo se confunde con la nada, el no-ser, el no-ser-con, por ejemplo. Digamos aquí entonces pecado para referirnos a esa culpabilidad ontológico-religiosa, flotante; y digamos delito, para, a diferencia, de ésta, hablar del incumplimiento de las leyes sociales o de los principios individuales. Una cosa es la religiosidad y otra cosa es la ética, aunque ésta halle su base o razón de ser en aquella. Lo infinito como transgresión: el deseo humano de significar. Si desfundamos la ética y le damos valor absoluto, renunciando a nuestra dimensión religiosa, no puede haber, tan siquiera, un verdadero proyecto político, teológico-político, pues definitivamente, nos habríamos desarraigado y atomizado: “Esa pulverización de la culpa en múltiples culpabilidades subjetivas vuelve a poner en cuestión en <<nosotros>> de la confesión de los pecados y pone de manifiesto la soledad de la conciencia culpable” (p. 263). El proyecto político no es necesario para habitar el Ser de la ontología sino que Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 ontexto, nos vamos despegando del temor a la muerte física para del temor a la muerte espiritual, tratando de evitarla y así el pecado ya no será el de la arbitrariedad del dolor sino el de la falta de “Esta vinculación entre la mancilla y el sufrimiento vivida en un <<temor y temblor>>, ha sido tanto más tenaz que, durante mucho oporcionó un esquema de racionalización, un primer esbozo de : si sufres, si estás enfermo, si fracasas, si mueres, es porque has l valor del sufrimiento como síntoma y detector de la mancilla se en valor explicativo, etiológico del mal moral (…) Por eso ha sido da menos que el cuestionamiento de esa primera racionalización y la que el Job babilónico y el Job hebreo fueron los admirables testigos, iar el mundo ético del pecado y el mundo físico del sufrimiento. Esa n ha sido una de las grandes fuentes de angustia de la conciencia pues fue necesario que el sufrimiento se tornase absurdo y so, para que el pecado, por su lado, accediese a su sentimiento te espiritual; a ese terrible precio el temor inherente a éste pudo e en temor a no amar suficientemente y disociarse del temor a sufrir, en suma, el temor a la muerte espiritual pudo escindirse del temor a ísica” (Ricoeur, 2011. p. 195). más allá del principio del placer. El proyecto político no es necesario para habitar el Ser de la ontología sino que cia entre el placer y la felicidad ha sido un tema clásico y recurrente En este contexto, nos vamos despegando del temor a la muerte física para ocuparnos del temor a la muerte espiritual, tratando de evitarla y así el pecado a impedir ya no será el de la arbitrariedad del dolor sino el de la falta de conexión: “Esta vinculación entre la mancilla y el sufrimiento vivida en un estado de <<temor y temblor>>, ha sido tanto más tenaz que, durante mucho tiempo, proporcionó un esquema de racionalización, un primer esbozo de causalidad: si sufres, si estás enfermo, si fracasas, si mueres, es porque has pecado; el valor del sufrimiento como síntoma y detector de la mancilla se convierte en valor explicativo, etiológico del mal moral (…) Por eso ha sido preciso nada menos que el cuestionamiento de esa primera racionalización y la crisis de la que el Job babilónico y el Job hebreo fueron los admirables testigos, para disociar el mundo ético del pecado y el mundo físico del sufrimiento. Esa disociación ha sido una de las grandes fuentes de angustia de la conciencia humana; pues fue necesario que el sufrimiento se tornase absurdo y escandaloso, para que el pecado, por su lado, accediese a su sentimiento propiamente espiritual; a ese terrible precio el temor inherente a éste pudo convertirse en temor a no amar suficientemente y disociarse del temor a sufrir, a fracasar, en suma, el temor a la muerte espiritual pudo escindirse del temor a la muerte física” (Ricoeur, 2011. p. 195). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 El proyecto político no es necesario para habitar el Ser de la ontología sino que Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) es necesario, porque el hombre entra en modo histórico al romper con la pura inocencia ontológica, para reparar el olvido del Ser: “la Biblia no habla nunca del pecado más que desde la perspectiva de la salvación que libra de él. Esta <<pedagogía>> del género humano hace que abunde el pesimismo de la caída con el fin de que sobreabunde el optimismo de la salvación” (p. 415). Buscar significado a nuestro nacimiento es como buscar el sentido de la vida y ya convinimos, si es que algo he logrado, que solo adelantaríamos algo invirtiendo la pregunta y empezando por buscar el sentido de la muerte. Aunque confusamente, lo hallamos en el amor sacrificial. Al invertir la pregunta no solo cambiamos el objeto de la vida por el objeto de la muerte sino que la inversión es total y cambiaremos la pregunta por una respuesta, pues la muerte, comentábamos, toma sentido al volverse contestataria, constituyéndose, así, como una respuesta. Para ser una respuesta, la muerte tiene el cometido de significar. De ahí que la voluntad de significar que acompaña al proyecto de nuestra muerte, se torne transgresión de nuestra finitud: “Lejos, pues, de advertir el estrechamiento de mi perspectiva afectiva estoy ante todo volcado en lo que hago; y lo que hago se intercala en la meta de lo que queda por hacer. Por consiguiente, en mi ingenuidad prerreflexiva, mi atención se dirige ante todo hacia la obra proyectada, hacia el prágma” (p. 69). Querer significar para la muerte es, de nuevo, cumplir con una función hermenéutica, pues “lo hermenéutico no quiere decir primeramente interpretar sino que, antes aún, significa el traer mensaje y noticia” (Heidegger, 2002. p. 91). © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 sociada a Nomads. La dicha: más allá del principio del placer. La diferencia entre el placer y la felicidad ha sido un tema clásico y recurrente La diferencia entre el placer y la felicidad ha sido un tema clásico y recurrent © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) en la historia de la filosofía. Se trata de una problemática central pues de ella depende la orientación y el sentido, así como todas las decisiones en torno a cómo y en qué y con qué ligar nuestra energía. Desde los antiguos griegos se sospecha que el mero placer, sin falta de ser condenable, no es la clave para mantener el aliento vital sino, en todo caso, de modo secundario o auxiliar. Fundamentalmente, ocurre, si no he sido fagocitada por un nihilismo exterminador, que en algún momento (o en varios), me doy cuenta de que el placer es algo que me pasa y no algo que hago y, siendo que una muerte con sentido y una vida propia y plena que la procure, pasan más por un disponer que por un padecer, me veo invitada a plantearme un más allá del principio del placer e incluso a hacer valer el derecho a existir de mi conciencia y a poder dignificarla, pues aun cuando el inconsciente es divino y me impulsa en la urgencia de la vida, cubriendo las lagunas de mi apercibimiento, dando motivos cuando no hay tiempo para razones o cuando hay que reinventar las razones, solo una conciencia, que es inaugural e irrenunciable para la condición humana, por muy defectuosa que sea, y por muchas trampas en las que me enrede, puede dar cuenta de ese mismo inconsciente y de cómo nos relacionamos con él y en él. Ciertamente, hay una búsqueda aparentemente activa del placer pero la búsqueda del placer suele ser, sobre todo si se alza soberana, un búsqueda desesperada y no una exploración esperanzada. Ya que debemos afrontar la muerte, opto por quererla significativa, y resulta que el placer por el placer solo nos puede matar de sobredosis o de un letal síndrome de abstinencia. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 La dicha: más allá del principio del placer. Sin embargo, la diversión es un alejamiento, un quitarse algo de encima y lanzarlo hasta donde ya no podamos verlo. Así, no resulta la diversión algo que podamos definir positivamente sino que la diversión es un tipo de negación. De ahí que comprendamos mejor a Pascal en su sospecha ante lo divertido, pues tal vez nos disponga únicamente a evadir algo, que cómodo o incómodo, nos es esencial. Puede que en lo divertido solo nos estemos perdiendo. Puede que esta enajenación que es la diversión, aparezca, en nuestra experiencia, interrumpida, casi siempre, por accesos de culpabilidad. Demasiadas veces creímos acríticamente, ser críticos con esa autocondena de la diversión, que se nos antojó externa o, mejor dicho, internalizada a partir de operadores externos (nuestros padres, nuestros tutores, nuestros profesores, nuestros curas, nuestras instituciones morales…) que se empeñaron en alojar un policía extraño en nuestro interior. Demasiadas veces le echamos la culpa al cristianismo, al pensamiento judeo-cristiano que nos hizo de cuna. En cambio, reflexionemos hoy y advirtamos que el sentimiento de culpa y la mala conciencia que nos asalta cuando nos divertimos, esa sensación de caos, de decadencia, de que algo desagradable va a seguirnos después, no es la voz de Cristo sino la de un dios mucho más antiguo, la del que reúne todo lo diverso en lugar de dispersarlo hasta la nada. Eso divino que nos conmina al pensar, al saber, a la verdad, nos advierte de que lo que hacemos por diversión, nos separa de la autenticidad de nuestras obras. Por si alguien se sintiera tentado de convocar al devenir del lenguaje y de cuestionar mi planteamiento, atestiguando el deslizamiento semántico de las palabras, debo repasar la conservación del significado original en nuestra propia expresividad popular, en esa que nos atraviesa cuando algo nos turba o nos conmueve. Decimos a los malos amantes: “Solo me quieres para divertirte”, y, con ello, ni los alagamos ni nos sentimos alagados. Queremos acusarlos de no estar con nosotros estando con nosotros sino simplemente no estando con otra cosa y, en el límite, no estando ni siquiera con nosotros, con lo que nos es esencial, pues dando la espalda a la realidad, ese amante nos está alejando a nosotros de él, de la realidad y de nosotros mismos ¿Qué clase de estupidez es el sexo por diversión o el baile por diversión o incluso el juego por diversión? La dicha: más allá del principio del placer. Por supuesto, la lógica del vicio y las aventuras y desventuras del incontinente y del lascivo son dignas de estudio y, tras la conformación de dicho hábito, se hallan cientos de incógnitas sobre la exaltación, la euforia y la desdicha, así como de notas acerca del mismísimo funcionamiento orgánico. Pero la vida recreativa es puro divertimento, es decir, pura distracción. El placer nos entretiene, tal vez, de aquello que deberíamos atender. Decía Pascal, como vimos, que la razón de todas nuestras desdichas es muy simple, es una desgracia natural: nuestra condición miserable, esa que consiste en ser frágiles y mortales. Somos desdichados porque, a pesar de nuestra miseria, el hombre quiere conocerse a sí mismo. De ahí que la diversión le parezca a Pascal una cierta “aversión por la verdad…” que podría hacernos más miserables aún, pues al conjunto de nuestros defectos se añadiría la falta de conocimiento, el disimulo, el autoengaño. Pensemos entonces, ¿qué hacemos solo por diversión o cuándo justificamos nuestros actos a través de la búsqueda de diversión? ¿A dónde nos lleva actuar solo por diversión? ¿No será la resaca de la diversión precisamente la desdicha de encontrarse ante la terrible idea de haber estado disimulando nuestra desgracia, nuestra debilidad, nuestra mortalidad y nuestra miseria? Resulta siempre oportuno, para comprender qué sea algo o qué opera ese algo en nosotros o hacia dónde nos lleva, acudir a la génesis de la palabra que nombra ese algo, pues las palabras no son casuales ni fugaces sino que como las antiguas rocas, sedimentan, conservan, estabilizan el iris de sensaciones, intenciones y rumbos destinados por ese primer nombrar. Puesto que no Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) hablamos como pensamos sino que pensamos como hablamos y puesto que el lenguaje está más allá de nuestra propia vida y más acá de nuestro propio alcance, sacar las palabras de nuestras expresiones, aislarlas e investigarlas, nos permite, mejor que ninguna otra cosa, comenzar con atino un análisis del drama de nuestra existencia, un viaje por nuestros deseos escondidos. La palabra “diversión” proviene del latín diversum, superlativo de la acción nombrada por el verbo divertere que significaba alejar. De hecho la base del término, vers, quiere decir voltear, volver la espalda. Así, entendamos, que cuando decimos que queremos divertirnos estamos diciendo que queremos alejarnos. A menudo creemos que la diversión une, aproxima entretejiendo un lazo afectivo. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 y la muerte, la ganancia y la pérdida... y la muerte, la ganancia y la pérdida... Diversión también significa distracción, en el lenguaje militar: se divierte al enemigo ¿Debemos, entonces, creer en la diversión? ¿Nos divierten los amigos o los enemigos? ¿De qué nos quieren distraer los que nos divierten y para qué sino para matarnos antes de que los matemos? En Más allá del principio del placer, Freud se pregunta por el placer y la repetición, mostrando la anterioridad de ésta respecto de aquel. A través de la observación de los juegos infantiles y de la especificidad de los sueños traumáticos, nos ofrece el lado más tanático de ese preámbulo del placer que es la repetición. Lo que tiene de negativo el placer, es decir, lo que le da su carácter de reverso, de compensación , más que de propuesta o don, es su génesis funcional. Cuando descubrimos que no repetimos porque nos place sino que nos place porque repetimos, abandonamos la pregunta por el placer y nos adentramos en esa otra más enigmática: la pregunta por la repetición ¿Por qué repito?, ¿no es cierto que, cuando hago balance de mis repeticiones, encuentro en ellas más ruina que riqueza?, es decir, ¿no pasa que más bien me lamento por “haber caído en los mismo” que celebro el “otra vez”?, pero ¿no es patente también que antes de la crónica de ese lamento anunciado, me abandoné a la gustosa sensación de familiaridad perdida, al “por fin vuelvo a sentir lo mismo”, aun cuando es lo mismo que no necesariamente acaba demasiado bien? ¿no me encuentro atrapada en eso que he definido como mis placeres, esperando que les suceda una dicha que nunca llega?, ¿por qué es placentero justo eso que me defrauda una y otra vez y que solo me deja con una sed de lo mismo que no me permite salir del bucle de decepción, de caida, de sinsentido (de pecado)? Parece que así es porque con el placer no realizo una auténtica búsqueda sino que escapo de algo, paradójicamente, por anticipación. Lo que Freud viene a descubrir es que en la compulsión, no repito algo que me agrada sino algo que temo que, angustioso para mí, ocurrirá igualmente. Lo angustioso solo es placentero en contraste con lo que asusta, con lo que dolorosamente sobresalta. Con la repetición anterior convertimos en esperado y controlado lo que, si no, sería, además de desagradable, imprevisto. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 La dicha: más allá del principio del placer. Es un no-sexo, pues no une ni llena sino que fractura y vacía hasta el sinsentido de la depresión postcoital. Es un no-baile, pues no nos permite desgarrarnos para celebrar nupcias con nuestro suelo, con nuestro compañero y con nuestro cielo. Es no-juego porque el verdadero jugador pone el alma en cada partida y envida su destino, saboreando el borde entre la vida Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) Con el placer, el dolor sabe a desahogo. Sin embargo, cuando finalmente la y la muerte, la ganancia y la pérdida... Repitiendo solo queremos estar preparados. Generamos angustia para evitar el pánico. Así, entiendo porqué en el placer siempre asoma un fondo masoquista: la auto-lesión hace de la lesión algo más indoloro. En un rulo algo complicado, preferimos ser artífices de nuestra propia desgracia que padecerla como imbéciles ingenuos. De modo tal, aparece la parodia: “no me abandonas; te echo”, “no me echas; me voy”. Con razón dice Marx que la historia se repite pero una vez como tragedia y otra como comedia. La comedia, la parodia, la farsa... las tenemos, ya en nuestro imaginario como más pobres y leves, pero, por eso mismo, como más llevaderas que la tragedia. Se trata de una condena mediante la risa (Bergson, 2008) o de tragarnos la desgracia con la lubrificación del juego. Con el placer, el dolor sabe a desahogo. Sin embargo, cuando finalmente la on el placer, el dolor sabe a desahogo. Sin embargo, cuando finalmente la Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) desgracia llega de fuera, por mucho que la hallamos anticipado, es decir, por mucho que nos hallamos esforzado en conseguir una anestésica habituación, nos ahoga. Por ello, cuando van asociados al placer, no queremos que se cumplan nuestros deseos. El objeto del placer acaba volviéndose insípido cuando lo solicitamos o, cayendo sobre nosotros, sin el consentimiento de nuestra voluntad, aparece como cruz y condena. Los placeres son, como ya popularmente sabemos, pequeñas muertes, y, por mucho que queramos eludir la muerte adelantándonos a ella, “la muerte siempre viene de fuera”16. Lo único que podemos hacer es tratar de que esta desposesión, este inevitable dar, signifique, conforme una respuesta, un haber apostado por una conciencia que diga algo, que ofrezca, al menos un motivo de reflexión. Frente a la desesperada persecución del placer, frente al siempre falaz intento de retener y agotarse en lo que, de suyo, ha de ser fugaz, puntual e insuficiente, la filosofía, como también la religión, instaron a maquinar un distinto modo de existencia, en el que el lugar del placer no fuera sino residual; un modo de existencia volcado hacia lo que tiene el poder de esenciar, de permanecer y de valer la pena. Pienso que salvar el alma sigue valiendo la pena. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 16 G. Deleuze: “Spinoza”. Le cours de Gilles Deleuze. http://www.webdeleuze.com/php/texte.php?cle=44&groupe=Spinoza&langue=3. y la muerte, la ganancia y la pérdida... Efectivamente es un nombre que le damos a la felicidad siempre y cuando queremos diferenciarla no solo del placer sino también de alegrías que bien pueden ser pasajeras; y siempre que queremos mentar un edificar en lugar de un padecer. El vocablo francés procede del latín “augurium”, algo así como aumento que los dioses insuflan a un emprendimiento. La palabra latina deriva del indoeuropeo “aweg” entendido también como augurio en sentido de reconstitución, concesión de autoría, de derecho de autor. Así, bonheur pone en relación la propia existencia, el propio proyecto, con el aval divino. Se trata de una potenciación, de una capacitación que los dioses me dan para reconstituirme en el obrar. Se me pone en la existencia pero, en mi empresa, me reconstituyo con el augurio divino. En esta reconstitución tomo derechos de autor. Lo que es importante destacar es el modo complejo y clásico en el que se nos permite entender el augurio divino. En él confluyen destino divino y obra propia. El augurio hace referencia a un vaticinio, a un desciframiento. Puedo comprender con legitimidad que la felicidad enraizada en el buen augurio consiste, en la buena interpretación de los signos, en un caminar descifrando las señales divinas. Se conceden derechos de autor para reconstituir y entiendo para reconstituir el destino y traer el mensaje. La felicidad es un hacer pero, sobre todo, es un saber decir lo que simbolizan los dioses. En castellano, se adjudica a esta felicidad particularmente ricoeuriana, el nombre de Dicha. De este modo conservaríamos la mayor proximidad, aun en la torsión del texto traducido. La palabra dicha viene del latín “dicta”, las cosas dichas. Si atendemos a la significación más original del verbo “dicere” del que proviene este nominativo neutro relacionado con la raíz indoeuropea “deik”, lo traducimos como indicar, señalar. Dicta se refería a las cosas indicadas por el destino de una persona al nacer. Fatum y dicta harían referencia al destino pero en el primer caso evolucionaría como destino fatídico y en el segundo como destino favorable. En cualquiera de los dos hilos nos encontramos con la fatalidad y solo por una especie de afección mediante, parece que sentimos esa fatalidad adversamente, como si resultara muy costoso u obstaculizado su camino; o favorablemente, con “el viento a favor”, como impulsados en la dirección que corresponde. y la muerte, la ganancia y la pérdida... Así, recortar la vida en función de la proximidad con el placer y la lejanía con el dolor, puede, a lo sumo, servir, para conservarse, dada una acepción moderada, o para perderse, si nos colocamos en el trazado del exceso. Lo primero es mera supervivencia y no es vivir conforme a las potencias de lo humano, dicho éticamente. Lo segundo nos señala un lugar común, una irrefrenable huida hacia adelante, hacia el irrecuperable instante cero, hacia las fondos... No sé si esta experiencia es necesaria para un reflote más brillante que la lineal retención o, si, por el contrario, una puede ocuparse de sí solo a pesar de tal hundimiento, más que inspirada por él. En cualquier caso, sabemos ya que estar perdida constituye la situación natural de quién transita el medio de las cosas, el medio de un bosque, de un océano, de un desierto, de cualquier meseta extendida entre el nacimiento y la muerte, entre la pregunta y la respuesta o entre el absurdo y la significación. Pero el encontrarse perdida no constituye la culminación de ningún viaje sino que siempre hay un norte hacia el que enderezar el rumbo. Hay un camino de virtud que algunos llamaron felicidad y otros ascesis y otros beatitud, en el que buscar alojamiento para cumplir con la propia esencia. Esta vez vamos a quedarnos con el nombre que Paul Ricoeur le da a este más allá del placer en el que encontramos esencia y transcendencia o, lo que es lo mismo, sentido, esa transgresión de la finitud mediante el deseo de significar: la dicha. Quedarnos con el nombre no es una cuestión superficial sino un motivo para especificar, para darle a Ricoeur la particularidad y distinción de su propuesta. Puede que tanto si traemos la eudaimonía de Aristóteles como la beatitud de Spinoza, la dicha de Ricoeur e incluso el temor y el temblor de Kierkegaard, estemos tratando con el amor intelectual a Dios. En cambio, quiero conceder a cada uno su originalidad e inclinación a la hora de tratarlo, pues son diferentes los acentos que ponen y las fascinaciones que producen. Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) Con razón, se traduce el término bonheur del francés de Ricoeur como dicha. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 y la muerte, la ganancia y la pérdida... Observamos que la dicha corresponde a una felicidad que implica un decir en el que somos destinados al nacer y y un decir con el que desciframos recorriendo el destino con el viento a favor, excitados, impelidos, atraídos con él hacia él. La dicha, el buen augurio, son la felicidad que construimos en los límites del destino. La felicidad consistiría, entonces en interpretarse dentro de la interpretación que hacemos de los símbolos de la naturaleza o de un texto sagrado. Más allá del placer, lo que da adecuada orientación a la vida es la dedicación a la interpretación de los signos, para poder dar significado y para poder decir (traer mensaje y noticia), al final, con la muerte: dar significado para significar. Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) De este modo me gustaría reformular el círculo hermenéutico. Este círculo da cuenta de la desproporción mencionada con anterioridad, entre la finitud de la perspectiva y la infinitud en la que se aloja el sentido. Por ello la hermenéutica de Ricoeur es ascética. Nuestro nacimiento es el acontecimiento de otro que nos da un carácter y, de camino a la muerte, para que aquel se “disuelva”, se transgreda, en el sentido, buscamos las señales para poder decir lo que nos dicen. Esta comprensión requiere una anticipación de la fe, una puesta en marcha esperanzada: “La <<desproporción>> entre el sentido y la perspectiva, entre el querer-decir y el mirar, entre el verbo y el punto de vista es como la célula melódica de todas las variaciones y de todos los desarrollos que culminan en la <<desproporción>> entre la dicha y el carácter” (Ricoeur, 2011. p. 82); ”Yo, que soy perspectiva finita, dilección por mi cuerpo, hábito e inercia, carácter soy capaz de concebir la idea del <<querer perfecto de un ser racional dotado al mismo tiempo de omnipotencia>>; o, por retomar otra expresión kantiana evocada más arriba, yo soy portador del <<destino supremo de la razón>>, conforme a la cual puedo <<continuar mi existencia>>. Esta idea del querer perfecto y del destino de la razón abren en mi deseo una profundidad infinita y lo convierten en el deseo de la dicha y no sólo en el deseo del placer” (p. y la muerte, la ganancia y la pérdida... 85); “Lo mismo que recojo indicios del estrechamiento de mi percepción - aunque sólo sea por la refutación de los demás-, recojo signos de estar destinado a la dicha. Se trata de experiencias privilegiadas, de momentos especiales en los que recibo la seguridad de ir en la dirección acertada; de pronto, el horizonte se despeja, ante mí se abren posibilidades ilimitadas; el sentimiento de lo <<inmenso>> responde entonces dialécticamente al sentimiento de lo <<estrecho>>” (p. 86); “El carácter es el origen cero de esta orientación de campo, la dicha es el término infinito de la orientación. Esta imagen hace comprender que la dicha no se da en ninguna experiencia; únicamente es designada en una conciencia de dirección. Ningún acto proporciona la dicha; pero los encuentros de nuestra vida más dignos de ser llamados <<acontecimientos>> indican la dirección de la dicha (…) Los acontecimientos que hablan de la dicha son aquellos que apartan los obstáculos, que descubren un amplio paisaje de existencia; el exceso de sentido, el excedente, lo inmenso, ésta es la señal de que estamos <<dirigidos- hacia>> la dicha” (p. 86); “<<Hay que comprender para creer, pero hay que creer para comprender. Este círculo no es un círculo vicioso,menos aún mortal; es un círculo lleno de vida y estimulante. Hay que creer para comprender: en efecto, el intérprete no se aproximará jamás a lo que dice su texto si no vive en el aura del sentido en cuestión...” (p. 485). Hermenéutica y amor: obrar en el encuentro. Como conclusión, solo quiero ofrecer un muy breve resumen de mi exposición que ayude a asentar una lógica de saltos temáticos o un panorama de asociaciones conceptuales, mostrando, a su vez, cómo mi escrito reivindica la observación atenta de la fe, la salvación y la dicha, tanto para alimentar el brío vital y dar un sentido a la muerte como para comprender adecuadamente la afectividad humana. A veces anhelamos la inocencia de la animalidad salvaje y desearíamos ser © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) despreocupadamente inconscientes. A veces, dándonos cuenta de que dicha vuelta es imposible, quisiéramos acogernos a una total superestructuración, para, del mismo modo, no tener que responder de nuestros actos y descansar en códigos arbitrarios, que se nos antojaran puramente estéticos en el olvido de su génesis. Por caminos contrarios, buscamos, así, descansar de la reflexión que nos agota y dejar que nuestro cuerpo funcione solo, conducido por una eficacia desconocida que nos permita escapar de la interrogación. Pero no por mucho tiempo logramos mantenernos en este equilibrio insospechado, pues de pronto, nos hallamos en situaciones inéditas que nos interpelan exigiendo decisiones, elecciones y tomas de partido. Entonces, solo un consciente y miserable abandono en la decadencia voluntaria, nos permitiría continuar, indolentes y crueles, en una existencia caótica sin proyectos ni interrogantes. Amar sin preguntar no es amar o no es amar humanamente, pues quien no quiere saber nada es que sobrevive sin interés alguno ya por la vida: “Si no se acepta la desproporción originaria del deseo vital y del amor intelectual (o de la dicha espiritual), se pierde totalmente la especificidad de la afectividad humana” (Ricoeur, 2011. p. 110) Respetar el misterio, dejarlo ser, es interrogarlo aun sin pretender despejarlo o despojarlo de su esencia, pues el misterio gusta de ocultarse pero también de dar señales. No es por abundancia que prescindamos del sentido, ni de auténtica vitalidad que nos sobre. Eludir la pregunta por el sentido de la vida solo muestra la cobardía de haberse acomodado en el fracaso anticipado. Exterminar sin más tal cuestionamiento equivale a renunciar torpemente de nuestra humana condición para mecerse en el frenesí de los placeres. Incluso si optamos por un nihilismo tal, más pronto que tarde, advertiremos que las resacas son cada vez más insoportables y nos veremos obligados a reorientarnos o a morir insignificantemente, a morir, tristemente, del todo, para todo y para todos. Por muy perdidos que nos encontremos, no queremos tal cosa. De hecho, la sensación de estar perdidos indica ya una búsqueda irrenunciable. En los paraísos artificiales también ansiamos la eternidad y eso no podemos negarlo. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 A veces anhelamos la inocencia de la animalidad salvaje y desearíamos ser Ya un pronto filosófico, ya un arrebato religioso, ya un ardor artístico, nos inclina a crear un sentido para la vida pero, en lo profundo, intuimos que ni creamos de la nada ni nos contentamos con meros divertimentos sino que creamos para reconstruir aquello que nos empuja y llama: el destino. Nos queremos destinados-a, pues a falta de tal sentimiento de destinación, no encontramos más motivo que nuestra propia mismidad y ese es un nombre de la soledad y la desconexión. Tal vez la primera de las situaciones inéditas y grandiosas a las que me refería hace un momento, esas que nos exigen una elección, un procesamiento, sea el encuentro con la muerte, pues es mi convencimiento que solo en el abismo incomprensible de la muerte, nuestra niña comienza a filosofar. Por ello propongo que la persecución del sentido de la vida es solo un modo amable y segundo de nuestra necesidad de explicarnos y reconciliarnos con la muerte. primer encuentro terrible con la muerte no nos impactaría si no fuera por el Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) amor que guardamos, porque ya en ese momento amamos. Puede que gracias a esa trágica experiencia sepamos verdaderamente del alcance de nuestro amor, de un amor que no se adecua sin más a la ausencia. No podemos decir, como avisa Heidegger, que la ausencia no es pues tanto es que duele. Aprender a vivir con aliento es aprender a vivir con ese dolor de la finitud que nos impele a transgredirla. Aprender a vivir con aliento es vivir más allá de la vida, hacia un más allá de esto actual que se fuga constantemente. Es por eso que no nos sirven los placeres sino que aspiramos a la dicha. A veces anhelamos la inocencia de la animalidad salvaje y desearíamos ser Y es por eso que no nos conformamos con amoríos menores sino que más allá de ellos o incluso en ellos, buscamos una conexión que nos permite ser algo más que nosotros mismos, que nos permita no terminar en los límites de nuestra superficie: “el placer acentúa y afianza mi arraigo orgánico en el mundo; magnifica el cariño que le tengo a la vida que me atraviesa y ese centro de perspectiva que soy; así pues, la perfección misma del placer es la que me ata a la vida, pues manifiesta que vivir no es una actitud más entre otras, sino la condición existencial de todas las demás; al afirmar alegremente aquello de <<primero vivir>>, el placer no deja de sugerir constantemente el aplazamiento de <<después filosofar>>. Y, no obstante, el placer es total, como la dicha; representa la dicha en el momento; pero precisamente esa contracción de la dicha en el momento es lo que amenaza con paralizar el dinamismo del obrar en la celebración del Vivir” (p. 112). Por suerte, nuestros cuerpos están animados, almados, y no terminan en su recortada figura ni en su limitada perspectiva sino que se expanden todo lo que se expande su potencia. Es ahí que la fantasía de conexión nos permite sentir el éxtasis y, así, repartiéndonos, prolongarnos en los otros, en lo otro. Sin embargo, dado que igual que topamos con la experiencia de la muerte, topamos con la experiencia y la angustia del desamor, solo la fe nos salva de un nuevo ensimismamiento. La fe es la confianza que persiste a las sucesivas confianzas quebradas. La fe es el más allá del duelo y la melancolía. Solo en ella realizamos la apertura a nuevos encuentros, bien puedan darse en la sociabilidad o en el retiro. Me gustaría, para terminar, rememorar la importancia de los ciclos y valorizar, también como acto de amor, el retiro, incluido el retiro definitivo de la muerte, si con él conseguimos responder, si logramos un retiro y una muerte contestatarios que signifiquen, aporten mensaje y noticia, en el regreso al hogar, tras la aventura del desciframiento. Hacer el amor es hacer hermenéutica, ya sea ésta inconclusa. El acto de amor acabado es aquel por el que, esperanzados, afrontamos la muerte, a condición de significar algo con ella, de redondear el obrar cumplido. © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 A veces anhelamos la inocencia de la animalidad salvaje y desearíamos ser Obramos en el encuentro pero, en el retiro, conseguimos encontrarnos con las voces que no se oyen entre el griterío y el alboroto, con las voces de los muertos que nos dejaron algo que leer, con las voces que forman ya parte de lo divino. Quizás incluso, con el retiro definitivo, propiciemos un momento de silencio entre el griterío y el alboroto, un momento para la reflexión. Nómadas. Revista Crítica de Ciencias Sociales y Jurídicas \| 45 (2015.1) © EMUI Euro-Mediterranean University Institute \| Universidad Complutense de Madrid \| ISSN 1578-6730 Asociada a Nomads. Mediterranean Perspectives \| EMUI_EuroMed University Salento \| ISSN 1889-7231 Bibliografía Bataille, Georges. El erotismo. Barcelona: Tusquets. 2007. Bergson, Henri. La risa. Madrid: Alianza. 2008. Deleuze, Gilles: Lógica del sentido. Barcelona: Paidós. 1989. Freud, Sigmund. Obras completas. Barcelona: RBA. 2006. Hume, David: Investigación sobre el entendimiento humano. Madrid: Istmo. 2004. Kierkegaard, Soren. Temor y temblor. Madrid: Tecnos. 1987. Kierkegaard, Soren. Las obras del amor. Salamanca: Sígueme. 2006. Kirk, G. S., Raven, J. E., Schofield, M. (1994) Los filósofos presocráticos. Madrid: Gredos. Klein, Melanie. Obras completas. Barcelona: RBA. 2006. Pascal, Blaise. Pensamientos. Madrid: Alianza. 2004. Ricoeur, Paul. Hermenéutica y acción. Buenos Aires: Prometeo Libros. 1988. Ricoeur, Paul. Fe y filosofía. Buenos Aires: Prometeo Libros.2008. Ricoeur, Paul. Finitud y culpabilidad. Madrid: Trotta. 2011. Ricoeur, Paul. Amor y justicia. Madrid: Trotta. 2011 (b). Bataille, Georges. El erotismo. Barcelona: Tusquets. 2007. Bataille, Georges. El erotismo. Barcelona: Tusquets. 2007. Bergson, Henri. La risa. Madrid: Alianza. 2008. Bergson, Henri. La risa. Madrid: Alianza. 2008. Deleuze, Gilles: Lógica del sentido. Barcelona: Paidós. 1989. Freud, Sigmund. Obras completas. Barcelona: RBA. 2006. Hume, David: Investigación sobre el entendimiento humano. Madrid: Istmo 2004. Hume, David: Investigación sobre el entendimiento humano. Madrid: Istmo. 2004. Hume, David: Investigación sobre el entendimiento humano. Madrid: Istmo. 2004. Kierkegaard, Soren. Temor y temblor. Madrid: Tecnos. 1987. Kierkegaard, Soren. Temor y temblor. Madrid: Tecnos. 1987. Kierkegaard, Soren. Las obras del amor. Salamanca: Sígueme. 2006. g g Kirk, G. S., Raven, J. E., Schofield, M. (1994) Los filósofos presocráticos. Madrid: Gredos. Klein, Melanie. Obras completas. Barcelona: RBA. 2006. Pascal, Blaise. Pensamientos. Madrid: Alianza. 2004. icoeur, Paul. Hermenéutica y acción. Buenos Aires: Prometeo Libros. 1988. Ricoeur, Paul. Hermenéutica y acción. Buenos Aires: Prometeo Libros. 1 Ricoeur, Paul. Fe y filosofía. Buenos Aires: Prometeo Libros.2008. icoeur, Paul. Fe y filosofía. Buenos Aires: Prometeo Libros.2008. Ricoeur, Paul. Finitud y culpabilidad. Madrid: Trotta. 2011. Ricoeur, Paul. Finitud y culpabilidad. Madrid: Trotta. 2011. Ricoeur, Paul. Amor y justicia. Madrid: Trotta. 2011 (b). Ricoeur, Paul. Amor y justicia. Madrid: Trotta. 2011 (b).
https://openalex.org/W4282917267	https://zenodo.org/records/6644599/files/post-print_Diamanti_ADMI.pdf	English	null	Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads	Advanced materials interfaces	2,022	cc-by	13,019	This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This is the peer reviewed version of the following article: Diamanti, E; Arana-Pena, S; Ramos- Cabrer, P; Comino, N; Carballares, D; Fernandez-Lafuente, R; Lopez-Gallego, F Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads. Adv. Mater. Interfaces 2022, which has been published in final form at 10.1002/admi.202200263 Thi i l b d f i l i d i h Wil T d This is the peer reviewed version of the following article: Diamanti, E; Arana-Pena, S; Ramos- Cabrer, P; Comino, N; Carballares, D; Fernandez-Lafuente, R; Lopez-Gallego, F Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads. Adv. Mater. Interfaces 2022, which has been published in final form at 10.1002/admi.202200263 Thi i l b d f i l i d i h Wil T d This is the peer reviewed version of the following article: Diamanti, E; Arana-Pena, S; Ramos- Cabrer, P; Comino, N; Carballares, D; Fernandez-Lafuente, R; Lopez-Gallego, F Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads. Adv. Mater. Interfaces 2022, which has been published in final form at 10.1002/admi.202200263 This is the peer reviewed version of the following article: Diamanti, E; Arana-Pena, S; Ramos- Cabrer, P; Comino, N; Carballares, D; Fernandez-Lafuente, R; Lopez-Gallego, F Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads. Adv. Mater. Interfaces 2022, which has been published in final form at 10.1002/admi.202200263 This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Intraparticle macromolecular migration alters the structure and function of proteins reversibly immobilized on porous microbeads leftheria Diamanti, Sara Arana-Peña, Pedro Ramos-Cabrer, Natalia Comino, Diego arballares, Roberto Fernandez-Lafuente and Fernando López-Gallego Eleftheria Diamanti, Sara Arana-Peña, Pedro Ramos-Cabrer, Natalia Comino, Diego Carballares, Roberto Fernandez-Lafuente and Fernando López-Gallego Dr. E. Diamanti, Prof. P. Ramos-Cabrer, Dr. Natalia Comino, Prof. F. López-Gallego Center for Cooperative Research in Biomaterials (CIC biomaGUNE) – Basque Research and Technology Alliance (BRTA) Paséo Miramón, 194, 20014 Donostia-San Sebastián, Spain. Prof. P. Ramos-Cabrer, Prof. F. López- Gallego IKERBASQUE, Basque Foundation for Science, Maria Diaz de Haro 3, 48013 Bilbao, Spain. Dr. S. Arana-Peña, Mr. Diego Carballares, Prof. R. Fernández-Lafuente Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain. Prof. R. Fernández-Lafuente Center of Excellence in Bionanoscience Research, External Scientific Advisory Academic, King Abdulaziz University, Jeddah 21589, Saudi Arabia. er of Excellence in Bionanoscience Research, External Scientific Advisory Academic, Abdulaziz University, Jeddah 21589, Saudi Arabia. E-mail: ediamantini@cicbiomagune.es, flopez@cicbiomagune.es E-mail: ediamantini@cicbiomagune.es, flopez@cicbiomagune.es Keywords: Protein immobilization, protein storage, bio-interfaces, single-particle analysis, lipases Keywords: Protein immobilization, protein storage, bio-interfaces, single-particle analysis, lipases Abstract. While migration of reversibly immobilized proteins across the volume of supports is investigated in conditions where an external force is applied or under fluid flow conditions, their passive migration upon sample storage and its effect on the protein functionality remain unexplored. Understanding such intraparticle macromolecular migration is essential to develop protein functionalized biomaterials with a longer life span. This work investigates the spatiotemporal migration of His-tagged immobilized fluorescent proteins inside porous agarose microbeads under different storage conditions. We develop a tool that assesses the intraparticle protein migration across the surface of the porous supports. Our studies reveal that protein migration takes place under different storage conditions. Differences in migration patterns between different proteins suggest that binding dynamics between proteins and their supports play a key role in their migration. We explore the effect of macromolecular migration on the functional and structural properties of bound proteins and enzymes. Therefore, we perform 1 single-particle measurements to understand how the migration process affects the functionality of immobilized enzymes. Evaluating protein migration and understanding the reason behind such phenomena allows gaining control over immobilization processes and design immobilization chemistries that either prevent or promote intraparticle macromolecular diffusion upon storage, depending on the desired final application. 2 2 1. Introduction Protein adsorption and immobilization on solid surfaces have been a scientific and industrial strive for the past decades to fabricate biomaterials with biotechnological applications (i.e., surface-based biosensors, medical devices, biomedical implants), industrial biocatalysts, or even protein arrays for drug screening[1–5]. In this context, biomolecules such as proteins have been successfully immobilized on a plethora of solid supports to enhance their functional properties[6]. Therefore, a deep understanding of the interactions between proteins and solid supports is crucial to gaining control over the immobilization process. Biophysical studies based on spectroscopy, microscopy, and molecular simulations evidence the effect of those interactions on protein conformation, stability, and functionality[6–8]. For instance, single- molecule studies unveiled how structural distortions (i.e., protein folding, spatial distribution, mass transport issues, etc.) of immobilized enzymes affect their functional properties[9,10]. In addition, the interactions between the support surface and the enzyme determine not only the initial enzyme structure but also the enzyme inactivation pathway[11]. Likewise, recently, our group demonstrated through single-particle studies that the spatial distribution of immobilized enzymes has a clear effect on their final catalytic performance[12,13]. 3 Under certain storage and usage conditions, proteins are known to suffer conformational changes that deteriorate the functionality of materials functionalized with proteins. For instance, enzymes and recognition proteins (i.e., antibodies) undergo inactivation processes that make them lose their catalytic or binding properties, respectively. The molecular causes of protein inactivation at the interface with solid materials are not yet fully understood. When proteins are immobilized on solid surfaces, we tend to assume that they are attached to a fixed position within the support, and their localization remains unchanged during both their storage and operational utilization. Microscopic protein mobility is yet disregarded in the stability test of biomaterials. In fact, protein migration across solid surfaces remains elusive for conventional 3 bulk studies, nevertheless, it is possible to follow that phenomenon through microscopy-based single-particle analysis. In the last decades, single-particle and single-molecule analysis unveiled valuable information about the intraparticle structure and functionality of immobilized proteins[14]. However, protein migration phenomena have been rarely investigated. Based on previous studies of macromolecular hindered diffusion[15] and on the hypothesis that proteins reversibly bound to solid porous surfaces can passively migrate, as well as that migration should cause structural and ultimately functional alterations in the immobilized proteins, there is a clear need in confirming and understanding this phenomenon. 1. Introduction While protein migration has been extensively investigated in phase liquid chromatography[16] or under conditions where an external force is applied (e.g. shear forces under flow) [17], the effect of passive migration on the functionality of immobilized proteins has not yet been explored. Protein migration triggered by either storage or operational conditions may alter the protein distribution along the support surface and indirectly affect the functional properties of the immobilized proteins. A deeper understanding of these migration processes will guide the preparation of materials functionalized with proteins with better performance for long-standing uses such as the utilization of heterogeneous biocatalysts in flow processes, inmunobiosensors in line, as well as the storage of protein delivery systems. In this work, we investigate if the spatiotemporal migration of immobilized proteins inside porous agarose-based microbeads under different storage conditions occurs and if the macromolecular migration affects the functional and structural properties of the bound proteins. To that aim, we have performed single-particle studies to understand the dynamics of biomolecules at the interface with solid materials[10,18–21]. As a model system, we selected the site-directed immobilization of His-tagged proteins on porous agarose microbeads functionalized with cobalt chelates[22–25]. In the latter, Co2+ provides interaction with two His residues in the Poly-His tag, while when there is a high enough density of these groups in the 4 4 support surface, additional enzyme-support weak interactions may be established (i.e., electrostatic, hydrophobic)[26]. In this work, we have developed a method to assess the migration of fluorescent proteins (i.e., GFP) or fluorophore-labeled proteins across the porous surface of the supports through analysis of confocal scanning laser microscopy (CSLM) images acquired upon storage at different conditions. Understanding of protein migration phenomena enabled us to design new immobilization chemistries to avoid the intraparticle macromolecular diffusion during storage and explain how protein migration affects enzyme functionality upon storage. 2. Results and Discussion 2.1. Protein migration under different conditions. For protein migration studies we immobilized two His-tagged proteins, green fluorescence protein (His-GFP) and mCherry fluorescence protein (His-mCherry) on porous agarose microbeads activated with cobalt- chelates (AG-Co2+), obtaining His-GFP@AG-Co2+ and His-mCherry@AG-Co2+, respectively. In these immobilizates, proteins are oriented toward their N-terminus through reversible coordination bonds between the imidazole rings of their His-tag and the cobalt chelates at the bead surface[22,23,25,27]. The interaction between His-tag proteins and the cobalt functionalized surfaces, although reversible, is very strong[28]. Therefore, protein lixiviation is not expected unless beads are incubated in presence of high imidazole concentrations or acidic pH. For quantitative determination of protein migration, we acquired CLSM images of samples stored under different conditions. Then the acquired images were processed to obtain a normalized fluorescence intensity radial profile of single beads using FIJI software[29] (see Experimental Section, Figure 1a and b). Subsequently, we developed a single-particle analysis algorithm that was implemented in the above-mentioned image processing program. The developed plugin automatically performs a Gaussian fit on the normalized fluorescence intensity radial profile of every single bead of the confocal image under analysis (Figure 1c). 5 Then, the plugin searches for the fitted data point that corresponds to the radius coordinate (𝑥50%) where fluorescence is at 50% of the maximum normalized fluorescence fitted peak (𝑦50%). Then, 𝑥50% is subtracted from the radius of the analyzed bead to obtain the infiltration distance (μm). Finally, protein migration is defined as the relative infiltration that mean the average infiltration distance divided by the average radius of the analyzed beads (n = 10) with a diameter that ranged from 75 – 100 μm, expressed as a percentage (%). Figure 1. Explanatory illustration of infiltration distance calculation. (a) Representation of fluorescence radial profile of a single microbead in a confocal fluorescence microscopy (CLSM) image. (b) Acquired normalized fluorescence intensity radial profile (R represents the particle radius) and (c) normalized fluorescence intensity radial profile (black solid line) together with the corresponding Gaussian fit (yellow dotted line) and explanatory illustration of infiltration distance estimation derived from the obtained fitted radial profile. 2. Results and Discussion 30 35 40 45 50 55 60 0 10 20 30 40 50 60 Normalized fluorescence intensity Radius (mm) radial profile Gaussian Fit CLSM image (a) Radial profile 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Normalized fluorescence intensity Radius (mm) radial profile (b) (c) 30 35 40 45 50 55 60 0 10 20 30 40 50 60 Normalized fluorescence intensity Radius (mm) radial profile Gaussian Fit 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Normalized fluorescence intensity Radius (mm) radial profile b) (c) CLSM image (a) Radial profile (b) (a) Figure 1. Explanatory illustration of infiltration distance calculation. (a) Representation of fluorescence radial profile of a single microbead in a confocal fluorescence microscopy (CLSM) image. (b) Acquired normalized fluorescence intensity radial profile (R represents the particle radius) and (c) normalized fluorescence intensity radial profile (black solid line) together with the corresponding Gaussian fit (yellow dotted line) and explanatory illustration of infiltration distance estimation derived from the obtained fitted radial profile. To study the passive protein migration under different conditions, we incubated the immobilizates His-GFP@AG-Co2+ and His-mCherry@AG-Co2+ in 25 mM phosphate buffer at pH 7 and 4 ºC (typical storage temperature) and 37 ºC (typical operational temperature) for one and 30 days. As expected from previous studies[30], His-mCherry (Figure 2a) was initially mainly localized at the outer surface of the AG-Co2+ microbeads. Surprisingly, His-mCherry significantly migrated toward the inner regions of the microbeads after one day of incubation at 37 ºC; the relative infiltration increased from 9% to 30% (Figure 2c). At a lower incubation temperature (4 ºC), the protein migration was rather slow as it required 30 days to reach a relative infiltration of 50% (Figure 2c). On the contrary, His-GFP migrated to a much lower extent than His-mCherry when incubated under the same conditions (Figure 2b and c). We 6 suggest that differences found in the migration of His-mCherry and His-GFP are related to other reversible interactions established with the support surface beyond the coordination with the His- tag. tag. Figure 2. Fluorescent confocal images of; (a) His-mCherry (red channel, λex: 561 nm) and (b) His-GFP (green channel, λex: 488 nm), both reversibly immobilized on AG-Co2+ porous microbeads (0 d) and after 1 d of incubation at 4 ºC and 37 ºC and after 30 days at 4 ºC. 2. Results and Discussion (c) Relative infiltration of reversibly immobilized His-mCherry (red bars) and His-GFP (green bars) toward the center of the microbead. Error bars in panel (c) represent the standard deviation from data obtained from 10 microbeads of similar size that ranged from 75 – 150 μm (n =10). 0d 1d / 4 ºC 1d / 37ºC 30d / 4 ºC 0 10 20 30 40 50 60 Relative Infiltration (%) His-mCherry His-GFP (a) (c) (b) His-mCherry His-GFP 0d 1d / 4 ºC 1d / 37 ºC 30d / 4 ºC 0d 1d / 4 ºC 1d / 37 ºC 30d / 4 ºC (a) (b) His-mCherry His-GFP 0d 1d / 4 ºC 1d / 37 ºC 30d / 4 ºC 0d 1d / 4 ºC 1d / 37 ºC 30d / 4 ºC (a) 0d (b) His-GFP 0d 1d / 4 ºC 1d / 37 ºC 30d / 4 ºC (b) 0d 1d / 4 ºC 1d / 37ºC 30d / 4 ºC 0 10 20 30 40 50 60 Relative Infiltration (%) His-mCherry His-GFP (c) 0d 1d / 4 ºC 1d / 37ºC 30d / 4 ºC 0 10 20 30 40 50 60 Relative Infiltration (%) His-mCherry His-GFP (c) (c) Figure 2. Fluorescent confocal images of; (a) His-mCherry (red channel, λex: 561 nm) 2 Figure 2. Fluorescent confocal images of; (a) His-mCherry (red channel, λex: 561 nm) and (b) His-GFP (green channel, λex: 488 nm), both reversibly immobilized on AG-Co2+ porous microbeads (0 d) and after 1 d of incubation at 4 ºC and 37 ºC and after 30 days at 4 ºC. (c) Relative infiltration of reversibly immobilized His-mCherry (red bars) and His-GFP (green bars) toward the center of the microbead. Error bars in panel (c) represent the standard deviation from data obtained from 10 microbeads of similar size that ranged from 75 – 150 μm (n =10). In order to decipher if intraparticle protein migration also occurs using other reversible immobilization chemistries like ionic adsorption, we immobilized His-mCherry on agarose beads activated with primary amines (AG-MANAE) which interact with the carboxyl groups (Asp and Glu) of the protein surface. 2. Results and Discussion We confirmed the intraparticle passive migration of this model fluorescent protein, ionically immobilized on AG-MANAE, upon 24 hours of incubation In order to decipher if intraparticle protein migration also occurs using other reversible immobilization chemistries like ionic adsorption, we immobilized His-mCherry on agarose beads activated with primary amines (AG-MANAE) which interact with the carboxyl groups (Asp and Glu) of the protein surface. We confirmed the intraparticle passive migration of this model fluorescent protein, ionically immobilized on AG-MANAE, upon 24 hours of incubation 7 at 37 ºC. Despite being initially immobilized at the outer surface of the beads, even forming protein patches (bright fluorescent spots), His-mCherry migrated to a more uniform distribution across the surface of the microbeads upon storage (Figure S1). 2.2 Study of protein-support binding dynamics To better understand how the interactions at the interface between the protein and the support surface affect the protein intraparticle migration, we studied the dynamics of the protein-surface interactions of His-mCherry and His-GFP with the AG-Co2+ supports through FRAP measurements. As reversible histidine-cobalt chelates interactions present low KD values[28], we suggest that the protein diffusion in FRAP experiments will be ruled by binding interactions rather than pure protein diffusion. Figure 3 shows incomplete fluorescence recoveries of the area submitted to photobleaching for both immobilized proteins indicating that a population of the immobilized proteins remains immobile at the bleached spot, whereas another population (mobile fraction) majorly contributes to the fluorescence recovery. We observed that the immobilized His-mCherry and His-GFP show an immobile fraction of 93% and 45%, respectively (Figure 3a and 3b). On one hand, the large fraction of immobile proteins supports the strong interactions between the His-tagged protein and the support surfaces [31,32]. On the other hand, the observed mobile fractions support the reversibility of the interaction, demonstrating the existence of a population of proteins that diffuse through the support surface explaining the migration process observed in Figure 2. To further comprehend the role of such mobile fraction in the migration process we studied the binding and dissociation dynamics of each protein. To this aim, we fitted the obtained FRAP curves (Figure 3a and b) with the full reaction-diffusion model represented in the Laplace space as described elsewhere[31] (see Experimental Section), which includes all possible behaviors that contribute to the fluorescence recovery within the bleached spot inside the porous support. Through fitting of FRAP curves, 8 we derived the 𝑘𝑜𝑛 and 𝑘𝑜𝑓𝑓 values (see Experimental Section) for the mobile fraction, which represents the rate constants that describe the rate of the protein binding-to (binding rate constant) and release-from (dissociation rate constant) the microbead surface, respectively. Figure 3. Fluorescence recovery after photobleaching (FRAP) measurements to study protein diffusion and binding dynamics. FRAP curves after photobleaching (top) together with the corresponding confocal fluorescent images (bottom) before, during and after photobleaching of; (a) His-mCherry (red channel, λex: 561 nm), and (b) His-GFP (green channel, λex: 488 m) immobilized on AG-Co2+ supports. Dots in FRAP curves represent the experimental data, while the solid black line corresponds to the full reaction-diffusion model fitting (see Experimental Section). 2.2 Study of protein-support binding dynamics The binding rate constant, 𝑘𝑜𝑛, dissociation rate constant, 𝑘𝑜𝑓𝑓 and pseudo- equilibrium constant 𝑘𝑜𝑛𝑘𝑜𝑓𝑓 ⁄ were calculated from FRAP analysis and shown on the top right of the FRAP graph. In the confocal fluorescent images (bottom), the red circular region of interest (ROI, 10 μm) represents the photobleached area and the green ROI represents the non- bleached area of the same size, used as a control. Scale bar, 20 μm. 0 1 2 3 4 5 6 0.0 0.2 0.4 0.6 0.8 1.0 AG-Co2+-His-GFP Full Reaction Model Fitting FRAP Recovery Time (min) 0 2 4 6 0.0 0.2 0.4 0.6 0.8 1.0 AG-Co2+-His-mCherry Full Reaction Model Fitting FRAP Recovery Time (min) Kon = 1.37 Koff = 0.31 Kon/Koff >> 1 Immobile Fraction Kon = 0.09 Koff = 0.7 Kon/Koff < 1 Immobile Fraction (a) (b) Pre-Bleach: 0 s Bleach: 2 sec Post-Bleach: 12 min His-mCherry Pre-Bleach: 0 s Bleach: 2 sec Post-Bleach: 6 min His-GFP 0 1 2 3 4 5 6 0.0 0.2 0.4 0.6 0.8 1.0 AG-Co2+-His-GFP Full Reaction Model Fitting FRAP Recovery Time (min) Kon = 1.37 Koff = 0.31 Kon/Koff >> 1 Immobile Fraction (b) (b) 0 2 4 6 0.0 0.2 0.4 0.6 0.8 1.0 AG-Co2+-His-mCherry Full Reaction Model Fitting FRAP Recovery Time (min) Kon = 0.09 Koff = 0.7 Kon/Koff < 1 Immobile Fraction (a) (a) Pre-Bleach: 0 s Bleach: 2 sec Post-Bleach: 6 min His-GFP Pre-Bleach: 0 s Bleach: 2 sec Post-Bleach: 12 min His-mCherry Figure 3. Fluorescence recovery after photobleaching (FRAP) measurements to study protein diffusion and binding dynamics. FRAP curves after photobleaching (top) together with the corresponding confocal fluorescent images (bottom) before, during and after photobleaching of; (a) His-mCherry (red channel, λex: 561 nm), and (b) His-GFP (green channel, λex: 488 m) immobilized on AG-Co2+ supports. Dots in FRAP curves represent the experimental data, while the solid black line corresponds to the full reaction-diffusion model fitting (see Experimental Section). The binding rate constant, 𝑘𝑜𝑛, dissociation rate constant, 𝑘𝑜𝑓𝑓 and pseudo- equilibrium constant 𝑘𝑜𝑛𝑘𝑜𝑓𝑓 ⁄ were calculated from FRAP analysis and shown on the top right of the FRAP graph. In the confocal fluorescent images (bottom), the red circular region of interest (ROI, 10 μm) represents the photobleached area and the green ROI represents the non- bleached area of the same size, used as a control. Scale bar, 20 μm. 2.2 Study of protein-support binding dynamics We found out that the mobile fraction of His-mCherry presents a much lower binding rate constant (𝑘𝑜𝑛= 0.09), than His-GFP (𝑘𝑜𝑛= 1.67), and accordingly the dissociation rate constant was greater for the mobile fraction of His-mCherry than that of His-GFP. These kinetic constants evidence that the mobile fraction of His-GFP binds stronger to the surface than the mobile fraction of His-mCherry (Figure 3a and b). In addition, the ratio of 𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 estimated for His-GFP (𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 = 5.3) was 40 times higher than that estimated for His-mCherry (𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 = 0.13). As 𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 represents a pseudo-equilibrium constant[31] and defines the We found out that the mobile fraction of His-mCherry presents a much lower binding rate constant (𝑘𝑜𝑛= 0.09), than His-GFP (𝑘𝑜𝑛= 1.67), and accordingly the dissociation rate constant was greater for the mobile fraction of His-mCherry than that of His-GFP. These kinetic constants evidence that the mobile fraction of His-GFP binds stronger to the surface than the mobile fraction of His-mCherry (Figure 3a and b). In addition, the ratio of 𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 estimated for His-GFP (𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 = 5.3) was 40 times higher than that estimated for His-mCherry (𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 = 0.13). As 𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 represents a pseudo-equilibrium constant[31] and defines the 9 ratio between the population of bound and unbound proteins inside one AG-Co2+ microbead, we suggest that the binding equilibrium of the mobile fraction of His-GFP is shifted toward the bound states, unlike the equilibrium of His-mCherry that is shifted toward the unbound states. Hence, the mobile fraction of the immobilized protein with a pseudo-equilibrium constant lower than 1 seems to be more prone to migrate, explaining the faster migration and higher relative infiltration of His-mCherry when compared with His-GFP. Despite both proteins being immobilized through their corresponding His-tags, each protein can establish additional weak and reversible interactions with the carrier surface that may contribute to the migration process. These weak interactions may rely on the number of native His sprinkled around the protein surface, and the net charge of the protein surface at the immobilization conditions. When inspecting the X-ray structure of both GFP (PDB ID: 2BP3) and mCherry (PDB ID: 2HQ5) (Figure 4), we found out that both fluorescence proteins have 4 His residues fairly exposed to the solvent (solvent accessible surface area, SASA > 100 Å2) besides their fused His-tags (Figure S2). 2.2 Study of protein-support binding dynamics These weak interactions may rely on the number of native His sprinkled around the protein surface, and the net charge of the protein surface at the immobilization conditions. When inspecting the X-ray structure of both GFP (PDB ID: 2BP3) and mCherry (PDB ID: 2HQ5) (Figure 4), we found out that both fluorescence proteins have 4 His residues fairly exposed to the solvent (solvent accessible surface area, SASA > 100 Å2) besides their fused His-tags (Figure S2). Figure 4. X-Ray crystallographic 3D structure of (a) His-mCherry (PDB ID: 2H5Q) and (b) His-GFP (PDB ID: 2BP3). In yellow are the fairly exposed His residues (SASA: solvent Figure 4. X-Ray crystallographic 3D structure of (a) His-mCherry (PDB ID: 2H5Q) and (b) His-GFP (PDB ID: 2BP3). In yellow are the fairly exposed His residues (SASA: solvent 10 accessible surface area > 100 Å2). The N-terminus where the tagged 6xHis polypeptide is fused is highlighted in blue. Tables under each protein show the corresponding distances (in Å) between each His residue and the N-terminus. In green, distances < 10 Å may allow two His to coordinate the cobalt chelates displayed at the support surface. In red, distances > 10 Å make impossible the coordination between the two His and the metal chelates at the support surface. However, His-GFP has 2 potential His clusters (His77/His231 and His25/His139) where the distance between two native His is less than 10 Å whereas His-mCherry has no native His close to another in the rage of a plausible interaction distance. In particular, the pair formed by His77 and His231 of His-GFP are separated by only 5.9 Å and located close to the N-terminus (< 15 Å) where the His-tag is fused. Furthermore, under the immobilization and storage pH (pH = 7), the net charge of His-GFP (calculated with Bluues server[33]) is slightly negative (-2.82), while the net charge of His-mCherry is almost zero (-0.01). Hence, His-GFP seems to have deprotonated Asp and Glu residues that may also interact electrostatically with the positively charged cobalt divalent ions present at the agarose surface. Thus, the negatively charged residues (i.e., Asp and Glu) and the uncharged His residues scattered in the surface of His-GFP can establish unspecific weak electrostatic and metal-imidazole interactions, with the carrier surface, respectively. 2.2 Study of protein-support binding dynamics Therefore, these native His clusters and the slightly negative charge of His-GFP may explain the higher 𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 ratio of its mobile fraction and its restricted migration across the inner support surface, when compared with His-mCherry that may establish weaker interactions with the AG-Co2+ surface. All in all, these results suggest that the dynamics of the migration process rely on several weak and non-specific surface interactions (i.e., electrostatic, metal-coordination) that are unique for each protein-support pair. However, His-GFP has 2 potential His clusters (His77/His231 and His25/His139) where the distance between two native His is less than 10 Å whereas His-mCherry has no native His close to another in the rage of a plausible interaction distance. In particular, the pair formed by His77 and His231 of His-GFP are separated by only 5.9 Å and located close to the N-terminus (< 15 Å) where the His-tag is fused. Furthermore, under the immobilization and storage pH (pH = 7), the net charge of His-GFP (calculated with Bluues server[33]) is slightly negative (-2.82), while the net charge of His-mCherry is almost zero (-0.01). Hence, His-GFP seems to have deprotonated Asp and Glu residues that may also interact electrostatically with the positively charged cobalt divalent ions present at the agarose surface. Thus, the negatively charged residues (i.e., Asp and Glu) and the uncharged His residues scattered in the surface of His-GFP can establish unspecific weak electrostatic and metal-imidazole interactions, with the carrier surface, respectively. Therefore, these native His clusters and the slightly negative charge of His-GFP may explain the higher 𝑘𝑜𝑛/𝑘𝑜𝑓𝑓 ratio of its mobile fraction and its restricted migration across the inner support surface, when compared with His-mCherry that may establish weaker interactions with the AG-Co2+ surface. All in all, these results suggest that the dynamics of the migration process rely on several weak and non-specific surface interactions (i.e., electrostatic, metal-coordination) that are unique for each protein-support pair. 2.3 Imidazole affects the intraparticle protein migration. To further explore the m 2.3 Imidazole affects the intraparticle protein migration. To further explore the migration of the reversibly immobilized proteins we incubated His-mCherry@AG-Co2+ and His- GFP@AG-Co2+ in presence of 10 mM imidazole to study the effects of binding competitors in the migration process. In Figure 5, CLSM images show that imidazole accelerates the migration process of both His-mCherry and His-GFP at 4 ºC (Figure 5a and b) compared to 11 the results observed in absence of imidazole (Figure 2a and b). Imidazole competes with the His-tag for the binding to the cobalt-chelates of the AG-Co2+supports, favoring the dissociation of both His-tagged fluorescence proteins. Even though the proteins migrate more easily inside the microbeads in presence of imidazole, SDS-PAGE analysis demonstrates that they are not released from the microbeads at concentrations as low as 10 mM (Figure S3a and b). In contrast, proteins were quantitatively eluted from the microbeads when incubated at 300 mM imidazole. Figure 5. Fluorescent confocal images of (a) His-mCherry (red channel, λex: 561 nm) and (b) His-GFP (green channel, λex: 488 nm) reversibly immobilized on AG-Co2+ porous microbeads (0 d) and after incubation for 1 day at 4 ºC in presence of 10 mM imidazole (abbreviated as I in figure legends). (a) (b) His-mCherry His-GFP 0d 1d + I / 4 ºC 0d 1d + I / 4 ºC (a) 0d (b) 0d Figure 5. Fluorescent confocal images of (a) His-mCherry (red channel, λex: 561 nm) and (b) His-GFP (green channel, λex: 488 nm) reversibly immobilized on AG-Co2+ porous microbeads (0 d) and after incubation for 1 day at 4 ºC in presence of 10 mM imidazole (abbreviated as I in figure legends). The weakening of the protein-surface interactions was supported by FRAP measurements (Figure S4, Supporting Information), where the mobile fraction of the immobilized His-GFP in presence of 10 mM of imidazole was 97%, much higher than in its absence (45%). In conclusion, imidazole led the immobilized proteins to colonize 100% of the bead radius indicating that a larger mobile fraction of the bound protein is more prone to migrate within the porous beads. The weakening of the protein-surface interactions was supported by FRAP measurements (Figure S4, Supporting Information), where the mobile fraction of the immobilized His-GFP in presence of 10 mM of imidazole was 97%, much higher than in its absence (45%). 2.3 Imidazole affects the intraparticle protein migration. To further explore the m In conclusion, imidazole led the immobilized proteins to colonize 100% of the bead radius indicating that a larger mobile fraction of the bound protein is more prone to migrate within the porous beads. 2.4 Irreversible immobilization halts protein migration. Once we observed protein migration under different storage conditions, we tried to prevent such migration through the irreversible immobilization of proteins. Irreversible immobilization was achieved through 2.4 Irreversible immobilization halts protein migration. Once we observed protein migration under different storage conditions, we tried to prevent such migration through the irreversible immobilization of proteins. Irreversible immobilization was achieved through 12 covalent bonds formed between the nucleophile and solvent exposed residues (Lys, His, Cys, or Tyr) at the protein surface and the epoxy groups at the surface of the agarose microbeads functionalized with both cobalt-chelates and epoxy groups (AG-Co2+/E, Figure 6a)[34,35]. Irreversible immobilization of the enzymes at the AG-Co2+/E surface was demonstrated by SDS-PAGE analysis (Figure S5) as bound proteins were not eluted from the support upon their incubation under denaturing conditions (100 ºC in Laemmli buffer). Reversibly and irreversibly immobilized proteins were incubated for 15 days at 4 ºC, (typical storage conditions). Remarkably, we observed that migration of His-mCherry was restrained, as after 15 days at 4 ºC His-mCherry remained immobilized mostly at the outer surface of the AG-Co2+/E microbeads (Figure 6b). The migration of His-mCherry irreversibly immobilized on AG- Co2+/E was dramatically restricted (Figure 6b and c), under conditions where reversibly immobilized His-mCherry colonized 100% of the microbeads radius (1 day at 37 ºC in presence of 10 mM of imidazole). Such behavior was also observed for His-GFP@AG-Co2+/E (Figure S6a and b, Supplementary Information). Regardless the small relative infiltration of both proteins compared to the one observed for reversibly immobilized proteins, this small relative infiltration is attributed to the fact that although through the presence of epoxy groups on the heterofunctional support of AG-Co2+/E we enable the formation of additional covalent bonds (besides His-tag coordination), in the crowded conditions where the His-tag proteins are immobilized (outer surface of the beads), there should still exist some fraction of proteins that would not have the space to interact with the epoxy groups and this small fraction would be able to then migrate towards the inner surface of the beads. 13 Figure 6. (a) Schematic representation of the site-selective protein immobilization on agarose porous microbeads activated with cobalt chelates for reversible protein immobilization (AG- Co2+) and cobalt chelates and epoxy groups for irreversible protein immobilization (AG- Co2+/E). (b) Fluorescent confocal images of His-mCherry (red channel, λex: 561 nm) reversibly and irreversibly immobilized on AG-Co2+ and AG-Co2+/E, respectively (0 d), after incubation for 15 days at 4 ºC and after incubation for 1 day at 37 ºC in presence of 10 mM of imidazole (abbreviated as I in figure legends). (c) Relative infiltration of reversibly and irreversibly immobilized His-mCherry toward the center of the microbeads. 2.4 Irreversible immobilization halts protein migration. Once we observed protein migration under different storage conditions, we tried to prevent such migration through the irreversible immobilization of proteins. Irreversible immobilization was achieved through Error bars in panel (c) represent the standard deviation of data obtained from 10 microbeads of similar size (75 – 100 μm) for each incubation condition (n =10). (b) 0d Reversible Irreversible (a) AG-Co2+ AG-Co2+/E (c) 1d + I / 37 ºC 15d / 4 ºC 0 1 15 30 0 25 50 75 100 Relative infiltration (%) Reversible Reversible + I Irreversible Irreversible + I 0 1 15 30 Days 0 1 15 30 0 1 15 30 (a) AG-Co2+ AG-Co2+/E (b) 0d Reversible Irreversible 1d + I / 37 ºC 15d / 4 ºC (b) (a) 0d Irreversible (c) (c) 0 1 15 30 0 25 50 75 100 Relative infiltration (%) Reversible Reversible + I Irreversible Irreversible + I 0 1 15 30 Days 0 1 15 30 0 1 15 30 Figure 6. (a) Schematic representation of the site-selective protein immobilization on agarose porous microbeads activated with cobalt chelates for reversible protein immobilization (AG- Co2+) and cobalt chelates and epoxy groups for irreversible protein immobilization (AG- Co2+/E). (b) Fluorescent confocal images of His-mCherry (red channel, λex: 561 nm) reversibly and irreversibly immobilized on AG-Co2+ and AG-Co2+/E, respectively (0 d), after incubation for 15 days at 4 ºC and after incubation for 1 day at 37 ºC in presence of 10 mM of imidazole (abbreviated as I in figure legends). (c) Relative infiltration of reversibly and irreversibly immobilized His-mCherry toward the center of the microbeads. Error bars in panel (c) represent the standard deviation of data obtained from 10 microbeads of similar size (75 – 100 μm) for each incubation condition (n =10). Likewise, CALB labeled with fluorescein was immobilized on agarose beads functionalized with octyl groups (AG-O), through reversible hydrophobic interactions, also migrated during storage. That migration could be similarly halted by immobilizing the labeled enzyme on agarose activated with both octyl and vinyl sulfone groups (AG-O/V) to establish irreversible bonds that will avoid the protein migration (Figure S7). Despite being irreversibly immobilized, CALB recovered 45% of the specific activity[36,37] of its free counterpart (Table Likewise, CALB labeled with fluorescein was immobilized on agarose beads functionalized with octyl groups (AG-O), through reversible hydrophobic interactions, also migrated during storage. That migration could be similarly halted by immobilizing the labeled enzyme on agarose activated with both octyl and vinyl sulfone groups (AG-O/V) to establish irreversible bonds that will avoid the protein migration (Figure S7). 2.4 Irreversible immobilization halts protein migration. Once we observed protein migration under different storage conditions, we tried to prevent such migration through the irreversible immobilization of proteins. Irreversible immobilization was achieved through Despite being irreversibly immobilized, CALB recovered 45% of the specific activity[36,37] of its free counterpart (Table 14 S1). Like the epoxy groups, the vinyl groups establish irreversible bonds with the nucleophilic and well exposed residues (Lys, His, Cys, or Tyr) of the protein surface. Therefore, we confirm that intraparticle enzyme migration is prevented by establishing irreversible covalent bonds between the protein and the support surface. 3. Effect of protein co-immobilization on the intraparticle protein migration. Interestingly, we observed that the migration pattern is altered when His-mCherry and His-GFP are co-immobilized either sequentially or simultaneously on AG-Co2+ supports (Figure 7a). When the fluorescent proteins were sequentially co-immobilized, being His-GFP first and followed by His-mCherry, and incubated for 30 days at 4 ºC, the migration of the latter was 19% less than when the same protein was individually immobilized on the same carrier and under the same incubation conditions (Figure 7b). In contrast, the subsequent immobilization of His-mCherry triggered the migration of His-GFP toward inner microbead regions after 30 days at 4º C, pushing this primarily immobilized protein to colonize a particle radius 12% deeper than that one colonized by the same protein individually immobilized on the same carrier and under the same conditions. When we either inverted the immobilization order; first His- mCherry and second His-GFP or mixed the two proteins (Figure 7a), the relative infiltration (Figure 7b) of both His-mCherry and His-GFP was similar to the one achieved when both enzymes were reversibly immobilized alone and stored under the same conditions (Figure 2). The different binding dynamics found for His-mCherry and His-GFP when immobilized on AG-Co2+ microbeads may explain the dependency of the migration pattern on the co- immobilization sequence. When the two proteins co-localize within the same local surface of the support, lateral forces may emerge among the proteins[17] altering the binding dynamics and thus the migration behavior of each protein. This insight is very relevant in the context of immobilized multi-enzyme systems. In these systems, the spatial distribution of the co- 15 immobilized enzymes may change during storage, altering the functionality of the multi- functional heterogeneous biocatalyst. Figure 7. (a) Fluorescent confocal images of His-GFP (green channel, λex: 488 nm) and His- mCherry (red channel, λex: 561 nm) reversibly co-immobilized in sequence or simultaneously on AG-Co2+ porous microbeads after incubation for 30 days at 4 ºC. (b) Calculated relative infiltration of reversibly co-immobilized His-GFP and His-mCherry toward the center of the microbeads at 0 and 30 days of incubation at 4 ºC. Protein X/Protein Y indicates the order of the co-immobilization sequence, whereas Protein X + Protein Y indicates that both enzymes were mixed and co-immobilized simultaneously. Error bars in panel (b) represent the standard deviation of data obtained from 10 microbeads of similar size (75 – 100 μm) for each incubation condition (n =10). 3. Effect of protein co-immobilization on the intraparticle protein migration. (a) GFP / mCherry GFP + mCherry mCherry / GFP His-GFP His-mCherry (b) His-GFP His-mCherry 0 25 50 75 100 Relative infiltration (%) His-GFP His-mCherry 0d 30d / 4 ºC GFP / mCherry mCherry / GFP GFP + mCherry His-GFP His-mCherry (b) His-GFP His-mCherry 0 25 50 75 100 Relative infiltration (%) His-GFP His-mCherry 0d 30d / 4 ºC GFP / mCherry mCherry / GFP GFP + mCherry His-GFP His-mCherry (a) GFP / mCherry His-GFP His-mCherry (b) (a) mCherry / GFP Figure 7. (a) Fluorescent confocal images of His-GFP (green channel, λex: 488 nm) and His- mCherry (red channel, λex: 561 nm) reversibly co-immobilized in sequence or simultaneously on AG-Co2+ porous microbeads after incubation for 30 days at 4 ºC. (b) Calculated relative infiltration of reversibly co-immobilized His-GFP and His-mCherry toward the center of the microbeads at 0 and 30 days of incubation at 4 ºC. Protein X/Protein Y indicates the order of the co-immobilization sequence, whereas Protein X + Protein Y indicates that both enzymes were mixed and co-immobilized simultaneously. Error bars in panel (b) represent the standard deviation of data obtained from 10 microbeads of similar size (75 – 100 μm) for each incubation condition (n =10). This co-migration phenomenon was also proved for the lipase B from Candida antarctica labeled with fluorescein (FITC-CALB) and lipase from Thermomyces lanuginosus labeled with Rhodamine B (RhB-TLL), which were reversibly co-immobilized via hydrophobic interactions on octyl agarose supports as described in Experimental Section (AG-O)[38,39]. We observed that both fluorescently labeled enzymes fully migrated after 1 day of storage at 37 ºC regardless of their initial spatial distribution (Figure S8). While TLL was hyperactivated; 2.8 times upon the immobilization on AG-O, CALB maintained its specific activity. However, none of the enzymes underwent activity changes upon the migration process as resulted after 1 day of incubation at 37º C (Table S1). This co-migration phenomenon was also proved for the lipase B from Candida antarctica labeled with fluorescein (FITC-CALB) and lipase from Thermomyces lanuginosus labeled with Rhodamine B (RhB-TLL), which were reversibly co-immobilized via hydrophobic interactions on octyl agarose supports as described in Experimental Section (AG-O)[38,39]. We observed that both fluorescently labeled enzymes fully migrated after 1 day of storage at 37 ºC regardless of their initial spatial distribution (Figure S8). While TLL was hyperactivated; 2.8 times upon the immobilization on AG-O, CALB maintained its specific activity. 3. Effect of protein co-immobilization on the intraparticle protein migration. However, none of the enzymes underwent activity changes upon the migration process as resulted after 1 day of incubation at 37º C (Table S1). 16 16 4. Effect of migration on functional properties of proteins. Having in hand that protein migration does occur under certain storage conditions we interrogated whether migration affects the functional properties (i.e., stability) of the migrated proteins. Figure 8 shows the changes in the Tryptophan (Trp) fluorescence intensity of soluble and immobilized His-GFP on AG-Co2+ after storage at 4 ºC for 30 days. These studies revealed that soluble His-GFP showed no changes in its Trp fluorescence over 30 days at 4 ºC, (Figure 8a), while the Trp fluorescence of the immobilized His-GFP was red-shifted (from 310 nm to 325 nm) (Figure 8b) at the same storage conditions. Such behavior may be associated with the migration of His-GFP, where the protein may suffer conformational changes during the migration process. On the contrary, those conformational changes were not observed for His- GFP irreversibly immobilized on AG-Co2+/E supports after 30 days of storage at 4 ºC, since the protein negligibly migrated across the carrier surface (Figure S9). Figure 8. Tryptophan fluorescence emission spectra of (a) soluble His-GFP and (b) immobilized His-GFP on AG-Co2+ supports at 0 days and after 30 days of incubation at 4 ºC. (a) (b) 320 340 360 380 400 420 440 0.0 0.2 0.4 0.6 0.8 1.0 Normalized fluorescence intensity Wavelength (nm) 0d 30d / 4 ºC 320 340 360 380 400 420 440 0.0 0.2 0.4 0.6 0.8 1.0 Normalized fluorescence intensity Wavelength (nm) 0d 30d / 4ºC (a) 320 340 360 380 400 420 440 0.0 0.2 0.4 0.6 0.8 1.0 Normalized fluorescence intensity Wavelength (nm) 0d 30d / 4ºC (b) 320 340 360 380 400 420 440 0.0 0.2 0.4 0.6 0.8 1.0 Normalized fluorescence intensity Wavelength (nm) 0d 30d / 4 ºC (b) (a) Figure 8. Tryptophan fluorescence emission spectra of (a) soluble His-GFP and (b) immobilized His-GFP on AG-Co2+ supports at 0 days and after 30 days of incubation at 4 ºC. 17 A recent study by Chaparro Sosa et al.[10] revealed that the distances ( > 0.5 μm) traveled by the proteins until finding their stable binding sites causes molecular friction with the support surface that increases the probability of protein unfolding. We observe similar results during the migration process as the proteins are structurally distorted while are passively colonizing the inner regions of the porous support. 4.1 Migration effects on the enzyme activity 18 To understand if the migration process also affects the catalytic properties of the immobilized enzymes, we evaluated the changes in the spatial distribution of an industrially relevant enzyme upon storage. The NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus (BsADH)[40] was labeled with RhB and reversibly immobilized on AG-Co2+ supports to follow its migration upon incubation for 1 day at 4 ºC and 37 ºC. CLSM images and radial profile analysis showed a very slight migration after 1 day at 4 ºC (Figure 9a and b). However, a more noticeable migration of His-BsADH-RhB was observed after 1 day of incubation at 37 ºC (Figure 9b). Like in His-GFP, we identified 2 His residues (H76 and H94) exposed to the solvent (solvent accessible surface area, SASA > 100 Å2) close enough to form an inter-subunit His-cluster that might coordinate the cobalt chelates of the carrier surface (Figure S10). Moreover, the net charge of the enzyme surface at the immobilization conditions (pH 7) is slightly negative (-1.38) (calculated with Bluues server[33]). Therefore, His-BsADH presents surface characteristics that explain the incomplete migration under the tested storage conditions. Subsequently, we evaluated whether the observed migration has an impact on the activity of the immobilized enzyme. To this aim, we performed enzyme activity studies through single- 18 particle measurements, which have shown to reveal valuable information on the enzyme activity at the sub-micrometer level[13]. activity at the sub-micrometer level[13]. Figure 9. (a) Fluorescent confocal images of His-BsADH labeled with RhB (red channel, λex: 561 nm) reversibly immobilized on AG-Co2+ porous microbeads (0 d) and after storage for 1 day at 4 ºC and 37 ºC. Yellow lines on top of the beads depict the radial cross-sections for one single bead that corresponds to the (b) normalized fluorescence intensity radial profile. (c) Enzyme specific activity as calculated through enzyme kinetics for each incubation conditions. Specific activity is defined as the activity units per enzyme concentration (U µM-1). Activity unit (U) is defined as the concentration of cofactor consumed per sec (μM sec-1). Activity refers to the decreasing of NADH fluorescence (fluorescent images) of a region of interest of a diameter of 100 μm in the interparticle space (bulk). Error bars in panel (c) represent the standard deviation from data obtained from from 10 ROIs (n =10). 4.1 Migration effects on the enzyme activity (a) (b) (c) 0d 1d / 4 ºC 1d / 37 ºC Bs-ADH-RhB 0d 1d / 4 ºC 1d / 37 ºC 0.0E+0 2.0E-4 4.0E-4 6.0E-4 8.0E-4 1.0E-3 Specific activity (U mM-1) 0 10 20 30 40 50 0.0 0.2 0.4 0.6 0.8 1.0 Normalized Integrated Density Radius (mm) 0d 1d / 4 ºC 1d / 37 ºC (a) 0d 1d / 4 ºC 1d / 37 ºC Bs-ADH-RhB (a) (c) 0d 1d / 4 ºC 1d / 37 ºC 0.0E+0 2.0E-4 4.0E-4 6.0E-4 8.0E-4 1.0E-3 Specific activity (U mM-1) (b) 0 10 20 30 40 50 0.0 0.2 0.4 0.6 0.8 1.0 Normalized Integrated Density Radius (mm) 0d 1d / 4 ºC 1d / 37 ºC (b) (c) 9. (a) Fluorescent confocal images of His-BsADH labeled with RhB (red channel, λex: Figure 9. (a) Fluorescent confocal images of His-BsADH labeled with RhB (red channel, λex: 561 nm) reversibly immobilized on AG-Co2+ porous microbeads (0 d) and after storage for 1 day at 4 ºC and 37 ºC. Yellow lines on top of the beads depict the radial cross-sections for one single bead that corresponds to the (b) normalized fluorescence intensity radial profile. (c) Enzyme specific activity as calculated through enzyme kinetics for each incubation conditions. Specific activity is defined as the activity units per enzyme concentration (U µM-1). Activity unit (U) is defined as the concentration of cofactor consumed per sec (μM sec-1). Activity refers to the decreasing of NADH fluorescence (fluorescent images) of a region of interest of a diameter of 100 μm in the interparticle space (bulk). Error bars in panel (c) represent the standard deviation from data obtained from from 10 ROIs (n =10). Figure 9. (a) Fluorescent confocal images of His-BsADH labeled with RhB (red channel, λex: 561 nm) reversibly immobilized on AG-Co2+ porous microbeads (0 d) and after storage for 1 day at 4 ºC and 37 ºC. Yellow lines on top of the beads depict the radial cross-sections for one single bead that corresponds to the (b) normalized fluorescence intensity radial profile. (c) Enzyme specific activity as calculated through enzyme kinetics for each incubation conditions. Specific activity is defined as the activity units per enzyme concentration (U µM-1). Activity unit (U) is defined as the concentration of cofactor consumed per sec (μM sec-1). 4.1 Migration effects on the enzyme activity Activity refers to the decreasing of NADH fluorescence (fluorescent images) of a region of interest of a diameter of 100 μm in the interparticle space (bulk). Error bars in panel (c) represent the standard deviation from data obtained from from 10 ROIs (n =10). Interestingly, in operando single-particle kinetics showed an increment of the specific activity 19 where we observed less passive migration. Remarkably, we found a positive correlation between the infiltration radius and the specific activity of His-BsADH, which meant that the specific activity upon 1 day at 37 ºC was much higher than upon incubation for 1 day at 4 ºC. This behavior was not observed in the case of His-BsADH irreversibly immobilized on AG- CO2+/E supports, where no migration was observed (Figure S11). These findings agree with a 19 recent work from our group where we showed that the kinetics of heterogeneous biocatalysts strongly depend on the spatial distribution and density of the immobilized enzymes[13]. Based on those recent insights we suggest that the intraparticle migration of His-BsADH leads to a lower enzyme density within the agarose porous microbeads that explains the higher activity of such immobilized enzyme upon migration. Despite the observed changes in activity of His- BsADH after incubation for 1 day at 4 ºC and 37 ºC, we did not observe changes in the proteins Trp fluorescence thus in its structure (Figure S12). recent work from our group where we showed that the kinetics of heterogeneous biocatalysts strongly depend on the spatial distribution and density of the immobilized enzymes[13]. Based on those recent insights we suggest that the intraparticle migration of His-BsADH leads to a lower enzyme density within the agarose porous microbeads that explains the higher activity of such immobilized enzyme upon migration. Despite the observed changes in activity of His- BsADH after incubation for 1 day at 4 ºC and 37 ºC, we did not observe changes in the proteins Trp fluorescence thus in its structure (Figure S12). In conclusion, enzyme migration as a result of temporal evolution influences both the conformational dynamics and enzyme activity that ultimately affected the functional properties of the immobilized biomacromolecules. 3. Conclusions By introducing irreversible bonds between the proteins and the support surfaces, we can effectively halt the protein migration process. Finally, we showed that the intraparticle migration process affects both the structure and the functionality of the immobilized but migrated proteins. This fact is relevant for enzymes whose specific activity depends on the enzyme concentration within the solid surface of the support. Hence, this work seeks to encourage the investigation of macromolecular migration on those systems that involve a protein bound to a porous material. We consider this process fundamental to understand the evolution structure and functional properties of protein functionalized materials upon their use. The insights herein reported can guide future in silico studies that model protein migration 3. Conclusions 20 In this work, we have developed a single-particle analysis tool based on CLSM image processing, that enabled us to follow the spatiotemporal migration of immobilized proteins inside 3D porous agarose-based microbeads. As a result, these studies allowed us to understand the dynamics of biomolecules at the interface with solid materials. As a model system, we explored the migration of His-tagged fluorescent proteins reversibly immobilized on porous agarose microbeads (AG-Co2+). Intraparticle protein migration is explained by an equilibrium between protein bound and unbound states governed by metal coordination but also electrostatic interactions. When other reversible immobilization chemistries (i.e., electrostatic or hydrophobic interactions) are involved, proteins also migrate but with different dynamics. We suggest that the migration dynamics are both protein and carrier specific, so the same protein 20 will migrate differently across two different carrier surfaces, and the migration of two different proteins will be different across the same carrier surface. Furthermore, the migration process is also affected by the incubation time and temperature (storage or operational conditions) as well as the presence of immobilization competitors. Under those conditions where the binding between the enzyme and the support surface is weakened, the migration process occurs faster. By introducing irreversible bonds between the proteins and the support surfaces, we can effectively halt the protein migration process. Finally, we showed that the intraparticle migration process affects both the structure and the functionality of the immobilized but migrated proteins. This fact is relevant for enzymes whose specific activity depends on the enzyme concentration within the solid surface of the support. Hence, this work seeks to encourage the investigation of macromolecular migration on those systems that involve a protein bound to a porous material. We consider this process fundamental to understand the evolution structure and functional properties of protein functionalized materials upon their use. The insights herein reported can guide future in silico studies that model protein migration processes across the surface of solid materials will migrate differently across two different carrier surfaces, and the migration of two different proteins will be different across the same carrier surface. Furthermore, the migration process is also affected by the incubation time and temperature (storage or operational conditions) as well as the presence of immobilization competitors. Under those conditions where the binding between the enzyme and the support surface is weakened, the migration process occurs faster. 4. Experimental Section Materials: Agarose microbeads (50-150 µm diameter) were purchased from Agarose Bead Technologies (Madrid, Spain). Octyl-Sepharose® CL-4B beads matrix was purchased from GE Healthcare (Alcobendas, Spain). Rhodamine B isothiocyanate mixed isomers (RhB), fluorescein isothiocyanate isomer I (FITC), iminodiacetic acid (IDA), albumin bovine serum standard (BSA), p-Nitrophenyl butyrate (p-NPB) and other reagents and solvents of analytical 21 grade were purchased from Sigma-Aldrich (St. Louis, IL, USA). Nicotinamide-adenine- dinucleotide reduced sodium salt (NADH) was purchased from GERBU Biotechnik GmbH (Heidelberg, Germany). Isopropyl-betha-D-thiogallactopiranoside (IPTG, 100%) was purchased from Fisher Bioreagents. Bradford protein assay dye reagent was purchased from BIORAD (Biorad. Hercules, CA, USA). Clear bottom black, white, and UV microplates (96- well) were purchased from avantor (2021 VWR International, LLC). u-Slides 8 well glass bottom was purchased from Ibidi (Planegg, Germany). Two different commercial liquid lipase formulations were used in this study: Lipozyme® CALB L (lipase B from Candida antarctica, CALB, 7.75 mg of protein per mL-1 of extract) and Lipozyme® TL 100 L (lipase from Thermomyces lanuginosus, TLL, 23.02 mg of protein per mL-1 of extract) were kindly donated by Novozymes (Alcobendas, Spain). SmakeSkin™ Dialysis Tubing, (10k MWCO) was purchased from Thermo scientific™. Supports used in this study. Functionalized agarose microbeads (AG) used for protein immobilization for migration studies were based on commercially cross-linked agarose beads 6BCL (particle size: 50 – 150 μm, average pore size: 112 nm) and cobalt-activated agarose microbeads 6BCL (AG-Co2+) (particle size: 50 – 150 μm, average pore size: 112 nm, 15 µmol of Co2+ x g carrier−1), purchased from ABT technologies (Madrid, Spain). For immobilization through enzyme ionic adsorption, 6BCL AG supports were activated with primary amines (AG- MANAE) as described elsewhere for MANAE (mono-aminoethyl-N-aminoethyl) supports[41] (particle size: 50 – 150 μm, average pore size: 112 nm). For irreversible immobilization of the proteins, the AG supports were functionalized with cobalt chelates and epoxy groups (AG- Co2+/E, 11 μmol of Co2+ x carrier−1 and 28 μmol of epoxides x carrier−1) as described elsewhere[42,43]. Octyl-Sepharose® CL-4B beads (AG-O) matrix was purchased from GE Healthcare (Alcobendas, Spain). For irreversible immobilization on (AG-O), the latter was functionalized with divinyl sulfone (AG-O/V) as described by Albuquerque et al.[37]. 22 Enzyme Expression and purification. Green fluorescent protein (GFP), the monomeric red fluorescent protein mCherry, and the alcohol dehydrogenase from Bacillus stearothermophilus (His-BsADH) with a poly-histidine tag at the N-terminus were heterogeneously expressed in Escherichia coli. The genes encoding all enzymes were optimized for E. 4. Experimental Section coli codon usages and synthesized by Genscript Biotech (Piscataway, NJ, USA). The synthetic genes were cloned into pET28b(+) using NdeI and XhoI restriction sites. DNA isolation, plasmid purification, restriction analysis, plasmid construction, and DNA sequencing were carried out by standard methods[44]. Briefly, the recombinant plasmids that harbor the gene that encodes His-GFP, His- mCherry, and His-BsADH were transformed into E. coli BL21 (DE3) chemical competent cells and cultivated under gently shaking at 37 ºC in 50 mL of LB medium supplemented with 30 µg mL−1 of kanamycin until the OD 600nm reached 0.6. At that point, the culture was induced with 1 mM IPTG for His-GFP, His-mCherry, and His-BsADH expression. After induction, the cells were grown at 37 ºC for 3 h in the case of His-GFP, His-mCherry, and His-BsADH. Finally, cells were harvested by centrifugation at 4000 g for 30 min at 4 ºC. Then, the cell pellet containing His-GFP, His-mCherry, and His-BsADH were resuspended in 25 mM of sodium phosphate buffer at pH 7 containing 50 mM NaCl and 10 mM imidazole. The resulting suspensions were sonicated and centrifuged and the supernatant containing the enzyme was purified through immobilized metal affinity chromatography (IMAC). All enzymes were eluted with elution buffer (50 mM Tris−HCl buffer containing 500 mM imidazole at pH 8). Eluted proteins were then filtered in a tangential ultrafiltration unit (10 kDa) to remove imidazole and exchange the buffer with 25 mM sodium phosphate at pH 7. TLL and CALB colorimetric assay in solution. Specific activity of soluble and immobilized TLL and CALB were measured through colorimetric assay measurements. Briefly, for free enzyme activity measurements, 200 μL of the reaction mixture containing 0.5 mM p-NPB as substrate in 50 mM Tris HCl pH 7.0 were incubated with 5 μL of free enzyme at 30 ºC. Increase 23 in absorbance caused by the release of p-nitrophenol during hydrolysis of p-NPB was monitored at 410 nm. In the case of immobilized enzymes, we followed the same procedure but instead of 5 μL of free enzyme we added 10 μL from the suspension of the biocatalyst (1:10). One unit of activity was defined as the amount of enzyme to hydrolyze 1 µmol of p-NPB per minute. Enzyme fluorescent labeling. Fluorescent labeling of His-BsADH and TLL with RhB and CALB with FITC was carried out as described elsewhere[45]. 4. Experimental Section Briefly, in the case of the commercial extract of the lipases, TLL and CALB a dialysis step (dialysis tube, 10 kDa) was carried out before the labeling process to eliminate any component that could interfere with the fluorophore labeling. Next, all enzymes were filtered in a tangential ultrafiltration unit (10 kDa) to exchange the buffer with 100 mM sodium bicarbonate at pH 8.5. The solution with the suitable buffer was mixed with the corresponding fluorophore (RhB or FITC dissolved in DMSO) at a 1:1 protein: fluorophore molar ratio and incubated for at least 1 h under gentle shaking at 25 ºC. The remaining unconjugated RhB or FITC were eliminated by filtering the enzyme solution in a tangential ultrafiltration unit (10 kDa) with 25 mM sodium phosphate at pH 7 until no fluorescence was detected in the filtered solution. Immobilization and co-immobilization of proteins on activated agarose supports. Pure His- GFP, His-mCherry, and RhB labeled His-BsADH (0.1 mg mL-1 in 25 mM sodium phosphate buffer at pH 7) were incubated with AG-Co2+ and AG-Co2+/E at a ratio of 1:10 (w:v) for 1 h at 25 ºC. Irreversibly immobilized proteins on AG-Co2+/E were incubated for 2h with phosphate buffer 100 mM at pH 8 and subsequently incubated overnight with 1 M Glycine at pH 8, to block any remaining epoxy groups. Finally, all supports were washed with 25 mM sodium phosphate buffer at pH 7. Fluorescently labeled lipases were reversibly immobilized on octyl activated agarose support (AG-O) by hydrophobic interactions, as described elsewhere[36,46]. Irreversible immobilization 24 24 of the lipases was done on AG-O/V. For reversible immobilization of fluorophore labeled lipases on AG-O supports, lipases were diluted in 50 mM Tris HCl at pH 7 (0.1 mg mL-1) and subsequently added to AG-O support at a ratio of 1:10 (w:v) for 1 h at 25 ºC. For irreversible immobilization of fluorophore labeled lipases on AG-O/V supports, lipases were diluted at 5 mM sodium acetate at pH 5.0 (0.1 mg mL-1) and subsequently mixed with the support AG-O/V support at a ratio of 1:10 (w:v) for 1 h at 25 ºC. After immobilization, the supports were filtered and washed with 50 mM sodium bicarbonate at pH 8 and then resuspended in 50 mM sodium bicarbonate at pH 8 and incubated (1:10 w:v ratio) for 4 h at 25 °C, to favor the enzyme-support covalent reaction. 4. Experimental Section Finally, the AG-O/V supports were filtered and incubated in 2 M of aspartic acid at pH 8 (1:10 w:v ratio) for 16 h at 25 ºC, to block any remaining Vs groups. The covalently immobilized and blocked biocatalysts were finally filtered and washed with 50 mM Tris HCl at pH 7. The immobilization yield of the herein used proteins and enzymes was calculated using Bradford’s method[47]. SDS-PAGE analysis. His-GFP and His-mCherry reversibly immobilized on AG-Co2+ supports were analyzed by SDS-PAGE (Figure S3, Supporting Information). Briefly, a 1:3 (w:v) suspension of support was incubated with 10 mM Imidazole for 1 h and then it was filtered and the filtered support, as well as the obtained flowthrough, were incubated with Laemmli buffer and boiled in a water bath for 5 min. Then, the samples were centrifuged at 10000 g and the supernatant was withdrawn and loaded in the SDS-PAGE gel and run as described in standard molecular biology protocols[44]. The gel was stained with Coomassie and imaged with a Gel Doc EZ Gel documentation system (BIORAD). Confocal laser scanning microscopy. Temporal evolution of the spatial organization of immobilized His-GFP, His-mCherry, and His-BsADH and lipases labeled with RhB and FITC were followed using confocal laser scanning microscopy with a ZEISS LSM 880 (Carl, Zeiss, 25 Germany). Confocal imaging was done using a 20x (0.8 NA) or 40x (oil, 1.2 NA) immersion objectives and excitation lasers of λex: 488 nm for His-GFP and CALB-FITC and λex: 561 nm for mCherry, and His-BsADH-RhB and TLL-RhB. Image processing of confocal images was done using FIJI[48]. Fluorescence recovery after photobleaching measurements (FRAP). FRAP measurements were performed with a ZEISS LSM 880 (Carl, Zeiss, Germany) equipped with an argon laser (488 nm and 561 nm lasers were used for the excitation of His-GFP and His-mCherry, respectively). FRAP measurements and analysis to extract His-GFP and His-mCherry fluorescence recovery curves were performed according to Axelrod et al.[49], Soumpasis et al.[50] and Lopez et al.[51]. The diameter of the bleached spot (round shaped) was 10 μm, and it was photobleached with an Argon laser (100% laser intensity) for 5 sec while at the same time a non-bleached spot of the same size was considered as a reference for further corrections of focus drift, bleaching and/or loss of intensity during image acquisition. 𝑞2 = ( 𝑝 𝐷𝑓) (1 + 𝑘𝑜𝑛 𝑝+ 𝑘𝑜𝑓𝑓), 4. Experimental Section To derive binding affinity coefficients of the His-tag fluorescent proteins toward the AG-Co2+ supports, we fitted the FRAP curves, with the full reaction-diffusion model represented in the Laplace space as described by Sprague et al.[31] (E ti (1)) (Equation (1)). 𝑓𝑟𝑎𝑝(𝑝) = 1 𝑝− 𝐹𝑒𝑞 𝑝 (1 −2𝐾1(𝑞𝑤)𝐼1(𝑞𝑤)) ∗ (1 + 𝑘𝑜𝑛 𝑝+ 𝑘𝑜𝑓𝑓) − 𝐶𝑒𝑞 𝑝+ 𝑘𝑜𝑓𝑓, (1) (1) 𝐹𝑒𝑞= 𝑘𝑜𝑓𝑓 𝑘𝑜𝑛+ 𝑘𝑜𝑓𝑓, (2) (2) (3) (4) 26 where 𝑓𝑟𝑎𝑝 ̅̅̅̅̅̅̅ is the mean relative fluorescence intensity in the bleach spot, 𝑝 is the Laplace variable that inverts to yield time, 𝐾1 and 𝐼1 are the first-order-modified Bessel functions of the first and second kind, respectively, 𝑤 is the radius of the bleach spot, 𝑘𝑜𝑛 and 𝑘𝑜𝑓𝑓 are the rate constants that represent the rate of the protein binding and dissociation from the AG-Co2+ surface, respectively. Feq (Equation 2) and Ceq (Equation 3) represent the unbound and bound protein concentrations at the equilibrium, respectively. 𝐷𝑓 (Equation 4) is the diffusion coefficient of the protein in the absence of binding. where 𝑓𝑟𝑎𝑝 ̅̅̅̅̅̅̅ is the mean relative fluorescence intensity in the bleach spot, 𝑝 is the Laplace variable that inverts to yield time, 𝐾1 and 𝐼1 are the first-order-modified Bessel functions of the first and second kind, respectively, 𝑤 is the radius of the bleach spot, 𝑘𝑜𝑛 and 𝑘𝑜𝑓𝑓 are the rate constants that represent the rate of the protein binding and dissociation from the AG-Co2+ surface, respectively. Feq (Equation 2) and Ceq (Equation 3) represent the unbound and bound protein concentrations at the equilibrium, respectively. 𝐷𝑓 (Equation 4) is the diffusion coefficient of the protein in the absence of binding. Protein migration studies through image processing and analysis. To evaluate the migration of proteins and enzymes inside the porous supports, where they are immobilized, as a result of temporal evolution, we acquired CLSM images at different time points, conditions of storage, and types of immobilizations, as shown in the main manuscript. Subsequently, the obtained micrographs were analyzed using FIJI software and the corresponding plugin for radial profile generation (developed by Paul Baggethun) to determine fluorescence profiles along the radius of single beads of similar size that ranged from 75 – 150 μm (n = 10, Figure 1). 4. Experimental Section In this work, we have developed and implemented an algorithm that is used to determine fluorescent molecules migration by calculating the relative infiltration as shown in Figure 1 and described in corresponding text in the main manuscript. Time-lapse fluorescence microscopy for real-time activity measurements at the single-particle level. The activity of His-BsADH immobilized on AG-Co2+, was studied at different time points of storage conditions under Cytation5 Cell Imaging Reader (BioTek Instruments) on a black clear bottom 96 well microplate. The sample was monitored with a Plan Fluorite 4X phase objective (0.13 N.A.) and coupled to an apotome grid WD (17 mm working distance). DAPI and RFP imaging filters were used for fluorescent imaging for monitoring the fluorescent cofactor NADH and RhB labeled His-BsADH, respectively. Brightfield channel was also 27 27 monitored to detect changes in the position of the microbeads during the experiment, avoiding any artifact that may interfere with the subsequent image analysis. Single-particle time-lapse experiments were performed as described elsewhere[13]. Briefly, a suspension of 1:400 (w:v) of His-BsADH@AG-Co2+ in 10 mM Tris-HCl at pH 7 was placed into the well under microscopic analysis. Then, NADH was added into the well at a concentration of 1 mM in 10 mM Tris-HCl at pH 7 and subsequently, redox reaction was triggered with 65 mM acetone in 10 mM Tris- HCl at pH 7 to measure the activity of His-BsADH. The fluorescence decay of the reaction was monitored every 8 sec through NADH time-lapse imaging in the DAPI channel until no fluorescence of NADH was detected. Further Image processing and analysis were performed with a FIJI plugin recently developed in our group[52], through which we quantified the relative fluorescence units (RFUs) of a selected circular area of a diameter of 100 μm in the space between the microbeads (bulk) at each time point from the acquired set of images. Likewise, we recorded the fluorescence corresponding to the RhB labeled His-BsADH of ROIs of more than 35 microbeads before starting the time-lapse experiment, to determine the enzyme concentration on one single bead. Obtained fluorescence intensity values were then divided by the volume of the corresponding ROI to calculate the integrated volumetric fluorescence of each cofactor at each time point and of each enzyme at time 0. Supporting Information Supporting Information Supporting Information is available from the Wiley Online Library or from the author. pp g Supporting Information is available from the Wiley Online Library or from the author. 4. Experimental Section Fluorescence standard curves were derived for fluorescently labeled His-BsADH and NADH cofactor using known concentrations of both and measured under the same conditions with the time-lapse experiments. Obtained calibration curves were used to quantify enzyme and cofactor concentration (μM) inside the particles before the reaction and during all reaction time points to finally derive the single-particle reaction courses. Time-courses were then fitted using originLab[53] to derive enzyme apparent kinetic parameters toward the confined phosphorylated cofactor from the single-particle analysis. The initial rate (Vo) of each reaction time-course was calculated from the slope obtained from the linear fitting of the experimental data points at the 28 28 Statistical analysis. The experimental methods where we carried out single-particle measurements to derive protein infiltration distance and enzyme activity involved statistical analysis of the corresponding data. Prior to statistical analysis our studies included fluorescence image acquisition of agarose beads where the fluorescent proteins or the fluorescently labelled enzymes were immobilized. Then, for the single-particle analysis, we performed image analysis to select the regions of interest (ROIs) which in our case where the agarose beads of 50 – 150 μm diameter. In the case of the calculation of the infiltration distance we analyzed at least 10 ROIs (n = 10) of similar size (diameter 75 – 100 μm) for each incubation time point. We have chosen beads of similar size as the relative infiltration in average is greater for smaller beads (50 – 75 μm) than for bigger beads (100 – 150 μm). Error bars in bar plots of relative infiltration data represent the standard deviation within a pool of relative infiltration data obtained for 10 beads as derived from the standard deviation function under Excel statistical functions. For FRAP measurements we bleached a ROI of 10 μm for at least 5 beads and we obtained mean recovery of fluorescence plots where we performed subsequent fitting to derive binding dynamics data. In the case of His-BsADH single-particle activity measurements we derived the enzyme activity data by performing analysis as described in the corresponding experimental and the error bars in bar plots of the corresponding data represent the standard deviation within a pool of activity data obtained for 10 different circular regions of interest of a diameter of 100 μm and are derived from the standard deviation function under Excel statistical functions. Acknowledgments This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Grant agreement No.818089)”. FLG acknowledges the funding of IKERBASQUE and the Spanish State Research Agency (AIE) (RTI2018-094398-B-I00). RFL thank the Ministerio de Ciencia e innovacion for the financial support ((project number CTQ2017-86170-R). This work was 29 supported by the Spanish Ministry of Science and Innovation under the Maria de Maeztu Units of Excellence Programme (MDM‐2017‐0720). supported by the Spanish Ministry of Science and Innovation under the Maria de Maeztu Units of Excellence Programme (MDM‐2017‐0720). The authors declare no conflict of interest Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff)) References [1] A. Ananth, M. Genua, N. Aissaoui, L. Díaz, N. B. Eisele, S. Frey, C. Dekker, R. P. Richter, D. Görlich, Small 2018, 14, 1. [2] J. M. Paloni, B. D. Olsen, ACS Appl. Polym. Mater. 2020, 2, 4481. [3] L. Betancor, F. López-Gallego, A. Hidalgo, N. Alonso-Morales, G. D.-O. C. Mateo, R. Fernández-Lafuente, J. M. Guisán, Enzyme Microb. Technol. 2006, 39, 877. [4] M. Romero-Fernández, F. Paradisi, Curr. Opin. Chem. Biol. 2020, 55, 1. [5] L. Berrade, A. E. Garcia, J. A. Camarero, Pharm. 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https://openalex.org/W3213872555	https://www.frontiersin.org/articles/10.3389/fcvm.2021.751910/pdf	English	null	Ideal Cardiovascular Health Metrics Modify the Association Between Exposure to Chinese Famine in Fetal and Cardiovascular Disease: A Prospective Cohort Study	Frontiers in cardiovascular medicine	2,021	cc-by	8,263	Xiong Ding 1†, Jinfeng Li 2†, Ying Wu 1, Peng Yang 3, Dandan Zhao 1, Xiaojie Yuan 4, Shuohua Chen 2, Xiaoyan Luo 5, Yun Li 1* and Shouling Wu 2* Alika Maunakea, University of Hawaii, United States Reviewed by: Zhenghe Wang, Southern Medical University, China Yuying Wang, Shanghai Ninth People’s Hospital, China Correspondence: Yun Li liyun8022@163.com Shouling Wu drwusl@163.com †These authors have contributed equally to this work and share ﬁrst authorship Reviewed by: Zhenghe Wang, Southern Medical University, China Yuying Wang, Shanghai Ninth People’s Hospital, China Reviewed by: Zhenghe Wang, Southern Medical University, China Yuying Wang, Shanghai Ninth People’s Hospital, China Reviewed by: Zhenghe Wang, Southern Medical University, China Yuying Wang, Shanghai Ninth People’s Hospital, China 1 School of Public Health, North China University of Science and Technology, Tangshan, China, 2 Department of Cardiology, Kailuan General Hospital, Tangshan, China, 3 Department of Neurosurgery, Afﬁliated Hospital of North China University of Science and Technology, Tangshan, China, 4 Department of Epidemiology and Biostatistics, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China, 5 Department of Emergency, Kailuan General Hospital, Tangshan, China Correspondence: Yun Li liyun8022@163.com Shouling Wu drwusl@163.com Background: No study has explored the modiﬁcation effect of ideal cardiovascular health metrics (ICVHMs) on the association between famine exposure and risk of cardiovascular disease (CVD) so far. We aim to examine the effect of ICVHMs on the association between exposure to famine early in life and the risk of CVD in adulthood. †These authors have contributed equally to this work and share ﬁrst authorship Methods: A total of 61,527 participants free of CVD were included in this study from the Kailuan Study. All participants were divided into three groups, included nonexposed, fetal-exposed, and childhood-exposed groups. Cox regression was used to estimate the effect of famine exposure and ICVHMs on CVD risk. Specialty section: This article was submitted to Cardiovascular Epidemiology and Prevention, a section of the journal Frontiers in Cardiovascular Medicine Received: 02 August 2021 Accepted: 27 September 2021 Published: 04 November 2021 Results: After a median of 13.0 (12.7–13.2) years follow-up, 4,814 incident CVD cases were identiﬁed. Compared with nonexposed participants, the CVD risk increased in participants with fetal famine exposure (hazard ratio [HR]: 1.21; 95% CI: 1.07– 1.37), but not in childhood famine-exposed participants. After stratifying by the number of ICVHMs, the increased CVD risk associated with fetal famine exposure was only observed in participants with less ICVHMs (≤2) (HR: 1.30; 95% CI: 1.11–1.52, P for interaction=0.008), but disappeared in those with three or more ICVHMs. ORIGINAL RESEARCH published: 04 November 2021 doi: 10.3389/fcvm.2021.751910 Xiong Ding 1†, Jinfeng Li 2†, Ying Wu 1, Peng Yang 3, Dandan Zhao 1, Xiaojie Yuan 4, Shuohua Chen 2, Xiaoyan Luo 5, Yun Li 1* and Shouling Wu 2* The modiﬁed effect of ICVHMs was sex speciﬁc (P for sex interaction = 0.031). Received: 02 August 2021 Accepted: 27 September 2021 Published: 04 November 2021 Keywords: fetal, ideal cardiovascular health metrics, China famine, cardiovascular disease, cohort study Edited by: Alika Maunakea, University of Hawaii, United States Edited by: Alika Maunakea, University of Hawaii, United States Xiong Ding 1†, Jinfeng Li 2†, Ying Wu 1, Peng Yang 3, Dandan Zhao 1, Xiaojie Yuan 4, Shuohua Chen 2, Xiaoyan Luo 5, Yun Li 1* and Shouling Wu 2* Citation: Ding X, Li J, Wu Y, Yang P, Zhao D, Yuan X, Chen S, Luo X, Li Y and Wu S (2021) Ideal Cardiovascular Health Metrics Modify the Association Between Exposure to Chinese Famine in Fetal and Cardiovascular Disease: A Prospective Cohort Study. Front. Cardiovasc. Med. 8:751910. doi: 10.3389/fcvm.2021.751910 Conclusions: Exposing to famine in the fetal period could increase the risk of CVD in late life; however, ICVHMs might modify the effect of famine exposure on CVD risk, especially in men. Conclusions: Exposing to famine in the fetal period could increase the risk of CVD in late life; however, ICVHMs might modify the effect of famine exposure on CVD risk, especially in men. November 2021 \| Volume 8 \| Article 751910 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 1 ICVHMs Modify Famine and CVD Ding et al. Famine Exposure and Severity p y Because the Chinese Famine occurred from 1959 to 1962, consistent with previous Chinese famine studies (4, 12, 16, 17), birth year was taken as the proxy variable of exposure to famine in this study. We deﬁned those born between January 1, 1959, and December 31, 1962, as fetal exposure group, those born between January 1, 1949, and December 31, 1958, as childhood-exposed group, and those born between January 1, 1963, and December 31, 1974, as a nonexposed group. As a large enterprise group, the employees in the Kailuan group were nationwide. Therefore, the severity of the famine was determined according to the excess death rate for each province, which was calculated as this rate change from the average level in 1956–1958 to the highest value in 1959–1962 (7). Based on the information, an excess mortality rate of 50% was used as a threshold value to deﬁne severely and less severely aﬀected areas in the present study. Data Collection We collected information on sociodemographic characteristics (e.g., age, gender, birth date, and education level), lifestyle factors (e.g., smoking, alcohol intake, salt intake, and physical activity), medical history (e.g., CVD, hypertension, diabetes mellitus, family history of CVD), and active treatments such as hypoglycemic, antihypertensive, and lipid-lowering medications through the self-reported questionnaire in the Kailuan Study since the baseline survey, as detailed previously (13). Education was classiﬁed as less than high school and high school or above. Drinking status was stratiﬁed into two levels: current or never/former. Weight and height were measured, and BMI was calculated as weight (kg)/height (m)2. Moreover, BP was measured on the left arm using an appropriate cuﬀsize after the participant had a rest in a chair for at least 5 min. The average values of at least two readings each of systolic and diastolic BPs were used for further analysis. Ideal cardiovascular health metrics (ICVHMs) were ﬁrst proposed in 2010 by the American Heart Association (8), which consisted of seven items, four behavioral metrics (nonsmoking, ideal body mass index [BMI], physical activity at goal levels, and a dietary pattern recommended) and three biological metrics (ideal levels of untreated blood pressure [BP], fasting blood glucose [FBG], and total cholesterol [TC]). Accumulating epidemiological evidence suggested that higher ICVHMs were associated with a lower risk of CVD (9), and all-cause death (10, 11). In 2020, Lu et al. (12) reported that ICVHMs might modify the association between famine exposure in early life and the risk of diabetes mellitus. However, to the best of our knowledge, there have been no studies to explore whether ICVHMs in later life modify the association between famine exposure and risks of CVD in adulthood. We conducted this prospective study in the Kailuan Study, an ongoing cohort enrolled approximately 100,000 participants beginning in 2006, with aims to examine the association between exposure to the Great Chinese Famine in early life and risk of CVD in adults and the modiﬁcation eﬀect of ICVHMs. The blood sample of each participant was collected on the morning of the survey after at least a 12-h fast. All samples were measured by a Hitachi 747 autoanalyzer at the central laboratory of the Kailuan General Hospital. The estimated glomerular ﬁltration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration creatinine equation (15). INTRODUCTION current study, data analysis was performed from baseline survey to December 31, 2019. current study, data analysis was performed from baseline survey to December 31, 2019. Cardiovascular disease (CVD), the most common noncommunicable disease and the leading cause of mortality globally, is an important contributor to the disease burden (1). Reducing the incidence of CVD will be helpful in lowering the burden of this disease and promoting health. Previous studies have demonstrated an association between famine exposure in early life and health eﬀects in adulthood, such as hypertension (2), diabetes mellitus (3), stroke (4, 5), myocardial infarction (MI) (4), and metabolic syndrome (4, 6). The Great Chinese Famine of 1959–1962 was one of the largest famines in human history (7), swiped almost all of the mainland of China. Individuals who were born around the period experienced various severity of famine in early life and were the high-risk populations of CVD. Modifying or reversing the eﬀect of famine exposure on CVD risk might help to reduce the incidence of CVD in these high-risk populations. In the present study, according to previous Chinese famine studies (4, 12), 65,105 participants born between January 1, 1949, and December 31, 1974, were recruited from the Kailuan Study. Participants (n = 1,060) who had a history of MI or stroke at the baseline survey were excluded. Moreover, we excluded participants (n = 2,518) who had incomplete data on health factors or behaviors, leaving 61,527 participants (47,549 men and 13,978 women) available for analyses (Supplementary Figure S1). Study Design and Population Study Design and Population The Kailuan Study (trial registration number ChiCTR-TNC- 11001489) is an ongoing longitudinal prospective study conducted in the Kailuan community in Tangshan, Republic of China. A total of 101,510 employees (including the retired, 81,110 men and 20,400 women, mean age = 51.9 y in 2006) of the Kailuan Group were invited and agreed to participate in the Kailuan Study between June 2006 and October 2007 (referred to as the “baseline survey”). Detailed study design and procedures have been described in previous studies (13, 14). All participants completed questionnaire assessments, clinical examinations, and laboratory tests in 11 hospitals responsible for the health care of the Kailuan general hospital. Participants then took physical examination every 2 years, and the incidences of chronic diseases (e.g., stroke, MI, cancer, et al.) were recorded annually. In the Frontiers in Cardiovascular Medicine \| www.frontiersin.org Ascertainment of ICVHMs Metrics Ascertainment of ICVHMs Metrics ICVHMs were adapted from the recommendations of American Heart Association (8): nonsmoking, BMI < 24 kg/m2, physical activity at goal (at least 80 min per week of moderate-intensity Frontiers in Cardiovascular Medicine \| www.frontiersin.org November 2021 \| Volume 8 \| Article 751910 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 2 ICVHMs Modify Famine and CVD Ding et al. physical activity), ideal diet (daily salt intake < 6 g) (18); TC< 5.2 mmol/L (untreated), BP < 120/80 mmHg (untreated), FBG< 5.6 mmol/L (untreated). Cardiovascular health status was then categorized as high (5–7 metrics), moderate (3–4 metrics), or low (0–2 metrics) levels (19). physical activity), ideal diet (daily salt intake < 6 g) (18); TC< 5.2 mmol/L (untreated), BP < 120/80 mmHg (untreated), FBG< 5.6 mmol/L (untreated). Cardiovascular health status was then categorized as high (5–7 metrics), moderate (3–4 metrics), or low (0–2 metrics) levels (19). (yes/no for each), and individual ICVHMs. Cochran-Armitage trend test was used to investigate the association between the increasing number of ICVHMs and the cumulative incidence of CVD. Missing covariates (drinking, eGFR, Hs-CRP, TG) will be imputed by means of multiple imputations using the Fully Conditional Speciﬁcation method computing 10 imputed datasets and to prevent case-wise deletion of missing data; 60,874 (98.9%) participants had complete data for all covariates. Ascertainment of Incident CVD Events The main outcome of this study was the ﬁrst occurrence of CVD events, including MI, severe coronary artery disease, intracerebral hemorrhagic stroke, and ischemic stroke. CVD events were deﬁned as described (20, 21). Brieﬂy, the Municipal Social Insurance Institution database and Hospital Discharge Register were linked to identify the incidence of CVD based on The International Statistical Classiﬁcation of Diseases and Related Health Problems 10th Revision (ICD-10) (I61 for intracerebral hemorrhagic stroke, I63 for ischemic stroke, and I21 for MI, I25.1 for coronary heart disease). These two databases covered all the Kailuan Study participants. For all suspected CVD cases, a panel of three experienced physicians, consisting of neurologists, cardiologists, and radiologists, reviewed the medical records, blind to exposure status. We deﬁned coronary artery disease and MI cases according to the Multinational Monitoring of Trends and Determinants in CVD criteria of WHO on basis of clinical symptoms and dynamic changes in cardiac enzymes and/or biomarker concentrations and electrocardiogram results. Severe coronary artery disease was deﬁned as coronary heart disease treated with coronary bypass surgery or stent placement. Incident stroke was diagnosed according to the WHO criteria, based on symptoms, neuroimages (from CT or MRI), and other diagnostic reports. Mortality data were collected from provincial vital statistics oﬃces, as described previously (14). To explore the modifying eﬀect of ICVHMs, we evaluated the association between famine exposure and CVD by strata of the individual ICVHMs items and the number of ICVHMs. Other ICVHMs items were adjusted when evaluating individual ICVHMs items. P-value was corrected for multiple testing via false discovery rate using the Benjamini-Hochberg method. To demonstrate potential interactions of famine exposure and ICVHMs on the development of CVD, we generated interaction terms using the cross products of famine exposure and ICVHMs in Cox regression models. To address speciﬁc questions on age diﬀerences between famine and postfamine births, an “age- balanced” method was used, in which both prefamine and postfamine births were combined as unexposed control subjects, as detailed previously (12, 22). We further conducted several sensitivity analyses. To assess the impact of imputing missing covariates, we repeated the main analyses in those without imputation. To test the robustness of our ﬁnding, we repeated the main analyses excluding those with antihypertensive, hypoglycemic, or lipid-lowering medications treatment, excluding those lost to follow-up, and excluding those who had CVD events or death within the ﬁrst 2 years of follow-up. Statistical Analyses All analyses were performed using SAS, version 9.4 (SAS Institute, Inc, Cary, NC). Two-sided values of P < 0.05 were regarded as signiﬁcant. Participant Characteristics A total of 61,527 participants (mean age 47.9 ± 6.6 y) were recruited in the present analyses, including 47,549 (77.3%) men and 13,978 (22.7%) women. There were 13.9% (n = 8,572), who were exposed to the Great Chinese Famine in the fetal stage. Compared with the nonexposed group (born between January 1, 1963, and December 31, 1974), participants with fetal famine exposure were more likely to be with a family history of CVD, or use antihypertensives or hypoglycemics, had higher BMI, TC, TG, systolic or diastolic BPs, FBG level, Hs-CRP level, had a greater proportion of individuals with ideal physical activity, as well as lower eGFR, education level, less proportion of individuals with other six individual ideal items, and less drinkers (Table 1). Supplementary Table S1 showed baseline information for the age-balanced group with combining of prefamine and postfamine births as the reference group. The baseline characteristics of the study population were described by famine exposure status. Continuous variables with normal distribution were expressed as means ± SDs and compared using one-way ANOVA analysis, while those with skewed distribution were expressed as medians and interquartile range and compared by Kruskal-Wallis test. Categorical variables were shown in proportions and compared by Pearson’s Chi- Square test. We computed the person-year of follow-up for each participant from the date of the baseline survey to the date of the CVD onset, death, loss to follow-up (n=2,156, 3.5%), or the end of follow-up (December 31, 2019), whichever came ﬁrst. The Cox regression model was used to predict the risk of CVD and to estimate the hazard ratio (HR) and 95% CI, after adjustments for age, sex, education attainment (less than high school, high school, or above), drinking (current, never/former), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 mL/min/1.73 m2), and high-sensitivity C-reactive protein (Hs-CRP) (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), triglycerides (TG) (<1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, using of antihypertensive, hypoglycemic, lipid-lowering medications Frontiers in Cardiovascular Medicine \| www.frontiersin.org Association Between Famine Exposure and CVD Furthermore, in the sex stratiﬁed analysis (Table 2) and age-balanced analysis (Supplementary Table S3), the results were similar. Association Between Famine Exposure and CVD Association Between Famine Exposure and CVD During a median of 13.0 (12.7–13.2) years follow-up, 4,814 incident CVD events were identiﬁed. Age of CVD onset was 49.5±4.3, 54.2±3.8, 60.8±4.4 for the nonexposed, fetal-exposed, or childhood-exposed group, respectively (Supplementary Table S2). As shown in Table 2, both the During a median of 13.0 (12.7–13.2) years follow-up, 4,814 incident CVD events were identiﬁed. Age of CVD onset was 49.5±4.3, 54.2±3.8, 60.8±4.4 for the nonexposed, fetal-exposed, or childhood-exposed group, respectively (Supplementary Table S2). As shown in Table 2, both the November 2021 \| Volume 8 \| Article 751910 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 3 ICVHMs Modify Famine and CVD Ding et al. TABLE 1 \| Baseline characteristics of participants according to famine exposure in early life. Nonexposed Famine exposure P value Fetal Childhood Number of participants 18,761 8,572 34,194 Age at baseline, years 39.6 ± 3.4 46.1 ± 1.3 52.8 ± 2.8 <0.001 Men 13,844 (73.8) 6,271 (73.2) 27,434 (80.2) <0.001 BMI, kg/m2 25.0 ± 3.5 25.1 ± 3.4 25.2 ± 3.3 <0.001 High school education or above 5,142 (27.4) 1,517 (17.7) 3,999 (11.7) <0.001 Current drinking 8,388 (44.7) 3,508 (40.9) 13,652 (39.9) <0.001 TC, mmol/L 4.9 ± 1.1 5.0 ± 1.1 5.0 ± 1.1 <0.001 TG, mmol/L 1.3 (0.9, 2.0) 1.3 (0.9,2.0) 1.2 (0.9, 2.0) <0.001 SBP, mmHg 123.3 ± 17.7 127.4 ± 19.1 131.9 ± 20.4 <0.001 DBP, mmHg 81.8 ± 11.8 83.7 ± 12.0 84.9 ± 11.9 <0.001 FBG, mmol/L 5.3 ± 1.3 5.5 ± 1.7 5.6 ± 1.7 <0.001 Hs-CRP, mg/L 0.6 (0.2, 1.5) 0.6 (0.2,1.6) 0.8 (0.3, 2.1) <0.001 eGFR, ml/min/1.73m2 87.9 (73.7, 105.4) 83.4 (70.3,99.2) 80.7 (68.3–95.5) <0.001 Family history of CVD 1,288 (6.9) 603 (7.0) 2,325 (6.8) 0.741 Severity famine exposed 1,156 (6.2) 665 (7.8) 2,320 (6.8) <0.001 Use of antihypertensive agent 779 (4.2) 583 (6.8) 3,723 (10.9) <0.001 Use of hypoglycemic medications 121 (0.6) 118 (1.4) 766 (2.2) <0.001 Use of lipid-lowering medications 82 (0.4) 35 (0.4) 274 (0.8) <0.001 Ideal BP 5,486 (29.2) 1,868 (21.8) 5,709 (16.7) <0.001 Ideal FBG 13,966 (74.4) 5,947 (69.4) 22,539 (65.9) <0.001 Ideal TC 12,254 (65.3) 5,006 (58.4) 19,324 (56.5) <0.001 Ideal BMI 7,711 (41,1) 3,364 (39.2) 12,643 (37.0) <0.001 Ideal smoking 11,333 (60,4) 5,129 (59.8) 20,107 (58.8) 0.001 Ideal salt intake 1,705 (9.1) 701 (8.2) 2,963 (8.7) 0.039 Ideal physical activity 1,145 (6.1) 625 (7.3) 4,859 (14.2) <0.001 No. Association Between Famine Exposure and CVD of ICVHMs <0.001 ≤2 7,312 (39.0) 3,968 (46.3) 16,403 (48.0) 3–4 9,481 (50.5) 4,059 (47.4) 15,926 (46.6) ≥5 1,968 (10.5) 545 (6.3) 1,865 (5.4) BMI, body mass index; TC, total cholesterol; TG, triglycerides; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; Hs-CRP, high- sensitivity C-reactive protein; eGFR, estimated glomerular ﬁltration rate; BP, blood pressure; ICVHMs, ideal cardiovascular health metrics. TABLE 2 \| HR (95% CI) for incident CVD according to famine exposure in early life. Nonexposed Famine exposure Fetal Childhood Total Case subjects/total number 737/18,761 632/8,572 3,445/34,194 IR, 1000 person-years 3.10 (2.89–3.34) 5.95 (5.51–6.44) 8.31 (8.04–8.59) Univariate model 1.00 (Reference) 1.93 (1.73–2.14) 2.71 (2.50–2.94) Age- and sex- adjusted model 1.00 (Reference) 1.25 (1.11–1.42) 1.04 (0.88–1.21) Multivariate model 1 1.00 (Reference) 1.27 (1.12–1.43) 1.05 (0.89–1.23) Multivariate model 2 1.00 (Reference) 1.21 (1.07–1.37) 1.00 (0.85–1.17) Sex P for interaction=0.051 Men Case subjects/total number 670/13,844 541/6,271 3,061/27,434 IR, 1,000 person-years 3.83 (3.55–4.14) 7.00 (6.43–7.61) 9.29 (8.97–9.62) Multivariate model 1.00 (Reference) 1.16 (1.02–1.33) 0.98 (0.83–1.15) Women Case subjects/total number 67/4,917 91/2,301 384/6,760 IR, 1,000 person-years 1.07 (0.84–1.36) 3.15 (2.57–3.87) 4.52 (4.09–4.99) Multivariate model 1.00 (Reference) 1.67 (1.17–2.40) 1.21 (0.74–1.96) HR, hazard ratio; CI, conﬁdence interval; CVD, cardiovascular disease; IR, incidence rate. Multivariate model 1: Adjusted for age, sex, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (<1.0, 1.0 ≤Hs-CRP ≤ 3.0, or >3.0 mg/L), eGFR (< 30, 30 ≤eGFR < 60, or ≥60 ml/min/1.73m2), and TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L). Multivariate model 2: Included covariates in multivariate model 1 and further adjusted for the severity of famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid-lowering medications (yes/no for each), and individual ideal cardiovascular health metrics. Multivariate model in men or women: Adjusted for age, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 ml/min/1.73m2), TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid-lowering medications (yes/no for each) and individual ideal cardiovascular health metrics. adjusting for covariates, the increased CVD risk remained signiﬁcant in fetal famine-exposed participants (HR: 1.21; 95% CI: 1.07–1.37), but not in childhood famine-exposed participants (HR: 1.00; 95% CI: 0.85–1.17). DISCUSSIONS In this large community-based prospective study, we observed that fetal exposure to the Great Chinese Famine was associated with an increased risk of CVD in adults. More importantly, ICVHMs may modify the association between fetal exposure to famine and the risk of CVD in adults. The eﬀect of fetal exposure to famine on the CVD risk only occurred in participants with less ICVHMs items but not in those with more ICVHMs items. The results not only emphasized the importance of adequate nutrition in early life but also suggested a healthy lifestyle in late life. The results are of great signiﬁcance for that experienced famine or malnutrition in early life to reduce the risk of CVD. In this large community-based prospective study, we observed that fetal exposure to the Great Chinese Famine was associated with an increased risk of CVD in adults. More importantly, ICVHMs may modify the association between fetal exposure to famine and the risk of CVD in adults. The eﬀect of fetal exposure to famine on the CVD risk only occurred in participants with less ICVHMs items but not in those with more ICVHMs items. The results not only emphasized the importance of adequate nutrition in early life but also suggested a healthy lifestyle in late life. The results are of great signiﬁcance for that experienced famine or malnutrition in early life to reduce the risk of CVD. The sex diﬀerence was further explored in stratiﬁed analysis. Due to the small sample size of participants with ≥5 ICVHMs items in men or women, we combined and divided men or women into two groups according to the number of ICVHMs: participants with less ICVHMs (≤2) items or more ICVHMs (≥3) items. Similar results were observed in men, that the association between fetal famine exposure and CVD risk was signiﬁcant in men with less ICVHMs (HR: 1.26, 95% CI: 1.07– 1.49) rather than those with three or more ICVHMs (HR: 1.05, 95% CI: 0.84–1.31). However, results were inverse in women, that the association between fetal famine exposure and CVD risk was signiﬁcant in women with three or more ICVHMs (HR: 1.65, 95% CI: 1.01–2.69) rather than those with less ICVHMs (HR: 1.58, 95% CI: 0.92–2.70). The interaction between sex and famine exposure was signiﬁcant (P = 0.031) (Table 4). Previous epidemiological studies have reported conﬂicting results regarding the association between famine exposure and CVD (23–25). Modiﬁed Effect of ICVHMs on the Association Between Famine Exposure and CVD The incidence of CVD according to famine exposure and the number of ICVHMs were displayed in Figure 1. There was a reverse correlation between the number of ICVHMs and the incidence of CVD. With the increasing number of incidences of CVD for fetal famine-exposed (5.95/1000 pys) or childhood famine-exposed (8.31/1000 pys) group were greater than that for the nonexposed group (3.10/1000 pys). After November 2021 \| Volume 8 \| Article 751910 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 4 Ding et al. ICVHMs Modify Famine and CVD FIGURE 1 \| Cumulative incidence of CVD according to famine exposure and the number of ICVHMs. FIGURE 1 \| Cumulative incidence of CVD according to famine exposure and the number of ICVHMs. rate analyses showed similar results (Supplementary Table S5). In age-balanced analysis, the risk estimates did not change dramatically (Supplementary Table S6). ICVHMs, the cumulative incidence of CVD decreased gradually for participants in nonexposed or fetal-exposed groups (all P for trend<0.001). After stratifying participants by the number of ICVHMs, we found that in participants with less ICVHMs (≤2), fetal famine exposure signiﬁcantly increased the CVD risk after adjusting for all covariates (HR: 1.30; 95% CI: 1.11–1.52, P for interaction=0.008), comparing with a nonexposed group (Table 3). However, we do not observe the association in participants with three or more ICVHMs (HR: 1.14; 95% CI: 0.92–1.40, HR: 0.89; CI: 0.37–2.11). In sensitivity analyses, consistent results were observed without imputing covariates, excluding participants using antihypertensive, hypoglycemic, or lipid-lowering medications, participants lost to follow-up, and who had events or death within 2 years of follow-up (Table 3). Similar results were observed in the age-balanced analysis (Supplementary Table S4). DISCUSSIONS In a median 10.1 years follow-up of 92,284 participants, in urban areas, compared with nonexposed births, famine births had a higher risk of cerebrovascular disease (HR 1.18; 95% CI: 1.09–1.28) (23). Similarly, in the present study, our results supported the association between famine exposure in early life and CVD risk. However, two studies (24, 25) from the Dutch Famine Birth Cohort have reported that prenatal exposure to the Dutch famine of 1944–1945 was not associated with the subsequent risk of stroke or coronary artery disease in middle-aged adults. Both these two studies were limited with a relatively small sample (∼3,000 participants), which might contribute to the diﬀerent results. In addition, compared with the Dutch famine (∼6 months), the Chinese Great Famine lasted longer (∼4 years) and had greater severity, aﬀected 600 million population and led to some 30 million premature deaths (7, 26). We further analyzed the association between famine exposure and CVD risk according to individual ICVHMs items. The associations between famine exposure and CVD risk were observed in participants with nonideal ICVHMs items, except for TC (in ideal TC) and smoking items (in both ideal and nonideal) (Table 5). Multiple testing via false discovery There were studies that demonstrated the preventive eﬀect of ICVHMs on CVD risk (19, 27). However, limited researches addressed the modiﬁed eﬀect of ICVHMs on the association between famine exposure and CVD risk. In the current study, November 2021 \| Volume 8 \| Article 751910 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 5 ICVHMs Modify Famine and CVD Ding et al. TABLE 3 \| Multivariable adjusted HR (95% CI) for incident CVD according to famine exposure and combined ICVHMs. Case subjects/total number IR, 1,000 person-years Nonexposed Famine exposure P for interaction Fetal Childhood No. of ICVHMs 0.008 ≤2 2,933/27,683 8.73 (8.42–9.05) 1.00 (Reference) 1.30 (1.11–1.52) 1.12 (0.92–1.37) 3–4 1,784/29,466 4.87 (4.65–5.10) 1.00 (Reference) 1.14 (0.92–1.40) 0.93 (0.72–1.21) ≥5 97/4,378 1.75 (1.43–2.13) 1.00 (Reference) 0.89 (0.37–2.11) 0.51 (0.16–1.60) Sensitivity analyses Without imputation No. of ICVHMs 0.008 ≤2 2,933/27,683 8.73 (8.42–9.05) 1.00 (Reference) 1.30 (1.11–1.52) 1.12 (0.92–1.37) 3–4 1,784/29,466 4.87 (4.65–5.10) 1.00 (Reference) 1.14 (0.92–1.40) 0.93 (0.72–1.21) ≥5 97/4,378 1.75 (1.43–2.13) 1.00 (Reference) 0.88 (0.37–2.11) 0.51 (0.16–1.60) Excluding 5,873 participants antihypertensive, hypoglycemic, or lipid-lowering medications users No. DISCUSSIONS of ICVHMs 0.049 ≤2 2,223/23,631 7.69 (7.37–8.01) 1.00 (Reference) 1.27 (1.07–1.51) 1.11 (0.88–1.40) 3–4 1,558/27,738 4.50 (4.28–4.73) 1.00 (Reference) 1.08 (0.87–1.34) 0.84 (0.63–1.10) ≥5 92/4,285 1.69 (1.38–2.08) 1.00 (Reference) 0.89 (0.37–2.12) 0.48 (0.15–1.52) Excluding 2,156 participants lost to follow up No. of ICVHMs 0.007 ≤2 2,933/26,707 9.07 (8.75–9.41) 1.00 (Reference) 1.29 (1.10–1.50) 1.11 (0.91–1.36) 3–4 1,784/28,423 5.06 (4.83–5.30) 1.00 (Reference) 1.12 (0.91–1.38) 0.92 (0.71–1.20) ≥5 97/4,241 1.80 (1.48–2.20) 1.00 (Reference) 0.88 (0.37–2.09) 0.50 (0.16–1.57) Excluding 321 participants who had events or death within 2 years of follow-up No. of ICVHMs 0.010 ≤2 2,872/27,512 8.55 (8.24–8.87) 1.00 (Reference) 1.30 (1.11–1.52) 1.14 (0.93–1.39) 3–4 1,750/29,327 4.78 (4.56–5.01) 1.00 (Reference) 1.15 (0.94–1.42) 0.96 (0.73–1.25) ≥5 95/4,367 1.71 (1.40–2.09) 1.00 (Reference) 0.87 (0.37–2.09) 0.47 (0.15–1.50) HR, hazard ratio; CI, conﬁdence interval; CVD, cardiovascular disease; ICVHMs, ideal cardiovascular health metrics; IR, incidence rate. Adjusted for age, sex, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 ml/min/1.73m2), TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid lowering medications (yes/no for each) Adjusted for age, sex, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 ml/min/1.73m2), TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid-lowering medications (yes/no for each). TABLE 4 \| Multivariable adjusted HR (95% CI) for CVD according to sex and famine exposure. Case subjects/total number IR, 1,000 person years Nonexposed Famine exposure P for interaction* Fetal Childhood Sex (men vs. women) 0.031 Men Number of ICVHMs ≤2 2,696/24,416 9.12 (8.78–9.47) 1.00 (Reference) 1.26 (1.07–1.49) 1.14 (0.93–1.41) ≥3 1,576/23,133 5.51 (5.24–5.79) 1.00 (Reference) 1.05 (0.84–1.31) 0.81 (0.61–1.08) Women Number of ICVHMs ≤2 237/3,267 5.85 (5.15–6.64) 1.00 (Reference) 1.58 (0.92–2.70) 0.83 (0.39–1.76) ≥3 305/10,711 2.24 (2.01–2.51) 1.00 (Reference) 1.65 (1.01–2.69) 1.51 (0.79–2.86) HR, hazard ratio; CI, conﬁdence interval; CVD, cardiovascular disease; IR, incidence rate; ICVHMs, ideal cardiovascular health metrics. Adjusted for age, sex, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 ml/min/1.73m2), TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid-lowering medications (yes/no for each). HR, hazard ratio; CI, conﬁdence interval; CVD, cardiovascular disease; ICVHMs, ideal cardiovascular health metrics; IR, incidence rate. Adjusted for age, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 ml/min/1.73m2), TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid-lowering medications (yes/no for each). P for interaction was between sex and famine exposure group DISCUSSIONS Fetal exposure to famine was associated with hyperglycemia and diabetes mellitus (3) in adulthood, which could be attenuated by ≥3 ICVHMs (12), and then might reduce the CVD risk. As is well known, higher pulse wave velocity was a risk factor for CVD. The number of ICVHMs items was inversely associated with pulse wave velocity, every one-point increase corresponded to a 0.09-m/s decrease in pulse wave velocity (33), which might be one potential mechanism for our results. Finally, the ICVHMs attained in mid- to late-life could improve the cardiovascular structure and function of blood vessels in late life (34), and subsequently reduce the incidence of CVD. we examined the eﬀect of ICVHMs on the association between famine exposure in early life and the risk of CVD in a prospective study. We, interestingly, found that the association between famine exposure and CVD risk only showed in participants with less ICVHMs items (≤2), not in those with ≥3 ICVHMs items, which suggested that the increased risk of CVD owing to famine exposure in early life might be modiﬁed by ICVHMs. Individual ICVHMs items also supported the modiﬁed eﬀects. There are few studies exploring the modiﬁed eﬀect of ICVHMs on the inﬂuence of famine exposure. Meng et al. (23) examined physical activity level, one of the ICVHMs items, in 92,284 participants born between 1954 and 1964, and concluded that the increased risk of CVD aﬀected by prenatal famine exposure was only shown in participants with lower physical activity levels, but not in higher ones. Ding et al. (28) demonstrated that a healthy lifestyle, such as adequate physical activity, may partially alleviate the adverse eﬀects of increasing TC in participants with prenatal or early postnatal exposure to famine. Lu et al. (12)reported that ICVHMs might modify the association between famine exposure in early life and the risk of diabetes mellitus. Sex diﬀerences of the associations between early life famine exposure and metabolic diseases were often reported. Similarly, we also observed a sex diﬀerence results in the present study. In men, not in women, more ICVHMs items (≥3) might mitigate the eﬀect of fetal famine exposure on the CVD risk (P for interaction = 0.031). Evidence showed that male usually was more susceptible to adverse environmental conditions, and female may be more adaptable to famine (35). Therefore, healthy cardiovascular health has a greater eﬀect on male. DISCUSSIONS Adjusted for age, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 ml/min/1.73m2), TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid-lowering medications (yes/no for each). P for interaction was between sex and famine exposure group. ABLE 4 \| Multivariable adjusted HR (95% CI) for CVD according to sex and famine exposure. November 2021 \| Volume 8 \| Article 751910 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 6 ICVHMs Modify Famine and CVD Ding et al. TABLE 5 \| Multivariable adjusted HR (95% CI) for incident CVD according to famine exposure and individual ICVHMs items. Case subjects/total number IR, 1,000 person-years Nonexposed Famine exposure Fetal Childhood BP Ideal 473/13,063 2.87 (2.62–3.14) 1.00 (Reference) 1.31 (0.91–1.91) 1.01 (0.61–1.67) Nonideal 4,341/48,464 7.32 (7.10–7.54) 1.00 (Reference) 1.21 (1.06–1.37) 1.01 (0.85–1.19) FBG Ideal 2,832/42,452 5.37 (5.18–5.57) 1.00 (Reference) 1.17 (0.99–1.37) 0.98 (0.80–1.20) Nonideal 1,982/19,075 8.58 (8.21–8.97) 1.00 (Reference) 1.31 (1.08–1.60) 1.08 (0.84–1.39) TC Ideal 2,457/36,584 5.42 (5.21–5.64) 1.00 (Reference) 1.26 (1.06–1.50) 1.02 (0.82–1.28) Nonideal 2,357/24,943 7.73 (7.42–8.05) 1.00 (Reference) 1.18 (0.99–1.40) 1.01 (0.81–1.27) BMI Ideal 1,353/23,718 4.59 (4.35–4.84) 1.00 (Reference) 1.11 (0.88–1.41) 0.92 (0.68–1.25) Nonideal 3,461/37,809 7.47 (7.22–7.72) 1.00 (Reference) 1.28 (1.10–1.47) 1.06 (0.88–1.28) Smoking Ideal 2,495/36,569 5.51 (5.30–5.73) 1.00 (Reference) 1.22 (1.03–1.45) 0.94 (0.75–1.18) Nonideal 2,319/24,958 7.59 (7.29–7.91) 1.00 (Reference) 1.24 (1.04–1.48) 1.11 (0.89–1.39) Salt intake Ideal 395/5,369 5.93 (5.38–6.55) 1.00 (Reference) 1.15 (0.73–1.79) 1.11 (0.63–1.94) Nonideal 4,419/56,158 6.39 (6.21–6.58) 1.00 (Reference) 1.23 (1.08–1.40) 1.01 (0.86–1.19) Physical activity Ideal 563/6,629 6.91 (6.36–7.51) 1.00 (Reference) 1.10 (0.69–1.74) 0.95 (0.55–1.64) Nonideal 4,251/54,898 6.28 (6.10–6.47) 1.00 (Reference) 1.23 (1.08–1.40) 1.01 (0.86–1.20) HR h d i CI ﬁd i l CVD di l di ICVHM id l di l h l h i IR i id BP bl d FBG f i bl d l TABLE 5 \| Multivariable adjusted HR (95% CI) for incident CVD according to famine exposure and individual ICVHMs items. Case subjects/total number IR, 1,000 person-years Nonexposed with higher BP, the major risk factor of CVD, in later life. A healthy lifestyle such as moderate physical exercise and a low-salt diet may lower BP (31), and eventually reduced the risk of CVD (32). HR, hazard ratio; CI, conﬁdence interval; CVD, cardiovascular disease; ICVHMs, ideal cardiovascular health metrics; IR, incidence rate; BP, blood pressure; FBG, fasting blood glucose; TC, total cholesterol; BMI, body mass index. Adjusted for age, sex, education attainment (less than high school, high school, or above), drinking (current, never/former), Hs-CRP (< 1.0, 1.0 ≤Hs-CRP≤3.0, or > 3.0 mg/L), eGFR (< 30, 30 ≤eGFR< 60, or ≥60 ml/min/1.73m2), TG (< 1.7, 1.7 ≤TG< 2.3, or ≥2.3 mmol/L), severity famine exposed, family history of CVD, use of antihypertensive, hypoglycemic, and lipid-lowering medications (yes/no for each), and individual ideal cardiovascular health metrics were mutually adjusted. Frontiers in Cardiovascular Medicine \| www.frontiersin.org DISCUSSIONS In addition, due to the traditional Chinese male-sex-preference culture (36), There are several potential mechanisms by which ICVHMs modify the association between famine exposure in early life and the risk of CVD in adulthood. Stein et al. (29, 30) found that maternal exposure during pregnancy to famine was associated November 2021 \| Volume 8 \| Article 751910 7 Ding et al. ICVHMs Modify Famine and CVD ACKNOWLEDGMENTS The authors thank the investigators who made this cohort study possible. SUPPLEMENTARY MATERIAL In conclusion, we found that fetal exposure to the Great Chinese Famine signiﬁcantly increased the risk of CVD in adulthood. However, ICVHMs might modify the eﬀect of famine exposure on CVD risk. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2021.751910/full#supplementary-material DATA AVAILABILITY STATEMENT female may suﬀer from severer famine and have a lower chance to survive than male after birth. But the female survivors might be healthier, and hence be less aﬀected by the environmental conditions, included ICVHMs. Moreover, lots of studies have conﬁrmed the cardiovascular protective eﬀect of estrogen, which might play a stronger eﬀect on cardiovascular health than ICVHMs in female. All these might contribute to the less protective eﬀect of ICVHMs in female. female may suﬀer from severer famine and have a lower chance to survive than male after birth. But the female survivors might be healthier, and hence be less aﬀected by the environmental conditions, included ICVHMs. Moreover, lots of studies have conﬁrmed the cardiovascular protective eﬀect of estrogen, which might play a stronger eﬀect on cardiovascular health than ICVHMs in female. All these might contribute to the less protective eﬀect of ICVHMs in female. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors. AUTHOR CONTRIBUTIONS XD, JL, YW, PY, DZ, XY, SC, XL, YL, and SW: writing—original draft. XD, JL, YW, PY, DZ, SC, and XL: investigation. XD, YW, XY, YL, and SW: writing— review and editing. XD, JL, XY, and SW: methodology. SC, XL, YL, and SW: project administration and funding. All authors contributed to the article and approved the submitted version. FUNDING This study was supported by the Natural Science Foundation of Hebei Province (H2021209018) and Tangshan Science and Technology Innovation Team Program (20130206D). ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee of the Kailuan General Hospital. The patients/participants provided their written informed consent to participate in this study. The strengths of the current study include a prospective cohort design with a large sample size, detailed information about lifestyle factors, and long follow-up time. The diagnosis of CVD was not self-reported but linked to the Municipal Social Insurance Institution database and Hospital Discharge Register for incidence of CVD. However, there were several limitations to our study. First, misclassiﬁcation of famine exposure may exist, which might underestimate the eﬀects of famine exposure. However, using the birth date of an individual to deﬁne famine exposure was the most common method in studies on the Great Chinese Famine (12, 17). Second, the ideal diet calculated by the AHA Diet Score should be calculated on the basis of consumption of whole grains, fruits, vegetables, ﬁsh, sodium, sweets, and sugary beverages. Although no detailed dietary composition data were collected in this study, our previous work found a strong association between higher salt intake and lower healthy eating scores. So, we use daily salt intake as a surrogate indicator of diet quality (37). Third, most of the participants were men (77.3%) in the current study; therefore, the observed results may not be generalizable to other populations. However, similar associations between the exposure to famine in early life and risks of CVD have been shown in other nationwide prospective cohort studies, suggesting the broad generalizable nature of the data (4, 23). 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 26. Cai Y, Feng W. Famine, social disruption, and involuntary fetal loss: evidence from Chinese survey data. Demography. (2005) 42:301–22. doi: 10.1353/dem.2005.0010 27. Ahmed A, Pinto Pereira SM, Lennon L, Papacosta O, Whincup P, Wannamethee G. Cardiovascular health and stroke in older British Men: November 2021 \| Volume 8 \| Article 751910 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 9
https://openalex.org/W4318917046	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0278931&type=printable	English	null	Natural history of incidentally diagnosed prostate cancer after holmium laser enucleation of the prostate	PloS one	2,023	cc-by	5,235	Results Among 2630 patients, 141 (5.4%) were diagnosed with IPCa after HoLEP. Pathologic T stage and magnetic resonance imaging results were highly associated with the physician’s primary treatment decision-making for IPCa. Active surveillance (AS) was performed in 80% of patients, of whom 90% underwent follow-up without intervention, while the remaining 10% underwent deferred active treatment with a median follow-up of 46.3 months due to International Society of Urological Pathology grade group upgrading or increasing core involvement percentage. Meanwhile, 20% of patients underwent immediate active treat- ment. With a median follow-up period of 88.3 months after treatment, only one of 25 patients had biochemical recurrence. Copyright: © 2023 Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The author(s) received no specific funding for this work. Editor: Wen-Wei Sung, Chung Shan Medical University, TAIWAN Received: August 17, 2022 Accepted: November 23, 2022 Published: February 2, 2023 OPEN ACCESS Citation: Han JH, Chung DH, Cho MC, Ku JH, Jeong CW, Kwak C, et al. (2023) Natural history of incidentally diagnosed prostate cancer after holmium laser enucleation of the prostate. PLoS ONE 18(2): e0278931. https://doi.org/10.1371/ journal.pone.0278931 PLOS ONE PLOS ONE RESEARCH ARTICLE Jang Hee Han1, Dae Hyuk Chung1, Min Chul Cho2, Ja Hyeon Ku1,3, Chang Wook Jeong1,3, Cheol Kwak1,3, Jae-Seung Paick4, Seung-June OhID1,3* Jang Hee Han1, Dae Hyuk Chung1, Min Chul Cho2, Ja Hyeon Ku1,3, Chang Wook Jeong1,3, Cheol Kwak1,3, Jae-Seung Paick4, Seung-June OhID1,3* 1 Department of Urology, Seoul National University Hospital, Seoul, South Korea, 2 Department of Urology, SMG-SNU Boramae Medical Center, Seoul, South Korea, 3 Department of Urology, Seoul National University College of Medicine, Seoul, South Korea, 4 Department of Urology, Mediplex Sejong Hospital, Seoul, South Korea * sjo@snu.ac.kr * sjo@snu.ac.kr a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Objectives There is no consensus on the management plan for incidental prostate cancer (IPCa) after holmium laser enucleation of the prostate (HoLEP). This study aims to investigate the natu- ral course of this disease and suggest appropriate treatment in real clinical practice. Methods The medical records of a prospective cohort of patients with LUTS/BPH who underwent HoLEP between July 2008 and December 2020 at Seoul National University Hospital were retrospectively reviewed. Patients who underwent HoLEP for palliative purpose of prostate cancer control were excluded. The natural history of IPCa was assessed by the clinician in a descriptive manner for each treatment option. Editor: Wen-Wei Sung, Chung Shan Medical University, TAIWAN Received: August 17, 2022 Accepted: November 23, 2022 Published: February 2, 2023 Copyright: © 2023 Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1. Introduction Benign prostatic hyperplasia (BPH) affects approximately 70% of men by the seventh decade of life in the United States; among them, approximately 1% of patients ultimately undergo active intervention [1], mostly transurethral endoscopic surgery, such as transurethral resec- tion of the prostate (TURP), and holmium laser enucleation of the prostate (HoLEP). Theoret- ically, HoLEP shares the surgical principle of enucleation, which allows complete removal of adenoma tissue in the transition zone. HoLEP is currently accepted as the gold standard for all prostate sizes [2, 3]. Prior to prostatectomy in patients with BPH with prostate-specific antigen (PSA) level ele- vation, one of the critical steps is the preoperative exclusion of prostate cancer (PCa). With the widespread use of PSA screening and the development of imaging techniques, such as multi- parametric prostate magnetic resonance imaging (mpMRI) [4], the incidence of incidental PCa (IPCa) detected in HoLEP seems to have decreased [5]. However, it still occurs in approx- imately 8% of cases in the final pathology examination [5]. Moreover, in some respects, clini- cians insist that the incidence of IPCa has increased compared to the previous TURP era due to removal of the entire transition zone along the surgical capsule [5, 6]. Despite the considerable incidence rate, there are no clinical guidelines regarding the man- agement of this group of patients for several reasons. First, most studies were conducted with a small number of patients, even when mixed with both TURP and HoLEP cases [5]. Second, most studies focused on the discovery of predictive factors for IPCa rather than its long-term oncological outcome, lacking data on the real-world IPCa management practice. Therefore, this study aimed to primarily focus on the natural history of IPCa after HoLEP. It attempted to assess how clinicians make their decisions and how each decision led to long- term oncological outcomes in a descriptive manner. 2.2 Patient population The medical records of consecutive patients with LUTS/BPH who underwent HoLEP from July 2008 to December 2020 at SNUH were obtained from our prospective BPH cohort. P ti t h d t H LEP f th lli ti f PC t l l d d The medical records of consecutive patients with LUTS/BPH who underwent HoLEP from July 2008 to December 2020 at SNUH were obtained from our prospective BPH cohort. Patients who underwent HoLEP for the palliative purposes of PCa control were excluded. Patients who underwent HoLEP for the palliative purposes of PCa control were excluded. Conclusions The incidence of IPCa after HoLEP was 5.4%, and among these, approximately 20% proceeded with immediate definitive therapy and an additional 6% ultimately received Competing interests: The authors have declared that no competing interests exist. 1 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PLOS ONE Natural history of incidental prostate cancer definitive therapy within a median of 4 years of AS but showed excellent oncological outcomes. definitive therapy within a median of 4 years of AS but showed excellent oncological outcomes. 2.1 Ethics approval and informed consent This study was approved by the Institutional Review Board (IRB) of Seoul National University Hospital (SNUH) (IRB no. 2201-084-1290). The requirement for informed consent was waived owing to the retrospective nature of the study. The study was performed in accordance with applicable laws and regulations, good clinical practice, and ethical principles provided in the Declaration of Helsinki. PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 3.1 Patient characteristics A total of 2630 patients who underwent HoLEP were included in this study. Their median PSA level was 2.7 ng/mL, and the mean prostate volume (PV) was 67.5 g. Among them, 141 (5.4%) were pathologically proven to have IPCa (Table 1). In the IPCa patient group, approxi- mately 30% of patients underwent preoperative prostate biopsy, while 13% of patients under- went concurrent prostate biopsy with HoLEP. The median PSA level was 3.3 ng/mL. Most patients were classified in the International Society of Urological Pathology (ISUP) grade group (GG) 1 (85.1%), followed by GG 2 (12.8%). Moreover, there were three patients (1.4%) who showed higher than GG 3. According to the D’Amico risk stratification, approximately 79% were classified in the very low to low risk group, while the remaining 21% belonged to the intermediate to high risk group. The median follow-up period was 48.9 months (Table 2). 2.3 Collected parameters We collected and reviewed the following items: patient’s demographic and clinicopathological data, including age at surgery, accompanying comorbidities, preoperative transrectal ultraso- nography, MRI findings, PSA level, digital rectal examination findings, preoperative prostate 2 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PLOS ONE Natural history of incidental prostate cancer biopsy results, perioperative findings (operative time, resection time, resection volume, used energy, intraoperative complications, and hospital stay), pathologic findings, follow-up data with prostate biopsy, prostate MRI, PSA level, treatment information of IPCa, and oncological outcomes. biopsy results, perioperative findings (operative time, resection time, resection volume, used energy, intraoperative complications, and hospital stay), pathologic findings, follow-up data with prostate biopsy, prostate MRI, PSA level, treatment information of IPCa, and oncological outcomes. 2.4 Statistical analysis All statistical analyses were performed using SPSS version 25 software (SPSS, version 25.0.0.2, IBM Corp., Armonk, NY, USA). A P-value < 0.05 was considered statistically significant, and all statistical tests were two-sided. PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 p ( q g ) PSA, prostate-specific antigen; PIN, prostate intraepithelial neoplasia; ASAP, atypical small acinar proliferation †Data are presented as median (interquartile range) PSA, prostate-specific antigen; PIN, prostate intraepithelial neoplasia; ASAP, atypical small acinar proliferation 3.2 Comparison of clinical features between T1a and T1b Of patients with IPCa, 125 (88.7%) had T1a, and 14 (9.9%) had T1b (tumor volume percentage was not annotated in two cases). Age, PSA level, and ISUP GG were not significantly different between the two groups. In T1b, total PV tended to be smaller (p = 0.069), and PSA density Table 1. Baseline characteristics. Total (n) 2630 Age (years) 69.8±7.3 BMI (kg/cm2) 24.2±3.1 PSA (ng/ml) † 2.7 [1.5, 5.1] PSA density (ng/ml2) † 0.05 [0.03, 0.07] Total prostate volume (TPV) 67.5±33.9 Transition zone volume (TZV) 38.7±25.9 Enucleated weight (g) 24.9±25.5 Enucleated volume / TPV (%) 33.9±27.9 Enucleated volume / TZV (%) 62.5±60.5 Pathology (n,%) Benign prostatic hyperplasia 2482(94.3) Prostate cancer 141 (5.4) PIN / ASAP 2 (0.1) Transitional cell carcinoma 2 (0.1) Other cancers 3 (0.1) †Data are presented as median (interquartile range) PSA, prostate-specific antigen; PIN, prostate intraepithelial neoplasia; ASAP, atypical small acinar proliferation https://doi.org/10.1371/journal.pone.0278931.t001 Table 1. Baseline characteristics. 3 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PLOS ONE Natural history of incidental prostate cancer Table 2. Characteristics of incidental prostate cancer. Total (n) 141 Age (years) 72.1±6.6 PSA (ng/ml) † 3.3 [1.8, 5.8] PSA density (ng/ml2) † 0.05 [0.03, 0.09] Total prostate volume 65.2±35.1 Prostate biopsy prior to HoLEP 42 (29.8) Concurrent Prostate biopsy with HoLEP 18 (12.7) T stage (n, %) T1a 125 (88.7) T1b 14 (9.9) ISUP Grade Group Grade Group 1 120 (85.1) Grade Group 2 18 (12.8) Grade Group 3 3 (1.4) Risk stratification Very low to Low risk 111 (78.7) Intermediate risk 29 (20.6) High risk 1 (0.7) Median follow up period (months) 48.9 [23.5, 77.9] †Data are presented as median (interquartile range) PSA, prostate specific antigen; HoLEP, holmium laser enucleation of the prostate htt //d i /10 1371/j l 0278931 t002 tended to be higher (0.07 vs 0.05, p = 0.054) with borderline significance compared to T1a. Patients with T1b were more likely to undergo active treatment than active surveillance (AS) (S1 Table). tended to be higher (0.07 vs 0.05, p = 0.054) with borderline significance compared to T1a. Patients with T1b were more likely to undergo active treatment than active surveillance (AS) (S1 Table). 3.4 Characteristics and oncological outcome of the active treatment group Among 25 patients, radical prostatectomy was the most commonly performed treatment (n = 18, 72%), followed by definitive radiation therapy (n = 5, 25%). Pathological findings showed that 89% of tumors had multifocality, and ISUP upgrading was observed in 27.8%. Meanwhile, no residual tumor was found in one patient. With a median follow-up period of 88.3 months after treatment, biochemical recurrence (BCR) occurred in one patient (S2 Table). 3.3 Comparison of clinical characteristics between two different decision groups: AS and immediate active treatment group Of 141 patients, 18 were lost to follow-up, leaving 123 patients for analysis. Among these, 98 patients (79.7%) underwent AS, whereas 25 patients (20.3%) underwent immediate active treatment. Age, PSA level, and ISUP GG were comparable between the two groups. Clinicians tend to choose active treatment when the tumor volume exceeds 5% of the total specimen (T1b) or when a tumorous condition is identified on postoperative mpMRI. Relatively low prostate volume or high PSA density was also associated with the physician’s intent to treat actively with borderline significance (Table 3). PLOS ONE Natural history of incidental prostate cancer Table 3. Clinician’s decision toward active surveillance or immediate treatment. Number of patients (n) Active Surveillance (N = 97) Active Treatment (N = 25) p-value Age (years) 71.7±6.4 71.4±6.6 0.808 PSA (ng/ml) † 3.3 [1.9, 5.3] 3.3 [1.9, 6.8] 0.800 PSA density (ng/ml2) † 0.05 [0.03, 0.07] 0.07 [0.10, 0.14] 0.097 Total prostate volume 68.7±38.1 54.7±25.2 0.085 T stage (n, %) <0.01 T1a 90 (94.7) 18 (72.0) T1b 5 (5.3) 7 (28.0) ISUP Grade Group 0.111 Grade Group 1 84 (86.6) 19 (76.0) Grade Group 2 12 (12.4) 4 (16.0) Grade Group 3 1 (1.0) 2 (8.0) Abnormal MRI (n, %) 36 (43.4) 20 (80.0) <0.01 †Data are presented as median (interquartile range) PSA, prostate-specific antigen Table 3. Clinician’s decision toward active surveillance or immediate treatment. months. The AS discontinuation group showed a higher ISUP GG and higher proportion of abnormal mpMRI findings. Moreover, the total PV tended to be smaller in the AS discontinu- ation group (p = 0.056), while PSA or PSA ratio (post-HoLEP/pre-HoLEP) did not differ between the two groups (Table 4). The most common reason for AS discontinuation was ISUP GG upgrading following prostate biopsy (n = 5, 55.6%), followed by increasing core involve- ment percentage (n = 3, 33.3%) (Table 5). PSA, prostate-specific antigen; AS, active surveillance https://doi.org/10.1371/journal.pone.0278931.t004 3.5 Characteristics of patients who discontinued AS Among patients who chose AS as primary therapy, 88 continued AS with a median follow-up period of 41.6 months, while nine discontinued AS after a median follow-up period of 46.3 4 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PSA, prostate-specific antigen; AS, active surveillance PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 4. Discussion Previous reports have defined IPCa as a low-grade, indolent disease that does not require sub- sequent intervention [1, 6–8]. Thus, most patients were considered reliable candidates for AS Table 4. Characteristics of patients who discontinued active surveillance. Number of patients (n) AS continue (N = 88) AS discontinue (N = 9) p-value Age (years) 71.9 [68.1, 76.1] 70.9 [67.9, 75.6] 0.486 PSA (ng/ml) 3.4 [1.9, 5.6] 2.8 [2.3, 4.4] 0.816 PSA ratio (post/pre) 0.27 [0.18, 0.53] 0.34 [0.26, 0.70] 0.111 PSA density (ng/ml2) 0.05 [0.03, 0.07] 0.06 [0.03, 0.10] 0.550 Total prostate volume 62.4 [48.4, 82.0] 50.9 [40.7,54.9] 0.056 T stage (n, %) 0.399 T1a 82 (95.3) 8 (88.9) T1b 4 (4.7) 1 (11.1) ISUP Grade Group (n,%) 0.039 Grade Group 1 77 (87.5) 7 (77.8) Grade Group 2 11 (12.5) 1 (11.1) Grade Group 3 0 (0.0) 1 (11.1) Follow-up period 41.6 [26.5, 67.6] 50.3 [37.7, 93.2] 0.087 Follow-up period 46.3 [24.7, 93.6] Until AS discontinue Table 4. Characteristics of patients who discontinued active surveillance. Table 4. Characteristics of patients who discontinued active surveillance. 5 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PLOS ONE Natural history of incidental prostate cancer Table 5. Reason for AS discontinuation (N = 9). Total (n) 9 ISUP Grade group upgrading GG1 ! GG2 2 (22.2) GG1 ! GG3 1 (11.1) GG1 ! GG5 1 (11.1) GG2 ! GG4 1 (11.1) Core involvement percentage 50% 3 (33.3) Abnormal MRI and PSA velocity increased 2 (22.2) Patient wants 1 (11.1) †Data are presented as median (interquartile range) PSA, prostate-specific antigen https://doi.org/10.1371/journal.pone.0278931.t005 Table 5. Reason for AS discontinuation (N = 9). [9, 10]. However, due to the small number of patients included in each study and relative short follow-up time with absence of subsequent treatment description (treatment plan was unknown for approximately 40% of previous studies [5]), no clinical consensus could be made. In the real-world setting, there are still controversies regarding the risk of progression of IPCa, which is the most appropriate management for these patients [5]. Moreover, several additional points emphasize the necessity of this study. Since the AS cri- teria have been established [11], there has been a trend of continuous increase in AS [12–14], based on the hypothesis that low-risk PCa is indolent and cancer-specific mortality is lower than other-cause mortality. PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 4. Discussion 6 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PLOS ONE Natural history of incidental prostate cancer Furthermore, as in a previous study [20], high PSA density was also associated with active treatment decisions, while PSA level did not show any difference, probably due to active exclu- sion of suspicious patients with PCa preoperatively. Interestingly, PCa volume percentage (pathologic T stage) had a greater impact than ISUP GG on decision making. The second objective was to demonstrate the characteristics of T1a and T1b. PCa is now being more widely screened preoperatively in the real-world setting with the help of PSA screening and mpMRI, thus increasing the prostate biopsy performance. For this reason, con- tradictory to the previous study by Capitanio et al., which showed small PV with high PSA in patients with T1b compared to patients with T1a [7], our cohort showed higher PSA density in T1b with borderline significance (p = 0.054), while having comparable results for PSA level and PV. Because of the small number of patients who underwent AS in T1b, we could not compare the prognosis between T1a and T1b patients who underwent AS. The third issue was to comprehensively describe the natural history of patients who under- went AS. Of 97 patients who primarily underwent AS, approximately 90% maintained AS with a median period of 41.6 months, while 10% discontinued AS after a median period of 46.3 months. This AS continuation and discontinuation ratio was comparable to that in the HAROW study, showing 12.1% of AS discontinuation [21], and these rates are lower than those reported from most AS trials with a general low-risk PCa population [10]. This indicates that the patient may be informed preoperatively that AS may be safely recommended, and after 4 years, with regular check-up, residual cancer may show progression, such as GG upgrading or increased core involvement. However, even in these cases, cancer may be safely controlled. Furthermore, it is interesting to note that a small prostate is associated with AS dis- continuation, and it is noteworthy that it is also known as a predictor of IPCa preoperatively in many studies [5]. The fourth issue was determination of whether the immediate active treatment group was truly worthy of active treatment rather than AS. For a median period of 7.5 years, all but one patient showed no recurrence. 4. Discussion However, these criteria are based on prostate biopsy results rather than on specimens acquired from minimally invasive endoscopic surgery. We still cannot con- firm whether we can apply the same AS criteria, based on a previous study that showed that transition zone PCa has a different biology than peripheral zone cancer [13, 15, 16]. Thus, we tried to comprehensively demonstrate the natural course of IPCa using prospectively regis- tered large population-based cohorts with long-term follow-up. In this study, we focused on several issues that have been less investigated in previous stud- ies. Our study cohort had relatively low PSA and PSA density levels compared with those in previous studies, indicating that PCa was more sensitively screened before surgery. For the similar reason, PSA reduction ratio at 6 months (PSA 6months/Pre-operative PSA) after sur- gery was not significantly different between IPCa and BPH patients (IPCa 0.33 [0.22, 0.58] vs BPH 0.29 [0.16, 0.57], p = 0.091). p The first issue we demonstrate was the descriptive feature that affects the clinician’s deci- sion to perform AS or active treatment in a real-world setting. We revealed that approximately 80% of patients with IPCa underwent AS, while 20% of patients underwent active treatment. We clearly demonstrated that clinical stage T1b disease and abnormal mpMRI results were associated with immediate active treatment (Table 3). To be more specific, among 25 patients of active treatment, 20 patients (80%) showed suspicious prostate cancer lesions (16 peripheral zone, 4 transitional zone) on prostate MRI at a median of 1.2 months after HoLEP surgery. Among them, 7 patients also harbored a higher pathological tumor burden (>5%). For the other 5 patients who did not show abnormal findings on MRI, most of them showed clinically significant PCa in either HoLEP specimen or prostate biopsy performed post-operatively, which may have led to immediate active treatment. This decision was supported by previous studies showing that T1b is an independent predictor of BCR and disease-specific mortality [17, 18] and that patients with undistinguishable lesions on mpMRI should be considered for AS because they have the least risk of residual cancer development after TURP [19]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 4. Discussion Based on the pathologic diagnosis, the tumor multifocality rate (88.9%) and HGPIN accompanying rate (61.1%) were considerably high, which was a distinct feature in this study compared to the previous studies [7, 22, 23]. This supports the innate characteristics of multifocality in PCa; thus, the message from this descriptive analysis is that we should essentially rule out the co-existence of peripheral zone cancer, which may harbor more aggressive features, using regular follow-up biopsy and mpMRI. This study had several limitations. First, this was a single-center study, which may not have reflect the heterogeneous treatment policy for IPCa. However, we believe that this study still has a great advantage in offering more conclusive information using the largest prospective registered database with a long follow-up period. Second, less than half of patients diagnosed with IPCa underwent prostate biopsy or prostate MRI before HoLEP. This is partly due to the rapidly increasing role of mpMRI in PCa diagnosis recently. However, we made our best efforts to exclude PCa by applying the PCa risk calculator preoperatively [24]. As a result, the IPCa incidence rate (5.4% in our cohort) was lower than the pooled incidence of 8% reported in a recent systematic review and meta-analysis [5]. Furthermore, patients with pathologically proven BPH and IPCa did not show a difference in PSA levels preoperatively in our cohort compared to previous studies showing higher PSA levels in IPCa [25], indicating well screened patients preoperatively. Moreover, many patients were referred from urology oncologists after excluding the possibility of PCa. Third, due to the limited information, we could not consider socioeconomic status in diagnosis of IPCa, which may have affected the diagnosis and treat- ment of IPCa. And fourth, patients who underwent AS did not follow a uniform protocol. Most of the patients underwent multiparemetric prostate MRI-based or both MRI and biopsy based surveillance. However, for a certain group of low risk patients (38.1%), PSA-only 7 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0278931 February 2, 2023 PLOS ONE Natural history of incidental prostate cancer surveillance was performed after confirmation of no existence of PCa on post-HoLEP follow- up biopsy (S3 Table). This study is meaningful in that it comprehensively demonstrated the long-term observed natural course of IPCa, which was further analyzed using each subsequent treatment method in the real-world setting. S3 Table. Active surveillance protocol in this study. (DOCX) Author Contributions Conceptualization: Jang Hee Han. Data curation: Jang Hee Han, Dae Hyuk Chung, Seung-June Oh. Formal analysis: Jang Hee Han, Dae Hyuk Chung, Seung-June Oh. Investigation: Dae Hyuk Chung, Seung-June Oh. Methodology: Jang Hee Han, Dae Hyuk Chung, Min Chul Cho, Ja Hyeon Ku, Chang Wook Jeong, Cheol Kwak, Jae-Seung Paick, Seung-June Oh. Project administration: Jang Hee Han, Dae Hyuk Chung, Min Chul Cho, Ja Hyeon Ku, Chang Wook Jeong, Cheol Kwak, Jae-Seung Paick, Seung-June Oh. Resources: Jang Hee Han, Dae Hyuk Chung, Min Chul Cho, Ja Hyeon Ku, Chang Wook Jeong, Cheol Kwak, Jae-Seung Paick, Seung-June Oh. Supervision: Seung-June Oh. 4. Discussion Overall, we revealed that, compared to the previous study, a higher number of patients with IPCa had an intermediate risk (approximately 21%). Patients who underwent AS through the long follow-up period, after approximately 4 years of AS, may be definitive treatment candidates due to ISUP GG increment or core percentage increment, which may also be safely treated (S1 Fig). 5. Conclusions We comprehensively demonstrated the long-term observed natural course of IPCa, which was further analyzed using each subsequent treatment method in the real-world setting. The inci- dence of IPCa after HoLEP was 5.4%, and among these, approximately 20% proceeded with immediate definitive therapy and an additional 6% ultimately received definitive therapy within the median of 4 years of AS but showed excellent oncological outcomes. S1 Dataset. (XLSX) S1 Dataset. (XLSX) S1 Fig. Kaplan-Meier curve of time to the active treatment for incidental prostate cancer after HoLEP surgery. (TIF) S1 Table. Comparison between T1a and T1b incidental prostate cancer. (DOCX) S2 Table. Characteristics and oncological outcome of the active treatment group. (DOCX) S3 Table. Active surveillance protocol in this study. (DOCX) S1 Fig. Kaplan-Meier curve of time to the active treatment for incidental prostate cancer after HoLEP surgery. (TIF) S1 Table. Comparison between T1a and T1b incidental prostate cancer. (DOCX) S2 Table. Characteristics and oncological outcome of the active treatment group. (DOCX) References 1. Bhojani N, Boris RS, Monn MF, Mandeville JA, Lingeman JE. 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Conceptualization: Jang Hee Han. Data curation: Jang Hee Han, Dae Hyuk Chung, Seung-June Oh. Data curation: Jang Hee Han, Dae Hyuk Chung, Seung-June Oh. Data curation: Jang Hee Han, Dae Hyuk Chung, Seung-June Oh. Formal analysis: Jang Hee Han, Dae Hyuk Chung, Seung-June Oh. Formal analysis: Jang Hee Han, Dae Hyuk Chung, Seung-June Oh. Investigation: Dae Hyuk Chung, Seung-June Oh. Methodology: Jang Hee Han, Dae Hyuk Chung, Min Chul Cho, Ja Hyeon Ku, Chang Wook Jeong, Cheol Kwak, Jae-Seung Paick, Seung-June Oh. Methodology: Jang Hee Han, Dae Hyuk Chung, Min Chul Cho, Ja Hyeon Ku, Chang Wook Jeong, Cheol Kwak, Jae-Seung Paick, Seung-June Oh. Project administration: Jang Hee Han, Dae Hyuk Chung, Min Chul Cho, Ja Hyeon Ku, Chang Wook Jeong, Cheol Kwak, Jae-Seung Paick, Seung-June Oh. Resources: Jang Hee Han, Dae Hyuk Chung, Min Chul Cho, Ja Hyeon Ku, Chang Wook Jeong, Cheol Kwak, Jae-Seung Paick, Seung-June Oh. 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https://openalex.org/W1918899533	https://epub.ub.uni-muenchen.de/34339/1/10.1038_ncomms7963.pdf	English	null	Subdiffractional focusing and guiding of polaritonic rays in a natural hyperbolic material	Nature communications	2,015	cc-by	7,433	ARTICLE Received 24 Dec 2014 \| Accepted 19 Mar 2015 \| Published 22 Apr 2015 Subdiffractional focusing and guiding of polaritonic rays in a natural hyperbolic material S. Dai1, Q. Ma2, T. Andersen2, A.S. Mcleod1, Z. Fei1, M.K. Liu1,3, M. Wagner1, K. W M. Thiemens5, F. Keilmann6, P. Jarillo-Herrero2, M.M. Fogler1 & D.N. Basov1 S. Dai1, Q. Ma2, T. Andersen2, A.S. Mcleod1, Z. Fei1, M.K. Liu1,3, M. Wagner1, K. Watanabe4, T. Taniguchi4, M. Thiemens5, F. Keilmann6, P. Jarillo-Herrero2, M.M. Fogler1 & D.N. Basov1 Uniaxial materials whose axial and tangential permittivities have opposite signs are referred to as indeﬁnite or hyperbolic media. In such materials, light propagation is unusual leading to novel and often non-intuitive optical phenomena. Here we report infrared nano-imaging experiments demonstrating that crystals of hexagonal boron nitride, a natural mid-infrared hyperbolic material, can act as a ‘hyper-focusing lens’ and as a multi-mode waveguide. The lensing is manifested by subdiffractional focusing of phonon–polaritons launched by metallic disks underneath the hexagonal boron nitride crystal. The waveguiding is revealed through the modal analysis of the periodic patterns observed around such launchers and near the sample edges. Our work opens new opportunities for anisotropic layered insulators in infrared nanophotonics complementing and potentially surpassing concurrent artiﬁcial hyperbolic materials with lower losses and higher optical localization. 1 Department of Physics, University of California, San Diego, La Jolla, California 92093, USA. 2 Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02215, USA. 3 Department of Physics, Stony Brook University, Stony Brook, New York 11794, USA. 4 National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan. 5 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA. 6 Ludwig-Maximilians-Universita¨t and Center for Nanoscience, 80539 Mu¨nchen, Germany. Correspondence and requests for materials should be addressed to D.N.B. (email: dbasov@physics.ucsd.edu). 1 NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. The upper band comprises o ¼ 1,370–1,610 cm 1 where the real part of et (the in-plane permittivity) is negative while that of ez is positive. In the lower band spanning o ¼ 746–819 cm 1, the signs of the permittivity components are reversed. Thus, the out-of-plane crystal vibrations enable the type I hyperbolic response, whereas the in-plane ones accounts for the type II behaviour. The momentum-frequency dispersion surface for the hyperbolic polaritons of the upper band resembles a ‘butterﬂy’ (Fig. 1c) composed of individual hyperbolas sketched in Fig. 1a. It can be contrasted with the ﬂat dispersion surfaces of longitudinal phonons typical for isotropic materials. Effectively, in hBN the longitudinal phonons are hybridized with the transverse ones by quasi-static Coulomb interaction mediated by large-k photons38. Because the hyperbolic response in hBN originates from the anisotropic phonons, in the following, the large-k hyperbolic polaritons are referred to as hyperbolic phonon polaritons (HP2). e 1 t k2 z þ e 1 z ðk2 x þ k2 yÞ ¼ 2po ð Þ2; ð1Þ ð1Þ where ez and etex ¼ ey are the axial and tangential permittivities, respectively. The hyperboloid is single-sheeted if ez40, eto0 (type II) and two-sheeted if ezo0, et40 (type I), see Fig 1a,b, respectively. In both cases, the slope of the propagation (group velocity) direction, which is orthogonal to the dispersion surface, asymptotically approaches tan yðoÞ ¼ i ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ et o ð Þ p ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ez o ð Þ p : ð2Þ ð2Þ The condition for achieving super-resolution is Im kzd ¼ (ktd)Im tan yB1. Hence, admissible Im ez, Im et scale algebraically rather than exponentially with the resolution k 1 t . p y t Directional propagation of hyperbolic polaritons along ‘resonance cones’ of apex angle y has been observed in a −10 0 10 1,350 1,550 1,515 cm–1 v kt v θ kz Type II =1,370–1,610 cm–1 Type I =746–819 cm–1 kt kz −5 −10 0 5 10 0.5 0.4 0.3 kt (105 cm–1) (cm–1) kz (105 cm–1) / Figure 1 \| Hyperbolic dispersion of hBN. (a) A sketch of the isofrequency curves for a type II HM, which is realized in the upper stop-band of hBN. The arrow indicates the polariton group velocity. (b) A similar sketch for the type I case, which is realized in the hBN lower stop-band. (c) The calculated dispersion surface of hBN polaritons. & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 O ne of the primary goals of nanophotonics is concentra- tion of light on scales shorter than the free-space wavelength l. According to the general principles of Fourier optics, this is only possible provided electromagnetic modes of large tangential momenta kt4o/(2p), normally evanescent, are nonetheless able to reach the focal plane (the x–y plane). Here o ¼ l 1 is the measure of frequency common in spectroscopy and kt ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ k2 x þ k2 y q . In devices known as superlenses1–6, this requirement is realized via resonant tunneling between the opposite sides of the structure. However, the tunneling is very sensitive to damping, for example, the magnitude of the imaginary part of the permittivity e of the superlens material7. The largest characteristic momentum that can pass through a superlens of thickness d can be found from the relation Im e e ktd. In this regard, hyperbolic media (HM)8,9 promise a signiﬁcant advantage as they support large-k hyperbolic polaritons that remain propagating rather than evanescent, so that the condition on damping is much softer (see below). The unusual properties of hyperbolic polaritons in HM8–20 stem from the dispersion of these modes that is described by the equation of a hyperboloid: magnetized plasma21,22, which behaves as a natural HM in the microwave domain. A major resurgence of interest to HM was prompted by their discussion in the context of artiﬁcial materials (metamaterials)23,24. Examples of such hyperbolic metamaterials include microstrip arrays, where directional propagation and focusing of hyperbolic polaritons have been experimentally observed25,26. Directional optical beams have been studied in planar25–28 and curved12,29 metamaterials made of alternating layers of metals and semiconductors. The work on non-planar structures12,29 was motivated by theoretical proposals of a hyperlens30–32, a device in which directional beams outgoing from a subdiffractional source enable optical magniﬁcation. However, improvement over the diffraction limit has so far been severely impeded by losses in constituent metals and imperfections of nanofabrication. Recent work33,34 identiﬁed hexagonal boron nitride (hBN) as a low-loss natural HM in the mid-infrared domain. This layered insulator has emerged as a premier substrate or a spacer for van der Waals heterostructures35,36. Light atomic masses, strong anisotropy and the polar band between B and N yield prominent optical phonon modes that create two widely separated stop- bands—spectral intervals where one of the principal values of the dielectric tensor is negative33,34,37. & 2015 Macmillan Publishers Limited. All rights reserved. The axes are the tangential momentum (kt), the axial momentum (kz) and the frequency (o, ranging from 1,370 to 1,515 cm 1). The colour represents the propagation angle y. The constant-frequency cut o ¼ 1,515 cm 1 is shown by the red line, to emphasize similarity with a. The dispersion of polaritons in a ﬁnite- thickness crystal (d ¼ 105 nm) is shown by the black lines to clarify their relation to Fig. 4a. v kt v θ kz Type II =1,370–1,610 cm–1 Type I =746–819 cm–1 kt kz −10 0 10 1,350 1,550 1,515 cm–1 −5 −10 0 5 10 0.5 0.4 0.3 kt (105 cm–1) (cm–1) kz (105 cm–1) / NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Results Subdiffractional focusing and imaging through hBN. In our experiments, efﬁcient excitation and detection of HP2 in hBN are accomplished with the help of optical antenna structures39,40. The antennas concentrate electric ﬁeld and bridge the large momentum mismatch between the free-space photons and the HP2. In our previous work33, we used for this purpose a sharp tip of an atomic force microscope (AFM) incorporated in our scattering-type scanning near-ﬁeld optical microscopy (s-SNOM) apparatus (Methods). Here, we additionally demonstrate the antenna and polariton-launching capabilities of Au disks patterned on a SiO2 substrate. The AFM topography image in Fig. 2a depicts Au disks of diameters (top to bottom) 1,000, 500 and 200 nm and thickness of about 50 nm. After the subsequent deposition of hBN crystals of thickness d ¼ 100–1,060 nm and lateral sizes up to 10 mm, these Au disks become encapsulated between hBN and SiO2. The hBN crystal remains essentially ﬂat, as veriﬁed by AFM. Below we present experimental results demonstrating that interaction of these disks with an incident infrared beam excites polaritons that travel across hBN and produce speciﬁc contrast patterns at the other surface. We show that the observed dependence of the near-ﬁeld images on the frequency and hBN thickness is the result of directional propagation of the polaritons along conical surfaces with frequency-tunable apex angle given by Equation (2). Thus, hBN Figure 1 \| Hyperbolic dispersion of hBN. (a) A sketch of the isofrequency Figure 1 \| Hyperbolic dispersion of hBN. (a) A sketch of the isofrequency curves for a type II HM, which is realized in the upper stop-band of hBN. The arrow indicates the polariton group velocity. (b) A similar sketch for the type I case, which is realized in the hBN lower stop-band. (c) The calculated dispersion surface of hBN polaritons. The axes are the tangential momentum (kt), the axial momentum (kz) and the frequency (o, ranging from 1,370 to 1,515 cm 1). The colour represents the propagation angle y. The constant-frequency cut o ¼ 1,515 cm 1 is shown by the red line, to emphasize similarity with a. The dispersion of polaritons in a ﬁnite- thickness crystal (d ¼ 105 nm) is shown by the black lines to clarify their relation to Fig. 4a. Figure 1 \| Hyperbolic dispersion of hBN. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 Focal spots of similar size 185–210 nm were observed in all other hBN crystals, with the thickness up to 1,050 nm (Supplementary Fig. 5). m We now elaborate on the formation of images in Fig. 2 recorded with our s-SNOM apparatus with the help of a model of HP2 propagation through a slab of hBN (Fig. 3a,c,d). Consider a perfectly thin metallic disk sandwiched between a slab of a HM of thickness d and an isotropic dielectric substrate. The system is subject to a uniform electric ﬁeld of frequency o and amplitude E0 in the x direction. An approximate solution for the total ﬁeld in this system can be found analytically (Supplementary Note 1). The corresponding distributions of the z-component of the ﬁeld Ez(x, y, z) in the two cross-sections, y ¼ 0 (the vertical symmetry plane) and z ¼ d 0 (just below the top surface of the hBN slab), are illustrated in Fig. 3c. These plots are computed for three representative radii of the disk using permittivity values at o ¼ 1,515 cm 1. The plots demonstrate a series of concentric high-intensity rings on the top surface, very similar to the data in Fig. 2b. The interpretation (Fig. 3a,c) is straightforward: the external ﬁeld polarizes the disk, which perturbs the adjacent HM (hBN in our case) and launches polaritons. The HP2 emission occurs predominantly at disk edges due to the high concentration of electric ﬁeld therein. Polaritonic rays propagate across the slab, maintaining a ﬁxed angle y with respect to the z axis: the ‘resonance cone’ direction18,21,22,25–28. Upon reaching the other slab surface, they undergo a total internal reﬂection with the reﬂected cone extending toward the bottom surface. The process repeats until eventually the ﬁeld vanishes because of radial A proposal for focusing of electromagnetic radiation via resonance-cone propagation in hyperbolic media was theoreti- cally discussed in the context of magneto-plasmas21. Experimental conﬁrmation of this idea in an artiﬁcial hyperbolic multi-layer was reported where l/6 focusing was deduced from examining the pattern of a polymerized photoresist behind a two-slit polaritonic launcher26. Here, using a natural hyperbolic slab (hBN crystal), we demonstrated the l/33 focusing in both spatial directions via out-coupling of polaritons with the infrared nano-probe. We stress that a distinction should be made between ‘focusing’ and ‘imaging.’ Focusing devices can be of both imaging and non-imaging type41 and both are important in applications. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 Figure 2b depicts an s-SNOM scan taken at the top surface of hBN of thickness d ¼ 395 nm at frequency o ¼ 1,515 cm 1 (l ¼ 6.6 mm). Here we plot the third harmonics of the scattering amplitude s(o) (Methods). In this image, each Au disk is surrounded by a series of concentric ‘hot rings’ of strongly enhanced nano-infrared contrast. The diameters of all the disks are much smaller than l (see also Fig. 2a), the smallest one being 200 nm ¼ l/33. The diameters of the hot rings can be larger, smaller or equal to those of the disks. The spacing between adjacent hot rings in the same sample increases with the infrared frequency but decreases with the sample thickness. We stress that images displayed in Fig. 2b could only be detected if the infrared wavelength falls inside the hyperbolic spectral regions. Outside of the hBN stop bands, no hot rings can be identiﬁed by the s-SNOM. In fact, the entire image is homogeneous, comprised of nothing but random noise, as illustrated by Fig. 2d for o ¼ 1,740 cm 1 (l ¼ 5.7 mm). The above model of image formations via HP2 yields a number of quantitative predictions that are in accord with our observa- tions. The scenario of oblique propagation implies that upon each roundtrip across the slab, the excitation front returns to the same surface displaced radially by the distance d ¼ 2 tanyðoÞ d: ð3Þ ð3Þ Accordingly, the radii of the ‘hot rings’ at the top surface of the slab are given by Accordingly, the radii of the ‘hot rings’ at the top surface of the slab are given by rn ¼ a þ n 1 2 j d j ; n¼ 0; 1; 2; . . . ð4Þ ð4Þ where a is the disk radius. The intensity of the rings is expected to decrease with \|n\|. Consistent with this formula, the smallest rings in Fig. 3c have the radius r0 ¼ \|a \|d\|/2\|. Particularly interesting is the case where the innermost ring shrinks to a single bright spot, r0 ¼ 0. Experimentally, we observed spots of diameter 200 nm (the full width at half maximum, see Supplementary Note 1), which corresponds to l/33 for Fig. 2b (top). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 0 75 nm Min Max Figure 2 \| Sub-diffractional focusing and imaging through an hBN crystal. (a) An AFM image of Au disks deﬁned lithographically on SiO2/Si substrate before hBN transfer. (b) Near-ﬁeld amplitude image of the top surface of a 395-nm-thick hBN at infrared laser frequency o ¼ 1,515 cm 1 (l ¼ 6.6 mm). The observed ‘hot rings’ are concentric with the Au discs. (c) Near-ﬁeld image of the same sample as in b at o ¼ 1,610 cm 1 (l ¼ 6.2 mm) where polaritons propagate almost vertically. (d) Near-ﬁeld image of the same sample at o ¼ 1,740 cm 1 (l ¼ 5.7 mm) showing complete homogeneity and lack of any distinct features. The colour scales for b–d are indicated in d. The scale bars in all panels are 1 mm long. 0 75 nm Figure 2 \| Sub-diffractional focusing and imaging through an hBN crystal. (a) An AFM image of Au disks deﬁned lithographically on SiO2/Si substrate before hBN transfer. (b) Near-ﬁeld amplitude image of the top surface of a 395-nm-thick hBN at infrared laser frequency o ¼ 1,515 cm 1 (l ¼ 6.6 mm). The observed ‘hot rings’ are concentric with the Au discs. (c) Near-ﬁeld image of the same sample as in b at o ¼ 1,610 cm 1 (l ¼ 6.2 mm) where polaritons propagate almost vertically. (d) Near-ﬁeld image of the same sample at o ¼ 1,740 cm 1 (l ¼ 5.7 mm) showing complete homogeneity and lack of any distinct features. The colour scales for b–d are indicated in d. The scale bars in all panels are 1 mm long. may emerge as a new standard bearer for mid-infrared nanophotonics by enabling devices for deeply subdiffractional propagation, focusing and imaging with tunable characteristics. spreading and/or damping. The role of the s-SNOM tip in imaging experiments in Fig. 2 is to out-couple HP2 ﬁelds at the top surface (Fig. 3a). The observed s-SNOM signal is roughly proportional to the amplitude of the electric ﬁeld immediately above the slab Ez(z ¼ d ¼ 0). (Note that it is related to the ﬁeld just inside the slab by a constant factor, Ez(z ¼ d þ 0) ¼ ez(o)Ez(z ¼ d 0).) 2 p p g , g g g Representative s-SNOM imaging data are shown in Fig. 2. Results (a) A sketch of the isofrequency curves for a type II HM, which is realized in the upper stop-band of hBN. The arrow indicates the polariton group velocity. (b) A similar sketch for the type I case, which is realized in the hBN lower stop-band. (c) The calculated dispersion surface of hBN polaritons. The axes are the tangential momentum (kt), the axial momentum (kz) and the frequency (o, ranging from 1,370 to 1,515 cm 1). The colour represents the propagation angle y. The constant-frequency cut o ¼ 1,515 cm 1 is shown by the red line, to emphasize similarity with a. The dispersion of polaritons in a ﬁnite- thickness crystal (d ¼ 105 nm) is shown by the black lines to clarify their relation to Fig. 4a. NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 2 ARTICLE & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 Our hBN device (Fig. 3a) is an example of the latter. h h ﬁbl d f d l Continuing with the veriﬁable predictions of our model, we note that Equations (3) and (4) indicate that the slope tan y of the resonance cone is uniquely related to the radii of the hot rings (Fig. 3a). To test this prediction, we analysed the images collected 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 Min Max x y z E0 a d r1 hBN SiO2 E0 Data Theory 0 1 2 3 4 5 \|tan \| (cm–1) 1,400 1,500 1,600 Figure 3 \| Image formation. (a) Imaging schematics. Under infrared illumination (green arrow), the polaritons were launched by the Au disk edges and propagate towards the hBN top surface where the near-ﬁeld images were recorded via the back-scattered infrared beam (green arrow). The propagation angle y can be inferred from the hot ring radius r1, hBN thickness d and disk radius a. (b) The tangent of the propagation angle y derived from imaging data for different hBN samples (symbols) and from Equation (2) (solid line). Squares, triangles, crosses and dots indicate data from hBN samples with thickness d ¼ 395, 984, 270 and 1,060 nm, respectively. (c) The distribution of the z-component of the electric ﬁeld in the analytical model (see text). The hot rings on the surfaces appear as a result of multiple reﬂections of polaritons launched at the disk edges. The ratio a/\|d\| ¼ 0.5, 0.25, 0.15 decreases from top to bottom. In the top picture, the smallest ring shrinks to a focal point. The blue arrow indicates the direction of electric ﬁeld E0 in simulation. (d) Similar to b for a/d ¼ 1.12 and (top to bottom) \|tan y\| ¼ 0.75, 0.375 and 0.01. Min Max x y z E0 a d r1 hBN SiO2 E0 Data Theory 0 1 2 3 4 5 \|tan \| (cm–1) 1,400 1,500 1,600 Figure 3 \| Image formation. (a) Imaging schematics. Under infrared illumination (green arrow), the polaritons were launched by the Au disk edges and propagate towards the hBN top surface where the near-ﬁeld images were recorded via the back-scattered infrared beam (green arrow). The propagation angle y can be inferred from the hot ring radius r1, hBN thickness d and disk radius a. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 1,400 1,500 1,600 1,450 1,550 (cm–1) 1 l=0 2 750 775 800 l=0 (cm–1) kt (105 cm–1) 0 1 3 2 a Real-space imaging of multiple guided polaritons in hBN. The outlined real-space picture has a counterpart in its conjugate momentum space. Mathematically, the resonance cones in the real space are coherent superpositions of an inﬁnite number of polariton modes of a slab. Such modes are characterized by quantized momenta, kz,l ¼ (p/d)(l þ a), labelled by integer index l 33. Here aB1 (in general, o-dependent) quantiﬁes the phase shift acquired at the total internal reﬂection from the slab surfaces. Per Equation (1), the tangential momenta of these modes are also quantized, Figure 4 \| Polariton frequency (x) – in-plane momentum (kt) dispersion relation for hBN. (a) The dispersion curves from Fig. 1c replotted as frequency (o) versus in-plane momenta (kt). The experimental data (squares) are obtained from the polariton reﬂection images near the sample edges (Fig. 5). (b) Same as a for the lower hBN stop-band (Supplementary Fig. 3). Thickness of hBN: 105 nm. kt;lðoÞ ’ cot y o ð Þkz;lðoÞ ¼ 2p d o ð Þ ½l þ a o ð Þ: ð5Þ ð5Þ Therefore, if several guided modes are excited simultaneously by a source, their superposition would produce beats with period 2p/Dkt in real space. This is precisely the spacing d between periodic revivals of the ‘hot rings’ (Equation (3) and Fig. 2). Thus, the multi-ring images and the existence of higher-order guided modes are complementary manifestations of the same fundamental physics. In our previous work33, we reported nano- imaging and nano-spectroscopic study of the lowest-momentum guided mode l ¼ 0 in hBN crystals. Below we present new results documenting the ﬁrst observation of the higher-order (up to three) guided modes in such materials by direct nano-infrared imaging. Therefore, if several guided modes are excited simultaneously by a source, their superposition would produce beats with period 2p/Dkt in real space. This is precisely the spacing d between periodic revivals of the ‘hot rings’ (Equation (3) and Fig. 2). Thus, the multi-ring images and the existence of higher-order guided modes are complementary manifestations of the same fundamental physics. In our previous work33, we reported nano- imaging and nano-spectroscopic study of the lowest-momentum guided mode l ¼ 0 in hBN crystals. NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 (b) The tangent of the propagation angle y derived from imaging data for different hBN samples (symbols) and from Equation (2) (solid line). Squares, triangles, crosses and dots indicate data from hBN samples with thickness d ¼ 395, 984, 270 and 1,060 nm, respectively. (c) The distribution of the z-component of the electric ﬁeld in the analytical model (see text). The hot rings on the surfaces appear as a result of multiple reﬂections of polaritons launched at the disk edges. The ratio a/\|d\| ¼ 0.5, 0.25, 0.15 decreases from top to bottom. In the top picture, the smallest ring shrinks to a focal point. The blue arrow indicates the direction of electric ﬁeld E0 in simulation. (d) Similar to b for a/d ¼ 1.12 and (top to bottom) \|tan y\| ¼ 0.75, 0.375 and 0.01. from samples of different hBN thicknesses and different Au disk diameters. For each of these, we determined the radius r1 of the ﬁrst-order ring and computed \|tan y\| ¼ (r1 a)/d as a function of the infrared frequency (Fig. 3a). As shown in Fig. 3b, all the data collapse toward a single smooth curve computed from Equation (2) using optical constants of hBN from ref. 33. Yet another prediction of the model: the polaritonic rays travel along the z axis provided that et(o) and therefore y(o) are vanishingly small. This condition is satisﬁed at o ¼ 1,610 cm 1 (Fig. 2c) where we observe almost 1:1 images of Au disks. Similar behaviour was observed when instead of the disks more complicated metallic shapes were imaged (Supplementary Fig. 5). Thus, the totality of our data establishes the notion of directional propagation of HP2 in hBN over macroscopic distances with a frequency-tunable slope (Fig. 3b). a b ... 1,400 1,500 1,600 1,450 1,550 (cm–1) 1 l=0 2 750 775 800 l=0 (cm–1) kt (105 cm–1) 0 1 3 2 Figure 4 \| Polariton frequency (x) – in-plane momentum (kt) dispersion relation for hBN. (a) The dispersion curves from Fig. 1c replotted as frequency (o) versus in-plane momenta (kt). The experimental data (squares) are obtained from the polariton reﬂection images near the sample edges (Fig. 5). (b) Same as a for the lower hBN stop-band (Supplementary Fig. 3). Thickness of hBN: 105 nm. a b ... NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications 15 Macmillan Publishers Limited All rights reserved NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 Below we present new results documenting the ﬁrst observation of the higher-order (up to three) guided modes in such materials by direct nano-infrared imaging. 2 In the last step, we have applied Equation (3). For illustration, the dispersion curves of such guided modes in the upper stop-band of hBN of thickness 105 nm are shown in Fig. 1c, where they are overlaid on the dispersion surface of bulk hBN. The same curves are replotted as o vs kt in Fig. 4a. In Fig. 4b the dispersion curves of the guided modes of lower stop-band are shown. An intriguing aspect of these curves is that their slope qo/qkt is positive (negative) in the upper (lower) band. This sign difference is a consequence of the opposite direction of the group velocity vector for the type I and type II cases, cf. Fig. 1a,b. Central to the connection between the resonance cones in the real space and the quantized momenta in the k-space is that these momenta form an equidistant sequence of period Dkt ¼ kt,l þ 1 kt,l ¼ 2p/d. To map the dispersion of HP2, we utilized hBN crystals on SiO2 substrate without any intervening metallic disks (Methods). 4 & 2015 Macmillan Publishers Limited. All rights reserved ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 hBN SiO2 1,420cm–1 1,410cm–1 1,390cm–1 1,400cm–1 SiO2 0 5 10 15 α β γ ζ 10–10 10–12 10–11 10–13 1,420cm–1 Max Min ζ' F (kt) kt (105 cm–1) 0 1 3 2 1,400 cm–1 1,400 cm–1 s () (a.u.) 0 500 1,000 1,500 2,000 γ' L (nm) β' Figure 5 \| Imaging of polariton waveguide modes near the hBN edges. (a) Experimental schematic is similar to Fig. 3a except that imaging here is performed near the edge of an unpatterned sample. (b) Near-ﬁeld amplitude image measured at 1,420 cm 1. The olive square indicates the area whose expanded view is shown in c–e). (c–e) Near-ﬁeld image of the area marked in b at several frequencies. hBN thickness in b e: 31 nm. (f) Near-ﬁeld image of 105-nm-thick hBN at 1,400 cm 1. The cyan dashed lines in b f indicate the hBN edges. Scale bar in b f, 300 nm. (g) Line traces perpendicular to the hBN edge. Trace a was extracted from the image in f. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 Traces b, g and z were obtained from the Fourier analysis of the trace a as described in the text. (h) The Fourier transform of trace a in g. SiO2 0 5 10 15 α β γ ζ 1,400 cm–1 s () (a.u.) 0 500 1,000 1,500 2,000 L (nm) 10–10 10–12 10–11 10–13 ζ' F (kt) kt (105 cm–1) 0 1 3 2 1,400 cm–1 γ' L (nm) β' Figure 5 \| Imaging of polariton waveguide modes near the hBN edges. (a) Experimental schematic is similar to Fig. 3a except that imaging here is performed near the edge of an unpatterned sample. (b) Near-ﬁeld amplitude image measured at 1,420 cm 1. The olive square indicates the area whose expanded view is shown in c–e). (c–e) Near-ﬁeld image of the area marked in b at several frequencies. hBN thickness in b e: 31 nm. (f) Near-ﬁeld image of 105-nm-thick hBN at 1,400 cm 1. The cyan dashed lines in b f indicate the hBN edges. Scale bar in b f, 300 nm. (g) Line traces perpendicular to the hBN edge. Trace a was extracted from the image in f. Traces b, g and z were obtained from the Fourier analysis of the trace a as described in the text. (h) The Fourier transform of trace a in g. Here the sharp tip of the s-SNOM serves as both the emitter and the detector of the polariton waves on the open surface of the hBN. As the tip is scanned toward the sample edge, distinct variations in the detected scattering amplitude s(o) are observed. Such variations are caused by passing over minima and maxima of the standing waves created by interference of the polaritons launched by the tip and their reﬂections off the sample edges (Fig. 5a). Representative data for the upper stop-band (the type II hyperbolic region) are shown in Fig. 5b–f, where we plot s(o) at various infrared frequencies. Speciﬁcally, the image presented in Fig. 5b exhibits oscillations with the period B1 m m extending parallel to the edge of a 31-nm-thick hBN crystal. While these oscillations are similar to those reported previously33, a high- resolution scan performed very close to the edge (the olive square) reveals additional oscillations occurring on a considerably shorter scale: down to hundreds of nanometres (Fig. 5c–e). Similar results have been obtained using many other samples. For example, Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 5f also shows short-scale oscillations near the edges co-existing with longer-range oscillations further away from the edge in the data collected for a thicker hBN crystal (d ¼ 105 nm). The remaining step in the analysis is to establish the connection of thus determined momenta kb, kg and kz and the momenta kt,l of the guided modes, Equation (5). This requires more care than in prior studies of single-mode waves in two- dimensional materials33,42–44. The interference patterns near the edge can be created by various combinations of the tip-launched waves (labeled by l) and edge-reﬂected waves (labeled by r). The total momentum of a particular combination is kt,l þ kt,r. If the mode index is conserved, l ¼ r, the set of possible periods narrows down to 2kt,l. This is consistent with our data obtained for several infrared frequencies (Fig. 4a), where the symbols indicate kb, kg and kz. These data are in a quantitative agreement with the calculated dispersion curves for the l ¼ 0, 1 and 2 polariton- guided waves in the upper stop-band. The analysis of polariton propagation length33 shows that the loss factor is as low as gB0.03 (Supplementary Fig. 4). Dispersion mapping in the lower band (746–819 cm 1) where no monochromatic lasers are available is discussed in Supplementary Fig. 3. Broad-band lasers used in an independent study by Li et al. have allowed to demonstrate focusing behaviour of hBN in this challenging frequency region45. To analyse the harmonic content of the measured s(o) quantitatively, we used the spatial Fourier transform (FT). An example shown in Fig. 5h is the FT of the line trace a from Fig. 5g. The three dominant peaks in the FT are marked with b’ (blue), g’ (magenta) and z’ (olive). These peaks have been deemed statistically signiﬁcant and their positions kb, kg and kz have been recorded for each of the traces studied. We reasoned that including additional weaker peaks into consideration may be unwarranted at this stage. Indeed, the gross features in the real- space trace a exceeding the noise level of B1 a.u. are accounted for by oscillations in the three partial traces b, g and z, which are obtained by the inverse FT of the shaded regions in Fig. 5h. NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. /ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. References 36. Fogler, M. M., Butov, L. V. & Novoselov, K. S. High-temperature super-ﬂuidity with indirect excitons in van der Waals heterostructures. Nat. Commun. 5, 4555 (2014). 1. Pendry, J. B. Negative refraction makes a perfect lens. Phys. Rev. Lett. 85, 3966–3969 (2000). 37. Xu, X. G. et al. 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The s-SNOM is based on a tapping-mode AFM illuminated by monochromatic quantum cascade lasers (QCLs) (www.daylightsolutions.com) and a broad-band laser source utilizing the difference frequency generation (www.lasnix.com)49. Together, these lasers cover a frequency range of 700–2,300 cm 1 in the mid-infrared. The nanoscale near-ﬁeld images were registered by pseudo-heterodyne interferometric detection module with AFM tapping frequency and amplitude around 250 kHz and 60 nm, respectively. To obtain the background-free images, the s-SNOM output signal used in this work is the scattering amplitude s(o) demodulated at the nth harmonics of the tapping frequency. We chose n ¼ 3 in this work. 26. Ishii, S., Kildishev, A. V., Narimanov, E., Shalaev, V. M. & Drachev, V. P. Sub-wavelength interference pattern from volume plasmon polaritons in a hyperbolic medium. Laser Photonics Rev. 7, 265–271 (2013). 27. Thongrattanasiri, S. & Podolskiy, V. A. 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The Au patterns of various lateral shapes and 50-nm thickness were fabricated on these wafers by electron beam lithography. The hBN microcrystals of various thicknesses were exfoliated from bulk samples synthesized under high pressure50. Such microcrystals were subsequently mechanically transferred onto either patterned or unpatterned parts of the substrates. 32. Lu, D. & Liu, Z. Hyperlenses and metalenses for far-ﬁeld super-resolution imaging. Nat. Commun. 3, 1205 (2012). 33. Dai, S. et al. Tunable phonon polaritons in atomically thin van der waals crystal of boron nitride. Science 343, 1125–1129 (2014). 34. Caldwell, J. D. et al. Methods E i Sub-diffraction, volume-conﬁned polaritons in the natural hyperbolic material, hexagonal boron nitride. Nat. Commun. 5, 5221 (2014). 35. Geim, A. K. & Grigorieva, I. V. Van der Waals heterostructures. Nature 499, 419–425 (2013). Discussion Data presented in Figs 2–5 demonstrate launching, long-distance waveguiding transport and focusing of electromagnetic energy in thin crystals of hBN. These phenomena are enabled by directional propagation of large-momentum polariton beams in this natural hyperbolic material. The sharpness of the attained focusing, l/33 at distances up to l/6 (Supplementary Fig. 5), in units of the free- space wavelength, surpasses all prior realizations of superlenses and hyperlenses. Remarkably, a simple addition of a circular metallic launcher transforms an hBN crystal into a powerful 5 ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 focusing19 device! The analysis presented in Supplementary Note 1 (Supplementary Equation 10) indicates that the size of the focal spot in our system is limited by the ﬁnite thickness B50 nm of Au disks. By using thinner disks, say 20 nm thick, one should be able to achieve focal spots as small as Bl/102, comparable to the spatial resolution of our nano-infrared apparatus. A fundamental advantage of using natural rather than artiﬁcial hyperbolic materials is the magnitude of the upper momentum cutoff. In a natural material such as hBN, this cutoff is ultimately set by interatomic spacing thus immensely enhancing the spatial resolution. In addition, we have shown that hBN can serve as a multi-mode waveguide for polaritons with excellent ﬁgure of merit: loss factor as small as gB0.03. These characteristics exceed the benchmarks46–48 of current metal-based plasmonics and metamaterials. The physics behind this fundamental advantage of phonon polaritons over plasmons in conducting media is in the absence of electronic losses in insulators. Applications of hBN for non-imaging focusing devices41, subdiffractional waveguides and nanoresonators34 readily suggest themselves45. Combining such elements together may lead to development of sophisticated nanopolaritonic circuits. 14. Argyropoulos, C., Estakhri, N. M., Monticone, F. & Alu, A. Negative refraction, gain and nonlinear effects in hyperbolic metamaterials. Opt. Express 21, 15037–15047 (2013). 15. Biehs, S. A., Tschikin, M. & Ben-Abdallah, P. Hyperbolic metamaterials as an analog of a blackbody in the near ﬁeld. Phys. Rev. Lett. 109, 104301 (2012). 16. Noginov, M. A. et al. Controlling spontaneous emission with metamaterials. Opt. Lett. 35, 1863–1865 (2010). 17. Guo, Y., Cortes, C. L., Molesky, S. & Jacob, Z. Broadband super-Planckian thermal emission from hyperbolic metamaterials. Appl. Phys. Lett. 101, 131106 (2012). yp pp y 18. Jacob, Z., Smolyaninov, I. I. & Narimanov, E. E. Broadband Purcell effect: yp pp y 18. Jacob, Z., Smolyaninov, I. I. & Narimanov, E. E. Broadband Purcell effect: radiative decay engineering with metamaterials. Appl. Phys. Lett. 100, 181105 18. Jacob, Z., Smolyaninov, I. I. & Narimanov, E. E. Broadband Purcell effect: radiative decay engineering with metamaterials. Appl. Phys. Lett. 100, 181105 (2012). radiative decay engineering with metamaterials. Appl. Phys. Lett. 100, 181105 (2012). 19. Smith, D. R., Schurig, D., Mock, J. J., Kolinko, P. & Rye, P. Partial focusing of radiation by a slab of indeﬁnite media. Appl. Phys. Lett. 84, 2244–2246 (2004). Acknowledgements D.N.B. acknowledges support from DOE-BES grant DE-FG02-00ER45799 and the Gordon and Betty Moore Foundation’s EPiQS initiative through Grant GBMF4533; research on polariton focusing is supported by AFOSR. Work at UCSD is supported by the Ofﬁce of Naval Research, AFOSR, NASA and The University of California Ofﬁce of the President. A.S.M. acknowledges support from an Ofﬁce of Science Graduate Research Fellowship from U.S. Department of Energy. P.J-H acknowledges support from AFOSR grant number FA9550-11-1-0225. How to cite this article: Dai, S. et al. Subdiffractional focusing and guiding of polaritonic rays in a natural hyperbolic material. Nat. Commun. 6:6963 doi: 10.1038/ncomms7963 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms7963 NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Additional information 49. Keilmann, F. & Amarie, S. Mid-infrared frequency comb spanning an octave based on an Er ﬁber laser and difference-frequency generation. J. Infrared Millimeter Terahertz Waves 33, 479–484 (2012). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 50. Watanabe, K., Taniguchi, T. & Kanda, H. Direct-bandgap properties and evidence for ultraviolet lasing of hexagonal boron nitride single crystal. Nat. Mater. 3, 404–409 (2004). Competing ﬁnancial interests: F.K. is one of the co-founders of Neaspec and Lasnix, producer of the s-SNOM and infrared source used in this work. The remaining authors declare no competing ﬁnancial interests. References Low-loss plasmonic metamaterials. Science 331, 290–291 (2011). 12. Liu, Z., Lee, H., Xiong, Y., Sun, C. & Zhang, X. Far-ﬁeld optical hyperlens magnifying sub-diffraction-limited objects. Science 315, 1686 (2007). 47. Tassin, P., Koschny, T., Kafesaki, M. & Soukoulis, C. A comparison of graphene, superconductors and metals as conductors for metamaterials and plasmonics. Nat. Photonics 6, 259–264 (2012). 13. Yang, X., Yao, J., Rho, J., Yin, X. & Zhang, X. Experimental realization of three- dimensional indeﬁnite cavities at the nanoscale with anomalous scaling laws. Nat. Photonics 6, 450–454 (2012). 48. Khurgin, J. B. & Boltasseva, A. Reﬂecting upon the losses in plasmonics and metamaterials. MRS Bull. 37, 768–779 (2012). NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com NATURE COMMUNICATIONS \| 6:6963 \| DOI: 10.1038/ncomms7963 \| www.nature.com/naturecommunications 6 & 2015 Macmillan Publishers Limited. All rights reserved. Author contributions All the authors were involved in designing the research, performing the research and writing the paper. 7
https://openalex.org/W4381884374	https://www.frontiersin.org/articles/10.3389/fonc.2023.1192489/pdf	English	null	Case Report: Two clinical cases of Wilms tumor comorbid to gingival fibromatosis in young children with constitutionally mutated REST	Frontiers in oncology	2,023	cc-by	4,329	OPEN ACCESS REVIEWED BY Huanmin Wang, Capital Medical University, China Andrew Murphy, St. Jude Children’s Research Hospital, United States Anastasiya S. Salomatina 1, Liudmila A. Yasko 1, Maria A. Kurnikova 1, Yulia M. Mareeva 1, Ruslan K. Abasov 1, Nina V. Gegeliya 1, Anna M. Mitrofanova 1, Natalia Y. Usman 1, Galina A. Novichkova 1 and Alexander E. Druy 1,2 1Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia, 2Research Institute of Medical Cell Technologies, Yekaterinburg, Russia CITATION Salomatina AS, Yasko LA, Kurnikova MA, Mareeva YM, Abasov RK, Gegeliya NV, Mitrofanova AM, Usman NY, Novichkova GA and Druy AE (2023) Case Report: Two clinical cases of Wilms tumor comorbid to gingival ﬁbromatosis in young children with constitutionally mutated REST. Introduction: Nephroblastoma (Wilms tumor (WT)) is an embryonal tumor accounting for >90% of pediatric renal cancers. About 10% of WTs harbor pathogenic germline mutations. The REST gene, classiﬁed as a putative tumor suppressor, is affected in 2% of WTs. High-throughput molecular methods facilitate advanced diagnostics of cancer. In addition to this, germline mutations in REST are also associated with familial gingival ﬁbromatosis (GFM). Reciprocally, none of the articles on RESTmut WT mentions GFM as a comorbid condition. This report provides unique evidence on the WT-GFM comorbidity in RESTmut carriers. Front. Oncol. 13:1192489. doi: 10.3389/fonc.2023.1192489 © 2023 Salomatina, Yasko, Kurnikova, Mareeva, Abasov, Gegeliya, Mitrofanova, Usman, Novichkova and Druy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Case presentation: Patient 1 (a 5-year-old boy with unilateral WT) is a proband, who has two healthy siblings. Patient 2 (a 4-year-old girl with bilateral WT) is a proband from in vitro fertilization (IVF) triplets, with a sister and brother without WT. We analyzed probands’ DNA extracted from peripheral blood leucocytes with a custom-targeted next-generation sequencing (NGS)-198 gene panel. The detected variants were checked in family members by Sanger sequencing. Patient 1 had a pathogenic germline mutation in REST: c.1035_1036insTA, p.(E346), as did his mother and both brothers. There were two other WT cases in this family (proband’s maternal uncles). TYPE Case Report PUBLISHED 22 June 2023 DOI 10.3389/fonc.2023.1192489 TYPE Case Report PUBLISHED 22 June 2023 DOI 10.3389/fonc.2023.1192489 Case Report: Two clinical cases of Wilms tumor comorbid to gingival ﬁbromatosis in young children with constitutionally mutated REST OPEN ACCESS EDITED BY Stefano Cairo, Champions Oncology, Inc., United States REVIEWED BY Huanmin Wang, Capital Medical University, China Andrew Murphy, St. Jude Children’s Research Hospital, United States CORRESPONDENCE Anastasiya S. Salomatina anastasiya.salomatina@fccho- moscow.ru RECEIVED 23 March 2023 ACCEPTED 30 May 2023 PUBLISHED 22 June 2023 CITATION Salomatina AS, Yasko LA, Kurnikova MA, Mareeva YM, Abasov RK, Gegeliya NV, Mitrofanova AM, Usman NY, Novichkova GA and Druy AE (2023) Case Report: Two clinical cases of Wilms tumor comorbid to gingival ﬁbromatosis in young children with constitutionally mutated REST. Front Oncol 13:1192489 OPEN ACCESS EDITED BY Stefano Cairo, Champions Oncology, Inc., United States REVIEWED BY Huanmin Wang, Capital Medical University, China Andrew Murphy, St. Jude Children’s Research Hospital, United States CORRESPONDENCE Anastasiya S. Salomatina anastasiya.salomatina@fccho- moscow.ru RECEIVED 23 March 2023 ACCEPTED 30 May 2023 PUBLISHED 22 June 2023 CITATION Salomatina AS, Yasko LA, Kurnikova MA, Mareeva YM, Abasov RK, Gegeliya NV, Mitrofanova AM, Usman NY, Novichkova GA and Druy AE (2023) Case Report: Two clinical cases of Wilms tumor comorbid to gingival ﬁbromatosis in young children with constitutionally mutated REST. F t O l 13 1192489 Frontiers in Oncology OPEN ACCESS Patient 2 had a pathogenic germline variant in REST: c.2668_2671del, p.(E891Pfs6), as well as her sister. The mutation was probably inherited from their deceased father, as he had gingival ﬁbromatosis. Family members with REST mutations from both families had gingival ﬁbromatosis. A somatic REST c.663C>A p.C221 mutation was identiﬁed in one patient with WT. At the moment both patients with WT are under dynamic observation without signs of the disease. Conclusion: Here, we describe two clinical cases of WT in nonrelated young children with germline-inactivating REST variants identiﬁed by next-generation 01 Frontiers in Oncology frontiersin.org Salomatina et al. 10.3389/fonc.2023.1192489 sequencing. Both patients present with familial gingival ﬁbromatosis, regarded as clinically useful comorbidity indicative of the tumor predisposition syndrome. The two cases illustrate Wilms tumor-gingival ﬁbromatosis comorbidity in carriers of germline-inactivated REST alleles previously identiﬁed as a predisposition factor for both conditions. Wilm’s tumor, REST (RE-1 silencing transcription factor), pediatric oncology, gingival ﬁbromatosis, next-generation sequence (NGS) Introduction predisposition can be suspected based on genitourinary malformations, bilateral primary lesions (encountered in up to 10% of WTs), and multifocal patterns, as well as the early onset and family history of renal tumors (4, 5). Nephroblastoma, a.k.a. Wilms tumor (WT), is embryonal-type renal cancer developing from pluripotent progenitor cells of primordial kidneys (1). High-throughput molecular methods facilitate the discovery of nonconventional tumorigenic mutations in solid tumors, including recently described CTR9, TRIM28, and REST variants in WT (6, 7). ‘Restrictive element-1 silencing transcription factor’ (REST) encodes a zinc-ﬁnger nuclear protein. Mahamdallie et al. (2015) (6) describe 11 germline-inactivating REST variants in patients with WT. However, initially, pathogenic variants in REST were associated with familial gingival ﬁbromatosis (GFM), as researchers mentioned in their studies (8). At an estimated prevalence of 0.7–1 per 100,000 in pediatric populations, WT accounts for 5% of pediatric cancer cases. About 90% of pediatric kidney tumors are WTs, and about 80% of WTs are diagnosed in under-5-year olds. The median age at diagnosis is 3.5 years (2, 3). Although WTs are mostly sporadic, 10% of them involve hereditary predisposition (4). Congenital genetic conditions associated with high risks of WT are listed in Box 1; clinical manifestations range from multiple congenital malformations to the lack of a distinguishable phenotype apart from cancer risks. WT Here, we report for the ﬁrst time WT comorbidity to GFM in nonrelated young children with constitutionally mutated REST. BOX 1 Wilms tumor (WT) predisposition syndromes. Most signiﬁcant in terms of prevalence and risks: WT1-associated syndromes: Denys–Drash syndrome (AD) (OMIM: 607102): WT1 (11p13) Frasier syndrome (AD) (OMIM: 607102): WT1 (11p13) Despite a degree of clinical dissimilarity attributed to different patterns of mutagenesis within WT1 coding region, these syndromes reveal a continuum of clinical signs qualifying them as a single nosological entity. PAX6 and WT1 deletion syndrome (11p13 del) a.k.a. KEYWORDS Wilm’s tumor, REST (RE-1 silencing transcription factor), pediatric oncology, gingival ﬁbromatosis, next-generation sequence (NGS) Introduction WAGR (Wilms tumor-aniridia-genital anomalies-retardation/plus obesity (WAGRO)) (OMIM 194072, 612469) Beckwith–Wiedemann syndrome (AD, uniparental disomy, epimutations mapped to 11p15.5, e.g., in CDKN1C) (OMIM: 616186, 604115, 600856) Other: Fanconi anemia (AR) (OMIM: 227650): BRCA2, PALB2 Trisomy 18 (Edwards syndrome) TP53-dependent tumor predisposition (Li-Fraumeni syndrome) (AD) (OMIM: 191170): TP53 GLOW syndrome (AD, SM) (OMIM: 618272): DICER1 Perlman syndrome (AR) (OMIM: 614184): DIS3L2 Bohring–Opitz syndrome (AD) (OMIM: 605039): ASXL1 Bloom syndrome (AR) (OMIM: 210900): BLM Simpson–Golabi–Behmel syndrome (XLR) (OMIM: 180849): GPC3, GPC4 Mulibrey (MUscle, LIver, BRain, and EYes) nanism (AR) (OMIM 605073): TRIM37 Mosaic variegated aneuploidy syndrome (AR) (OMIM: 257300): BUB1B, TRIP13 PIK3CA-related overgrowth spectrum (SM) (OMIM 613089, 612918, 602501): PIK3CA WT, RESTmut-associated (AD) (OMIM: 616806) WT, CTR9mut-associated (AD) WT, TRIM28mut-associated (AD) Int. J. Cancer: 146, 1010–1017 (2020). NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Wilms Tumor (Nephroblastoma) Version 1.2022—12 April 2022 Human Molecular Genetics, Volume 29, Issue R2, 1 October 2020, Pages R138–R149. Inheritance patterns: AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive; SM, somatic mosaicism. Denys–Drash syndrome (AD) (OMIM: 607102): WT1 (11p13) Frasier syndrome (AD) (OMIM: 607102): WT1 (11p13) Frasier syndrome (AD) (OMIM: 607102): WT1 (11p13) Despite a degree of clinical dissimilarity attributed to different patterns of mutagenesis within WT1 coding region, these syndromes reveal a continuum of clinical signs qualifying them as a single nosological entity. g y g g g y PAX6 and WT1 deletion syndrome (11p13 del) a.k.a. WAGR (Wilms tumor-aniridia-genital anomalies-retardation/plus obesity (WAGR 612469) Beckwith–Wiedemann syndrome (AD, uniparental disomy, epimutations mapped to 11p15.5, e.g., in CDKN1C) (OMIM: 616186, 604115, 600856 Other: Fanconi anemia (AR) (OMIM: 227650): BRCA2, PALB2 Fanconi anemia (AR) (OMIM: 227650): BRCA2, PALB2 Trisomy 18 (Edwards syndrome) TP53-dependent tumor predisposition (Li-Fraumeni syndrome) (AD) (OMIM: 191170): TP53 GLOW syndrome (AD, SM) (OMIM: 618272): DICER1 Perlman syndrome (AR) (OMIM: 614184): DIS3L2 Bohring–Opitz syndrome (AD) (OMIM: 605039): ASXL1 Bloom syndrome (AR) (OMIM: 210900): BLM Simpson–Golabi–Behmel syndrome (XLR) (OMIM: 180849): GPC3, GPC4 Mulibrey (MUscle, LIver, BRain, and EYes) nanism (AR) (OMIM 605073): TRIM37 Mosaic variegated aneuploidy syndrome (AR) (OMIM: 257300): BUB1B, TRIP13 PIK3CA-related overgrowth spectrum (SM) (OMIM 613089, 612918, 602501): PIK3CA WT, RESTmut-associated (AD) (OMIM: 616806) WT, CTR9mut-associated (AD) WT, TRIM28mut-associated (AD) Int. J. Cancer: 146, 1010–1017 (2020). NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Wilms Tumor (Nephroblastoma) Version 1.2022—12 April 2022 Human Molecular Genetics, Volume 29, Issue R2, 1 October 2020, Pages R138–R149. BOX 1 Wilms tumor (WT) predisposition syndromes. KEYWORDS Salomatina et al. Introduction Inheritance patterns: AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive; SM, somatic mosaicism. Trisomy 18 (Edwards syndrome) y y TP53-dependent tumor predisposition (Li-Fraumeni syndrome) (AD) (OMIM: 191170): TP53 mor predisposition (Li-Fraumeni syndrome) (AD) (OMIM: 191170): TP53 GLOW syndrome (AD, SM) (OMIM: 618272): DICER1 Mulibrey (MUscle, LIver, BRain, and EYes) nanism (AR) (OMIM 605073): TRIM37 WT, RESTmut-associated (AD) (OMIM: 616806) Inheritance patterns: AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive; SM, somatic mosaicism. 02 Frontiers in Oncology frontiersin.org Salomatina et al. 10.3389/fonc.2023.1192489 Clinical case descriptions target panel (Roche, USA) of genes listed in Supplementary Table S1. The analysis revealed a REST (RefSeq NM_005612) c.1035_1036insTA, p.E346 heterozygous mutation qualiﬁed as pathogenic according to the American College of Medical Genetics (ACMG) guidelines. Subsequent genetic testing identiﬁed this constitutional variant in the patient’s mother and his two brothers, 7 years and 19 months old, both of whom presented without tumors but with signs of gum hyperplasia at the time of this writing. target panel (Roche, USA) of genes listed in Supplementary Table S1. The analysis revealed a REST (RefSeq NM_005612) c.1035_1036insTA, p.E346* heterozygous mutation qualiﬁed as pathogenic according to the American College of Medical Genetics (ACMG) guidelines. Subsequent genetic testing identiﬁed this constitutional variant in the patient’s mother and his two brothers, 7 years and 19 months old, both of whom presented without tumors but with signs of gum hyperplasia at the time of this writing. Patient 1 The boy, 4 years old, was born from a second pregnancy by a second full-term delivery. Early postnatal development was normal. A scheduled ultrasound examination after the appendectomy identiﬁed a mass in the lower pole of the right kidney. The patient received 4-week actinomycin D and vincristine (AV) neoadjuvant chemotherapy under the SIOP-RTSG UMBRELLA 2016 protocol, followed by surgical treatment. The tumor was identiﬁed as “nephroblastoma, regressive type, histologically intermediate risk group, local stage 1, 99.5% necrosis.” The residual vital tumor comprised small, solitary blastemal foci. Postoperatively, Pt 1 received 4 weeks of AV adjuvant chemotherapy. The patient has completed speciﬁc treatment and is under dynamic observation for 19 months without signs of the disease. To test for the additional tumorigenic event(s), the residual vital foci of blastemal tissue were microdissected from histological slides for DNA extraction, followed by Klenow-assisted whole-genome ampliﬁcation. NGS (QIAseq, Qiagen, Germany) at an average depth of 4,197 × identiﬁed somatic REST c.663C>A, p.C221* mutation at a 6% allele frequency read 9,176 times. The list of genes included in the NGS panel for somatic sequencing is presented in Supplementary Table S1. Thus, the tumor harbored two stop mutations in REST, one germline and one somatic, apparently in transconﬁguration (one mutation per copy), leading to the loss of REST protein in tumor cells. The patient has a burdened family history: one maternal uncle of the proband died from a renal tumor at the age of 3; a maternal aunt died of an unknown cause as a child; another maternal uncle and a maternal cousin–uncle underwent surgery for kidney masses at the age of 1.5–2 years. Clinical examination revealed hyperplasia of the maxillary gums typical of GFM (Figure 1A). Similar hyperplastic changes are observed in the mother (Figure 1B) and both brothers of the patient. The family tree is shown in Figure 1C. Patient 2 The girl, 3 years old, was born from the sixth pregnancy (IVF- induced, triple) by surgical delivery on gestation week 31. The neonatal period was complicated by bacterial pneumonia, hypoxic– ischemic CNS damage, and muscular dystonia syndrome. The The burdened family history suggested a hereditary cancer predisposition in the patient. Peripheral blood DNA was analyzed by high-throughput NGS using a tumor predisposition syndrome A B C FIGURE 1 Gingival ﬁbromatosis in patient 1 (A) and his mother (B); the family tree of patient 1 (C). B C FIGURE 1 Gingival ﬁbromatosis in patient 1 (A) and his mother (B); the family tree of patient 1 (C). FIGURE 1 Gingival ﬁbromatosis in patient 1 (A) and his mother (B); the family tree of patient 1 (C). 03 03 Frontiers in Oncology frontiersin.org Salomatina et al. 10.3389/fonc.2023.1192489 10.3389/fonc.2023.1192489 patient’s triplet sister is under observation for cerebral palsy; the other sibling (a boy) is healthy. The patient has no family history of cancer. Clinical examination revealed hyperplasia of the maxillary gums typical of GFM in the patient and her triplet sister (Figures 2A, B). The patient’s father, who died of a cause nonrelated to cancer, as well as their paternal grandfather, reportedly had similar changes in the upper jaw gums. The family tree is shown in Figure 2C. A scheduled ultrasound examination of abdominal organs revealed a mass in the left kidney. Additional examination revealed lesions in the other kidney. The patient received AV neoadjuvant chemotherapy followed by bilateral nephron-sparing surgery. Histological assessment of the left kidney specimen revealed foci of diffuse perilobar nephroblastomatosis as well as renal mass identiﬁed as “nephroblastoma, regressive type, 90% necrosis.” The residual vital tumor was presented with 90% blastemal and 10% epithelial components. The right kidney tumor was entirely blastemal without signs of response to treatment. The patient was diagnosed with “bilateral nephroblastoma, stage V.” She had a histologically intermediate-risk group of the left kidney, local stage 1, and a histologically high-risk group of the right kidney, local stage 1. The surgical margins of resection for each specimen were microscopically negative. Postoperatively, the patient received 27 weeks of adjuvant chemotherapy of actinomycin D, vincristine, and doxorubicin (AVD). The patient has completed speciﬁc treatment and has been under dynamic observation for 17 months without signs of the disease. The bilateral manifestation of tumors correlates with hereditary predisposition. Conclusion Germline-inactivating REST variants were for the ﬁrst time associated with WT by Mahamdallie et al. (2015) describing a total of 11 such mutations in four nonrelated pedigrees with familial WT and nine patients with no family history of kidney tumors. Seven of 14 identiﬁed familial carriers had kidney tumors, which corresponds to a 50% life-long risk of renal cancer. In two cases, molecular examination revealed a second genetic event in REST, supporting its putative role as a suppressor in WT. Noteworthy, of the 11 identiﬁed genetic variants, 10 affected the DNA-binding domain of the REST protein (6). The two cases illustrate Wilms tumor-gingival ﬁbromatosis comorbidity in carriers of germline-inactivated REST alleles previously identiﬁed as a predisposition factor for both conditions. In our opinion, such comorbidities warrant immediate molecular testing for constitutional mutations in REST, with extremely important implications in terms of cancer risks. Moreover, clinical signs of gingival ﬁbromatosis can justify ultrasound examination of the kidneys on a guideline basis in pediatric patients. One of our patients, patient 1, presented with a second, somatic, event in the REST coding sequence, apparently affecting the germline-intact copy of the gene. Such “double-hit” patterns feature REST as a tumor suppressor with the capacity of a single driver when double-inactivated. Patient 2 preserved the wild-type copy but presented with a missense DROSHA c.3439G>A, p. E1147K mutation previously characterized as truly somatic in WT (12). This second event was identiﬁed in residual tumor of the left kidney comprising epithelial foci but absent in blastema- only specimens. Despite the lack of comprehensive evidence on the pathogenetic role of REST in Wilms tumor, the loss-of-function nature of the variants implicates it as a putative suppressor. This assumption is supported by a second event affecting the germline-intact copy of REST in a signiﬁcant proportion of such tumors. Patient 2 NGS of peripheral blood DNA performed using the “tumor predisposition syndromes” panel (Supplementary Table S1) revealed the REST c.2668_2671del, p.E891Pfs6 heterozygous mutation qualiﬁed as pathogenic. Subsequent genetic testing identiﬁed this constitutional variant in the patient’s sister. Somatic NGS revealed only a missense variant in DROSHA (RefSeq NM_013235.5) c.3439G>A, p. E1147K, albeit for the left kidney tumor only. A search for copy number anomalies based on somatic NGS data revealed no extensive deletions in REST for tumors in both kidneys. Thus, the case presented a paternally inherited inactivating event in the REST coding sequence unaccompanied by somatic changes to the second, wild-type copy of the gene. A B C FIGURE 2 Gingival ﬁbromatosis in patient 2 (A) and her sister (B); family tree of patient 2 (C). A B C FIGURE 2 Gingival ﬁbromatosis in patient 2 (A) and her sister (B); family tree of patient 2 (C). FIGURE 2 Gingival ﬁbromatosis in patient 2 (A) and her sister (B); family tree of patient 2 (C). 04 04 Frontiers in Oncology frontiersin.org Salomatina et al. 10.3389/fonc.2023.1192489 Ethics statement The studies involving human participants were reviewed and approved by D. Rogachev Center Independent Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identiﬁable images or data included in this article. Familial GFM is a benign hyperplasia of ﬁbrous tissues in the gingivae and periodontium. This genetically diverse condition has FIGURE 3 REST protein with zinc-ﬁnger DNA-binding domain and repressor domains RD1 and RD2. Germline mutations identiﬁed in patients 1 and 2 are marked with black pins; mutations (germline and somatic) identiﬁed in patient 1 are labeled in red. FIGURE 3 REST protein with zinc-ﬁnger DNA-binding domain and repressor domains RD1 and RD2. Germline mutations identiﬁed in patients 1 and 2 are marked with black pins; mutations (germline and somatic) identiﬁed in patient 1 are labeled in red. Discussion been associated with aberrations/mutations mapping to the SOS1 gene, 5q13-q22 (OMIM #605544), 2p23.3-p.22.3 (OMIM #609955), and 11p15 (OMIM #6011010). The REST gene has been implicated as well: Bayram et al. (2017) describe three nonrelated families with GFM caused by three different heterozygous mutations in the last exon of REST, identiﬁed by whole-exome sequencing of peripheral blood DNA. Importantly, the article mentions no kidney tumors in any of the affected individuals (11 cases in total) (8). The REST gene (OMIM 600571) encodes a Krüppel-type transcription factor with a zinc-ﬁnger DNA-binding domain and two repressor domains that interact with transcription apparatus and chromatin remodeling complexes (Figure 3). The protein inhibits the activation of neuron-speciﬁc genes in nonneural cells through chromatin remodeling (9, 10). Reciprocally, none of the articles on RESTmut WT mention GFM as a comorbid condition. To the best of our knowledge, this report provides unique evidence on the WT-GFM comorbidity in RESTmut carriers. REST has been implicated as a putative tumor driver—either oncogene or suppressor, depending on the particular cancer type. Increased expression of REST in low-grade gliomas has been correlated to poor overall survival (11), which implicates it as a putative oncogene or disease progression marker in low- grade gliomas. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. According to Maciaszek et al. (2020), about 2% of WTs, regardless of histological type, harbor a germline pathogenic mutation in REST. The median age at diagnosis for such cases is 3 years (within the range of 0.5–6 years) (7). REST variants identiﬁed by us in two patients have not been described previously. Similarly, with the majority of WT-associated REST variants, these are loss-of-function mutations affecting the DNA- binding domain. frontiersin.org Author contributions FIGURE 3 REST protein with zinc-ﬁnger DNA-binding domain and repressor domains RD1 and RD2. Germline mutations identiﬁed in patients 1 and 2 are marked with black pins; mutations (germline and somatic) identiﬁed in patient 1 are labeled in red. AS, AD, MK, LY, and YM: conceptualized and drafted the initial manuscript. AS, AD, and MK: paper compilation and research. All authors contributed to the article and approved the submitted version. 05 Frontiers in Oncology frontiersin.org Salomatina et al. 10.3389/fonc.2023.1192489 Salomatina et al. Publisher’s note The molecular-genetic study was funded by the Foundation for Support and Development in the Field of Pediatric Hematology, Oncology, and Immunology “Science for Children.” All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2023.1192489/ full#supplementary-material The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 9. Lunyak VV, Burgess R, Prefontaine GG, Nelson C, Sze SH, Chenoweth J, et al. Corepressor-dependent silencing of chromosomal regions encoding neuronal genes. Science (2002) 298(5599):1747–52. doi: 10.1126/science.1076469 10. Magin A, Lietz M, Cibelli G, Thiel G. RE-1 silencing transcription factor-4 (REST4) is neither a transcriptional repressor nor a de-repressor. Neurochem Int (2002) 40(3):195–202. doi: 10.1016/s0197-0186(01)00091-2 8. Bayram Y, White JJ, Elcioglu N, Cho MT, Zadeh N, Gedikbasi A, et al. REST ﬁnal-Exon-Truncating mutations cause hereditary gingival ﬁbromatosis. Am J Hum Genet (2017) 101(1):149–56. doi: 10.1016/j.ajhg.2017.06.006 11. Wang G, Yang X, Qi M, Li M, Dong M, Xu R, et al. Systematic analysis identiﬁes REST as an oncogenic and immunological biomarker in glioma. Sci Rep (2023) 13 (1):3023. doi: 10.21203/rs.3.rs-1569719/v1 12. Torrezan GT, Ferreira EN, Nakahata AM, Barros BD, Castro MT, Correa BR, et al. Recurrent somatic mutation in DROSHA induces microRNA proﬁle changes in wilms tumour. Nat Commun (2014) 5:4039. doi: 10.1038/ncomms5039 7. Maciaszek JL, Oak N, Nichols KE. Recent advances in wilms' tumor predisposition. Hum Mol Genet (2020) 29(R2):R138–49. doi: 10.1093/hmg/ddaa091 6. Mahamdallie S, Hanks S, Karlin K, Kristen L, Zachariou A, Perdeaux R, et al. Mutations in the transcriptional repressor REST predispose to wilms tumor. Nat Genet (2015) 47:1471–4. doi: 10.1038/ng.3440 7. Maciaszek JL, Oak N, Nichols KE. Recent advances in wilms' tumor predisposition. Hum Mol Genet (2020) 29(R2):R138–49. doi: 10.1093/hmg/ddaa091 8. Bayram Y, White JJ, Elcioglu N, Cho MT, Zadeh N, Gedikbasi A, et al. REST ﬁnal-Exon-Truncating mutations cause hereditary gingival ﬁbromatosis. Am J Hum Genet (2017) 101(1):149–56. doi: 10.1016/j.ajhg.2017.06.006 9. Lunyak VV, Burgess R, Prefontaine GG, Nelson C, Sze SH, Chenoweth J, et al. Corepressor-dependent silencing of chromosomal regions encoding neuronal genes. Science (2002) 298(5599):1747–52. doi: 10.1126/science.1076469 10. Magin A, Lietz M, Cibelli G, Thiel G. RE-1 silencing transcription factor-4 (REST4) is neither a transcriptional repressor nor a de-repressor. Neurochem Int (2002) 40(3):195–202. doi: 10.1016/s0197-0186(01)00091-2 11. Wang G, Yang X, Qi M, Li M, Dong M, Xu R, et al. Systematic analysis identiﬁes REST as an oncogenic and immunological biomarker in glioma. Sci Rep (2023) 13 (1):3023. doi: 10.21203/rs.3.rs-1569719/v1 12. Torrezan GT, Ferreira EN, Nakahata AM, Barros BD, Castro MT, Correa BR, et al. Recurrent somatic mutation in DROSHA induces microRNA proﬁle changes in wilms tumour. Nat Commun (2014) 5:4039. doi: 10.1038/ncomms5039 3. Chu A, Heck JE, Ribeiro KB, Brennan P, Boffetta P, Bufﬂer P, et al. Wilms’ tumour: a systematic review of risk factors and meta-analysis Vol. 24. Paediatric and Perinatal Epidemiology (2010) p. 449–69. doi: 10.1111/j.1365-3016.2010.01133.x 4. Liu EK, Suson KD. Syndromic wilms tumor: a review of predisposing conditions, surveillance and treatment. Transl Androl Urol (2020) 9(5):2370–81. doi: 10.21037/tau.2020.03.27 5. Rivera MN, Haber DA. Wilms’ tumour: connecting tumorigenesis and organ development in the kidney Vol. 5. Nature Publishing Group (2005) p. 699–712. doi: 10.1038/nrc1696 1. Erginel B, Vural S, Akin M, Karadağ ÇA, Sever N, Yıldız A, et al. Wilms’ tumor: a 24-year retrospective study from a single center. Pediatr Hematol Oncol (2014) 31 (5):409–14. doi: 10.3109/08880018.2014.930767 2. Nelson MV, van den Heuvel-Eibrink MM, Graf N, Dome JS. New approaches to risk stratiﬁcation for wilms tumor. Curr Opin Pediatr (2021) 33(1):40–8. doi: 10.1097/ mop.0000000000000988 3. Chu A, Heck JE, Ribeiro KB, Brennan P, Boffetta P, Bufﬂer P, et al. Wilms’ tumour: a systematic review of risk factors and meta-analysis Vol. 24. Paediatric and Perinatal Epidemiology (2010) p. 449–69. doi: 10.1111/j.1365-3016.2010.01133.x 4. Liu EK, Suson KD. Syndromic wilms tumor: a review of predisposing conditions, surveillance and treatment. Transl Androl Urol (2020) 9(5):2370–81. doi: 10.21037/tau.2020.03.27 5. Rivera MN, Haber DA. Wilms’ tumour: connecting tumorigenesis and organ development in the kidney Vol. 5. Nature Publishing Group (2005) p. 699–712. doi: 10.1038/nrc1696 6. Mahamdallie S, Hanks S, Karlin K, Kristen L, Zachariou A, Perdeaux R, et al. Mutations in the transcriptional repressor REST predispose to wilms tumor. Nat Genet (2015) 47:1471–4. doi: 10.1038/ng.3440 References 1. Erginel B, Vural S, Akin M, Karadağ ÇA, Sever N, Yıldız A, et al. Wilms’ tumor: a 24-year retrospective study from a single center. Pediatr Hematol Oncol (2014) 31 (5):409–14. doi: 10.3109/08880018.2014.930767 7. Maciaszek JL, Oak N, Nichols KE. Recent advances in wilms' tumor predisposition. Hum Mol Genet (2020) 29(R2):R138–49. doi: 10.1093/hmg/ddaa091 2. Nelson MV, van den Heuvel-Eibrink MM, Graf N, Dome JS. New approaches to risk stratiﬁcation for wilms tumor. Curr Opin Pediatr (2021) 33(1):40–8. doi: 10.1097/ mop.0000000000000988 6. Mahamdallie S, Hanks S, Karlin K, Kristen L, Zachariou A, Perdeaux R, et al. Mutations in the transcriptional repressor REST predispose to wilms tumor. Nat Genet (2015) 47:1471–4. doi: 10.1038/ng.3440 06 Frontiers in Oncology frontiersin.org
https://openalex.org/W4220897275	https://bmccancer.biomedcentral.com/track/pdf/10.1186/s12885-022-09382-x	English	null	Mendelian randomization study of circulating lipids and biliary tract cancer among East Asians	BMC cancer	2,022	cc-by	7,116	Abstract Background: Associations of High-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, total cholesterol (CHL), and triglyceride (TRG) concentrations with risk of biliary tract cancer (BtC) were conflict- ing in observational studies. We aim to investigate the causal link between circulating lipids and BtC using genetic information. Methods: Single nucleotide polymorphisms of the four circulating lipids (n = 34,421) and BtC (418 cases and 159,201 controls) were retrieved from two independent GWAS studies performed in East Asian populations. Two-sample univariate and multivariate Mendelian Randomization (MR) analyses were conducted to determine the causal link between circulating lipids and BtC. Results: No significant horizontal pleiotropy was detected for all circulating lipids according to the MR-PRESSO global test (P = 0.458, 0.368, 0.522, and 0.587 for HDL, LDL, CHL, and TRG, respectively). No significant evidence of het- erogeneity and directional pleiotropy was detected by the Cochran’s Q test and MR-Egger regression. Univariate MR estimates from inverse variance weighting method suggested that one standard deviation (1-SD) increase of inverse- normal transformed HDL (OR = 1.38, 95% CI 0.98–1.94), LDL (OR = 1.46, 95% CI 0.96–2.23), and CHL (OR = 1.34, 95% CI 0.83–2.16) were not significantly associated with BtC risk. Whereas 1-SD increase of inverse-normal transformed TRG showed a significantly negative association with BtC risk (OR = 0.48, 95% CI 0.31–0.74). In multivariate MR analy- ses including all the four lipid traits, we found that 1-SD increase of LDL and TRG was significantly associated with elevated (OR = 1.32, 95% CI 1.04–2.01) and decreased (OR = 0.54, 95% CI 0.42–0.68) risk of BtC, respectively. Conclusion: Circulating lipids, particularly LDL and TRG, may have roles in the development of BtC. However, the results of this study should be replicated in MR with larger GWAS sample sizes for BtC. Keywords: Biliary tract cancer, Mendelian randomization, Lipid, HDL, LDL, Triglyceride, Cholesterol Jun Wang1†, Jinke Zhuge2†, Dongxu Feng1†, Bo Zhang1, Jianying Xu3, Dongkang Zhao4, Zhewei Fei1, Xia Huang1* and Wenjie Shi5* Jun Wang1†, Jinke Zhuge2†, Dongxu Feng1†, Bo Zhang1, Jianying Xu3, Dongkang Zhao4, Zhewei Fei1, Xia Huang1* and Wenjie Shi5* Jun Wang1†, Jinke Zhuge2†, Dongxu Feng1†, Bo Zhang1, Jianying Xu3, Dongkang Zhao4, Zhewei Fei1, Xia Huang1* and Wenjie Shi5* © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction Biliary tract cancer (BtC) constitutes approximately 3% of gastrointestinal malignancies with poor progno- sis and involves a spectrum of invasive adenocarcino- mas, including cholangiocarcinoma (cancers arising in the intrahepatic, perihilar, or distal biliary tree), and gallbladder carcinoma [1, 2]. The incidence of BtC var- ies across the world: the highest incidence rate was *Correspondence: shxiahuang@sina.com; wenjie.shi@uni-oldenburg.de †Jun Wang, Jinke Zhuge and Dongxu Feng contributed equally to this work. 1 Department of General Surgery, Xinhua Hospital Chongming Branch, 25 Nanmen Road, ShanghaiChongming 202150, China 5 University Hospital for Gynecology, Pius-Hospital, University Medicine Oldenburg, 12 Georg Street, 26121 Oldenburg, Germany Full list of author information is available at the end of the article Wang et al. BMC Cancer (2022) 22:273 https://doi.org/10.1186/s12885-022-09382-x © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. BMC Cancer (2022) 22:273 Page 2 of 9 measured by standard biochemical methods [16]. In the GWAS of circulating lipids, 34,421 participants from China, Japan, Korea, Philippines, and Singapore were included. The participants were genotyped using com- mercially available Affymetrix or Illumina genome-wide genotyping arrays, and the genotype data were then imputed to HapMap Project Phase II reference panel. Quality control criteria implemented in each population, including variant call rate and Hardy–Weinberg equilib- rium (HWE). The GWAS details have been shown else- where [16]. Briefly, in GWAS of circulating lipids, age, age2, sex, and other study-specific covariates (e.g., princi- pal components, sample recruitment sites) were adjusted in a linear regression model. The levels of circulating lipids (mg/dL) have been normal-inverse transformed in the GWAS. A meta-analysis for associations between the four lipid traits and ~ 2.4 million variants were then per- formed by two independent analysts, each using Stouffer sample-size weighted fixed effects meta-analysis imple- mented in METAL. observed in East Asia and Latin America [2, 3]. In devel- oped countries, BtC was rarely diagnosed in clinical practice. The varied BtC incidences in different regions were due to different underlying risk factors. Previous studies have demonstrated that a set of hepatic con- ditions including hepatic inflammation, fibrosis, and cirrhosis are risk factors for intrahepatic cholangio- carcinoma [4]. Methods y g We collected the GWAS summary data of circulating lipids from the Asian Genetic Epidemiology Network (AGEN; https://blog.nus.edu.sg/agen/). AGEN is a con- sortium of genetic epidemiology studies of type 2 dia- betes and cardiovascular disease related phenotypes including HDL, LDL, CHL, and TRG conducted among East Asian populations [16]. Plasma lipid levels were GWAS summary statistics of biliary tract cancer y y To ensure the concordance of ancestry of study partici- pants, in this study, we retrieved the GWAS summary data of BtC from Biobank Japan (BBJ) [17]. BBJ is a pro- spective genome biobank that collaboratively collected DNA and serum samples from 12 medical institutions in Japan, managed by the Institute of Medical Science, the University of Tokyo. BBJ has recruited approximately 260,000 participants, mainly of Japanese ancestry. All study participants had been diagnosed with one or more of 47 target diseases, among which the BtC was identified using ICD-10 codes of C22.1 and C23 and ICD-9 codes of 155 and 159.3. The BBJ participants were genotyped with the Illumina HumanOmniExpressExome BeadChip or a combination of the Illumina HumanOmniExpress and HumanExome BeadChips [18]. The genotype data were then imputed with 1000 Genome Project Phase 3 version 5 genotype and Japanese whole-genome sequenc- ing data (n = 1037). Variants with an imputation qual- ity < 0.7 were excluded, resulting in a total of 13,530,797 variants analyzed in the GWAS. For BtC, 418 cases and 159,201 controls that were East Asian ancestry were included (https://pheweb.jp/pheno/BtC). A generalized linear model that performed in SAIGE (version 0.37) was applied to conduct BtC GWAS, where age, age2, sex, age × sex, age2 × sex, and the top 20 principal compo- nents were adjusted. On the other hand, chronic irritation or inflammation of the gallbladder and cholelithiasis are deemed to be associated with a higher risk of gallbladder carcinoma [5–7]. Additionally, hyperlipidemia was also reported to associate with BtC development even after adjustment for body-mass index (BMI), diabetes, hyper- tension, and alcohol drinking [8, 9]. Hyperlipidemia is characterized by high serum lev- els of total cholesterol (CHL), triglycerides (TRG), low- density lipoprotein cholesterol (LDL), and low level of high-density lipoprotein cholesterol (HDL). Previous observational studies suggested a role for the circulating lipids in biliary carcinogenesis. For example, Andreotti et al. reported that participants in the lowest quintile of serum HDL level had a 16.8-fold risk of BtC [9]. Another case–control study from China also suggested that serum levels of lipids were significantly associated with BtC risk [10]. However, the findings from observational studies might be subject to the inherent defects of this type of study design, namely residual confounding and reverse causality. So far, there has been no randomized clinical trial to assess the effect of statin use on BtC development. In this case, Mendelian randomization (MR) analysis could serve as a good surrogate. MR leveraging genetic data is less susceptible to such biases due to the fact that alleles are randomly assigned during meiosis and ger- mline genetic variants are unaffected by disease process [11]. So far, MR analysis has been widely used to infer the causality between exposures and outcomes [12–14]. The findings of MR studies were of importance not only for the discovery of disease biomarkers, but also for the ther- apeutic and prophylactic strategies of diseases [15]. Nev- ertheless, the association of circulating lipids with BtC risk has not been determined by MR analysis. Herein, we conducted a two-sample MR analysis to address this need. Genetic instrumental variables We conducted a series of quality control steps to select eligible instrumental SNPs of circulating lipids. First, we extracted SNPs showing association with lipid levels at Wang et al. BMC Cancer (2022) 22:273 Page 3 of 9 Page 3 of 9 the traditional GWAS threshold (P < 5 × 10–8). Second, we performed a clumping process (R2 < 0.01; window size = 10,000 kb) based on the linkage disequilibrium (LD) estimates from the East Asian samples in 1000 genomes project. Among those pairs of SNPs that had LD estimate above the specified threshold (0.01), we only retained the SNP that had the lower P value. Third, SNPs with a minor allele frequency < 1% were removed. Next, we extracted the statistics (i.e., beta coefficient and stand- ard error) regarding the above selected SNPs from the BtC GWAS summary. If a particular requested SNP was absent in the BtC GWAS, we retrieved the data of a SNP proxy that had LD estimate R2 > 0.8 with the requested SNP. The effects of ambiguous SNPs with inconsistent alleles and palindromic SNPs with ambiguous strand were either corrected or directly excluded in the sub- sequent two-sample MR analysis. The methodological details of MR analysis were presented elsewhere [19, 20]. the traditional GWAS threshold (P < 5 × 10–8). Second, we performed a clumping process (R2 < 0.01; window size = 10,000 kb) based on the linkage disequilibrium (LD) estimates from the East Asian samples in 1000 genomes project. Among those pairs of SNPs that had LD estimate above the specified threshold (0.01), we only retained the SNP that had the lower P value. Third, SNPs with a minor allele frequency < 1% were removed. Next, we extracted the statistics (i.e., beta coefficient and stand- ard error) regarding the above selected SNPs from the BtC GWAS summary. If a particular requested SNP was absent in the BtC GWAS, we retrieved the data of a SNP proxy that had LD estimate R2 > 0.8 with the requested SNP. The effects of ambiguous SNPs with inconsistent alleles and palindromic SNPs with ambiguous strand were either corrected or directly excluded in the sub- sequent two-sample MR analysis. The methodological details of MR analysis were presented elsewhere [19, 20]. pleiotropy was presented. Second, we tested the between- SNP heterogeneity using inverse variance weighting (IVW) method based on the SNPs that retained after pleiotropy correction. Genetic instrumental variables The Cochran’s Q statistic was used to check for the presence of heterogeneity. In this step, we removed the SNPs with P < 1.00 in MR-PRESSO analysis if the heterogeneity was significant (P value of Cochran’s Q statistic < 0.05). Third, we conducted MR analysis using IVW method. We obtained the IVW esti- mate by meta-analyzing the SNP specific Wald estimates using multiplicative random effects. Given the small case number in the BtC GWAS, we calculated the statisti- cal power for MR analysis using mRnd website (https:// shiny.cnsgenomics.com/mRnd/) [21]. We also conducted a set of sensitivity analyses using MR-Egger regression, weighted median, and weighted mode methods. The MR-Egger regression is based on the InSIDE (INstru- ment Strength Independent of Direct Effect) assump- tion and consists of three parts: (i) a test for directional pleiotropy, (ii) a test for a causal effect, and (iii) an esti- mate of the causal effect [22]. The weighted median and weighted mode methods are more robust than IVW and MR-Egger methods if more than 50% of SNPs are inva- lid instruments [23, 24]. Finally, “leave-one-out” analysis Mendelian randomization analysishl The flowchart and schematic representation of MR anal- ysis is shown in Fig. 1. First, we tested the horizontal pleiotropy using MR-PRESSO global test and removed the outliers (i.e., SNPs with P < 0.05) if the horizontal Fig. 1 Flow chart (A) and schematic representation (B) of Mendelian randomization analysis in this study Fig. 1 Flow chart (A) and schematic representation (B) of Mendelian randomization analysis in this study Fig. (B) Wang et al. BMC Cancer (2022) 22:273 Wang et al. BMC Cancer (2022) 22:273 Page 4 of 9 was conducted to detect the influential SNPs. To interro- gate the presence of reverse causation, we conducted MR analyses in which the BtC was set as exposure and lipids were set as outcomes. In this analysis, we used a P value threshold < 5 × 10–5 to select the genetic instruments due to there was no SNP reached the traditional GWAS threshold. A total of 42 variants were obtained after data clumping. instrumental variable bias. No significant horizontal plei- otropy was detected for all circulating lipids according to the MR-PRESSO global test (P = 0.458, 0.368, 0.522, and 0.587 for HDL, LDL, CHL, and TRG, respectively). The results of assessment of heterogeneity and direc- tional pleiotropy are shown in Table 1. No significant evidence of heterogeneity and pleiotropy was detected by the Cochran’s Q test and MR-Egger regression, suggest- ing the variants that included in MR analysis are valid instruments.hf Considering the correlations among circulating lipids, we also performed a multivariable MR (MVMR) analysis including all of the four lipid traits to obtain the causal estimates (Fig. 1B). MVMR is an extension of MR that allows for the causal effects of multiple exposures on an outcome to be estimated [25]. MVMR estimates the “direct” causal effects of each exposure included in the estimation on the outcome, conditional on the other exposures included in the model [26]. MVMR is particu- larly useful when examining the causal effects of several exposures that are correlated with each other. We also incorporated BMI into the MVMR to examine the poten- tial mediation of obesity on association between lipids and BtC risk. The summary genetic data of BMI from East Asians were retrieved from IEU OpenGAWS pro- ject (https://gwas.mrcieu.ac.uk/datasets/bbj-a-1/). All statistical analyses were implemented using TwoSam- pleMR and MRPRESSO packages in R program (v 3.6.3). P value < 0.05 was considered statistically significant. Mendelian randomization analysishl The estimated effect sizes of the SNPs on both the exposures (HDL, LDL, CHL, and TRG) and outcome (BtC) are displayed in scatter plots (Fig. 2). The fitted lines denoting association between SNP effects on expo- sure and on outcome, based on different methods, were in the same direction, albeit the nuances of slopes. This concordance connotes the robustness of our MR esti- mates. MR estimates from IVW method suggested that one standard deviation (1-SD) increase of inverse-normal transformed HDL (OR = 1.38, 95% CI 0.98–1.94), LDL (OR = 1.46, 95% CI 0.96–2.23), and CHL (OR = 1.34, 95% CI 0.83–2.16) were not significantly associated with BtC risk (Table 1; Fig. 3). Whereas 1-SD increase of inverse- normal transformed TRG showed a significantly negative association with BtC risk (OR = 0.48, 95% CI 0.31–0.74). We have calculated 80% power in our MR studies to show an OR of 1.56 for HDL, 1.75 for LDL, 1.59 for CHL, and 0.74 for TRG respectively. As such, we are underpow- ered to study effects smaller than these ORs. The IVW- based MR estimates were further validated in other three methods. An exception was found for weighted median methods, in which 1-SD increase of inverse-normal transformed LDL and CHL levels were significantly associated with an increased risk of BtC. We observed a non-significant association between 1-SD increase of inverse-normal transformed TRG level and BtC risk Results After the quality control processes, we included 26, 19, 23, 10 variants in MR analysis for HDL, LDL, CHL, and TRG, respectively (Supplementary Tables S1-4). The mean F statistics for every instrument-exposure asso- ciation were greater than 10 in our study (F = 21.2, 13.4, 18.4, and 10.5 for HDL, LDL, CHL, and TRG, respec- tively), demonstrating the small possibility of weak Table 1 Association of circulating lipids with biliary tract cancer risk according to different methods HDL high density lipoprotein, LDL low density lipoprotein, CHL cholesterol, TRG triglyceride HDL LDL CHL TRG Inverse variance weighted OR (95%CI) 1.38 (0.98, 1.94) 1.46 (0.96, 2.23) 1.34 (0.83, 2.16) 0.48 (0.31, 0.74) Q statistics (P value) 24.6 (0.431) 20.6 (0.298) 25.5 (0.274) 5.9 (0.662) MR-egger OR (95%CI) 2.10 (0.99, 4.46) 1.36 (0.70, 2.63) 1.50 (0.50, 4.50) 0.46 (0.19, 1.12) Q statistics (P value) 23.3 (0.444) 20.5 (0.247) 25.5 (0.228) 5.9 (0.556) Intercept (P value) -0.048 (0.274) 0.009 (0.804) -0.009 (0.842) 0.004 (0.946) Weighted median OR (95%CI) 1.55 (0.94, 2.58) 1.74 (1.02, 2.97) 2.16 (1.15, 4.07) 0.46 (0.27, 0.76) Weighted mode OR (95%CI) 1.73 (0.99, 3.02) 1.75 (0.97, 3.14) 2.51 (0.98, 6.40) 0.48 (0.27, 0.83) Table 1 Association of circulating lipids with biliary tract cancer risk according to different methods HDL LDL CHL Wang et al. BMC Cancer (2022) 22:273 Wang et al. BMC Cancer Page 5 of 9 Fig. 2 Scatter plots for Mendelian randomization analyses of the causal effect of circulating lipids on biliary tract cancer in initial practice. A, HDL; B, LDL; C, cholesterol; D, triglyceride. The slope of each line corresponding to the estimated MR effect per method Fig. 2 Scatter plots for Mendelian randomization analyses of the causal effect of circulating lipids on biliary tract cancer in LDL; C, cholesterol; D, triglyceride. The slope of each line corresponding to the estimated MR effect per method and TRG, respectively; Supplementary Table S5). How- ever, the results might be subject to weak instrument bias in this analysis due to the low F-statistics.h according to MR-Egger method, although this associa- tion was significant according to other three methods (Table 1; Fig. 3). The forest plots of “leave-one-out” analy- ses were shown in Supplementary Figs. 1–4. No poten- tially influential SNP was found for HDL, CHL, and TRG. Results In contrast, we found that the association between 1-SD increase of inverse-normal transformed LDL level and BtC risk was statistically significant if removing a variant (rs10119). No significant association was detected in MR analysis when examining the BtC effect on levels of circu- lating lipids (F = 4.1, 5.2, 3.8, and 3.5 for HDL, LDL, CHL, The overlap among genetic instruments of circulating lipids was shown in Fig. 4A. We observed that a total of 11 variants were shared between LDL and CHL, whereas for other pairs of lipids, the shared variants were less than 5. We conducted a MVMR analysis to further vali- date the association between genetically predicted levels of circulating lipids and BtC risk. MVMR analysis esti- mated that 1-SD increase of inverse-normal transformed Wang et al. BMC Cancer (2022) 22:273 Page 6 of 9 LDL was significantly associated with elevated risk of BtC (OR = 1.32, 95% CI 1.04–2.01). On the contrary, 1-SD increase of inverse-normal transformed TRG was signifi- cantly associated with decreased risk of BtC (OR = 0.54, 95% CI 0.42–0.68) (Fig. 4B). We also performed pairwise all of the three models, we found that TRG were con- sistently associated with a decreased BtC risk (Supple- mentary Figure S5). Likewise, we observed an inverse relationship between TRG level and BtC risk in MVMR analysis in which we further incorporated BMI (Supple- Fig. 3 The causal effects of circulating lipids on biliary tract cancer from Mendelian randomization analyses based on four methods. Error bars denote 95% confidence interval of the odds ratio estimates Fig. 4 The causal effects of circulating lipids on biliary tract cancer from multivariate Mendelian randomization analyses. A, overlap of genetic instruments among the four lipids; B, causal estimates from multivariate Mendelian randomization analysis. Error bars denote 95% confidence interval of the odds ratio (OR) estimates Fig. 3 The causal effects of circulating lipids on biliary tract cancer from Mendelian randomization analyses based on four methods. Error bars denote 95% confidence interval of the odds ratio estimates Fig. 3 The causal effects of circulating lipids on biliary tract cancer from Mendelian randomization analyses based on four methods. Error bars denote 95% confidence interval of the odds ratio estimates Fig. 3 The causal effects of circulating lipids on biliary tract cancer from Mendelian randomization analyses based on four methods. Error bars denote 95% confidence interval of the odds ratio estimates Fig. Discussion reported that participants with the highest quintile of triglycerides (≥ 160 mg/dl) had a 40%, 90%, and 4.8-fold increase in the risk of biliary stones, gallbladder cancer, and bile duct cancer, respectively, compared to the reference group (third quintile: 90–124 mg/dl) [9]. By contrast, Borena et al. found that there was no significant association between serum triglyceride level and gallbladder can- cer [36]. The inconsistences might be ascribed to several reasons: (i) different study design and study participants; (ii) lipid measurement methods; (iii) lipid levels varied with times even in the same person; and (iv) inadequate adjustment for confounders. Given the inherent limita- tions of observational study, results from studies using genetic information might be an optimal complement for observational studies. For instance, Andreotti et al. reported that genetic variants in the lipid metabolism pathway (e.g., T allele of LDLR rs1003723) contribute to the risk of biliary tract stones and cancers, particularly of the bile duct [37]. Xu et al. reported that variants in a lipid metabolism-related gene (ABCG8 rs11887534) was also associated with an increased risk of BtC [8]. How- ever, these studies conventionally investigated effect of a single genetic variant on BtC risk alone. The additive effect of other variants was not taken into account. Although some of our results are seemed to be con- tradictory with the generally accepted association, these findings were further validated in sensitivity analyses that with different assumptions. The genetic instruments that we used in the current study were free of weak instru- mental variable bias and therefore could serve as strong indicators for circulating lipid levels. Furthermore, to ensure the robustness of results, we constructed a frame work of MR analysis to avoid the influences of heteroge- neity and pleiotropy. The detected links between lipids and BtC risk are clues for future studies, although our study lacks ability to provide more explanations regard- ing the main findings. gi g The limitations of our study should be noted here. First, our results were based on genetic data from East Asian populations, which limited the possibility of extrapo- lation to other populations. Second, the exposure and outcome studies in two-sample MR analysis should not involve overlapping participants. The participants in BBJ and AGEN might to some extent overlapped. However, Japanese participants only accounted for approximate 7% of AGEN population. Discussion populations owing to BtC were more commonly diag- nosed in populations in East Asian countries and there was lack of large scale GWAS of BtC in other popula- tions [2]. Herein, we tested associations between a total of four lipids and BtC risk leveraging MR analyses with a set of genetic variants as instruments. Paradoxical to the observational studies that reported high levels of serum triglycerides and low level of HDL were associated with risk of BtC [9, 38], we observed an inverse association between genetically determined level of triglyceride and BtC risk, whereas no significant association between HDL level and BtC risk was detected. Moreover, multi- variable MR results suggested that genetically high LDL level was associated with an increased risk of BtC. This association, to our knowledge, was rarely reported in previous studies. In a cross-sectional study, the authors reported a putatively “U-shaped” association between LDL level and BtC risk [9]. In this study, using several MR methods, we tested for a causal relationship between circulating lipid traits and BtC risk. Our results suggested that genetically elevated TRG concentration was associated with a decreased risk of BtC. Multivariable MR analysis revealed that geneti- cally elevated LDL level was associated with an increased risk of BtC, although this result did not detect in con- ventional MR analysis. Our findings were deemed to be robust due to no pleiotropy and heterogeneity was detected and were highly consistent with that of sensitiv- ity analyses. Biliary tract system plays important roles in many met- abolic processes that are critical for the maintenance of body homeostasis [27, 28]. For example, lipid metabo- lism was reported to closely associate with biliary tract (including gallbladder) [29]. Therefore, it is reasonable to assume that damage in this organ may have a reflection in blood lipids. In other word, alterations of circulating lipid levels may suggest an injury in biliary tract. Indeed, a set of epidemiological studies have reported associa- tion of circulating lipid levels with biliary diseases [9, 30, 31]. However, the reported associations were not con- sistent between studies. For instance, results from pre- vious studies of total CHL and LDL with gallstones are conflicting, with some studies reporting inverse, positive, and null associations [31–35]. Andreotti et al. Results 4 The causal effects of circulating lipids on biliary tract cancer from multivariate Mendelian randomization analyses. A, overlap of genetic instruments among the four lipids; B, causal estimates from multivariate Mendelian randomization analysis. Error bars denote 95% confidence interval of the odds ratio (OR) estimates Fig. 4 The causal effects of circulating lipids on biliary tract cancer from multivariate Mendelian randomization analyses. A, overlap of genetic instruments among the four lipids; B, causal estimates from multivariate Mendelian randomization analysis. Error bars denote 95% confidence interval of the odds ratio (OR) estimates all of the three models, we found that TRG were con- sistently associated with a decreased BtC risk (Supple- mentary Figure S5). Likewise, we observed an inverse relationship between TRG level and BtC risk in MVMR analysis in which we further incorporated BMI (Supple- mentary Figure S6). LDL was significantly associated with elevated risk of BtC (OR = 1.32, 95% CI 1.04–2.01). On the contrary, 1-SD increase of inverse-normal transformed TRG was signifi- cantly associated with decreased risk of BtC (OR = 0.54, 95% CI 0.42–0.68) (Fig. 4B). We also performed pairwise MVMR analysis between TRG and other three lipids. In Wang et al. BMC Cancer (2022) 22:273 Wang et al. BMC Cancer (2022) 22:273 Page 7 of 9 Abbreviations BtC Bili t t BtC: Biliary tract cancer; HDL: High-density lipoprotein cholesterol; LDL: Low-density lipoprotein (HDL) cholesterol; CHL: Cholesterol; TRG: Triglyceride; MR: Mendelian Randomization; GWAS: Genome-wide association study; IVW: Inverse variance weighting. Received: 6 July 2021 Accepted: 4 March 2022 Received: 6 July 2021 Accepted: 4 March 2022 References Additional file 1: Table S1.The genetic instruments used in Mendelian analysis for high-density lipoproteincholesterol. Table S2. The gene- ticinstruments used in Mendelian analysis for low-density lipoprotein cholesterol.Table S3. The genetic instrumentsused in Mendelian analysis for total cholesterol. Table S4. The genetic instruments used in Mendelian analysis fortriglyceride. Table S5. Associationof biliary tract cancer with levels of circulating lipids according todifferent. Figure S1. The forestplot of leave-one-out analysis for high-density lipoprotein cholesterol. Figure S2. The forest plot ofleave-one-out analysis for low-density lipoprotein cholesterol. Figure S3. The forest plot of leave-one-outanalysis for total cholesterol. FigureS4. The forest plot of leave-one-out analysis for triglyceride. Figure S5. Results of pairwisemultivariable Mendelian randomization analysis. Figure S6. Results of multivariable Mendelian randomizationanalysis. 1. Tella SH, Kommalapati A, Borad MJ, Mahipal A. Second-line therapies in advanced biliary tract cancers. Lancet Oncol. 2020;21(1):e29–41. 1. Tella SH, Kommalapati A, Borad MJ, Mahipal A. Second-line therapies in advanced biliary tract cancers. Lancet Oncol. 2020;21(1):e29–41. 2. Valle JW, Kelley RK, Nervi B, Oh DY, Zhu AX. Biliary tract cancer. Lancet (London, England). 2021;397(10272):428–44. 2. Valle JW, Kelley RK, Nervi B, Oh DY, Zhu AX. Biliary tract cancer. Lancet (London, England). 2021;397(10272):428–44. 3. Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, Znaor A, Bray F. Global Cancer Observatory: Cancer Today. Lyon, France: International Agency for Research on Cancer. 2020. Available from: https://gco.iarc.fr/ today , accessed Jun 16, 2021.. 3. Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, Znaor A, Bray F. Global Cancer Observatory: Cancer Today. Lyon, France: International Agency for Research on Cancer. 2020. Available from: https://gco.iarc.fr/ today , accessed Jun 16, 2021.. 4. Massarweh NN, El-Serag HB. Epidemiology of Hepatocellular Car- cinoma and Intrahepatic Cholangiocarcinoma. Cancer Control. 2017;24(3):1073274817729245. 5. Lowenfels AB, Lindström CG, Conway MJ, Hastings PR. Gallstones and risk of gallbladder cancer. J Natl Cancer Inst. 1985;75(1):77–80. 6. Hsing AW, Gao YT, Han TQ, Rashid A, Sakoda LC, Wang BS, Shen MC, Zhang BH, Niwa S, Chen J, et al. Gallstones and the risk of biliary tract can- cer: a population-based study in China. Br J Cancer. 2007;97(11):1577–82. Ethics approval and consent to participate This study only used publicly available data. No original data were collected. Ethical approval for each of the studies included in the investigation can be found in the original publications. All methods were carried out in accordance with relevant guidelines and regulations. In conclusion, according to both univariate and multi- variate MR estimates, genetically determined higher tri- glyceride level is associated with lower risk of BtC. On the contrary, genetically elevated LDL concentration is associated with higher risk of BtC according to multivari- ate MR estimate. Our findings suggest that circulating lipids may have roles in the development of BtC and have potentials to be prediagnostic biomarkers for BtC. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. Discussion Third, the genetic data of BtC were derived from GWAS with limited number of cancer cases, which might introduce bias into GWAS results due to unbalanced case–control ratios. Larger GWAS will allow for more precision in the estimates of SNPs used as instruments in future MR. Fourth, our estimates might also subject to the inherent pitfalls of MR analysis such as selection bias [39]. Genetic variants which are related to specific phenotypes might also related to participation. For example, participants with high polygenic risk score for the circulating lipids might be more likely to drop- out in the cohort because they might be more susceptible to diseases such as chronic cardiovascular disease than those have low genetic risk of lipid traits. Moreover, the f In our study, we retrieved the GWAS summary statis- tics regarding blood lipid traits and BtC from East Asian Wang et al. BMC Cancer (2022) 22:273 Page 8 of 9 MR estimates might be confounded by other unobserved environmental factors [40]. For example, in our study, we cannot correct the effect of lipid-lowering medicine, and the circulating levels of lipids are susceptible to transi- tory fluctuations due to many reasons. These potential factors might bias the GWAS results of circulating lipids. Finally, all the results from IVW method were underpow- ered (< 80%), although we conducted a rigorous quality- control process. Further investigations with larger sample size on associations between circulating lipids and BtC risk are needed. Availability of data and materials The datasets generated and/or analysed during the current study are available in the following repository: GWAS summary data of circulating lipids were available on the Asian Genetic Epidemiology Network website (AGEN; https://blog.nus.edu.sg/agen/). GWAS summary data of biliary tract cancer were available on IEU Open GWAS project website (https://pheweb.jp/pheno/BtC). The datasets generated and/or analysed during the current study are available in the following repository: GWAS summary data of circulating lipids were available on the Asian Genetic Epidemiology Network website (AGEN; https://blog.nus.edu.sg/agen/). GWAS summary data of biliary tract cancer were available on IEU Open GWAS project website (https://pheweb.jp/pheno/BtC). Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12885-022-09382-x. Competing interests Th h d l h The authors declare that they have no competing interests. Acknowledgements Not applicable 7. Bowlus CL, Lim JK, Lindor KD. AGA Clinical Practice Update on Surveil- lance for Hepatobiliary Cancers in Patients With Primary Sclerosing Chol- angitis: Expert Review. Clin Gastroenterol Hepatol. 2019;17(12):2416–22. 7. Bowlus CL, Lim JK, Lindor KD. AGA Clinical Practice Update on Surveil- lance for Hepatobiliary Cancers in Patients With Primary Sclerosing Chol- angitis: Expert Review. Clin Gastroenterol Hepatol. 2019;17(12):2416–22. Authors’ contributions 8. Xu HL, Cheng JR, Andreotti G, Gao YT, Rashid A, Wang BS, Shen MC, Chu LW, Yu K, Hsing AW. Cholesterol metabolism gene polymorphisms and the risk of biliary tract cancers and stones: a population-based case- control study in Shanghai. Chin Carcinogen. 2011;32(1):58–62. JW, XH, and WS conceived the idea for the study. JW, JZ and DF obtained the genetic data. JW, JZ, DF, and BZ performed the data analyses. JW, JZ and BZ interpreted the results of the data analyses. JW, JZ and DF wrote the manuscript. JX, DZ, ZF, XH, and WS read and approved the final manuscript. control study in Shanghai. Chin Carcinogen. 2011;32(1):58–6 9. Andreotti G, Chen J, Gao YT, Rashid A, Chang SC, Shen MC, Wang BS, Han TQ, Zhang BH, Danforth KN, et al. 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Publisher’s Note Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 30. Morán S, Duque-López MX, Salmerón-Castro J, Rodríguez-Leal G, Martínez-Salgado H, Uribe M. Association between serum concentration of apolipoproteins A-I and B with gallbladder disease. Arch Med Res. 2003;34(3):194–9. 31. Wang J, Shen S, Wang B, Ni X, Liu H, Ni X, Yu R, Suo T, Liu H. Serum lipid levels are the risk factors of gallbladder stones: a population-based study in China. 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https://openalex.org/W2746276356	https://hal.inria.fr/hal-01674857/document	English	null	Designing an Open Architecture for the Creative Industry	IFIP advances in information and communication technology	2,017	cc-by	3,358	To cite this version: Christian Zinke, Michael Becker, Stephan Klingner. Designing an Open Architecture for the Cre- ative Industry. 18th Working Conference on Virtual Enterprises (PROVE), Sep 2017, Vicenza, Italy. pp.696-703, 10.1007/978-3-319-65151-4_62. hal-01674857 Distributed under a Creative Commons Attribution 4.0 International License Designing an Open Architecture for the Creative Industry Christian Zinke, Michael Becker and Stephan Klingner Institute for Applied Informatics at the University of Leipzig, Hainstraße 11, Leipzig, Germany {zinke\|becker\|klingner}@infai.org Institute for Applied Informatics at the University of Leipzig, Hainstraße 11, Leipzig, Germany {zinke\|becker\|klingner}@infai.org Abstract. Companies in the Culture and Creative Industry are characterized by highly networked value chains. However, this value network lacks a profound support in terms of information technology and structure resulting in time- consuming and error-prone manual labor. To overcome these challenges, we conceptualize an application framework for creative industries. In particular a software architecture design and a data design will be proposed with the help of a design science research approach. The goal of the resulting application framework is to support the digital transformation of companies in the creative industry towards collaborative networks. Keywords: Music Business, Framework, Design Science Research, Collaborative Networks. Keywords: Music Business, Framework, Design Science Research, Collaborative Networks. Keywords: Music Business, Framework, Design Science Research, Collaborative Networks. HAL Id: hal-01674857 https://inria.hal.science/hal-01674857v1 Submitted on 3 Jan 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction The Culture and Creative Industry (CCI) is an emerging sector, with progressively increased significance. In 2014 the core sector of CCI had 4.4% of total GDP (558 billion Euro) and 3.8% of total EU workforce [1]. The current trend of digitalization leads to significant changes in the value chain of CCI, especially in music business [2– 5]. For example, through digitization music production does no longer require a recording studio and new digital distribution channels like music and video streaming portals weaken the influence of major labels [3]. While these developments are headed by major companies, small enterprises lack accurate digital support within their value chain. This became a significant challenge for the domain as recent studies show [6]: g g  The majority of companies in CCI are very small and small enterprises (99 %)  An average of 4.33 employee per enterprise renders an efficient division of work impossible.  Cooperation and highly interactive networks are a substantial part of the value chain (93 % of all enterprises) Thus, the CCI in general and music business in particular, are already highly networked. According to Ewig’s [7] collaboration business transformation pyramid the described small enterprises can be classified as a network on organizational structure level. This is challenged by a lack of information structure and technology, which 678 C. Zinke et al. includes the absence of unified and standardized exchange formats within the domain. Due to the wide variety of data formats, manual consolidation of data sets is necessary, which is time-consuming and error-prone. Thus, SMEs in music business have a data- based information structure – which means that there is no cross-functional information flow [8]. In addition, conducted questionnaires and workshops show high usage of proprietary software (like Excel) as well as a lot of manual work, which can be categorized as a very low information technology maturity (see Figure 1). Though the dimensions of Ewig’s business collaboration pyramid have interaction effects with each other, the remainder of this paper focuses on information structure and information technology due to space limitations. We aim at conceptualizing an Information System (IS) and, thus, for our paper organizational questions are of minor importance. Analytically, small enterprises are on a preliminary stage to (goal-oriented) collaborative network organizations (CNOs) [9], lacking suitable information technology. Among others the forms of CNOs are virtual organizations or virtual enterprises [10]. Fig. 1 Introduction 1 Maturity of small enterprises within music business, according to Ewig [7] Fig. 1 Maturity of small enterprises within music business, according to Ewig [7] The huge value network of the music industry and the resulting huge amount of requirements are challenging. Resulting mass-customization, from single worker (local workspace) via enterprise level (server) to collaborative network (cloud and web services), require a very flexible approach. However, the small stakeholders in music business lack the time, money and software solutions that fit their specific needs. Thus, open and scalable solutions are necessary to support the transformation process to CNOs. In summary, SME in music business need improved information structure and technology in order to evolve to a CNO (virtual network). Both will be addressed within this paper with the help of a design science research approach. The leading research question is, how to design an IS architecture to support digital transformation of SME in music industry? 679 Designing an Open Architecture for the Creative Industry 2 Methodology The presented paper is conducted through an IS Design Science Research approach. The major task and result of design science research is to create an artefact, which is “by definition, a purposeful IT artifact created to address an important organizational problem” [11]. We will focus on the design of a solution for the given problem. The detailed requirements – briefly described in chapter 1 – are derived from workshops and expert interviews with stakeholders of the music industry as well as analysis of available documentations (e.g. from label management or DDEX). Following common design science research methodologies for IS, the next step from requirements is the design of a possible solution, which will be presented next [12]. As a research in process this work is limited to a conceptual model as an artefact and cannot provide a concrete implementation or an evaluation [12]. Further on, the proposed concept will be used to iterate and collect additional requirements for real world application in order develop requirements that supports technical decisions on costs, complexity, performance and applicability. In order to evolve the focused dimensions of information structure and technology in music business, the paper focuses on Software Architecture Design and Data Design [13]. Data Design [13]. 1 http://dublincore.org/ 3 Design Amongst other, Software Architecture Design can be defined as “a high-level abstraction of software systems” [14]. The presented requirements for such a software system architecture can be summarized as follows:  Scalable from local applications to network solutions  Support the digital exchange within music business value chain Support the digital exchange within music business  Flexible enhancement As shown in Fig. 2, the proposed architecture of the framework consists of a runtime environment providing horizontal services. On top of the general environment, the installation of various small applications (so-called apps) shall be possible in order to support the individual value chain for each stakeholder within the value network. Each app provides specific functionalities that are required by one or more stakeholders. The app management layer is responsible for integrating apps into the runtime environment. The apps are managed by the framework through simple policies (e.g. naming conventions, meta-data for apps (Dublin Core1), data access rules, dependencies) [15]. The management layer ensures the unified and standardized structuring of apps. In order to enable data exchange between different software systems and the platform interfaces are needed. Interfaces to other software systems are the first step moving to the integration stage on information technology level of the business collaboration pyramid [7]. However, the apps should be integrated on data side through common data schemas and protected through access rules. 1 http://dublincore.org/ 680 2 http://wiki.dbpedia.org/ 3 https://github.com/LinkedBrainz/MusicBrainz-R2RML 3 https://github.com/LinkedBrainz/MusicBrainz-R2RML 2 http://wiki.dbpedia.org/ Designing an Open Architecture for the Creative Industry 681 suitable because heterogeneous data sources can be used across the value network, which will boost production efficiency of the small enterprises. However, Linked Data may lack in performance and practicability. Fig. 3 MUWI-App architecture User Interface Meta-data Dependencies Name Internal Data Processing Services LD Data Schema LD Data Source Platform Version Interfaces Fig. 3 MUWI-App architecture The proposed framework allows to create flexible apps for specific purposes, which access and enhance information and knowledge within the network (on server or via internet) easily due to the linked data approach. On information technology side, the approach goes beyond the integration level, because it is not about integration of existing tools, it will combine information from various sources and supports information flows all over the value network. Due to the linked data approach, the provided information are stored as graphs and new knowledge can be easily discovered and extracted [20]. Thus, the approach has the potential to boost the music business to a knowledge level of information structure and network level on information technology side. C. Zinke et al. The message exchange layer enables the communication between different apps. Therefore, this layer may use the Music Business Ontology (MBO) [16] which specifies a unified message format. Based on this format, apps can access data provided by the data storage layer. The unified format allows for transformation of company-specific formats and, thus, reduces transaction costs. Every company might maintain its own local installation of the framework. In addition to these local installations, a service directory containing apps has to be established. While an internal service directory only contains dedicated apps, an external directory which is available via internet connection, provides access to third party apps. Fig. 2 Architecture to support small enterprises in music business Fig. 2 Architecture to support small enterprises in music business We will use the term framework, which consist of a set of elements and its relations, including economical, organizational and software-technical elements. An application framework is such a framework for other software artefacts (apps). An app is the smallest executable software artefact, which provides a specific functionality or service to the user. The proposed architecture of the app is described in Figure 3. In particular, an app has two main components: the user interface and the internal data processing services. In order to ensure compatibility to the platform three information are mandatory: a name (conforming to naming conventions), meta-data (see Dublin Core) and the dependencies. The dependencies are important to ensure functionality of the apps. In order to do so, we suggest that the used interfaces (e.g. to ERP or CRM systems), Linked Data (LD) Data Schema, LD Data Sources (e.g. SPARQL endpoints) and the platform version need to be described. Besides software architecture, data design is an important aspect in transformation of companies towards collaborative networks. As stated above, open and scalable solutions are required and, thus, a linked data approach is proposed. Linked data is an approach applied for creating and distributing structured data on the Web with the help of RDF (Resource Description Framework) graphs. In order to make data accessible and enable automated processing, the Linked Data approach extents the vision of Semantic Web [17, 18]. Big players like BBC (British Broadcasting Corporation) already use this technology (with DBPedia2 and MusicBrainz3) [19]. Linked data is Designing an Open Architecture for the Creative Industry 4 Related Work This work has a strong link to software ecosystem (SE) research, which understands SE as “a set of actors functioning as a unit and interacting with a shared market for software and services, together with the relationships among them. These relationships are frequently underpinned by a common technological platform or market and operate through the exchange of information, resources and artefacts.” [21]. The MUWISTAR framework is a kind of a software ecosystem, and further a two-sided market solution, which addresses developers as well as customers [22]. Thus, the framework is a kind of a hub – an exchange platform [23], with many-to-many relations within the software supply chain [24]. While research on software ecosystem focuses on reports on procedures [25], the current work concentrates on software and data design. The framework allows for using apps as a kind of software component or plugin. However, the research is also linked to software component research. There is a huge variety of components definitions and approaches, e.g. [26, 27]. By lack of space, we only introduce a small selection of software component literature. According to Heineman and Councill “[a] software component is a software element that conforms 682 C. Zinke et al. to a component model and can be independently deployed and composed without modification according to a composition standard.” [28]. The definition is extended by two other definitions: the component model and the software component infrastructure [27, 28]. The component model specifies the composition and interaction [28] – also called bindings [29]. The infrastructure ensures the compliant performance of the components [28], also described as system platform [29], which is the presented application framework. Software components approaches are quite similar to plug-in architectures, with practical differences in simplicity of configuration and administration [27]. Designing an Open Architecture for the Creative Industry Designing an Open Architecture for the Creative Industry Acknowledgments. This work has been done within the frame of the joint research project “MUWISTAR” which was funded by the German Ministry of Education and Research (Grant Number: 01IS16018B) under the supervision of the PT-DLR. The authors thank for the funding. References 1. Forum D’avignon: The economic contribution of the creative industries to EU GDP and employment. http://www.forum-avignon.org/sites/default/files/editeur/2014-Oct- European-Creative-Industry-GDP-Jobs-full-Report-ENG.pdf (2014) 1. Forum D’avignon: The economic contribution of the creative industries to EU GDP and employment. http://www.forum-avignon.org/sites/default/files/editeur/2014-Oct- European-Creative-Industry-GDP-Jobs-full-Report-ENG.pdf (2014) p y p p ( ) 2. Bauckhage, T.: Das Ende vom Lied. Zum Einfluss der Digitalisierung auf die internationale Musikindustrie. Stuttgart, 113f (2002) g , ( ) 3. Kusek, D., Leonhard, G., Lindsay, S.G.: The future of music. Manifesto for the digital i l ti B kl P B t (2005) g , ( ) 3. Kusek, D., Leonhard, G., Lindsay, S.G.: The future of music. Manifesto for the digital music revolution. Berklee Press, Boston (2005) g ( 3. Kusek, D., Leonhard, G., Lindsay, S.G.: The fut music revolution. Berklee Press, Boston (2005) music revolution. Berklee Press, Boston (2005) 4. Moreau, F.: The Disruptive Nature of Digitization: The Case of the Recorded Music Industry. International Journal of Arts Management 15(2), 18–31 (2013) 5. Bourreau, M., Moreau, F., Gensollen, M.: The Digitization of the Recorded Music Industry. Impact on Business Models and Scenarios of Evolution. SSRN Journal (2008). doi: 10.2139/ssrn.1092138 6. Bertschek, I., Ohnemus, J., Erdsiek, D., Kimpeler, S., Rammer, C., Shala, E.: Monitoringbericht 2016: Ausgewählte wirtschaftliche Eckdaten der Kultur-und Kreativwirtschaft. Langfassung. ZEW-Gutachten und Forschungsberichte (2017) 7. Ewig, M.: Der Transformationsprozess zum collaborative business. Eine strategische, organisatorische und informatorische Betrachtung. Schriften zur Wirtschaftsinformatik, Bd. 17. Lang, Frankfurt am Main, New York (2006) 8. Ehrenberg, D., Ewig, M.: Collaborative Business — eine Herausforderung für die Wirtschaftsinformatik. In: Fink, K., Ploder, C. (eds.) Wirtschaftsinformatik als Schlüss zum Unternehmenserfolg, pp. 43–71. DUV, Wiesbaden (2006) 9. Camarinha-Matos, L.M., Afsarmanesh, H.: On reference models for collaborative networked organizations. International Journal of Production Research (2008). doi: 10.1080/00207540701737666 10. Camarinha-Matos, L.M., Afsarmanesh, H.: Collaborative networks. A new scientific discipline. J Intell Manuf (2005). doi: 10.1007/s10845-005-1656-3 12. Peffers, K., Tuunanen, T., Rothenberger, M.A., Chatterjee, S.: A Design Science Research Methodology for Information Systems Research. J. of Management Information Systems 24(3), 45–77 (2008) 13. Chatterjee, S., Hevner, A.: Design Research in Information Systems. Theory and Practice, 1st edn. Integrated series in information systems, vol. 22. Springer US, Berlin (2010) 14. Solms, F.: Software Architecture Design (2014) 15. Weinreich, R., Sametinger, J.: Component models and component services: Concepts and principles. Component-Based Software Engineering: Putting Pieces Together, 22–64 (2001) 16. Schumacher, F., Gey, R., Klingner, S.: Semantics for the music industry. 5 Conclusion and Further Work The progress of digitalization led to various challenges in the music business. While in the past the focus was on the substitution of physical distribution of music with digital alternatives, with the ascension of streaming and downloads today's challenges lie in the collection, processing and analyzing of data. To master the data, adequate software support is required. Consequently, the paper proposes a concept for a software architecture and data design as a solution for the current challenges for the music business. This solution aims at providing a powerful, extendable software support, suitable also for the various individual companies and small and medium enterprises (SMEs). However, also organizational questions are important and one of the dimensions of collaborative networks, which have been also addressed in further works. Yet this architecture is still mainly in the conceptual phase, so that various proposed technical approaches might be subject to changes to better suit the needs of the music business domain. As the concepts represent the best option from a theoretical perspective, it might be possible that requirements occur during implementation and practical evaluation, leading to architectural or technical changes. For example domain- or technology-specific parameters can lead to the substitution of the semantic web approach with common data format approaches, since technical decisions are always a trade-off between costs, complexity, performance and applicability. The technically most advanced solution does not inevitably represent the best approach. Therefore, the priority during the implementation is to create an easy usable and affordable system suiting the needs for the multitude of small and medium enterprises in the music business. This might require a revision of the technical concepts outlined in the paper. Currently we are working on a first implementation of the presented concept as an open source solution. After developing a first prototype a workshop based evaluation with domain experts will follow. In the long run, we aim to establish a framework community within CCI, aiming at developers as well as users. At this moment the work is still in process and there are no technical artifacts developed yet. Following Gregor and Hevner [30], the solution maturity of the presented case is low, while the domain maturity is high – thus our solution have research opportunities and knowledge contribution for the domain (improvement). 683 References In: Sack, H., Filipowska, A., Lehmann, J., Hellmann, S. 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https://openalex.org/W4367053409	https://www.frontiersin.org/articles/10.3389/fgene.2023.1131182/pdf	English	null	Paget’s disease: a review of the epidemiology, etiology, genetics, and treatment	Frontiers in genetics	2,023	cc-by	9,582	OPEN ACCESS Babajan Banaganapalli1,2, Ibrahim Fallatah 1, Fai Alsubhi1, Preetha Jayasheela Shetty3, Zuhier Awan 4, Ramu Elango1,2* and Noor Ahmad Shaik1,2* Babajan Banaganapalli1,2, Ibrahim Fallatah 1, Fai Alsubhi1, Preetha Jayasheela Shetty3, Zuhier Awan 4, Ramu Elango1,2* and Noor Ahmad Shaik1,2* Babajan Banaganapalli1,2, Ibrahim Fallatah 1, Fai Alsubhi1, Preetha Jayasheela Shetty3, Zuhier Awan 4, Ramu Elango1,2* and Noor Ahmad Shaik1,2* Babajan Banaganapalli1,2, Ibrahim Fallatah 1, Fai Alsubhi1, Preetha Jayasheela Shetty3, Zuhier Awan 4, Ramu Elango1,2* and Noor Ahmad Shaik1,2* 1Department of Genetic Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia, 2Princess Al-Jawhara Al-Brahim Center of Excellence in Research of Hereditary Disorders, King Abdulaziz University, Jeddah, Saudi Arabia, 3Department of Biomedical Sciences, College of Medicine, Gulf Medical University, Ajman, United Arab Emirates, 4Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia Paget’s disease of bone (PDB) is the second most prevalent metabolic bone disorder worldwide, with a prevalence rate of 1.5%–8.3%. It is characterized by localized areas of accelerated, disorganized, and excessive bone production and turnover. Typically, PDB develops in the later stages of life, particularly in the late 50s, and affects men more frequently than women. PDB is a complex disease inﬂuenced by both genetic and environmental factors. PDB has a complex genetic basis involving multiple genes, with SQSTM1 being the gene most frequently associated with its development. Mutations affecting the UBA domain of SQSTM1 have been detected in both familial and sporadic PDB cases, and these mutations are often associated with severe clinical expression. Germline mutations in other genes such as TNFRSF11A, ZNF687 and PFN1, have also been associated with the development of the disease. Genetic association studies have also uncovered several PDB predisposing risk genes contributing to the disease pathology and severity. Epigenetic modiﬁcations of genes involved in bone remodelling and regulation, including RANKL, OPG, HDAC2, DNMT1, and SQSTM1, have been implicated in the development and progression of Paget’s disease of bone, providing insight into the molecular basis of the disease and potential targets for therapeutic intervention. Although PDB has a tendency to cluster within families, the variable severity of the disease across family members, coupled with decreasing incidence rates, indicates that environmental factors may also play a role in the pathophysiology of PDB. The precise nature of these environmental triggers and how they interact with genetic determinants remain poorly understood. TYPE Review PUBLISHED 26 April 2023 DOI 10.3389/fgene.2023.1131182 TYPE Review PUBLISHED 26 April 2023 DOI 10.3389/fgene.2023.1131182 Paget’s disease of bone, genetics factors, environmental factors, osteoclast (OCs), SQSTM1 OPEN ACCESS Fortunately, majority of PDB patients can achieve long-term remission with an intravenous infusion of aminobisphosphonates, such as zoledronic acid. In this review, we discuss aspects like clinical characteristics, genetic foundation, and latest updates in PDB research. Paget’s disease: a review of the epidemiology, etiology, genetics, and treatment OPEN ACCESS EDITED BY Luis Corral-Gudino, Universidad de Valladolid, Spain REVIEWED BY Javier Del Pino, University of Salamanca, Spain Sher Zaman Saﬁ, Mahsa University, Malaysia *CORRESPONDENCE Ramu Elango, relango@kau.edu.sa Noor Ahmad Shaik, nshaik@kau.edu.sa RECEIVED 24 December 2022 ACCEPTED 17 April 2023 PUBLISHED 26 April 2023 CITATION Banaganapalli B, Fallatah I, Alsubhi F, Shetty PJ, Awan Z, Elango R and Shaik NA (2023), Paget’s disease: a review of the epidemiology, etiology, genetics, and treatment. Front. Genet. 14:1131182. doi: 10.3389/fgene.2023.1131182 Front. Genet. 14:1131182. doi: 10.3389/fgene.2023.1131182 Epidemiology of PDB PDB is the second most prevalent disorder of bone remodeling, after osteoporosis, despite it being asymptomatic in many with variable late age of onset. The incidence of PDB varies depending on the population studied and the diagnostic criteria used, but it is generally considered to be a rare disease. The global prevalence of PDB ranges from 1.5% to 8.3%, highest in Europeans living in the United Kingdom, followed by Australia, New Zealand, North America, France, Germany, Spain and Italy. Conversely, it appears rare among Scandinavians, Africans, Asians, and non- European immigrants living in Europe (Vallet and Ralston, 2016; Nebot Valenzuela and Pietschmann, 2017; Abdulla et al., 2018). Among Middle East Arabians in southern Israel revealed a 1% prevalence of PDB, comparable to southern Europe. In Saudi Arabia, Iran, Iraq, and Turkey, few isolated case reports of PDB have been recorded (Alshaikh et al., 2011; Merashli and Jawad, 2015; Michou and Orcel, 2019). The above epidemiological data conﬁrms that there is marked geographical variation in the occurrence of PDB. But whether this is linked to genetic susceptibility of speciﬁc ethnic or racial population groups, and/or potential environmental inﬂuences like diet (mineral and vitamin deﬁciencies), lifestyle (tobacco smoking) exposure to the pollutants (lead), and an infection (paramyxovirus) is unclear. FIGURE 1 Illustrates the major skeletal locations affected by PDB: “The disorder predominantly affects the axial skeleton, with the highest incidence observed in the pelvis (70%), femur (55%), lumbar spine (53%), cranium (42%), and tibia (32%). To provide a visual representation of the most commonly affected areas in PDB, these locations are highlighted in red." FIGURE 1 Illustrates the major skeletal locations affected by PDB: “The disorder predominantly affects the axial skeleton, with the highest incidence observed in the pelvis (70%), femur (55%), lumbar spine (53%), cranium (42%), and tibia (32%). To provide a visual representation of the most commonly affected areas in PDB, these locations are highlighted in red." areas. In rare cases, pathological fractures may occur. Leontiasis ossea, a condition in which the facial bones become deformed, can also occur. The most severe consequences of PDB include osteosarcomas and other sarcomas (chondrosarcoma, ﬁbrosarcoma), though their incidence is less than 1% (Makaram and Ralston, 2021). Biochemical assessment of PDB Paget’s disease of bone (PDB) can be diagnosed through clinical, radiographic, and biochemical assessments. Biochemical assessment involves measuring serum alkaline phosphatase (ALP) and urinary N-telopeptide (NTx) levels. ALP is an enzyme produced by osteoblasts, responsible for bone formation. In PDB, bone formation increases, leading to elevated ALP levels (Gennari et al., 2019). However, ALP levels can also be elevated in other conditions, such as liver disease or during pregnancy, making additional tests necessary for diagnosis conﬁrmation (Seton, 2013; Gennari et al., 2019). In contrast, NTx is a speciﬁc marker of bone resorption, where osteoclasts break down bone tissue. In PDB, there is an increase in both bone formation and resorption, leading to elevated NTx levels. However, NTx levels can be affected by other factors, such as age, gender, and menopausal status, necessitating interpretation in the context of clinical ﬁndings (Delmas, 1999). Additional tests, including bone scans and Epidemiology of PDB In addition, PDB primarily affects the skeletal system, with certain regions being more prone to its impact than others (Makaram et al., 2020), as seen in Figure 1, the axial skeleton is the most commonly affected area (Figure 2). COPYRIGHT COPYRIGHT © 2023 Banaganapalli, Fallatah, Alsubhi, Shetty, Awan, Elango and Shaik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 01 Frontiers in Genetics frontiersin.org Banaganapalli et al. 10.3389/fgene.2023.1131182 Introduction FIGURE 1 Illustrates the major skeletal locations affected by PDB: “The disorder predominantly affects the axial skeleton, with the highest incidence observed in the pelvis (70%), femur (55%), lumbar spine (53%), cranium (42%), and tibia (32%). To provide a visual representation of the most commonly affected areas in PDB, these locations are highlighted in red." Paget’s disease of bone (PDB) is a chronic and progressive bone disease that is characterized by bone pain, deformities, and fractures. The term “osteitis deformans” was ﬁrst coined by Sir James Paget, an English physician, in 1877 (Paget, 1877). However, evidence of the disease dates back to 3,000 years, as suggested by lesions resembling PDB found in dinosaur vertebrae from the late Paleozoic to the middle Mesozoic periods (Haridy et al., 2019; Gennari et al., 2022). PDB is characterized by abnormal activation of osteoclasts, which leads to improper bone resorption and compensatory osteogenic sclerosis (Tuck, 2020). The disease is associated with increased bone remodeling and mass, with abnormal osteoclast activity leading to increased metabolic osteolytic activity while osteoblasts produce bone normally (Alonso et al., 2017). Frontiers in Genetics frontiersin.org Clinical manifestations The classical PDB usually appears at the age of forty and is rarely diagnosed before the age of ﬁfty, with a slight male predominance (Vallet and Ralston, 2016). In around 70% of patients, PDB is asymptomatic and is typically discovered incidentally through elevated alkaline phosphatase (ALP) values (Kravets, 2018). When symptoms are present, the most frequent symptom is bone pain (73.8%), followed by morphological conditions (18.1%), hearing loss (7.9%), and pathological fractures (5.7%) (Ralston and Albagha, 2018; Michou and Orcel, 2019). Bone deformities, such as bowing of the legs, skull enlargement, and kyphosis, may also occur. Hearing loss, vision problems, and headaches can result from cranial nerve involvement. Increased blood ﬂow to the bones can cause warmth and redness in affected 02 frontiersin.org Banaganapalli et al. 10.3389/fgene.2023.1131182 FIGURE 2 A summary of the diagnosis, management, and treatment approaches to Paget’s disease of bone. FIGURE 2 A summary of the diagnosis, management, and treatment approaches to Paget’s disease of bone. GURE 2 summary of the diagnosis, management, and treatment approaches to Paget’s disease of bone. signiﬁcant osteolytic regions in the skull. Early-stage PDB may show primarily lytic lesions, while older lesions tend to have a mixed sclerotic and lytic appearance. Late-stage PDB is characterized by sclerotic lesions, enlarged and distorted bones, and distinct radiographic patterns (Gennari et al., 2019; Ralston et al., 2019). The damaged bone enlargement in diameter is a distinguishing feature of PDB. A set of plain X-rays and bone scintigraphy are used to evaluate PDB, but bone scintigraphy may be negative in some cases. The presence of aberrant trabeculae, irregular cementation lines, increased vascularity, and an increase in the number and size of osteoclasts are the most distinctive ﬁndings (Alonso et al., 2017). Since genetic or bone biomarkers alone may lack sensitivity, combining many diagnostic markers is preferable to detect PDB at early stages and in asymptomatic cases. The PDB phenotype may be detected more accurately by integrating a screen for SQSTM1 gene mutations, followed by either a gene panel or a combination of genetic and biochemical tests (Guay-Bélanger et al., 2016). Approximately 70% of PDB patients have no symptoms, making early diagnosis challenging (Merchant et al., 2009). imaging studies, may also be useful in PDB diagnosis to assess the extent of the disease and risk of fractures. Clinical manifestations Serum procollagen type 1 amino-terminal peptide (P1NP), osteocalcin, and bone-speciﬁc ALP (BALP) are more sensitive biochemical markers for bone production, while peptides of the collagen type I cross-linking domains, including NTx or CTX, are more speciﬁc for bone resorption. Despite the characteristic elevations in ALP and NTx levels in individuals with PDB, these markers are not speciﬁc to the condition (Cook and Wall, 2021). Thus, diagnostic conﬁrmation and treatment response monitoring require interpretation in the context of clinical ﬁndings and other diagnostic tests (Alonso et al., 2017). Frontiers in Genetics Genetic factors in PDB Multiple lines of evidence suggest that inherited factors play an important role in PDB. The cumulative risk for developing PDB for a ﬁrst-degree relative of an individual with PDB is estimated to be 9%, compared to 2% for those with unaffected relatives (Gennari et al., 2019). Recent evidence suggests that up to 40% of the PDB patients have a positive family history of PDB related symptoms or diseases. (Morales-Piga et al., 1995; Gennari et al., 2019; Gennari et al., 2022). At least one-third of patients have an autosomal dominant inheritance pattern with higher penetrance with age, risk in ﬁrst-degree relatives will be as high as 50% (Ralston and Albagha, 2018; Gennari et al., 2019; Gennari et al., 2022). Though ~40% of PDB patients had a positive family history for the illness, exclusion of undiagnosed, asymptomatic individuals makes it hard to determine the true incidence of familial disease (Morales-Piga et al., 1995). Intriguingly, the proportion of patients with a family history varies signiﬁcantly between countries, ranging from approximately 5% in the Netherlands to 50% in the French- Canadian population. Between 12% and 15% of PDB patients in the United Kingdom and Italy have a family history (Merlotti et al., 2005; Langston et al., 2010). The marked ethnic differences in PDB prevalence support a strong genetic basis (Morales-Piga et al., 1995; Hocking et al., 2000; Morissette et al., 2006). Etiopathogenesis The primary cause of PDB is believed to be the abnormal activation of osteoclasts, leading to improper bone resorption and compensatory osteogenic sclerosis. This results in increased bone remodeling and mass, with the osteoclasts being larger in size, number, and with more nuclei compared to normal cells (Albagha, 2015; Gennari et al., 2019). This increased metabolic osteolytic activity leads to bone destruction, but the normal osteoblasts continue to produce new bone. The result is structurally abnormal and weakened bones that are prone to fractures and deformities (Gennari et al., 2019). It is worth noting that while the activation of osteoclasts is the primary cause of PDB, the etiology of this activation is not yet fully understood. However, PDB is largely considered a multifactorial disease due to the combination of genetic and environmental factors contribute to its disease pathology. The genetic basis of PDB is complex and involves multiple genes. One of the most commonly implicated genes is SQSTM1, which encodes a multifunctional p62 protein (Shaik et al., 2021). Mutations in this gene have been found in up to 50% of people with familial PDB (Appelman-Dijkstra and Papapoulos, 2018). The p62 protein plays a crucial role in autophagy, a process involved in removing damaged cellular components. When this function is compromised, it can lead to the accumulation of damaged proteins in bone cells. The mutations in the SQSTM1 gene are responsible for both sporadic (10%–15%) and familial (25%–40%) forms of PDB. Studies have shown that the Pro392Leu mutation is present in about 46% of familial PDB cases of French-Canadian origin from Quebec, while two more mutations were found in a family of predominantly British background. In both studies, the P392L mutation was identiﬁed in 16% and 8.9% of sporadic PDB patients (Hocking et al., 2002; Laurin et al., 2002). Although SQSTM1 mutations are typically heterozygous, rare instances of homozygosity have also been reported (Rea et al., 2009). Over 30 distinct missense or truncating SQSTM1 mutations have been detected in up to 50% of familial and 20% of sporadic PDB cases in diverse populations (Gennari et al., 2019). In Hungary, about 21.95% of PDB patients carry the common p. Pro392Leu mutation in the SQSTM1 gene (Donáth et al., 2021). Radiological presentations in PDB Radiographic changes can help diagnose Paget’s disease of bone (PDB). Indicators of increased bone resorption include a decrease in bone density, wedge-shaped bone resorption in long bones, and 03 frontiersin.org 10.3389/fgene.2023.1131182 10.3389/fgene.2023.1131182 Banaganapalli et al. However, 15%–40% of patients have a positive family history, and ﬁrst-degree relatives have a higher risk of developing PDB. Screening with a serum alkaline phosphatase test every two to 3 years is recommended for at-risk family members (Seton, 2013). Paget’s disease can lead to complications such as bone deformities, fractures, osteoarthritis, and an increased risk of developing bone cancer (Sabharwal et al., 2014). For instance, patients with PDB have an elevated chance of developing osteosarcoma, and despite its rarity (0.3% of PDB individuals), vast majority of osteosarcomas (OS) diagnosed in adulthood occur in patients with PDB. Similarly, there have been observation of families in which PDB is accompanied by giant cell tumours (Makaram and Ralston, 2021). Early detection and treatment are crucial in preventing these complications and improving patient outcomes. Radiographs should be taken to conﬁrm the diagnosis (Ralston et al., 2019), and targeted genetic testing can be offered for at-risk family members (Seton, 2013; Guay-Bélanger et al., 2016). a decoy receptor, binds to RANKL to prevent RANK binding. Thereby, OPG suppresses the differentiation of osteoclasts. RANKL is expressed in the marrow stroma and on osteoblasts, when RANK binds on osteoclast precursors, it increases osteoclast proliferation and differentiation. It leads to the activation of a variety of downstream signaling pathways, including the nuclear factor kB (NF-kB), protein kinase B, c-jun N-terminal kinase, p38 mitogen- activated protein kinase and ERK pathways (Figure 3) (Tekkesin et al., 2011; Bellido et al., 2014). Frontiers in Genetics Molecular factors in PDB Normal adult skeleton remodeling involves osteoclasts destroying bone and osteoblasts generating new bone tissue at locations of past bone resorption (Kenkre and Bassett, 2018). The remodeling cycle is highly controlled and stereotypical, with ﬁve overlapping phases of activation, resorption, reversal, formation, and termination in cortical and trabecular bone respectively, within a period of 120–200 days. Osteocytes orchestrate the bone remodeling process by controlling osteoclast and osteoblast differentiation, and hence bone resorption and synthesis (Bellido et al., 2014). Several genes have been linked to osteoclast differentiation and activation which in turn leads to bone resorption (Albagha, 2015; Chen et al., 2018; Ralston and Albagha, 2018). At the cellular level, normal bone remodeling is regulated by the receptor activator of nuclear factor kappa B (NF-kB) ligand (RANKL)/receptor activator of NF-kB (RANK)/Osteoprotegerin (OPG) system, which also controls the production and activity of osteoclasts (Kenkre and Bassett, 2018; Bolamperti et al., 2022). OPG, 04 frontiersin.org 10.3389/fgene.2023.1131182 Banaganapalli et al. FIGURE 3 RANKL/RANK/OPG signaling pathway: RANKL is a receptor activator of nuclear factor kappa-B ligand, that is expressed by osteoblasts. OPG is also expressed by osteoblasts, it can bind to and inhibit RANKL and act as a protector against bone loss. RANKL/RANK/OPG signaling pathway: RANKL is a receptor activator of nuclear factor kappa-B ligand, that is expressed by osteoblasts. OPG is also expressed by osteoblasts, it can bind to and inhibit RANKL and act as a protector against bone loss. Genotype-phenotype correlation studies have suggested that patients with nonsense mutations causing partial translation of UBA domain, have more severe and extensive disease than patients with missense mutations (Gennari et al., 2010; Visconti et al., 2010). Missense mutations are mostly restricted to exons 7 (29.41%) and 8 (70.59%) of the SQSTM1 gene (Shaik et al., 2021). However, even among patients with the same mutation and within the same family, clinical heterogeneity is reported (Laurin et al., 2002) (Gennari et al., 2010). Also somatic mutations in the SQSTM1 gene have been reported in sporadic papillary osteosarcoma (Merchant et al., 2009). In around 5% of PDB patients, somatic mutation in SQSTM1 (P392L) was observed in monocyte lineage only. Interestingly, PDB patients with this somatic mutation had a milder bone phenotype than those with the same mutation as a germline mutation (Guay-Bélanger et al., 2015). Molecular factors in PDB In germline mutations, the defect is present in every cell, while somatic mutations are seen in a subset of cells only, leading to variable expression of normal and abnormal proteins with reduced function. This might explain the milder phenotype in the somatic mutation carriers. Although SQSTM1 mutations are detected in about 50% of familial cases from various countries, their occurrence is relatively low in sporadic cases. Genome-wide association studies have revealed seven unique potential loci that account for ~13% of the family risk of PDB in SQSTM1-negative individuals (Albagha et al., 2010; Albagha et al., 2011; Database, 2022). TNFRSF11A gene revealed several insertions at the exon 1, resulting in the duplication of amino acid sequences in the RANK signal peptide (Hughes et al., 2000). Until now, many heterozygous in-frame tandem duplications of varied length, resulting in longer RANK signal peptide, have been reported, with the majority are related to uncommon PDB-like illnesses (Ralston and Taylor, 2019). This gene encodes a protein belonging to the TNF-receptor superfamily. This receptor can interact with many TRAF family members, thus activating NF- kappa B and MAPK8/JNK. This receptor is also a key osteoclast development mediator. The receptor activator of NFkB (RANK) is encoded by the TIFRSF11A gene. The ZNF687 gene encodes for C2H2 zinc ﬁnger protein that may play a role in bone differentiation and development. Mutation in ZNF687 gene was identiﬁed for the ﬁrst time in large Italian family many affected family members by whole exome analysis, followed by other studies conﬁrms it as causal gene for PDB (Divisato et al., 2016; Scotto di Carlo et al., 2020). The ZNF687 gene mutation was also associated with polyostotic PDB, pagetic osteosarcomas and in undifferentiated pagetic sarcoma tissue as well (Scotto di Carlo et al., 2020). For instance, minority of PDB patients develop malignant giant cell tumors (GCTs) of the bone (PDB/GCTs) with an early onset (Wei et al., 2021). ZNF687 is the only gene currently proven to cause PDB/GCT; however, Proﬁlin 1 gene have recently been identiﬁed as the cause of early-onset Paget’s disease of bone with GCT in Italian and Chinese patients (Wei et al., 2021). Meanwhile, individuals with mutations in the ZNF687 gene experience severe PDB at multiple sites. Molecular factors in PDB In vitro studies have revealed that ZNF687 mutant osteoclasts can have up to 150 nuclei, a ﬁnding unique to PDB patients with ZNF687 mutations (Divisato et al., 2016; Scotto di Carlo et al., 2020). Knock-in mouse model of ZNF687 mutation supports the crucial role it plays in the PDB development and its potential oncogenic property by having high incidence of hepatocellular carcinomas (Russo et al., 2023). Frontiers in Genetics frontiersin.org Other PDB-associated genes The genome wide scans have revealed several susceptibility loci for PDB and related syndromes (Makaram and Ralston, 2021; Gennari et al., 2022). Most of these gene loci are linked to osteoclast differentiation or function. These genetic loci are identiﬁed by few rare and common genetic variants, which collectively increase the PDB risk. Table 1 shows the list of different genetic loci and corresponding genes involved in the predisposition of individuals to PDB (Albagha et al., 2010; Albagha et al., 2011; Albagha, 2015). The TNFRSF11A gene mutations were ﬁrst reported in isolated cases of PDB and other PDB-like illnesses, such as Familial expansile osteolysis (FEO) and Expansile skeletal hyperphosphatasia (ESH). Mutation of the Complete TNFRSF11B gene deletion is associated with JPD (Juvenile Paget Disease) (Scotto di Carlo et al., 2020; Naot et al., 2014). Also, partial TNFRSF11B gene resulting in loss of a conserved aspartate residue at codon 192 was also identiﬁed (Middleton-Hardie et al., 2006). Nevertheless, TNFRSF11B mutations have not yet been discovered in classical PDB, and the 8q24 locus did not emerge as a susceptibility gene in GWAS. 05 frontiersin.org Banaganapalli et al. 10.3389/fgene.2023.1131182 TABLE 1 PDB-predisposing risk genes with chromosomal location, encoded proteins, function, and diseases distinct from PDB (PDB-related disorders). Chr. Gene Protein Description/function PDB-related disorders References 5q35.3 SQSTM1 p62 This gene encodes a multifunctional protein that binds ubiquitin and activates NF-kB. Mutation in this gene causes sporadic and familial bone Paget disease. - Hocking et al. (2001), Laurin et al. (2001), Laurin et al. (2002) 18q21 TNFRSF11A RANK Greater NF-KB signaling activation in vitro correlates with higher disease severity in vivo. Familial expansile osteolysis (FEO), Expansile skeletal hypophosphatasia (ESH) Cody et al. (1997), Good et al. (2001), Nakatsuka et al. (2003) 1q21.3 ZNF687 C2H2 zinc ﬁnger protein The protein that is encoded by this gene may have a signiﬁcant function in the differentiation and development of bones. PDB, pagetic osteosarcomas and in undifferentiated pagetic sarcoma Divisato et al. (2016), Scotto di Carlo et al. (2020) 8q24.12 TNFRSF11B OPG Decoy receptor that regulates bone resorption negatively. Juvenile PDB Scotto di Carlo et al. (2020) 8q22 DCSTAMP (TM7SF4) TM7SF4 Fusion of osteoclast precursors to develop mature osteoclasts with multiple nuclei. - Kukita et al. (2004), Yagi et al. (2005), Albagha et al. (2011) 10p13 OPTN Optineurin Key regulator of osteoclast survival and development. ~60% increase the risk of developing the disease - Hocking et al. Other PDB-associated genes (2001) 1p13 CSF1 M-CSF Primary controller of osteoclast survival and development - Tsurukai et al. (2000). 9p13.3 VCP p97 Protein degradation, intracellular membrane fusion, DNA repair and replication, cell cycle control, and NF-kappa B pathway activation. Inclusion body myopathy with PDB and frontotemporal dementia syndrome (IBMPFD) Kovach et al. (2001), Watts et al. (2004) 6p21.31 FKBP5 Cis-trans prolyl isomerase The encoded protein participates in the regulation of the immune system and fundamental physiological processes such as protein folding and transportation. - Bouwmeester et al. (2004), Lu et al. (2017) 6p21 HLA HLA PDB1 locus seem to play minor role in development of PDB - Fotino et al. (1977), Tilyard et al. (1982), Good et al. (2001) 2q36 Unknown Unknown The putative locus on chromosome 2q36 showed linkage under a heterogeneity model but not a homogeneity model. - Hocking et al. (2001) 7q33 NUP205 nucleoporin 205 kDa It encodes nucleoporin 205 kDa, a transport- related component of the nuclear pore. However, its function in bone remains uncertain. - Lin et al. (2004). 15q24 PML Promyelocytic leukemia protein Osteoclast differentiation, survival, and resorption (mice) - Lin et al. (2004). 14q32 RIN3 Rab and Ras interactor 3 Vesicular trafﬁcking, especially expressed in osteoclasts - Saito et al. (2002), Kajiho et al. (2003), Albagha et al. (2011) There is some indication that common polymorphisms at the TNFRSF11B gene may predispose women, but not men, to classical PDB; however, this must be validated by a large-scale investigation (Solomon, 1979; Beyens et al., 2007). This gene, encodes Osteoprotegerin (OPG) protein, belongs to the TNF- receptor superfamily; an osteoblast-secreted decoy receptor that regulates bone resorption negatively. Also, this protein controls osteoclast development and function by inhibiting the stimulatory effects of RANKL on osteoclast differentiation (Makaram and Ralston, 2021; Gennari et al., 2022). The mutant OPG is unable to block osteoclastic resorption in a bone culture system, demonstrating that it is a loss-of- function mutation (Chong et al., 2003). The DCSTAMP gene (Dendrocyte Expressed Seven Transmembrane Protein, also known as TM7SF4), encodes for a seven-pass transmembrane protein and is expressed on cells that is involved in osteoclastogenesis, immunological activity, and myeloid differentiation. In addition, the fusing of osteoclast precursors into mature osteoclasts is facilitated by the DCSTAMP protein. During the osteoclast formation, expression of DCSTAMP is crucial. Frontiers in Genetics frontiersin.org Epigenetics in PDB In the last two to three decades, researchers have revealed that epigenetic modiﬁcations can inﬂuence the development of Paget’s disease (Leach et al., 2001; Galson and Roodman, 2014). Epigenetic alterations are chemical changes that occur in DNA and the proteins that bind to it without altering the actual DNA sequence. Environmental factors such as food, stress, pollutants, and infections can all inﬂuence these alterations, which can affect gene expression. Recent research has discovered various epigenetic changes that contribute to the development of PDB. For example, low DNA methylation of the RANKL gene promoter region was discovered in Paget’s disease patients compared to healthy controls, resulting in higher RANKL expression and associated bone resorption (Gennari et al., 2022). Similarly, increasing DNA methylation of the OPG gene promoter was linked to lower OPG expression, which accelerates bone resorption. Another important epigenetic component in PDB is histone modiﬁcation. Histones are proteins that wrap DNA into nucleosomes, which are structural units. Histone acetylation and deacetylation are important gene expression regulators (Xu et al., 2020). Hypomethylation of histone H3 lysine 4 (H3K4me3) and hypermethylation of histone H3 lysine 9 (H3K9me3) have been linked to enhanced bone resorption. Factors like aging and viral infections are also known to play a role in PDB epigenetic alterations. Aging is a well-known risk factor for PDB, and studies have indicated that epigenetic changes associated with aging, such as increased DNA methylation, inﬂuence to the disease development (Saul and Kosinsky, 2021) and (Maatallah et al., 2021). PDB has also been related to viral infections, such as the measles virus, and studies have shown that the virus can modify the epigenetic landscape of bone cells, contributing to the disease development. DNA methylation and histone modiﬁcation play important roles in bone resorption and creation, while environmental variables including aging and viral infections also contribute to epigenetic alterations (Diboun et al., 2021; Diboun et al., 2022). Therefore, epigenetic changes play a signiﬁcant role in the development of Paget’s disease. With a greater understanding of these changes, researchers may be able to develop novel treatments that target the epigenetic abnormalities caused by Paget’s disease and aid in preventing the disease’s progression. The chromosome 1p13 hosts a recombination hotspot in the vicinity of the CSF1 gene. Epigenetics in PDB Notably, CSF1 exclusively encodes M-CSF, a cytokine that plays a pivotal role in regulating osteoclastogenesis and modulating the activity and survival of macrophages (Tsurukai et al., 2000; Bouyer et al., 2007). It is a strong candidate for PDB susceptibility based on its function (Tsurukai et al., 2000). The function of CSF1 in the etiology of PDB is supported by the observation that individuals with PDB have elevated blood levels of M-CSF (Neale et al., 2002). Variations in this gene that predispose to PDB is still unknown, although it is hypothesized that they may cause PDB via enhancing osteoclast production through CSF1 activity. A predisposing variation for PDB in CSF1 was found by a GWAS analysis (Albagha et al., 2010) and conﬁrmed by a follow-up study the same group (Albagha et al., 2011). In parallel, a missense mutation in the CSF1 gene was identiﬁed in JPD (Donáth et al., 2015). Moreover, the SNPs linked with PDB are situated upstream of the gene in a region rich in regulatory elements, suggesting that their inﬂuence on control of CSF1 expression; however, the precise mechanism by which these variations predispose to PDB is yet to be determined (Neale et al., 2002). VCP gene, encodes valosin-containing protein, which is a member of the AAA (+) ATPase family of chaperone-like proteins. It is a multifunctional protein involved in several intracellular processes including NF-KB signaling, DNA repair, and autophagy. Mutations in the VCP gene were discovered as the cause of the autosomal dominant Inclusion Body Myopathy with PDB and Frontotemporal Dementia (IBMPFD), characterized by skeletal defects identical to the classical PDB (Kovach et al., 2001; Watts et al., 2004; Columbres et al., 2023) The VCP mutational effects include a modulatory inﬂuence on the NF-KB signaling pathway due to VCP’s involvement in the degradation of phosphorylated IkB (Woodman, 2003; Tresse et al., 2010). In addition, there is evidence that protein-coding mutations of VCP may arise infrequently in people with classical PDB who lack other components of IBMPFD syndrome; however, there is no conclusive data that common mutations at the VCP gene make the individuals susceptible to PDB (Albagha et al., 2014). Furthermore, syndromic IBMPFD linked with VCP mutations has been categorized as one of the multisystem proteinopathies, in which neurological and muscular abnormalities sometimes accompany the PDB (Taylor, 2015). Epigenetics in PDB Moreover, Myopathy occurs in up to 90% of IBMPFD families, whereas PDB and dementia have been identiﬁed in 43% and 37% of cases, respectively (Ralston and Taylor, 2019). Other PDB-associated genes Variants in the genes that predispose to PDB may increase DCSTAMP expression, leading to the formation of massive, multinucleated, granulocytic osteoclasts (Kukita et al., 2004; Yagi et al., 2005; Albagha et al., 2011). The OPTN gene encodes the protein optineurin, which has coiled-coil structures; highly expressed cytoplasmic protein with 06 frontiersin.org Banaganapalli et al. 10.3389/fgene.2023.1131182 physiological processes including protein folding and transport (Bouwmeester et al., 2004; Lu et al., 2017) (Zheng et al., 2021). physiological processes including protein folding and transport (Bouwmeester et al., 2004; Lu et al., 2017) (Zheng et al., 2021). many physiological activities, including NFB signaling, autophagy, and innate immunity (Zhu et al., 2007; Wild et al., 2011). Previous investigations have found a negative regulatory function for OPTN in osteoclast formation by altering in vitro and in vivo NF-kB and interferon signaling in mouse models (Obaid et al., 2015) (Silva et al., 2018a; Silva et al., 2018b). Frontiers in Genetics frontiersin.org Role of environmental factors Environmental factors are frequently proposed as an explanation for the variability in Paget’s disease epidemiology. For instance, river water contamination by arsenic in a pesticide used to treat cotton bales may have contributed to the high frequency of PDB in Lancashire during the 1970s. Because the nuclear inclusion bodies in osteoclasts appear to reﬂect viral nucleocapsids of one of the paramyxoviruses, it has been hypothesized that viral infections may be one of the causes of PDB (Visconti et al., 2017). Other environmental factors, such as Another gene reported to be associated with the PDB is FKBP5 gene. Mutation in a Chinese family with PDB and supported by mutant mouse model. The functional study of the mutant mouse shows hyperresponsive osteoclast precursor cells to RANK with signiﬁcantly high bone resorbing function (Liu and Zhang, 2015). The FKBP5 gene is a member of the immunophilin protein family, which is involved in immunoregulation and in fundamental 07 frontiersin.org 10.3389/fgene.2023.1131182 10.3389/fgene.2023.1131182 Banaganapalli et al. environmental impact. Genetic studies have changed our understanding of the pathophysiology of PDB and uncovered novel genes and signaling networks that govern the development and activity of osteoclasts. Despite these advances, the environmental triggers of PDB remain poorly understood. It is unknown why PDB attacks some bones but not others. In addition to gaining a better biological understanding of PDB, it will be useful for elucidating disease processes in other illnesses that affect the skeleton in a targeted manner. Now, gene expression and epigenetics studies highlight that PDB shares some targetable biomarkers, however, the development of targeted molecular drugs for PDB patients is lacking. Therefore, molecular novel drug targets must be identiﬁed using a complete molecular diagnostic proﬁle so personalized medicine and clinical management can be achieved in the coming decades. cigarette smoke and wood stove smoke during childhood, have also been linked to Paget’s disease. These exposures have decreased due to changes in lifestyle over the time (Audet et al., 2017). Lifestyle factors such as diet and physical activity also play a role in the development of PDB. A diet that is high in saturated fats and low in calcium and vitamin D can contribute to the development of the disease, as can a sedentary lifestyle. Exercise and a diet that is rich in calcium and vitamin D can help to prevent the development of PDB and slow the progression of the disease (Numan et al., 2019). Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Funding This research work was funded by institutional Fund Projects under grant no. (IFPRP: 388-140-1442). Treatment of PDB BB and NS, conceptualization; BB, IF, and FA data curation; funding acquisition; BB, RE NS, supervision; BB, IF, and FA: validation; BB, visualization; BB, IF, FA, PS, RE, and NS: writing original draft; BB, RE, and NS, writing review and editing. PDB does not have a speciﬁc treatment. However, some drugs can signiﬁcantly improve the clinical symptoms and disease management. Bisphosphonates are the most used and FDA-approved drugs for treating PDB, and nitrogen-containing bisphosphonates have been found to be more effective than non-nitrogen-containing ones (Ralston et al., 2019). Patients with moderate-to-severe Paget’s disease of bone (PDB) have achieved successful outcomes with either a 6-month daily dose of alendronate (40 mg) or a 2-month daily dose of risedronate (30 mg). In approximately 60%–70% of cases, treatment normalized the ALP levels, and some patients were able to maintain biochemical remission for over a year (Gennari et al., 2009). Zoledronic acid is the most potent nitrogen-containing bisphosphonate and is the preferred choice for treating PDB due to its extended bone retention and intermittent dosing (Reid, 2016). However, its potential renal toxicity must be considered (Reid, 2016; Gennari et al., 2019). Oral alendronate or risedronate can also normalize alkaline phosphatase (ALP) levels in patients with moderate-to-severe PDB, but they may cause esophageal irritation and upper gastrointestinal tract (GI) discomfort. If patients cannot tolerate bisphosphonates, calcitonin is a safe, well-studied drug that can help lower the metabolic activity of pagetic bone and relieve GI discomfort (Reid, 2016). Surgery may be necessary in severe osteoarthritis cases but requires careful planning and preventative bisphosphonate medication. 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https://openalex.org/W2063592545	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0106255&type=printable	English	null	MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays	PloS one	2,014	cc-by	9,086	Abstract A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin- fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers. Received April 20, 2014; Accepted August 1, 2014; Published September 3, 2014 Copyright: 2014 Powers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the National Institutes of Health/National Cancer Institute grants R21CA137704 and R01CA135087, and the state of South Carolina SmartState Endowed Research program to R.R.D. Additional resources and support were from the Biorepository & Tissue Analysis Shared Resource, Hollings Cancer Center (P30 CA138313), and by the South Carolina Clinical & Translational Research (SCTR) Institute (UL1 RR029882 & UL1 TR000062) for R.R.D. This work was supported by grants R01 CA120206 and U01 CA168856 from the National Cancer Institute (NCI), the Hepatitis B Foundation, and an appropriation from The Commonwealth of Pennsylvania to A.M. This work was supported by a grant from the National Institutes of Health/National Cancer Institute, U01CA168896, to B.B.H. Thomas W. Powers1, Benjamin A. Neely1, Yuan Shao2, Huiyuan Tang5, Dean A. Troyer3, Anand S. Mehta4, Brian B. Haab5, Richard R. Drake1,2* 1 Department of Cell and Molecular Pharmacology and Experimental Therapeutics and MUSC Proteomics Center, Medical University of South Carolina, Charleston, South Carolina, United States of America, 2 Hollings Cancer Center Biorepository and Tissue Analysis Resource, Medical University of South Carolina, Charleston, South Carolina, United States of America, 3 Departments of Pathology and Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia, United States of America, 4 Drexel University College of Medicine, Department of Microbiology and Immunology and Drexel Institute for Biotechnology and Virology, Doylestown, Pennsylvania, United States of America, 5 Laboratory of Cancer Immunodiagnostics, Van Andel Research Institute, Grand Rapids, Michigan, United States of America Abstract The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors confirm that Anand S. Mehta is a current member of the Editorial Board. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria. * Email: draker@musc.edu treatment results in the formation of methylene bridges between the amino acids of the proteins, complicating further analysis by mass spectrometry. There has been continued progress in improving extraction methods of trypsin digested peptides from FFPE tissues in recent years, in parallel with improved high resolution sequencing analysis of peptides by mass spectrometry [3,4]. Incorporation of multiple FFPE tumor tissue cores in a tissue microarray (TMA) format also has proven to be effective for immunohistochemistry analysis of potential biomarker candi- dates [5], and TMAs are increasingly being used for validation of alterations in protein expression associated with emerging genetic mutation phenotypes and transcriptional profiling studies [6,7]. The main advantages of experiments performed with TMAs are the ability to include multiple cores from the same subject September 2014 \| Volume 9 \| Issue 9 \| e106255 Editor: Surinder K. Batra, University of Nebraska Medical Center, United States of America Citation: Powers TW, Neely BA, Shao Y, Tang H, Troyer DA, et al. (2014) MALDI Imaging Mass Spectrometry Profiling of N-Gly Embedded Clinical Tissue Blocks and Tissue Microarrays. PLoS ONE 9(9): e106255. doi:10.1371/journal.pone.0106255 Introduction Tissues obtained from surgeries or diagnostic procedures are most commonly preserved in formalin-fixed paraffin-embedded (FFPE) tissue blocks. These tissues are fixed in formalin and processed as paraffin-embedded tissue blocks. The embedding process preserves the cellular morphology and allows tissues to be stored at room temperature, causing FFPE fixation to be used by many tissue banks and biorepositories [1,2]. For cancer biomarker discovery, FFPE tissues are particularly attractive because they are archived for years and are much more widely available than cryopreserved tissue. When combined with clinical outcomes, FFPE tissues are a rich source of samples for biomarker discovery and validation in retrospective studies. While the fixation method has many benefits, the formalin September 2014 \| Volume 9 \| Issue 9 \| e106255 September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org 1 MALDI MS Imaging of Glycans in FFPE Tissues tumors, improved sample throughput, statistical relevance and multiplexed analysis of diverse molecular targets [5,8]. Thus, it is possible to place up to 100 samples with duplicates and controls on a single slide. When correlated with associated clinical outcomes, this provides a powerful method for biomarker discovery and validation while minimizing reagent use and assuring that each core in the TMA is treated under identical conditions. Materials The glycan standard NA2 was obtained from ProZyme (Hayward, CA). Trifluoroacetic acid, sodium hydroxide, dimethyl sulfoxide, iodomethane and a-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Sigma-Aldrich (St. Louis, MO). HPLC grade methanol, ethanol, acetonitrile, xylene and water were obtained from Fisher Scientific (Pittsburgh, PA). ITO slides were purchased from Bruker Daltonics (Billerica, MA) and Tissue Tack microscope slides were purchased from Polysciences, Inc (Warrington, PA). Citraconic anhydride for antigen retrieval was from Thermo Scientific (Bellefonte, PA). Recombinant Peptide N- Glycosidase F (PNGaseF) from Flavobacterium meningosepticum was expressed and purified as previously described [25]. It is well documented that malignant transformation and cancer progression result in fundamental changes in the glycosylation patterns of cell surface and secreted glycoproteins [9–11]. Glycosylation of proteins are post-translational modifi- cations most commonly involving either N-linked addition to asparagine residues or O-linked additions to serine or threonine residues. Current approaches to evaluate glycosylation changes generally involve bulk extraction of glycans and glycoproteins from tumor tissues for analysis by mass spectrometry or antibody array platforms, however, this disrupts tissue architec- ture and distribution of the analytes. Broad affinity carbohy- drate binding lectins and a small number of glycan antigen antibodies can be used to target glycan structural classes in tissues, but not individual glycan species. Additionally, these detection methods for global alterations in glycosylation requires staining on many adjacent tissue sections, making large scale assessments on many samples difficult, expensive and time consuming. There are only a few reported studies examining glycosylation related changes of proteins or glycolipids in FFPE cancer tissues [12–14], and these focus primarily on determining the levels of the protein carriers or glycosyltransferases through immunostaining. FFPE Tissues and TMA All human tissues used were de-identified and determined to be not human research classifications by the respective Institutional Review Boards at MUSC and Van Andel. Mouse kidneys were excised from euthanized C57BL/6 mice and immediately placed in 10% formalin prior to processing for routine histology and paraffin embedding. Mice were housed in an Institutional Animal Care and Use Committee-approved small animal facility at MUSC, and tissues obtained were harvested as part of approved projects unrelated to glycan tissue imaging. A liver TMA was purchased from BioChain consisting of 16 cases of liver cancer in duplicates, and one adjacent non-tumor tissue for each case. Tissues were from 14 male and two female patients with an average age of 47.5 with a range of 33 to 68 years old, with additional information provided in Table S1. A de-identified prostate tumor FFPE block, stored for 10 years representing a Gleason grade 6 (3+3)/stage T2c adenocarcinoma from a 62 year old Caucasian male, was obtained from the Hollings Cancer Center Biorepository at the Medical University of South Carolina. A pathologist confirmed the presence of approximately 10% prostate cancer gland content in the sample. A de-identified large- cell undifferentiated pancreatic carcinoma FFPE tissue section with low CA19-9 staining was obtained from the Van Andel Institute Biospecimen Repository. For each section analyzed, histological analysis and staining with hematoxylin and eosin (H & E) were performed. One potential approach to assess glycan changes in tissues is matrix-assisted laser desorption/ionization imaging mass spec- trometry (MALDI-IMS). This technique has been used to directly profile multiple protein [15,16], lipid [17,18] and drug metabolite [19–21] in tissue, generating molecular maps of the relative abundance and spatial distribution of individual analytes linked to tissue histopathology. MALDI-IMS analysis of peptides following trypsin digestion of FFPE TMAs have also been reported [22–24]. Recently, our group reported a MALDI-IMS method workflow to directly profile N-linked glycan species in fresh/frozen tissues [25]. Adapting this method for the analysis of N-glycans in FFPE tissues would serve to extend the application of the technique to larger retrospective sample sets and TMAs. September 2014 \| Volume 9 \| Issue 9 \| e106255 Analysis of Formalin-Fixed Mouse Kidneys and Human Cancer Tissues Mouse kidney tissues were fixed in formalin and used as an initial model system to develop MALDI-IMS glycan imaging workflows for FFPE tissues. These tissues were chosen due to the availability of reference glycan structures and spectra (Consortium for Functional Glycomics; www.functionalglycomics.org), and previous MALDI-IMS glycan imaging data from our laboratory for fresh/frozen tissue analysis [21]. A summary workflow schematic is provided (Figure 1). Tissues were cut at 5 microns, deparaffinized and rehydrated in sequential xylene/ethanol/water rinses, followed by antigen retrieval in citraconic anhydride pH 3. The rehydrated tissues were sprayed with PNGaseF, incubated for glycan release, sprayed with CHCA matrix, and then analyzed by MALDI-IMS. While all data shown herein uses CHCA, 2,5- dihydroxybenzoic acid (DHB) matrix could also be used success- Collision-Induced Dissociation of N-linked Glycans Glycan standards were spotted on a stainless steel MALDI plate using CHCA matrix and desiccated to yield a homogenous layer. Tissues were prepared as previously described for MALDI imaging of FFPE tissues. 10 spectra of 1000 laser shots with a laser frequency of 1000 Hz were averaged for each spectra provided. The collision energy varied between 60–70V. Permethylation of Tissue Extracted N-glycans PNGaseF sprayed mouse kidney tissue slides were incubated for 2 hr at 37uC; 50 mL water was applied on top of the tissue and incubated for 20 minutes to extract the released native N-glycans. The water was removed from the tissue, and then concentrated under vacuum by centrifugation. Permethylation was performed as described [25], and glycans analyzed by MALDI. Masses detected in the permethylation experiments were searched against the permethylated glycan database provided by the Consortium for Functional Glycomics (www.functionalglycomics.org). TMA Statistics Mass spectra from TMA tissue Regions of Interest (ROIs) representing each tissue core were exported directly from FlexImaging and analyzed using an in-house workflow. The peak lists were first deconvoluted followed by calculating the mean peak intensity of points in each ROI, resulting in a monoisotopic peak list corresponding to signal intensity in each region. Comparison of tumor versus non-tumor was accomplished with a Wilcoxon rank sum test. Individual peaks were also evaluated to discriminate between tumor and non-tumor using receiver operator character- istic curves. Figure 1. Schematic of the methodology for imaging N-glycans from FFPE tissues. Prior to enzyme application, FFPE blocks are cut at 5 mm, incubated, deparaffinized and undergo antigen retrieval. PNGaseF is then applied and the slide is incubated before MALDI-IMS. The data is then linked with histopathology either on the same tissue slice or a serial tissue slice. doi:10.1371/journal.pone.0106255.g001 MALDI MS Imaging of Glycans in FFPE Tissues MALDI MS Imaging of Glycans in FFPE Tissues Figure 1. Schematic of the methodology for imaging N-glycans from FFPE tissues. Prior to enzyme application, FFPE blocks are cut at 5 mm, incubated, deparaffinized and undergo antigen retrieval. PNGaseF is then applied and the slide is incubated before MALDI-IMS. The data is then linked with histopathology either on the same tissue slice or a serial tissue slice. doi:10.1371/journal.pone.0106255.g001 using a Solarix dual source 7T FTICR mass spectrometer (Bruker Daltonics) (m/z = 690–5000 m/z) with a SmartBeam II laser operating at 1000 Hz, a laser spot size of 25 mm. Images of differentially expressed glycans were generated to view the expression pattern of each analyte of interest using FlexImaging 4.0 software (Bruker Daltonics). Following MS analysis, data was loaded into FlexImaging Software focusing on the range m/ z = 1000–4000 and reduced to 0.95 ICR Reduction Noise Threshold. Observed glycans were searched against the glycan database provided by the Consortium for Functional Glycomics (www.functionalglycomics.org). Glycan structures were generated by Glycoworkbench [26] and represent putative structures determined by combinations of accurate m/z, CID fragmentation patterns and glycan database structures. Washes for Deparaffinization and Rehydration In this report, we describe the application of MALDI-IMS glycan imaging to various formalin-fixed tissues. Formalin-fixed mouse kidney tissues were used to optimize antigen retrieval, PNGaseF digestion and glycan detection conditions for MALDI- IMS. This was followed by N-glycan analysis of clinical FFPE tissue blocks from prostate and pancreatic cancers, as well as a commercial tissue microarray of hepatocellular carcinoma (HCC). Glycan identity was confirmed by on-tissue collision-induced dissociation (CID) and off-tissue permethylation analysis. An optimized MALDI-IMS workflow is presented that allows routine simultaneous analysis of 30 or more glycans per FFPE tissue, including TMA formats. The approach is amenable to any FFPE tissue, and represents an additional molecular correlate assay for use with the TMA format. Furthermore, depending on the construction of the TMA and targeted tumor type, the approach has the potential to identify novel glycan biomarker panels for cancer detection and prognosis. To our knowledge, this represents the first instance of using MALDI-IMS to profile N-glycans in FFPE tissue blocks or TMAs. Tissue and TMA blocks were sectioned at 5 mm and mounted on positively charged glass slides measuring 25675 mm, compat- ible with the Bruker slide adaptor plate. The slides were heated at 60uC for 1 hr. After cooling, tissue sections were deparaffinized by washing twice in xylene (3 minutes each). Tissue sections were then rehydrated by submerging the slide twice in 100% ethanol (1 minute each), once in 95% ethanol (one minute), once in 70% ethanol (one minute), and twice in water (3 minutes each). Following the wash, the slide was transferred to a coplin jar containing the citraconic anhydride buffer for antigen retrieval and the jar was placed in a vegetable steamer for 25 minutes. Citraconic anhydride (Thermo) buffer was prepared by adding 25 mL citraconic anhydride in 50 mL water, and adjusted to pH 3 with HCl. After allowing the buffer to cool, the buffer was exchanged with water five times by pouring out K of the buffer and replacing with water, prior to replacing completely with water on the last time. The slide was then desiccated prior to enzymatic digestion. Tris buffer pH 9–10 was also effective, but citraconic anhydride buffer was used for all experiments in this study. September 2014 \| Volume 9 \| Issue 9 \| e106255 September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org 2 September 2014 \| Volume 9 \| Issue 9 \| e106255 N-glycan MALDI-IMS An ImagePrep spray station (Bruker Daltonics) was used to coat the slide with a 0.2 ml aqueous solution of PNGaseF (20 mg total/ slide) as previously described [25]. Adjacent control tissue slices lacking PNGaseF were generated by covering them with a glass slide during the spraying process. Following application of PNGaseF, slides were incubated at 37uC for 2 hr in a humidified chamber, then dried in a desiccator prior to matrix application. a- Cyano-4-hydroxycinnamic acid matrix (0.021 g CHCA in 3 ml 50% acetonitrile/50% water and 12 mL 25%TFA) was applied using the ImagePrep sprayer. Released glycan ions were detected September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org 3 MALDI MS Imaging of Glycans in FFPE Tissues Figure 2. MALDI-IMS of N-Glycans on Mouse Kidney Tissue. Two mouse kidneys were sliced at 5 um prior to proceeding with the MALDI-IMS workflow. One tissue was covered with a glass slide during PNGaseF application to serve as an undigested control tissue. An average annotated spectra from the tissue that received PNGaseF application is provided (a). Tissue regions were assessed by H&E stain (b). The labeled peaks correspond to native N-glycans that have been reported for the mouse kidney on the Consortium for Functional Glycomics mouse kidney database. Two of these ions were selected and their tissue localization was assessed. Hex4dHex2HexNAc5 at m/z = 1996.7 (c) is located in the cortex and medulla while Hex5dHex2HexNAc5 m/z = 2158.7 (d) is more abundant in the cortex of the mouse kidney. An overlay image of these two masses is also shown (e), as well as the corresponding image from untreated PNGaseF control tissues (f). doi:10.1371/journal.pone.0106255.g002 Figure 2. MALDI-IMS of N-Glycans on Mouse Kidney Tissue. Two mouse kidneys were sliced at 5 um prior to proceeding with the MALDI-IMS workflow. One tissue was covered with a glass slide during PNGaseF application to serve as an undigested control tissue. An average annotated spectra from the tissue that received PNGaseF application is provided (a). Tissue regions were assessed by H&E stain (b). The labeled peaks correspond to native N-glycans that have been reported for the mouse kidney on the Consortium for Functional Glycomics mouse kidney database. Two of these ions were selected and their tissue localization was assessed. N-glycan MALDI-IMS An overlay of the MALDI-IMS images for these two ions from the PNGaseF treated sections (Figure 2e) and the control tissue (Figure 2f) demonstrates that these two ions are released by PNGaseF. In control kidney tissues that were only sprayed with aqueous PNGaseF solution lacking enzyme, or tissue slices that were not processed by antigen retrieval plus and minus PNGaseF digestion, only matrix ions or paraffin/formalin polymer were detected (data not shown). A summary glycan image panel of 28 glycan ions detected in these kidneys, sodium adducts and observed/expected m/z values is provided in Figure S1. Additionally, N-glycans were extracted from the tissue following on-tissue PNGaseF digestion, permethy- lated and analyzed by MALDI. A representative spectra from this analysis is provided in Figure S2. These permethylated values were also compared with MALDI reference spectra for mouse kidney glycans from the Consortium for Functional Glycomics. The imaged glycan ions were correlated to the reference spectra glycans, illustrated in Figure S3, and could be matched to all 28 glycan species highlighted in the reference spectra. pancreatic cancer and one for prostate cancer. A section of human pancreatic cancer tissue of complex histology was processed, incubated with PNGaseF and glycans detected by MALDI-IMS (Figure 3). Different N-glycans were detected that could distin- guish between non-tumor, tumor, tumor necrotic and fibrocon- nective tissue regions. A representative glycan image overlay of four m/z values that correspond to the sodium adducts of potential N-glycan species is shown in Figure 3a, each representing a specific region of the tissue (Figure 3b). A glycan of m/z = 1891.80 (red)/Hex3dHex1HexNAc6 was detected primarily in the non- tumor region of the pancreas, while a glycan of m/z = 1743.64 (blue)/Hex8HexNAc2 was predominant in the tumor region of the tissue. A region of desmoplasia surrounding the tumor region, an area of increased extracellular matrix proteins and myofibro- blast-like cells resulting in a dense fibrous connective tissue [27], is represented by a glycan of m/z = 1809.69 (green)/Hex5dHex1- HexNAc4. A region of tumor necrosis is represented by a different glycan of m/z = 1663.64 (orange)/Hex5HexNAc4. Additional examples of tissue distributions of other individual glycan species are shown in Figure 3c. A human prostate tissue block containing both tumor and non- tumor gland regions was also analyzed by MALDI-IMS. N-glycan MALDI-IMS Hex4dHex2HexNAc5 at m/z = 1996.7 (c) is located in the cortex and medulla while Hex5dHex2HexNAc5 m/z = 2158.7 (d) is more abundant in the cortex of the mouse kidney. An overlay image of these two masses is also shown (e), as well as the corresponding image from untreated PNGaseF control tissues (f). doi:10.1371/journal.pone.0106255.g002 fully for N-glycan imaging of FFPE tissues. As shown in Figure 2, there were multiple ions detectable only in the tissue incubated with PNGaseF that were not present in the control tissue with no PNGaseF application. Different glycans were distributed across the cortex or medulla regions. For example, a Hex4dHex2Hex- NAc5 ion (m/z = 1996.74) is present in the cortex and medulla (Figure 2c), while a Hex5dHex2HexNAc5 glycan (m/z = 2158.76) is more specific to the cortex (Figure 2d). An overlay of the MALDI-IMS images for these two ions from the PNGaseF treated sections (Figure 2e) and the control tissue (Figure 2f) demonstrates that these two ions are released by PNGaseF. In control kidney tissues that were only sprayed with aqueous PNGaseF solution lacking enzyme, or tissue slices that were not processed by antigen retrieval plus and minus PNGaseF digestion, only matrix ions or paraffin/formalin polymer were detected (data not shown). A summary glycan image panel of 28 glycan ions detected in these kidneys, sodium adducts and observed/expected m/z values is provided in Figure S1. Additionally, N-glycans were extracted from the tissue following on-tissue PNGaseF digestion, permethy- lated and analyzed by MALDI. A representative spectra from this analysis is provided in Figure S2. These permethylated values were also compared with MALDI reference spectra for mouse kidney glycans from the Consortium for Functional Glycomics. The imaged glycan ions were correlated to the reference spectra glycans, illustrated in Figure S3, and could be matched to all 28 glycan species highlighted in the reference spectra. fully for N-glycan imaging of FFPE tissues. As shown in Figure 2, there were multiple ions detectable only in the tissue incubated with PNGaseF that were not present in the control tissue with no PNGaseF application. Different glycans were distributed across the cortex or medulla regions. For example, a Hex4dHex2Hex- NAc5 ion (m/z = 1996.74) is present in the cortex and medulla (Figure 2c), while a Hex5dHex2HexNAc5 glycan (m/z = 2158.76) is more specific to the cortex (Figure 2d). September 2014 \| Volume 9 \| Issue 9 \| e106255 N-glycan MALDI-IMS A heterogeneous N-glycan distribution reflective of the tissue histology was observed, and as an example of stroma and gland distributions, two glycan ions and two sub-regions within the tissue are highlighted in Figure 4. Distribution of glycans of m/ z = 1663.56 (Hex5HexNAc4) and m/z = 1850.65 (Hex4dHex1- HexNAc5) are shown in Figure 4b and 4c. A higher resolution We next assessed whether the method was compatible with two representative archived pathology FFPE tissue blocks, one for September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org 4 MALDI MS Imaging of Glycans in FFPE Tissues maging analysis was done for selected regions as marked in nel with the H&E images (Figure 4d–4f) highlighting is present in both the stroma and glands, while m/z = 1663.56 is predominantly located in the stroma An overlay of these two ions 3. MALDI-IMS of a Human Pancreas FFPE Tissue Block. An FFPE block of pancreatic tissue from a human patient was cut at 5 um prior elected for MALDI-IMS. Histopathology found four unique regions in the H&E of this tissue block. The tissue block contained tumor tissue, mor tissue, fibroconnective tissue representing desmoplasia surrounding the tumor tissue, and necrotic tissue (b). MALDI-IMS was able to ish these four regions based off of specific ions after MALDI-IMS. M/z = 1891.80 (red) is found in the non-tumor (NT) region of the pancreas responds to Hex3dHex1HexNAc6, while m/z = 1743.64 (blue) represents Hex8HexNAc2 and is predominant in the tumor region (T) of the esmoplasia (DP) is represented by m/z = 1809.69 (green) corresponding to Hex5dHex1HexNAc4. In the region where necrosis was identified /z = 1663.64 (orange) was elevated corresponding to Hex5HexNAc4. Image spectra were acquired at 200 mm raster. (c). Representative al glycan images for the pancreatic FFPE tissue slice. 371/journal.pone.0106255.g003 Figure 3. MALDI-IMS of a Human Pancreas FFPE Tissue Block. An FFPE block of pancreatic tissue from a human patient was cut at 5 um prior to and selected for MALDI-IMS. Histopathology found four unique regions in the H&E of this tissue block. The tissue block contained tumor tissue, non-tumor tissue, fibroconnective tissue representing desmoplasia surrounding the tumor tissue, and necrotic tissue (b). MALDI-IMS was able to distinguish these four regions based off of specific ions after MALDI-IMS. September 2014 \| Volume 9 \| Issue 9 \| e106255 N-glycan MALDI-IMS An archived FFPE block of prostate tissue from a human patient was cut at 5 mm and prepared for MALDI-IMS glycan analysis, (a). H&E image. A global glycan imaging experiment performed with a raster of 225 mm demonstrated a heterogeneous expression of two glycan ions (b). at m/z = 1663.56 and (c). m/z = 1850.65. Stromal versus gland distribution were further assessed in a high resolution experiment at 50 mm raster (d–f). Column (d) indicates a 26 amplification of the H&E, and distribution of the same two glycans are shown at this magnification for m/z = 1663.56 (red) and m/z = 1850.65 (green), and an overlay image. Column (e) (enlargement of upper region shown in d). and (f) (enlargement of lower region shown in d), show two highlighted regions of stroma and glands enhanced at 106 resolution, with the same colors and glycans shown for column (d). doi:10.1371/journal.pone.0106255.g004 Figure 4. MALDI-IMS of a Human Prostate FFPE Tissue Block. An archived FFPE block of prostate tissue from a human patient was cut at 5 mm and prepared for MALDI-IMS glycan analysis, (a). H&E image. A global glycan imaging experiment performed with a raster of 225 mm demonstrated a heterogeneous expression of two glycan ions (b). at m/z = 1663.56 and (c). m/z = 1850.65. Stromal versus gland distribution were further assessed in a high resolution experiment at 50 mm raster (d–f). Column (d) indicates a 26 amplification of the H&E, and distribution of the same two glycans are shown at this magnification for m/z = 1663.56 (red) and m/z = 1850.65 (green), and an overlay image. Column (e) (enlargement of upper region shown in d). and (f) (enlargement of lower region shown in d), show two highlighted regions of stroma and glands enhanced at 106 resolution, with the same colors and glycans shown for column (d). doi:10.1371/journal.pone.0106255.g004 N-glycan MALDI-IMS M/z = 1891.80 (red) is found in the non-tumor (NT) region of the pancreas and corresponds to Hex3dHex1HexNAc6, while m/z = 1743.64 (blue) represents Hex8HexNAc2 and is predominant in the tumor region (T) of the tissue. Desmoplasia (DP) is represented by m/z = 1809.69 (green) corresponding to Hex5dHex1HexNAc4. In the region where necrosis was identified (TN), m/z = 1663.64 (orange) was elevated corresponding to Hex5HexNAc4. Image spectra were acquired at 200 mm raster. (c). Representative individual glycan images for the pancreatic FFPE tissue slice. doi:10.1371/journal.pone.0106255.g003 is present in both the stroma and glands, while m/z = 1663.56 is predominantly located in the stroma. An overlay of these two ions depicts the stroma as an orange color, demonstrating the presence tissue imaging analysis was done for selected regions as marked in the panel, with the H&E images (Figure 4d–4f) highlighting stroma and gland substructures. In both instances, m/z = 1850.65 September 2014 \| Volume 9 \| Issue 9 \| e106255 September 2014 \| Volume 9 \| Issue 9 \| e106255 September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 MALDI MS Imaging of Glycans in FFPE Tissues Figure 4. MALDI-IMS of a Human Prostate FFPE Tissue Block. An archived FFPE block of prostate tissue from a human patient was cut at 5 mm and prepared for MALDI-IMS glycan analysis, (a). H&E image. A global glycan imaging experiment performed with a raster of 225 mm demonstrated a heterogeneous expression of two glycan ions (b). at m/z = 1663.56 and (c). m/z = 1850.65. Stromal versus gland distribution were further assessed in a high resolution experiment at 50 mm raster (d–f). Column (d) indicates a 26 amplification of the H&E, and distribution of the same two glycans are shown at this magnification for m/z = 1663.56 (red) and m/z = 1850.65 (green), and an overlay image. Column (e) (enlargement of upper region shown in d). and (f) (enlargement of lower region shown in d), show two highlighted regions of stroma and glands enhanced at 106 resolution, with the same colors and glycans shown for column (d). doi:10.1371/journal.pone.0106255.g004 MALDI MS Imaging of Glycans in FFPE Tissues of both red and green, while the glands are predominantly green. The distribution of other representative individual glycan ions is On-tissue Glycan Fragmentation and Structural Composition Figure 4. MALDI-IMS of a Human Prostate FFPE Tissue Block. On-tissue Glycan Fragmentation and Structural Composition For comparison, a Hex5HexNAc4 (m/z = 1663.6) purified standard (also termed NA2) was spotted on a stainless steel MALDI target plate and fragmented by CID, generating a robust fragmentation pattern of glycans for this ion as previously reported by Harvey et al [28]. The same glycan ion was abundant in pancreatic tissue after PNGaseF release of N-glycans (Figure 3) and was selected for CID. As shown in Figure 5b, the CID fragmentation pattern of m/z 1663.6 in pancreatic tissue was the same as the N-glycan standard, confirming detection of NA2 directly in pancreatic tissue (Figure 3). Mass shifts due to loss of On-tissue Glycan Fragmentation and Structural Composition of both red and green, while the glands are predominantly green. The distribution of other representative individual glycan ions is provided in Figure S4, including the distribution of high-mannose glycan species (Man5–Man9) associated with the heterogeneous tumor region in this tissue. The glycan structures identified by imaging of the FFPE tissue blocks were assigned based on the comparison to permethylated species, glycan reference databases and previous studies [24]. An on-tissue approach to further verify N-glycan structures was done PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org September 2014 \| Volume 9 \| Issue 9 \| e106255 6 MALDI MS Imaging of Glycans in FFPE Tissues Figure 5. Comparison of the Fragmentation Pattern of a Glycan Standard with the same Ion on Tissue. (a). A representative MALDI spectra for native N-linked glycans from pancreatic cancer FFPE tissue. (b). NA2 glycan standard (m/z = 1663.6) was fragmented using CID, revealing a variety of cleavages across glycosidic bonds as demonstrated in the spectrum (a). When the same ion was fragmented on the pancreatic tissue, the fragmentation pattern was the same, verifying that we were detecting Hex5HexNAc4 in the human pancreas. doi:10.1371/journal.pone.0106255.g005 f f Figure 5. Comparison of the Fragmentation Pattern of a Glycan Standard with the same Ion on Tissue. (a). A representative MALDI spectra for native N-linked glycans from pancreatic cancer FFPE tissue. (b). NA2 glycan standard (m/z = 1663.6) was fragmented using CID, revealing a variety of cleavages across glycosidic bonds as demonstrated in the spectrum (a). When the same ion was fragmented on the pancreatic tissue, the fragmentation pattern was the same, verifying that we were detecting Hex5HexNAc4 in the human pancreas. doi:10.1371/journal.pone.0106255.g005 individual sugar ions were detected, such as Hex (resulting in m/ z = 1502.5), HexNAc (resulting in m/z 1460.5), and Hex + HexNAc (resulting in m/z = 1298.5) (Figure 5b). An ion at m/ z = 712.2, which has been previously characterized [28] as the sodium adduct of Hex3HexNAc1, was also detected. The structures of 13 other glycan ions were confirmed using this CID approach, and additional fragmentation data and spectra are provided in Figures S5 & S6. using collision-induced dissociation (CID) directly on the human pancreatic tissue. Released native glycans from pancreatic cancer FFPE tissues were used as a source for on-tissue CID analysis, and a representative MALDI spectra of these glycans is shown in Figure 5a. Glycan MALDI-IMS of a Hepatocellular Carcinoma Tissue Microarray Interestingly 78 (94%) of the significantly different ions were elevated in tumor cores. After cross-referencing this list of 176 ions with glycans presented in this paper and our previous study [21], 26 N-glycans of high-confidence structure determinations were selected, listed in Table 1. Of these 26 known glycans, ion intensities of 13 species were significantly different in tumor and normal tissue (p,0.05), and 21 were increased in tumor relative to normal. FlexImaging was then used to demonstrate the distribu- tion and relative ion intensities of each glycan across the TMA (images provided in Figure S7). Additionally, ROC curves were used to evaluate how well each of the glycan ion intensities discriminates tumor versus non-tumor. Of the 176 identified ions, 61 had area under the ROC curve (AuROC).0.80, indicating they are strong classifiers. For two glycans at m/z = 2393.95 (Hex7HexNAc6) and m/z = 1743.64 (Hex8HexNAc2), both had an AuROC.0.80 and a p-value,0.05, with m/z 2393.95 being elevated in tumor tissue and m/z 1743.64 being elevated in non- tumor tissue, as demonstrated by the log2-fold change value (tumor/non-tumor) (Figure 6). In the overlay (Figure 6b) tumor Glycan MALDI-IMS of a Hepatocellular Carcinoma Tissue Microarray Glycan MALDI-IMS was done as described for the other FFPE tissues, and imaging data for two representative glycan ions at m/ z = 2393.92 (Hex7HexNAc6) and m/z = 1743.62 (Hex8Hex- NAc2) are shown in Figure 6. Analysis of the cumulative MALDI spectra and detected ions for each tissue core were processed and compared using an in-house bioinformatic workflow followed by statistical analysis. Of the 176 identified ions from the HCC TMA, 132 were increased in tumor cores, and 83 ions had a p-value, 0.05. Interestingly 78 (94%) of the significantly different ions were elevated in tumor cores. After cross-referencing this list of 176 ions with glycans presented in this paper and our previous study [21], 26 N-glycans of high-confidence structure determinations were selected, listed in Table 1. Of these 26 known glycans, ion intensities of 13 species were significantly different in tumor and normal tissue (p,0.05), and 21 were increased in tumor relative to normal. FlexImaging was then used to demonstrate the distribu- tion and relative ion intensities of each glycan across the TMA (images provided in Figure S7). Additionally, ROC curves were used to evaluate how well each of the glycan ion intensities discriminates tumor versus non-tumor. Of the 176 identified ions, 61 had area under the ROC curve (AuROC).0.80, indicating they are strong classifiers. For two glycans at m/z = 2393.95 (Hex7HexNAc6) and m/z = 1743.64 (Hex8HexNAc2), both had an AuROC.0.80 and a p-value,0.05, with m/z 2393.95 being elevated in tumor tissue and m/z 1743.64 being elevated in non- tumor tissue, as demonstrated by the log2-fold change value (tumor/non-tumor) (Figure 6). In the overlay (Figure 6b) tumor commercially available hepatocellular carcinoma (HCC) TMA (BioChain) consisting of samples from 16 individual patients, with two tumor tissue cores and one non-tumor tissue core per patient (Figure 6). Additional patient data are provided in Table S1. Glycan MALDI-IMS was done as described for the other FFPE tissues, and imaging data for two representative glycan ions at m/ z = 2393.92 (Hex7HexNAc6) and m/z = 1743.62 (Hex8Hex- NAc2) are shown in Figure 6. Analysis of the cumulative MALDI spectra and detected ions for each tissue core were processed and compared using an in-house bioinformatic workflow followed by statistical analysis. Of the 176 identified ions from the HCC TMA, 132 were increased in tumor cores, and 83 ions had a p-value, 0.05. Glycan MALDI-IMS of a Hepatocellular Carcinoma Tissue Microarray The ability to perform N-glycan analysis on FFPE tissues potentially enables the analysis of multiple FFPE tissue cores in a TMA format. Initial experiments were performed using a September 2014 \| Volume 9 \| Issue 9 \| e106255 September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 7 MALDI MS Imaging of Glycans in FFPE Tissues Figure 6. N-Glycan Imaging of a Liver TMA. A liver TMA purchased by BioChain consisting of 2 tumor tissue cores and one normal tissue core from 16 patients was imaged (200 mm raster). The H&E (a) provides the TMA location (red letters and numbers) and classifies whether the row is tumor (green bar) or non-tumor (red bar). M/z = 2393.95 (c) and m/z 1743.64 (d) were able to distinguish between hepatocellular carcinoma and uninvolved liver tissue. An overlay of these ion demonstrates that m/z = 2393.95 is elevated in tumor tissue and m/z = 1743.64 is elevated in normal tissue (b). Statistical data for these two ions is provided in Table 1. doi:10.1371/journal.pone.0106255.g006 Figure 6. N-Glycan Imaging of a Liver TMA. A liver TMA purchased by BioChain consisting of 2 tumor tissue cores and one normal tissue core from 16 patients was imaged (200 mm raster). The H&E (a) provides the TMA location (red letters and numbers) and classifies whether the row is tumor (green bar) or non-tumor (red bar). M/z = 2393.95 (c) and m/z 1743.64 (d) were able to distinguish between hepatocellular carcinoma and uninvolved liver tissue. An overlay of these ion demonstrates that m/z = 2393.95 is elevated in tumor tissue and m/z = 1743.64 is elevated in normal tissue (b). Statistical data for these two ions is provided in Table 1. doi:10.1371/journal.pone.0106255.g006 tissue is predominantly green and non-tumor tissue is predomi- nantly red, confirming results from our statistical analysis. This data, although from limited numbers of samples, demonstrates the potential ability of a panel of glycans to be used to accurately discriminate cell types or outcomes on a TMA by MALDI-IMS. commercially available hepatocellular carcinoma (HCC) TMA (BioChain) consisting of samples from 16 individual patients, with two tumor tissue cores and one non-tumor tissue core per patient (Figure 6). Additional patient data are provided in Table S1. Discussion Multiple N-linked glycans can be directly profiled from FFPE tissue blocks and TMAs while maintaining intact architecture. The basic methodology, which mirrors that of MALDI-IMS analysis of peptides in FFPE tissues and TMAs [18–20], requires deparaffi- nization and antigen retrieval prior to PNGaseF application. The ability to adapt the N-glycan imaging method originally designed for fresh/frozen tissues [25] to encompass FFPE tissue and TMA blocks increases the scope and speed of glycan-based studies that can be performed in tissues. In initial studies of formalin-fixed mouse kidney slices, the MALDI-IMS workflow successfully identified all 28 of the glycans in the mouse kidney database provided by the Consortium for Functional Glycomics. Many of the structures of these glycans were verified by permethylation (Figure S2) and CID experiments (Figures S5 & S6). As observed with the mouse brain [25], these glycans were not homogenously present across the entire mouse kidney slice, but were either predominantly located in the cortex, or distributed across the cortex and medulla (Figure S1). This unique distribution of N- glycans associated with tissue sub-structure or disease status was also observed in human pancreas and prostate tissue slices. In the pancreas, an overlay of four different glycans was able to map the normal pancreas tissue, tumor pancreas tissue, a region of desmoplasia, and a necrotic region (Figure 3a). Similarly, an September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org 8 MALDI MS Imaging of Glycans in FFPE Tissues Table 1. Comparison of Tumor and Non-Tumor Glycans Detected in Hepatocellular Carcinoma Tissue Microarrays. m/z AuROC log2-fold change p-val 1866.76 0.895 3.045 ,.001 2393.95 0.879 1.819 ,.001 2378.01 0.869 3.495 ,.001 1743.64 0.831 20.967 ,.001 1850.78 0.827 1.953 ,.001 1257.42 0.821 0.780 ,.001 1501.60 0.813 2.223 0.001 2686.02 0.764 4.316 0.003 1905.71 0.742 20.572 0.007 1298.44 0.742 0.910 0.007 2012.82 0.734 1.165 0.010 2320.89 0.707 2.146 0.022 2540.03 0.707 1.493 0.022 3271.15 0.657 2.004 0.082 2028.74 0.657 0.360 0.082 1647.62 0.649 0.683 0.099 1581.57 0.635 20.190 0.135 2174.89 0.633 0.598 0.141 1976.71 0.617 0.398 0.197 2100.77 0.597 0.295 0.266 1954.79 0.593 20.114 0.307 1419.48 0.577 20.076 0.400 1809.67 0.548 0.973 0.598 1485.54 0.488 0.149 0.902 1663.66 0.490 0.255 0.920 1282.46 0.494 0.703 0.991 A list of 26 monoisotopic ions that were identified in the HCC TMA were cross-referenced against a library of known glycan m/z values listed in this paper or our previous paper (21). Discussion The ability of individual glycan ions to distinguish tumor tissue from non-tumor tissue were assessed using AuROC, log2-fold changes (tumor/non- tumor), and p-values. Localization of m/z = 2393.95 and m/z = 1743.62 (highlighted in yellow) are depicted in Figure 6. doi:10.1371/journal.pone.0106255.t001 A list of 26 monoisotopic ions that were identified in the HCC TMA were cross-referenced against a library of known glycan m/z values listed in this paper or our previous paper (21). The ability of individual glycan ions to distinguish tumor tissue from non-tumor tissue were assessed using AuROC, log2-fold changes (tumor/non- tumor), and p-values. Localization of m/z = 2393.95 and m/z = 1743.62 (highlighted in yellow) are depicted in Figure 6. doi:10 1371/journal pone 0106255 t001 A list of 26 monoisotopic ions that were identified in the HCC TMA were cross-referenced against a library of known glycan m/z values listed in this paper or our previous paper (21). The ability of individual glycan ions to distinguish tumor tissue from non-tumor tissue were assessed using AuROC, log2-fold changes (tumor/non- tumor), and p-values. Localization of m/z = 2393.95 and m/z = 1743.62 (highlighted in yellow) are depicted in Figure 6. doi:10.1371/journal.pone.0106255.t001 overlay of two glycans could distinguish between prostate stroma and glands (Figure 4). observed in the average spectra of the mouse kidney tissue after PNGase application (Figure 2a) from m/z = 1250–1300, 1450– 1500, and 1650–1700. An additional key to distinguishing polymer peaks is the analysis of spectra from the non-PNGaseF treated control tissues. Particularly for the TMA format, the ion selection program that we report can detect and account for polymer peaks relative to glycan ions. These polymer peaks seem to vary in terms of intensity compared to N-glycan ions depending on what tissue is being used. It is possible that this variation is a function of different formalin formulations, variations is tissue processing (i.e. amount of time in formalin), storage time or variations in the tissue itself [1,2]. These considerations will be further monitored and evaluated as more glycans from FFPE tissues are analyzed. In general, the peak intensities of PNGaseF-released glycans in the FFPE tissues seems to be more intense than that obtained with fresh/frozen tissue sections. This may be a result of the more extensive heating and washing steps required in the deparaffiniza- tion and rehydration steps. September 2014 \| Volume 9 \| Issue 9 \| e106255 Figure S6 CID of N-Glycans from Human Pancreas Tissue II. (TIF) Figure S7 Images From Ions Corresponding to N- Glycans. The Ions identified in Table 1 were viewed in FlexImaging Software. (TIF) Table S1 Patient Data Summary for Hepatocellular Carcinoma TMA from Biochain. (TIF) Author Contributions Conceived and designed the experiments: TWP BAN DAT ASM BBH RRD. Performed the experiments: TWP BAN YS HYT. Analyzed the data: TWP BAN DAT ASM BBH RRD. Contributed reagents/materials/ analysis tools: YS ASM BAH RRD. Contributed to the writing of the manuscript: TWP BAN DAT ASM BBH RRD. Figure S1 Panel of Mouse Kidney N-Glycans. Ions detected in the kidney with enzyme application were compared to the control tissue. Ions that were only observed in the tissue following PNGaseF application were compared to the glycans found in the mouse kidney database on the Consortium for Discussion Our data analysis identified a total of 176 ions in the tissue, with the majority of significantly different ions being increased in HCC relative to non-tumor tissue, including 21 known or previously identified glycans. It is unclear how this trend of increased glycan levels relates specifically to tumor related biochemical changes, though the role of glycosylation in tumor development is well documented. Future work will also focus on determining the identity of the remaining ions to distinguish other glycan species from the aforementioned polymer peak contami- nants. Functional Glycomics. The panel provides the glycan species, the projected mass for the sodium adduct, and our observed mass for the sodium adduct. (TIF) Figure S2 Permethylation of Mouse Kidney N-Glycans. Mouse kidney N-glycans were extracted from the imaging slide after PNGaseF application and digestion. Glycans were dried down and underwent permethylation as previously described. The permethylated m/z values were then compared to the permethy- lation data from the Consortium for Functional Glycomics mouse kidney database (www.functionalglycomics.org). (TIF) 13. Chen CY, Jan YH, Juan YH, Yang CJ, Huang MS, et al. (2013) Fucosyltransferase 8 as a functional regulator of nonsmall cell lung cancer. Proc Natl Acad Sci U S A 110; 630–635. Figure S3 Panel of Mouse Kidney N-Glycans Linked to Known Glycan Database. (TIF) Figure S4 Individual N-Glycans from Prostate Cancer FFPE Tissue. The orange ovals highlight the areas of heterogeneous tumor for high mannose glycans. (TIF) Currently, MALDI-IMS provides a new approach to effectively visualize and evaluate N-glycan localization in tissue sections. It does not solve the known limitations of MALDI analysis of underivatized glycans like loss of sialic acids, nor does it provide anomeric linkage information for N-glycan structure. Established tandem mass spectrometry methods of glycan extraction, modi- fication and fragmentation are more capable of providing this structural information. Combining the glycan tissue maps generated by MALDI-IMS to target regions of interest for further tandem mass spectrometry analysis of glycans could be a new synergistic approach to more effectively identify tumor-associated glycans and glycoproteins in situ. Use of other glycosidases like sialidase, as we have previously reported [25], or fucosidases, could further extend the utility of the combined methods. Overall, the ability to effectively profile N-glycans on FFPE tissue blocks and TMAs provides new opportunities to evaluate glycan profiles associated with disease status. Figure S5 CID of N-Glycans from Human Pancreas Tissue I. (TIF) Discussion It is this increased detection sensitivity that facilitated CID fragmentation of N-glycans directly from the tissue (Figure 5b, Figures S5 & S6). Under the conditions used, CID generated mainly fragments across the glycosidic bonds, which were useful in characterizing that the structure was an intact hexose or HexNAc. This did not provide any information regarding anomeric linkages between sugar residues. The amount of fragmentation observed was directly related to the relative intensity of each parent N-glycan ion, and inversely related to the mass of the parent ion observed. This is typified by the extensive fragmentation of two glycans of m/z = 1663.50 and m/ z = 1809.64 (Figure 5b, Figures S5 & S6). In relation to potential cancer diagnostic applications, the most significant aspect to developing a method to image N-glycans on FFPE tissue blocks could be the ability to use TMAs for high- throughput glycan-based experiments. Not only does the method increase the number of tumor samples that can be analyzed in one experiment, but it could also be used to compare the glycans detected in a TMA core versus the larger source FFPE tissue. N- glycan MALDI-IMS of the HCC TMA (Figure 6) is provided as an example, but we have already obtained initial glycan profiling data from TMAs representing prostate, kidney, lung, breast, colon One drawback to using FFPE tissues is residual polymer from the paraffin block adjacent to the tissue. Detection of this polymer is more predominant in the lower mass range of the imaging runs, and can overlap with potential glycan masses, complicating detection and further statistical analysis. This polymer can be September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org 9 MALDI MS Imaging of Glycans in FFPE Tissues Functional Glycomics. The panel provides the glycan species, the projected mass for the sodium adduct, and our observed mass for the sodium adduct. (TIF) and pancreatic cancers. In the HCC TMA, a statistically significant increase in tetra-antennary N-glycan (m/z = 2393.95) and decrease in Man-8 glycan (m/z = 1743.64) was detected in HCC cores compared to adjacent non-tumor tissue (Figure 6 and Table 1). The tetra-antennary N-glycan has been previously demonstrated to be elevated in HCC compared to matched adjacent non-tumor tissue by Mehta et al. [29]. Continued investigations will be performed on whether these two ions can distinguish between matched HCC and non-tumor tissues in other HCC TMAs. 14. Kobayashi M, Nakayama J (2010) Immunohistochemical analysis of carbohy- drate antigens in chronic inflammatory gastrointestinal diseases. Methods Enzymol 479; 271–289. 15. Chaurand P, Norris JL, Cornett DS, Mobley JA, Caprioli RM (2006) New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry. J Proteome Res 5; 2889–2900. References 1. Thompson SM, Craven RA, Nirmalan NJ, Harnden P, Selby PJ, et al. 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Proteomics 12: 1045–1058. 12. van Cruijsen H, Ruiz MG, van der Valk P, de Gruijl TD, Giaccone G (2009) Tissue micro array analysis of ganglioside N-glycolyl GM3 expression and signal transducer and activator of transcription (STAT)-3 activation in relation to dendritic cell infiltration and microvessel density in non-small cell lung cancer. BMC Cancer 9:180. 4. Wis´niewski JR, Dus´ K, Mann M (2013) Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins. Proteomics Clin Appl 7; 225–233. p pp 5. Takikita M, Chung JY, Hewitt SM (2007) Tissue microarrays enabling high- throughput molecular pathology. Curr Opin Biotechnol 18: 318–325. 13. Chen CY, Jan YH, Juan YH, Yang CJ, Huang MS, et al. (2013) Fucosyltransferase 8 as a functional regulator of nonsmall cell lung cancer. Proc Natl Acad Sci U S A 110; 630–635. 6. Franco R, Caraglia M, Facchini G, Abbruzzese A, Botti G (2011) The role of tissue microarray in the era of target-based agents. Expert Rev Anticancer Ther 11; 859–869. 14. Kobayashi M, Nakayama J (2010) Immunohistochemical analysis of carbohy- drate antigens in chronic inflammatory gastrointestinal diseases. Methods Enzymol 479; 271–289. 7. Hewitt SM (2006) The application of tissue microarrays in the validation of microarray results. Methods Enzymol 410: 400–415. 15. Chaurand P, Norris JL, Cornett DS, Mobley JA, Caprioli RM (2006) New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry. J Proteome Res 5; 2889–2900. 8. MALDI MS Imaging of Glycans in FFPE Tissues MALDI MS Imaging of Glycans in FFPE Tissues kinase/extracellular signal-regulated kinase kinase kinase 2 discriminates cancer from uninvolved prostate tissue. Clin Cancer Res 15; 5541–5551. 23. Quaas A, Bahar AS, von Loga K, Seddiqi AS, Singer JM, et al. (2013) MALDI imaging on large-scale tissue microarrays identifies molecular features associated with tumour phenotype in oesophageal cancer. Histopathology 63; 455–462. 17. Berry KA, Hankin JA, Barkley RM, Spraggins JM, Caprioli RM, et al. (2011) MALDI imaging of lipid biochemistry in tissues by mass spectrometry. Chem Rev 111; 6491–6512. p yp p g p gy 24. Casadonte R, Caprioli RM (2011) Proteomic analysis of formalin-fixed paraffin- embedded tissue by MALDI imaging mass spectrometry. Nat Protoc 6; 1695– 1709. 18. Chaurand P, Cornett DS, Angel PM, Caprioli RM (2011) From whole-body sections down to cellular level, multiscale imaging of phospholipids by MALDI mass spectrometry. Mol Cell Proteomics 10: O110.004259. 25. Powers TW, Jones EE, Betesh LR, Romano PR, Gao P, et al. (2013) Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues. Anal Chem 85; 9799–9806. 19. Castellino S, Groseclose MR, Wagner D (2011) MALDI imaging mass spectrometry: bridging biology and chemistry in drug development. Bioanalysis 3; 2427–2441. 26. Ceroni A, Maass K, Geyer H, Geyer R, Dell A, et al. (2008) GlycoWorkbench: A Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans, J Proteome Res 7; 1650–1659. 20. Cornett DS, Frappier SL, Caprioli RM (2008) MALDI-FTICR imaging mass spectrometry of drugs and metabolites in tissue. Anal Chem 80; 5648–5653. 27. Shi C, Washington MK, Chaturvedi R, Drosos Y, Revetta FL, et al. (2014) Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction. Lab Invest 94; 409–421. 21. Nilsson A, Fehniger TE, Gustavsson L, Andersson M, Kenne K, et al. (2010) Fine mapping the spatial distribution and concentration of unlabeled drugs within tissue micro-compartments using imaging mass spectrometry. PLoS One; e11411. 28. Harvey DJ (2005) Structural determination of N-linked glycans by matrix- assisted laser desorption/ionization and electrospray ionization mass spectrom- etry. Proteomics 5; 1774–1786. 22. Groseclose MR, Massion PP, Chaurand P, Caprioli RM (2008) High- throughput proteomic analysis of formalin-fixed paraffin-embedded tissue microarrays using MALDI imaging mass spectrometry. Proteomics 8 3715– 3724. y 29. Mehta A, Norton P, Liang H, Comunale MA, Wang M, et al. References Camp RL, Neumeister V, Rimm DL (2008) A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers. J Clin Oncol 26; 5630–5637. 8. Camp RL, Neumeister V, Rimm DL (2008) A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers. J Clin Oncol 26; 5630–5637. 16. Cazares LH, Troyer D, Mendrinos S, Lance RA, Nyalwidhe JO, et al. (2009) Imaging mass spectrometry of a specific fragment of mitogen-activated protein PLOS ONE \| www.plosone.org 10 September 2014 \| Volume 9 \| Issue 9 \| e106255 September 2014 \| Volume 9 \| Issue 9 \| e106255 MALDI MS Imaging of Glycans in FFPE Tissues (2012) Increased levels of tetraantennary N-linked glycan but not core fucosylation are associated with hepatocellular carcinoma tissue. Cancer Epidemiol Biomarkers Prev 21; 925–933. September 2014 \| Volume 9 \| Issue 9 \| e106255 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 11
https://openalex.org/W2901305164	https://www.matec-conferences.org/10.1051/matecconf/201823201036/pdf	English	null	The Effective Sleep Scheduling in Wireless Opportunistic Networks	MATEC web of conferences	2,018	cc-by	4,224	The Effective Sleep Scheduling in Wireless Opportunistic Networks Wenzao Li1, Zhan Wen1,a,Jianwei Liu1, Shuang Xiao1, Xi Wu2 ,Yue Cao3, Jiliu Zhou2 Wenzao Li , Zhan Wen , ,Jianwei Liu , Shuang Xiao , Xi Wu ,Yue Cao , Jiliu Zhou 1 College of Communication Engineering, Chengdu University of Information Technology, Chengdu 610225, China 2 School of Computer Science, Chengdu University of Information Technology, Chengdu 610225, China 3 Department of Computer and Information Sciences, Northumbria University, Newcastle, UK 1 College of Communication Engineering, Chengdu University of Information Technology, Chengdu 610225, China 2 School of Computer Science, Chengdu University of Information Technology, Chengdu 610225, China 3 Department of Computer and Information Sciences, Northumbria University, Newcastle, UK Abstract. One of the purposes of Internet of Things (IoT) is to reach more deeper perception. For this purpose, the efficient energy consumption is necessary among intelligent devices that make up the part of Opportunistic Networks (ONs). It is irrational for an ONs without any sleep scheduling because of awful user experience. We explore a sleeping schedule which is based on duty cycling for mobile devices to reduce energy consumption of ONs. To see how schedule affects the performance of ONs, we took a series of simulations and the results indicated that the sleeping schedule is an efficient method for prolonging the network life time in ONs. The successful delivery ratio can increase two to three times when factor T୰ equal 0.2. We also observed that the network matrices are acceptable, and the network survival time can be extended effectively in ONs. includes a number of mobile devices. These devices run numerous programs and own various battery specifications. It is difficult to estimate the energy efficiency due to complex power supply conditions. The main devices in IoT technology are various portable devices, which have been performed for many applications and services. And these devices are used for not only one task in our daily life, then the energy consumption for short-distance communication is ambiguity for users. To assess and optimize the network energy efficiency, the energy consumption model and a sleep scheduling are needed in such complex situation. 1 Introduction Recently, various of intelligent devices (mobile phone, smart bands or tablets) widespread appear in our daily life. And these devices are equipped short-range wireless communication module. Therefore it can provide the enhanced wireless communication ability for different application systems[1]. ONs are based on Delay Tolerant Networks (DTNs) technology. It consists of sensors with short-range communication ability, the message transmits by store-and it attracts many researches in the context of Device-to-Device(D2D) communication in 5G[2, 3]. Especially, city areas become important places of data generation and consumption. In social environments, ONs network can enrich the integrated ecosystem which is create by IoT technology[4]. The ubiquitous wireless application systems play pivotal roles in Smart City, which is established by various of devices such as smart vehicles, personal electronics and wireless infrastructures. These devices always carried by human so they were given the mobility feature. Most of mobile devices exchange the message by encounter devices nearby. Therefore, the mobile devices can enhance the performance of seamless connectivity among data islands and depth of perceptive information in IoT. Then, many mobile sensor networks applied mobile devices or vehicles to build for emerging applications and data collection system. So, an obvious question arises, the energy of mobile devices is always supplied by battery, and the energy is limited during a period of time. But obviously, ONs communication network structure With the consideration to the network level and energy efficiency in ONs, the main research contributions are listed as follows: (1) The energy consumption model in opportunistic translation is described. (2) The network lifetime and network performances are effective promote by a sleep scheduling which is usually discussed as duty cycle in WSN. The rest of this paper is organized as follows. Section 1 describes the related work. Then we describe the energy consumption model for the device to transmit messages to another device nearby in ON networks in section 2. The section 3 gives the simulation environment and results. Finally, we give a conclusion in section 4. a Corresponding author:Zhan Wen wenzhan@cuit.edu.cn © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). MATEC Web of Conferences 232, 01036 (2018) EITCE 2018 MATEC Web of Conferences 232, 01036 (2018) EITCE 2018 https://doi.org/10.1051/matecconf/201823201036 thor:Zhan Wen wenzhan@cuit.edu.cn EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 censes/by/4.0/). 2 Related Works But these adaptive duty-cycling methods should be automatic synchronized by global clock and the inter-contact intervals of pair node. In precious research, the part of scan energy is the largest proportion in ONs, then the sleeping scheduling can prolong the network life time. Also, the message transmission can consume the nodes’ limited energy, the literature[10, 11] also discuss routing method for energy consumption. They limited the message copies in DTN routing, and then energy conservation is achieved by this way. The human-carried communication devices, which mainly consist of ONs in urban scenario, always take on variety of application tasks such as navigation, socializing, and etc. Generally, the inefficient energy consumption of devices will decrease the user experience although each device has opportunities for energy replenishment in human daily life. The ONs, as a data transmission way, is a function for the mobile intelligent device. Thus, the energy of ONs should be used as little as possible due to the limited electric capacity[12]. The ONs always be structured in urban areas which is different from other sensing areas. Hence the energy saving mechanism is increasingly important in urban based ONs. From the above, this paper presents the duty-cycling based sleeping schedule for ONs in urban environments that aims to improve the energy efficiency. This sleeping scheduling not only prolongs the network lifetime but also gives benchmarks for follow-up sleeping strategy. 3 D t li M th d f ON i U b 2 Related Works Generally speaking, mobile devices are mainly various smart phones or other portable equipments. The mobile devices need neighbor scanning and transmitting MATEC Web of Conferences 232, 01036 (2018) EITCE 2018 https://doi.org/10.1051/matecconf/201823201036 The common discussed duty-cycling method is widely used in Wireless Sensor Networks (WSN) because of the limited energy of nodes. The sleeping scheduling aims to improve the energy efficiency and extend the survival time of nodes. ONs are widely discussed with the rapid development of the personal intelligent mobile devices. The main different between WSN and ONs is the mobility of nodes. Generally, node carries the packets at the moving status and transmits the packets during the encounter opportunities in ONs. Thus, nodes should continue scanning the nodes which is covered by the transmitting range in such multi-hop wireless networks. Because of this working mechanism, the discovering behavior will exhaust the limited energy continuously. continuously in ONs. However, the energy consumption of mobile devices depends on the encounter opportunity. Obviously, contact discovery need a response message in the routing process, and this phenomenon is frequently happened in high node density areas. The massive multi-hop transmitting and transmitting among devices will lead to excessive energy consumption, which will give participants some unpleasant experience. In such case, the energy efficiency should be considered in ONs. In ONs, the energy consumption mainly occurs from scan device nearby, scan response behavior and transmitting messages[5]. Y. Dou describe a contact model[6] which sets the contact probing interval for the energy conservation. But the energy efficiency depends on the routing method, thus the scanning factor was ignored under the circumstance. In literature[7], B.J. Choi also described the energy saving mechanism. The fundamental problem is that the listening mode spend more than 95% percent of total energy for neighbor searching. Thus, most of the energy consumption is not efficient for the data transition. Generally, a sleep schedule includes wakeup method. For example, mobile nodes may equip low power radio module to wake up the device, named nodes. As the well-known IEEE 802.11 PSM protocol[8], can make the mobile nodes turn on and turn off for energy saving. It can be described as duty cycling in such single hop networks. The sleeping behaviors depend on the prediction of contact opportunity. Zeng F. et al.[9] also focus on the listening behavior and propose an adaptive scheduling mechanism which based on self-similarity feature. 3.1 Energy Model for ONs Generally, the energy model of ONs is described by literature[13]. From the literature, the energy consumption of DTN consists mainly of three parts, scan energy 𝐸௦, scan response energy 𝐸௥௘௦ and transmission energy 𝐸௧௥. Then the energy consumption model can be represented as equation (1). 𝐸௖= 𝐸௦+ 𝐸௥௘௦+ 𝐸௧௥ (1) (1) As previously mentioned, the 𝐸௦ is a big part of the energy consumption in ONs. The 𝐸௥௘௦ is the energy consumption for that when a node response for a scanning message, thus it depends on the contact probability. Then the inter-contact time is defined to describe the encounter intermittent time of node[14]. Generally, the ONs is a sparse node network, thus the inter-contact time among nodes are relatively long time period. If we assume a time period 𝑡 for a node 𝑖. The energy consumption 𝐸௖ (௜)(𝑡) can be represented by equation (2): 𝐸௖ (௜)(𝑡) = ௧ ௧ᇲ𝐸௦+ 𝑇௠(𝑡)𝐸௥௘௦+ 𝑇௡(𝑡)𝐸௧௥ (2) (2) ௧ Where the 𝑡ᇱ presents the scanning interval time. The 𝑇௠(𝑡) presents the cumulative number of responses to another scanning message during the time period 𝑡. In a similar way, the 𝑇௡(𝑡) presents the cumulative number of packet transmission. In the actual situation, the mobile intelligent devices (mobile phone, smart bracelet and etc.) play the role of mobile nodes, which could take on multiple tasks. For instance, the intelligent devices can be used for calls or navigation. Therefore, the energy consumption of ONs is a part of the total energy consumption for a single node. It’s a hard problem to estimate the energy consumption due to the using habit, power supplement, computational power and etc. that is to say, the 𝐸௖ (௜) ∈𝑆 where 𝑆 include the operation system energy consumption, personal application energy consumption and etc. Meanwhile, the energy supplement cannot be foreseen. So, we ignore the standby energy consumption and we use the equation (2) to describe the energy consumption states for ONs in metropolis areas. The mobile node should have continuous scanning for the purpose of neighbor node discovering. The neighbor node will response the discovering signal and it will consumes some transmitting energy. Then, the message will be transmitted after a pair of nodes finish a connection and the node will expend the data transmitting energy which 3.2 Duty Cycling Method for Energy Saving Duty Cycling methods are widely discussed in WSN for energy saving[15]. The sensors are in turn-off and turn-on status. When it is in turn off status, the transceiver of sensor is turn off and the sensor cannot take part in any communication tasks. Generally, the turn-on and turn-off statuses are spread regularly on the time axis. The work duty cycling of a sensor can be described as figure 1. Fig.2 The status of swapping of a node in duty cycling method in ONs Fig.1 The status of swapping of a node in duty cycling method in WSN Fig.2 The status of swapping of a node in duty cycling method in ONs Fig.2 The status of swapping of a node in duty cycling method in ONs Fig.1 The status of swapping of a node in duty cycling method in WSN The sleeping time period in ONs are established based on the global clock synchronization in application layer. If the short range communication technology use the Bluetooth, one approach is adopt Bluetooth clock to synchronized the slaves by master or layer-2 techniques [17, 18]. Each node is awaked during the time point 𝑇𝑠𝑠_𝑜𝑛 and 𝑇𝑠𝑠_𝑜𝑓𝑓. When nodes moving in a communication range, the synchronization statue ensure they are in Turn-on statue. In this sleeping schedule, the mobile nodes will stop neighbor searching during 𝑇𝑠_𝑜𝑓𝑓 time period. And the duty cycle 𝑇௥ can be calculated by equation (3). From figure 1, node 𝑖 switch working status 𝑇𝑠_𝑜𝑛 time period and 𝑇𝑠௢௙௙ time period for the purpose of prolonging the node’s survival time. The method can reduce the energy consumption obviously. The working duty ratio means how long can it works in each time period ∆𝑡. And there is adaptive duty cycling method for energy saving[16], it should have a node wake-up mechanism. 4 Duty Cycling as Sleeping Schedule in ONs 𝑇௥= ்௦_௢௡ ∆௧ (3) 𝑇௥= ்௦_௢௡ ∆௧ (3) The main difference between WSN and ONs is that nodes have mobility in opportunistic networks. Therefore, duty cycling method should face some roadblocks. (1) There is no timely information can be received for entire network, thus the turn-on and turn-off status should consider the clock synchronization for all nodes. The data transmission required a pair of awake nodes which is in each communication range. (2) The sleeping schedule should increase the average latency of delivery because of the fewer contact times and unforeseeable encounter opportunity in ONs. However, the sleeping schedule is necessary for ONs in urban areas due to some reasons. (1) The ONs main consist of intelligent mobile devices, it is a sparse network essentially. Then, the relatively long inter-contact time surely will bring on superabundant 𝐸௦, which should decrease the energy efficiency and short the survival time of nodes. (2) The delivery ratio is one of most important key metrics in ONs, a suitable efficient sleeping schedule can improve the delivery ratio in limit energy circumstances. Where ∆𝑡 equal to 𝑇𝑠_𝑜𝑛 plus the 𝑇𝑠_𝑜𝑓𝑓 time duration. 3 Duty-cycling Method for ONs in Urban Areas 3 Duty-cycling Method for ONs in Urban Areas 2 2 MATEC Web of Conferences 232, 01036 (2018) EITCE 2018 https://doi.org/10.1051/matecconf/201823201036 present by 𝑇௡(𝑡). Obvious, in a sparse wireless network, the scan energy spends a large partition of energy 𝐸௖. Therefore, the duty-cycling approach is an effective sleeping scheduling for improving energy efficiency in metropolis. has social characteristics, the nodes will have longer moving pause periods. For instances with this, people rest at home or working in office. But the data transmission opportunities are based on the mobility of nodes, it is more necessary to design a sleeping schedule in ONs. It is similar to duty cycling using in WSN, each node has turn-on and turn-off status in ONs. But it is only to application layer in an intelligent device. In order to ensure the contact nodes in the same status, the duty cycling units consider synchronized in entire network. 5 Simulation and results In figure 5, the experiment results indicate that the 0.2 Tr is not well, the performance of 0.2 Tr and 1 Tr for overhead ratio is similar. And it is acceptable in ONs. Network overhead ratio can reflect the invalid in ONs. Because it runs with multiple copies routing strategy, the higher value signifies the greater network pressure. It can result the message drop ratio in ONs. In figure 5, the experiment results indicate that the 0.2 Tr is not well, the performance of 0.2 Tr and 1 Tr for overhead ratio is similar. And it is acceptable in ONs. Fi 3 Th dit i ON ti Fig.6 The successful delivery ratio with various Tr over time From figure 6, the 0.2 Tr shows the best performance in successful delivery ability in ONs. 0.4 Tr and 0.6 Tr show that the successful delivery ratio decreased by the more working time in ONs with limited energy. And the sleeping schedule with 0.2 Tr can improve the successful delivery ratio two to three times than ONs with none sleeping schedule. The results indicate that the suitable sleeping mechanism can increase the delivery ability in mobile sensor networks. The main cause is that ONs is a sparse network in urban areas, thus a lot of energy is used for neighbor scanning. Therefore, in the case of energy constrain, the suitable sleeping schedule can effectively prolong the network life time. Fig.6 The successful delivery ratio with various Tr over time Fig. 3 The energy expenditure in ONs over time Fig. 3 The energy expenditure in ONs over time It shows that the 0.2 is the best in energy efficiency. The network exhausts the energy after 108 hours. If the network runs with no any sleeping schedule or working at 0.8 𝑇௥, the network will dead at less than 48 hours. This network energy efficiency is half of 0.2 𝑇௥. The 0.4 𝑇௥ shows the second best in ONs. It shows that the 0.2 is the best in energy efficiency. The network exhausts the energy after 108 hours. If the network runs with no any sleeping schedule or working at 0.8 𝑇௥, the network will dead at less than 48 hours. This network energy efficiency is half of 0.2 𝑇௥. The 0.4 𝑇௥ shows the second best in ONs. 5 Simulation and results In order to improve the reality of ONs scenario, we choose the Working Day Movement Model (WDM) and The Opportunistic Network Environment (The ONE) simulator. The Helsinki map is selected for urban areas. In order to observe the effect of this sleeping schedule in ONs, we choose the message flooding method for message routing. The simulation scenario is established by table 1. y Tab. 1 The simulation scenario Groups Type Nodes A,B,C,D Pedestrian 40 for each a,b,c,d Bus 4 for each T,P,Q Tram 2 for each The key parameters of the sleeping schedule are list as table 2. In ONs, the mobile communication devices are carried by person or assembled in car. Then, the mobility pattern 3 3 MATEC Web of Conferences 232, 01036 (2018) MATEC Web of Conferences 232, 01036 (2018) EITCE 2018 https://doi.org/10.1051/matecconf/201823201036 means that nodes in turn-off status result in the message transmitting stagnation. Fig.5 The overhead ratio with various Tr over time Network overhead ratio can reflect the invalid in ONs. Because it runs with multiple copies routing strategy, the higher value signifies the greater network pressure. It can result the message drop ratio in ONs. In figure 5, the experiment results indicate that the 0.2 Tr is not well, the performance of 0.2 Tr and 1 Tr for overhead ratio is similar. And it is acceptable in ONs. means that nodes in turn-off status result in the message transmitting stagnation. Tab. 2 the key parameters of the sleeping schedule Parameter name Value ∆𝑡 5 seconds 𝐸௦ 1/3.0105 percent 𝐸௥௘௦ 1/3.0105 percent 𝐸௥௘௦ 2/3.0*105 percent Fig.5 The overhead ratio with various Tr over time We chose the 𝑇௥ as 1,0.2, 0.4, 0.6 and 0.8 respectively, and duty cycling equal one means the sleeping schedule is invalid. The Epidemic routing is selected for ONs running, because it can transmit messages when nodes encounter in right condition. Then we carried out multiple simulations with the sleeping schedule. In order to observe energy efficiency, the energy expenditure of nodes and the key network indicators are important for a sleeping schedule. The energy expenditure with different 𝑇௥ in ONs are shown in figure 3. Fig.5 The overhead ratio with various Tr over time Network overhead ratio can reflect the invalid in ONs. Because it runs with multiple copies routing strategy, the higher value signifies the greater network pressure. It can result the message drop ratio in ONs. 5 Simulation and results Fig.6 The successful delivery ratio with various Tr over time Fig.4 The successful delivery ratio with various Tr over time In ONs, the average latency presents the average spend time on successful delivered messages. For a data transmission network, the shorter latency the better application system is based on ONs technology. It is clear in figure 4, the Tr=0.2 shows the highest latency in this scenario. The 0.8 and 1 show the relatively low latency. It g y From figure 6, the 0.2 Tr shows the best performance in successful delivery ability in ONs. 0.4 Tr and 0.6 Tr show that the successful delivery ratio decreased by the more working time in ONs with limited energy. And the sleeping schedule with 0.2 Tr can improve the successful delivery ratio two to three times than ONs with none sleeping schedule. The results indicate that the suitable sleeping mechanism can increase the delivery ability in mobile sensor networks. The main cause is that ONs is a sparse network in urban areas, thus a lot of energy is used for neighbor scanning. Therefore, in the case of energy constrain, the suitable sleeping schedule can effectively prolong the network life time. Fig.4 The successful delivery ratio with various Tr over time Fig.4 The successful delivery ratio with various Tr over time In ONs, the average latency presents the average spend time on successful delivered messages. For a data transmission network, the shorter latency the better application system is based on ONs technology. It is clear in figure 4, the Tr=0.2 shows the highest latency in this scenario. The 0.8 and 1 show the relatively low latency. It Fig.4 The successful delivery ratio with various Tr over time In ONs, the average latency presents the average spend time on successful delivered messages. For a data transmission network, the shorter latency the better application system is based on ONs technology. It is clear in figure 4, the Tr=0.2 shows the highest latency in this scenario. The 0.8 and 1 show the relatively low latency. It 7 Conflicts of Interest The authors declare that there are no conflicts of interest regarding the publication of this paper. 13. Keränen, A., J. Ott, and T. Kärkkäinen. The ONE simulator for DTN protocol evaluation. in International Conference on Simulation TOOLS and Techniques. 2009. 6 Conclusions ONs as one of DTN, it has more application prospects in IoT. It differs from the traditional wireless sensor networks due to the mobility of nodes. Then the sleeping schedule should be redesigned by deployment scenario. 4 https://doi.org/10.1051/matecconf/201823201036 MATEC Web of Conferences 232, 01036 (2018) MATEC Web of Conferences 232, 01036 (2018) EITCE 2018 10. Li, Y., et al. Optimal Opportunistic Forwarding Policies for Energy-Constrained Delay Tolerant Networks. in IEEE International Conference on Communications. 2010. Duty cycling method has effect in ONs, thus this paper discussed the sleeping schedule based on duty cycle method in ONs. The results indicated that, except the less-than-ideal average latency, the sleeping schedule can optimize the energy consumption and increase the successful delivery ration for ONs in urban areas. It is also important that it can serve as a benchmark for further similar researches. Duty cycling method has effect in ONs, thus this paper discussed the sleeping schedule based on duty cycle method in ONs. The results indicated that, except the less-than-ideal average latency, the sleeping schedule can optimize the energy consumption and increase the successful delivery ration for ONs in urban areas. It is also important that it can serve as a benchmark for further similar researches. 11. Li, Y., et al. Performance Evaluation of Routing Schemes for Energy-Constrained Delay Tolerant Networks. in IEEE International Conference on Communications. 2012. 12. Tan, D.N., et al. Mobile charging and data gathering in multiple sink Wireless Sensor Networks: How and why. in International Conference on System Science and Engineering. 2017. Acknowledgements The authors would like to thank the reviewers for their insightful feedback and valuable suggestions. This work was financially supported by the Science and Technology Department of Sichuan Province, Fund of Science and Technology Planning (No. 2018JY0290). 14. Luo, G., et al., Exploiting intercontact time for routing in delay tolerant networks. European Transactions on Telecommunications, 2013. 24(6): p. 589–599. 15. Bonomi, S., et al. FAROES: Fairness And Reliability using Overlay Expenseless Set-out for duty-cycle optimization in WSN. 2011. References 16. Aliouat, Z. and Z. Aliouat. Improved WSN Life Time Duration through Adaptive Clustering, Duty Cycling and Sink Mobility. in International Conference on Information Management and Engineering. 2016. 1. Wu, Y., S. Deng, and H. Huang, Performance analysis of hop‐limited epidemic routing in DTN with limited forwarding times. International Journal of Communication Systems, 2015. 28(15): p. 2035-2050. 17. Ringwald, M. and K. Romer. Practical time synchronization for Bluetooth Scatternets. in International Conference on Broadband Communications, Networks and Systems, 2007. Broadnets. 2007. 2. Lau, G., et al., Context-aware RAON middleware for opportunistic network. Pervasive & Mobile Computing, 2017. 41. 3. Dede, J., et al., Simulating Opportunistic Networks: Survey and Future Directions. IEEE Communications Surveys & Tutorials, 2017. 18. Wâhslén, J., I. Orhan, and T. Lindh. Local Time Synchronization in Bluetooth Piconets for Data Fusion Using Mobile Phones. in International Conference on Body Sensor Networks. 2011. 4. Cuka, M., et al., Implementation and performance evaluation of two fuzzy-based systems for selection of IoT devices in opportunistic networks. Journal of Ambient Intelligence and Humanized Computing, 2018: p. 1-11. 5. Kaur, S., A Review of Energy Consumption on DTN Routing Protocols. 6. Dou, Y., F. Zeng, and W. Li. Energy-Efficient Contact Detection Model in Mobile Opportunistic Networks. in International Conference on Wireless Algorithms, Systems, and Applications. 2017. Springer. 7. Choi, B.J. and X. Shen, Adaptive Asynchronous Sleep Scheduling Protocols for Delay Tolerant Networks. 2011: IEEE Educational Activities Department. 1283-1296. 8. Chumchu, P., An extension to IEEE 802.11 power save mode for NS-3. 2015: p. 799-804. 9. Zeng, F., et al., Efficient Listening and Sleeping Scheduling Mechanism Based on Self-Similarity for Duty Cycle Opportunistic Mobile Networks. Information, 2017. 8(3): p. 87. 5 5
https://openalex.org/W1977610488	https://europepmc.org/articles/pmc2634961?pdf=render	English	null	HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3	PloS one	2,009	cc-by	7,588	Abstract doi:10.1371/journal.pone.0004363 Editor: Andreas Bergmann, UT MD Anderson Cancer Center, United States of America Received November 6, 2008; Accepted December 23, 2008; Published February 10, 2009 Copyright: 2009 Gupta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants DK54733, DK60521, DA11190, DA 11806 and K02-DA13926 to LNW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: weixx009@umn.edu Citation: Gupta P, Ho P-C, Ha SG, Lin Y-W, Wei L-N (2009) HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3. PLoS ONE 4(2): e4363. doi:10.1371/journal.pone.0004363 Editor: Andreas Bergmann, UT MD Anderson Cancer Center, United States of America Received November 6, 2008; Accepted December 23, 2008; Published February 10, 2009 Copyright: 2009 Gupta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2009 Gupta et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants DK54733, DK60521, DA11190, DA 11806 and K02-DA13926 to LNW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: weixx009@umn.edu HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3 Pawan Gupta2, Ping-Chih Ho1, Sung Gil Ha1, Yi-Wei Lin1, Li-Na Wei1* 1 Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 2 Institute of Microbial Technology, Chandigarh, India Pawan Gupta2, Ping-Chih Ho1, Sung Gil Ha1, Yi-Wei Lin1, Li-Na Wei1* Pawan Gupta2, Ping-Chih Ho1, Sung Gil Ha1, Yi-Wei Lin1, Li-Na Wei1* 1 Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 2 Institute of Microbial Technology, Chandigarh, India Citation: Gupta P, Ho P-C, Ha SG, Lin Y-W, Wei L-N (2009) HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3. PLoS ONE 4(2): e4363. doi:10.1371/journal.pone.0004363 Abstract TR2 is an orphan nuclear receptor specifically expressed in early embryos (Wei and Hsu, 1994), and a transcription factor for transcriptional regulation of important genes in stem cells including the gate keeper Oct4 (Park et al. 2007). TR2 is known to function as an activator (Wei et al. 2000), or a repressor (Chinpaisal et al., 1998, Gupta et al. 2007). Due to the lack of specific ligands, mechanisms triggering its activator or repressor function have remained puzzling for decades. Recently, we found that all-trans retinoic acid (atRA) triggers the activation of extracellular-signal-regulated kinase 2 (ERK2), which phosphorylates TR2 and stimulates its partitioning to promyelocytic leukemia (PML) nuclear bodies, thereby converting the activator function of TR2 into repression (Gupta et al. 2008; Park et al. 2007). Recruitment of TR2 to PML is a crucial step in the conversion of TR2 from an activator to a repressor. However, it is unclear how phosphorylated TR2 is recruited to PML, an essential step in converting TR2 from an activator to a repressor. In the present study, we use both in vitro and in vivo systems to address the problem of recruiting TR2 to PML nuclear bodies. First, we identify histone deacetylase 3 (HDAC3) as an effector molecule. HDAC3 is known to interact with TR2 (Franco et al. 2001) and this interaction is enhanced by the atRA- stimulated phosphorylation of TR2 at Thr-210 (Gupta et al. 2008). Secondly, in this study, we also find that the carrier function of HDAC3 is independent of its deacetylase activity. Thirdly, we find another novel activity of atRA that stimulates nuclear enrichment of HDAC3 to form nuclear complex with PML, which is ERK2 independent. This is the first report identifying a deacetylase-independent function for HDAC3, which serves as a specific carrier molecule that targets a specifically phosphorylated protein to PML NBs. This is also the first study delineating how protein recruitment to PML nuclear bodies occurs, which can be stimulated by atRA in an ERK2-independent manner. These findings could provide new insights into the development of potential therapeutics and in understanding how orphan nuclear receptor activities can be regulated without ligands. Citation: Gupta P, Ho P-C, Ha SG, Lin Y-W, Wei L-N (2009) HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3. PLoS ONE 4(2): e4363. Introduction We previously demonstrated that HDACs 3 and 4 interact constitutively and directly with the orphan nuclear receptor TR2 via its DNA-binding domain [27]. We also reported that ERK2-phosphorylated TR2 is recruited to PML nuclear bodies (PML NBs) for its subsequent small ubiquitin- like modification (SUMOylation) and function as a potent transcriptional repressor [32,33]. An important question that remains to be answered is how phosphorylated TR2 is facilitated to the PML NBs. [29]. It is presumed that most of the pathways involving HDACs require deacetylase activity [13] but this has not been demon- strated conclusively [30]. We previously demonstrated that HDACs 3 and 4 interact constitutively and directly with the orphan nuclear receptor TR2 via its DNA-binding domain [27]. We also reported that ERK2-phosphorylated TR2 is recruited to PML nuclear bodies (PML NBs) for its subsequent small ubiquitin- like modification (SUMOylation) and function as a potent transcriptional repressor [32,33]. An important question that remains to be answered is how phosphorylated TR2 is facilitated to the PML NBs. We then investigated the relationship between Thr-210 phosphorylation or Lys-238 SUMOylation and the recruitment of TR2 to endogenous HDAC3. Both WT FLAG-TR2 and the phosphomimetic and deSUMOylated FLAG-210CP+K238R TR2 double mutant associated effectively with HDAC3 (Fig. 1C). However, the ability of TR2 to associate with HDAC3 was abolished completely in the FLAG-210CN+K238R double mutant defective for both phosphorylation and SUMOylation. As predicted, the ability of TR2 to associate with PML mirrored the pattern of TR2 association with HDAC3. Thus, phosphory- lation on Thr-210, but not deSUMOylation on Lys-238, triggers effective association of TR2 with HDAC3 and PML. TR2 is specifically expressed in early embryos [34], and is a transcription factor for transcriptional regulation of important genes in stem cells including the gate keeper Oct4 [32]. It is known to function as an activator [35], or a repressor [36,37]. Due to the lack of specific ligands, mechanisms triggering its activator or repressor function remains puzzling for decades. Recently, our finding of all- trans retinoic acid (atRA) triggered phosphorylation of TR2 stimulates its partitioning to PML nuclear bodies, which converts the activator function of TR2 into a repressor [32,33], prompted us to examine how phosphorylated TR2 is recruited to PML, an essential step in converting TR2 from an activator to a repressor. In vitro protein–protein interaction tests were undertaken to determine if the association of TR2 to the effector molecule HDAC3 was direct or indirect (Fig. The effect of specific TR2 phosphorylation on its interaction with HDAC3 and PML Our recent report [33] showed that site-specific phosphorylation of TR2 at Thr-210 modulates its association with the effector molecules PCAF and RIP140, albeit indirectly by increased SUMOylation of TR2 at Lys-238. Because HDAC3 binds to TR2 at the hinge region that encompasses Thr-210 [27], we assessed its ability to mediate recruitment of Thr-210-phosphorylated TR2 to PML using a two-hybrid interaction test (Fig. 1A). The wild type (WT) TR2 interacted effectively with HDAC3 [27]. This interaction was enhanced significantly in the phosphomimetic mutant TR2 (210CP; ThrRGlu [38,39]), but was abolished in the phosphory- lation-negative mutant TR2 (210CN; ThrRAla). Furthermore, although the SUMOylation-defective mutant TR2 (K238R; Lys- RArg) exhibited a basal level of interaction with HDAC3 similar to that of WT TR2, a double mutant containing both the phos- phomimetic and the SUMOylation-negative mutations (210CP+K238R) behaved like the phosphomimetic CP210 TR2 in terms of its ability to interact with HDAC3. In contrast, a constitutive TR2 double mutant negative for both phosphorylation and SUMOylation (210CN+K238R) could not effectively interact with HDACs. These data suggest that the interaction of TR2 with HDAC3 is phosphorylation-dependent but SUMOylation-indepen- dent. This phosphorylation-dependent interaction was verified using pharmacological agents to activate or inactivate ERK (Fig. 1B). A mitogen-activated protein kinase (MAPK)/ERK activator sphingo- sine-1-phosphate (S-1-P) increased the association of TR2 with HDAC3, whereas addition of an ERK inhibitor 3-(2-aminoethyl)5- ([4-ethoxyphenyl]methylene)-2,4-thiazolidinedione HCl (AMTZD) completely abolished the effect. The role of HDAC3’s deacetylase activity in facilitating TR2/ PML colocalization was examined using the deacetylase activity inhibitor TSA (Fig. 2B, upper panel). Interestingly, blocking the deacetylase activity of HDAC3 did not affect the atRA-triggered TR2/PML colocalization, suggesting that the carrier role of HDAC3 was independent of its deacetylase activity. However, TSA is known for many non-target effects [41], including modulation of global gene expression. We therefore sought to validate the results of these pharmacological studies by using a dominant negative mutant of HDAC3 (Ser-424RAla) which is specifically defective in its deacetylase activity [42]. There was no apparent difference between the WT and deacetylase-negative mutant in the ability of atRA to stimulate the association of TR2 with PML (Fig. 2B, lower panel). This confirms a deacetylase-independent chaperone role for HDAC3 in stimulating TR2 localization to PML. Immunohistochemistry was also conducted to monitor the distribution of endogenous TR2 and PML NBs in a gain- or loss- of-HDAC3-expression system (Fig. 2C). A functional role for HDAC3 in targeting phosphorylated TR2 to PML Because the association of TR2 with HDAC3/PML was related directly to phosphorylation on TR2 at Thr-210, and Thr-210 phosphorylation was a direct result of atRA stimulation, we monitored the role of endogenous HDAC3 in mediating atRA- triggered TR2/PML colocalization [32,33]. SiRNA knockdown of HDAC3 (Fig. 2A, panel 4) effectively (90%) blocked the atRA- triggered association of TR2 with PML (panel 1). This was similar to the efficiency achieved by TR2 knockdown (92%; panel 3). However unlike the HDAC3 knockdown, the TR2 knockdown did not affect complex formation between HDAC3 and PML. This suggests that TR2 does not alter the direct binding of HDAC3 to PML, as reported previously [40]. However, it supports the hypothesis that HDAC3 functions as a carrier or chaperone in the mobilization of TR2 to PML. Introduction 1D). TR2 was expressed and purified as a GST fusion protein. GST pull-down assays of WT and CP TR2 were carried out using in vitro-transcribed and translated HDAC3 or PML. WT or CP TR2 did not interact with PML (Fig. 1D, right panel), but weak interactions between HDAC3 and WT TR2 were detected; this effect was enhanced in the CP mutant (Fig. 1D, left panel). These data are consistent with the results of the cell-based assay, further supporting a phosphorylation-dependent, direct interaction between TR2 and HDAC3. This suggests that HDACs might function as a chaperone for the association of TR2 with PML, the target site for TR2 SUMOylation and its conversion into a repressor. In the present study we demonstrate that the interaction of HDAC3 with TR2 can be stimulated by phosphorylation of TR2 at a specific ERK2 target. Furthermore, HDAC3 serves to target this specifically phosphorylated TR2 to PML NBs for its subsequent SUMOylation. Importantly, this novel function of HDAC3 is independent of its deacetylase activity. Finally, a novel ERK2-independent activity of atRA is identified, which stimulates translocation and nuclear enrichment of HDAC3 to form nuclear complex with PML. Results The effect of specific TR2 phosphorylation on its interaction with HDAC3 and PML The effect of specific TR2 phosphorylation on its interaction with HDAC3 and PML Introduction complexes [23–25]. A thorough characterization of the structural and functional properties of HDAC3 identified a nonconserved region in its carboxy-terminal region that is required for histone deacetylation and transcriptional repression [13,26]. It was suggested that this carboxy-terminal domain acts in concert with the putative catalytic domain of the protein. In addition, a nuclear export signal is present in the central portion of the molecule (amino acids [aa] 180– 330), and a nuclear localization signal is present in the carboxy- terminal region (aa 313–428) [26]. Histone deacetylases (HDACs) are assigned to three distinct classes based on their sequence similarity to the yeast RPD3 (class I), HDA1 (class II), or Sir2 (NAD-dependent) proteins [1–3]. Class I includes HDACs 1, 2, 3, 8, and possibly 11 [1,4]. Class II includes HDACs 4, 5, 6, 7, 9, and 10 [1]. HDACs generally are found in large, multiprotein corepressor complexes that are targeted to chromatin by sequence-specific DNA-binding proteins and are involved in repressing transcription [5,6]. These DNA-binding proteins include nuclear receptors, the E-box binding proteins, and the methylcytosine-binding protein MeCP2 [6–8]. HDACs and histone acetyl transferases play crucial roles in regulating histone acetylation to regulate gene transcription [9–13]. Although HDAC3 functions primarily in histone deacetylation [9–13], a growing list of its nonhistone substrates suggests a role for HDAC3 in biological processes beyond transcriptional repression [13,27–29]. Studies have suggested that HDAC3 associates with complexes that are not directly involved in regulating genome activity [30], but it is not known if formation of these complexes requires its deacetylase activity. HDAC3 has been shown to function as a carrier/bridging molecule, targeting RbAp48 to the retinoblastoma protein [31]. HDAC3 also localizes to the mitotic spindle and is required for kinetochore–microtubule attachment Although the different classes of HDACs share a certain degree of sequence homology, they exhibit different specificities in various systems [14–17]. Class I HDACs function in developing embryos and carcinomas [18–22]. HDAC3, a class I HDAC [13], is usually found in corepressor complexes such as N-CoR, SMRT and RIP140 PLoS ONE \| www.plosone.org February 2009 \| Volume 4 \| Issue 2 \| e4363 1 February 2009 \| Volume 4 \| Issue 2 \| e4363 HDAC3 for Shuttling TR2 HDAC3 for Shuttling TR2 [29]. It is presumed that most of the pathways involving HDACs require deacetylase activity [13] but this has not been demon- strated conclusively [30]. The effect of specific TR2 phosphorylation on its interaction with HDAC3 and PML Without atRA (control cells), TR2 was only minimally (20%) colocalized with endogenous PML NBs. In the atRA-treated culture, 60% of the cells showed colocalization, as reported previously [33]. In a gain-of-function system that acquired ectopic expression of HDAC3, TR2 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org February 2009 \| Volume 4 \| Issue 2 \| e4363 2 HDAC3 for Shuttling TR2 Figure 1. Phosphorylation of Thr-210 but not SUMOylation of Lys-238 enhances association of HDAC3 with TR2 full-length (WT/Mut) constructs. A mammalian version of the two-hybrid system was used to HDAC3 and TR2 full-length (WT and phospho and/or SUMO Mut) constructs in COS-1 cells. Asterisks den Student’s t test; , P,0.01). (B) The association of endogenous TR2 with HDAC3 in P19 cells was examined in th activation (ERK2-Ac) or inhibition (ERK2-In). (C) P19 cells were transiently transfected (0.5 mg/ml, 16 h) wit negative SUMOylation mutation in combination with phosphomimetic (210CP+K238R) or negative phosp Association with HDAC3 was examined by coimmunoprecipitation. (D) GST pull-down assay of WT/MT TR2 wi and Thr-210 phosphomimetic mutant proteins were expressed and purified from E. coli and incubated wi translated (TNT) HDAC3 and PML (upper panels). Interacting proteins were resolved by SDS–PAGE and analyz Half of the TNT protein sample was taken as TNT input (bottom panels). HDAC3 interaction with TR2 was incre Thr-210 (left panel), whereas PML showed no interaction with WT or phosphomimetic TR2 (right panel). doi:10.1371/journal.pone.0004363.g001 Figure 1. Phosphorylation of Thr-210 but not SUMOylation of Lys-238 enhances association of TR2 with HDAC3. (A) Interaction of HDAC3 with TR2 full-length (WT/Mut) constructs. A mammalian version of the two-hybrid system was used to examine in vivo interactions between HDAC3 and TR2 full-length (WT and phospho and/or SUMO Mut) constructs in COS-1 cells. Asterisks denote significant differences (two-tailed Student’s t test; , P,0.01). (B) The association of endogenous TR2 with HDAC3 in P19 cells was examined in the presence of reagents triggering ERK2 activation (ERK2-Ac) or inhibition (ERK2-In). (C) P19 cells were transiently transfected (0.5 mg/ml, 16 h) with FLAG-tagged WT TR2, FLAG-tagged negative SUMOylation mutation in combination with phosphomimetic (210CP+K238R) or negative phosphorylation mutations (210CN+K238R). Association with HDAC3 was examined by coimmunoprecipitation. (D) GST pull-down assay of WT/MT TR2 with effectors. Recombinant GST–TR2 WT and Thr-210 phosphomimetic mutant proteins were expressed and purified from E. coli and incubated with 35S-labeled in vitro-transcribed and translated (TNT) HDAC3 and PML (upper panels). The effect of specific TR2 phosphorylation on its interaction with HDAC3 and PML (C) Immunostaining of endogenous TR2 and PML in control cells, or cells treated with atRA (0.1 mM) for 6 h in the context of gain or loss of HDAC3 expression. Nuclei were stained with DAPI. Large colocalized TR2/PML puncta in stimulated cells are marked with arrows. Right panel: Statistical analysis of the percentage of cells with colocalized TR2 and PML puncta (positive) among the total cells (positive+negative). doi:10.1371/journal.pone.0004363.g002 g colocalization with PML was enhanced in both the presence and interacts with HDAC3. Figure 2. HDAC3’s role as a chaperone in TR2 partitioning to PML is independent of its deac partitioning to PML. HDAC3 and TR2 were silenced by RNAi in embryonic stem cells and their com coimmunoprecipitation. (B) HDAC3 chaperoning of TR2 to PML independent of deacetylase activity. T ability to modulate atRA-triggered TR2 and PML colocalization (upper panel). Dominant negative modulation of atRA-triggered association of TR2 with PML (lower panel). (C) Immunostaining of endo treated with atRA (0.1 mM) for 6 h in the context of gain or loss of HDAC3 expression. Nuclei were s puncta in stimulated cells are marked with arrows. Right panel: Statistical analysis of the percentage o (positive) among the total cells (positive+negative). doi:10.1371/journal.pone.0004363.g002 Figure 2. HDAC3’s role as a chaperone in TR2 partitioning to PML is independent of its deacetylase activity. (A) HDAC3 modulates TR2 partitioning to PML. HDAC3 and TR2 were silenced by RNAi in embryonic stem cells and their complex formation with PML was monitored by coimmunoprecipitation. (B) HDAC3 chaperoning of TR2 to PML independent of deacetylase activity. TSA (a deacetylase inhibitor) was tested for its ability to modulate atRA-triggered TR2 and PML colocalization (upper panel). Dominant negative HDAC3 was compared to WT HDAC3 for modulation of atRA-triggered association of TR2 with PML (lower panel). (C) Immunostaining of endogenous TR2 and PML in control cells, or cells treated with atRA (0.1 mM) for 6 h in the context of gain or loss of HDAC3 expression. Nuclei were stained with DAPI. Large colocalized TR2/PML puncta in stimulated cells are marked with arrows. Right panel: Statistical analysis of the percentage of cells with colocalized TR2 and PML puncta (positive) among the total cells (positive+negative). doi:10.1371/journal.pone.0004363.g002 interacts with HDAC3. To verify this model, we manipulated the experimental system with regard to two critical elements: atRA and ERK2 (Fig. 3). In the presence of an ERK2 inhibitor (AMTZD), formation of the TR2–HDAC3 complex was reduced (Fig. The effect of specific TR2 phosphorylation on its interaction with HDAC3 and PML Interacting proteins were resolved by SDS–PAGE and analyzed by autoradiography (upper panels). Half of the TNT protein sample was taken as TNT input (bottom panels). HDAC3 interaction with TR2 was increased when TR2 was phosphorylated at Thr-210 (left panel), whereas PML showed no interaction with WT or phosphomimetic TR2 (right panel). doi:10.1371/journal.pone.0004363.g001 Figure 1. Phosphorylation of Thr-210 but not SUMOylation of Lys-238 enhances association of TR2 with HDAC3. (A) Interaction of HDAC3 with TR2 full-length (WT/Mut) constructs. A mammalian version of the two-hybrid system was used to examine in vivo interactions between HDAC3 and TR2 full-length (WT and phospho and/or SUMO Mut) constructs in COS-1 cells. Asterisks denote significant differences (two-tailed Student’s t test; *, P,0.01). (B) The association of endogenous TR2 with HDAC3 in P19 cells was examined in the presence of reagents triggering ERK2 activation (ERK2-Ac) or inhibition (ERK2-In). (C) P19 cells were transiently transfected (0.5 mg/ml, 16 h) with FLAG-tagged WT TR2, FLAG-tagged negative SUMOylation mutation in combination with phosphomimetic (210CP+K238R) or negative phosphorylation mutations (210CN+K238R). Association with HDAC3 was examined by coimmunoprecipitation. (D) GST pull-down assay of WT/MT TR2 with effectors. Recombinant GST–TR2 WT and Thr-210 phosphomimetic mutant proteins were expressed and purified from E. coli and incubated with 35S-labeled in vitro-transcribed and translated (TNT) HDAC3 and PML (upper panels). Interacting proteins were resolved by SDS–PAGE and analyzed by autoradiography (upper panels). Half of the TNT protein sample was taken as TNT input (bottom panels). HDAC3 interaction with TR2 was increased when TR2 was phosphorylated at Thr-210 (left panel), whereas PML showed no interaction with WT or phosphomimetic TR2 (right panel). doi:10.1371/journal.pone.0004363.g001 February 2009 \| Volume 4 \| Issue 2 \| e4363 PLoS ONE \| www.plosone.org 3 HDAC3 for Shuttling TR2 Figure 2. HDAC3’s role as a chaperone in TR2 partitioning to PML is independent of its deacetylase activity. (A) HDAC3 modulates TR2 partitioning to PML. HDAC3 and TR2 were silenced by RNAi in embryonic stem cells and their complex formation with PML was monitored by coimmunoprecipitation. (B) HDAC3 chaperoning of TR2 to PML independent of deacetylase activity. TSA (a deacetylase inhibitor) was tested for its ability to modulate atRA-triggered TR2 and PML colocalization (upper panel). Dominant negative HDAC3 was compared to WT HDAC3 for modulation of atRA-triggered association of TR2 with PML (lower panel). The effect of specific TR2 phosphorylation on its interaction with HDAC3 and PML 3A, top panel, lane c). Furthermore, because atRA phosphorylates TR2 through the ERK pathway [33], it was unable to rescue complex formation (lane d). The direct interaction between HDAC3 and PML [40] was increased slightly by atRA treatment (panel 2, lane b), but ERK2 inhibition did not affect this interaction (lane d). In contrast, the atRA-triggered association of TR2 with PML (panel 3) was completely blocked by inhibiting ERK2 activity (lane d). Taken together, our data suggest two independent pathways. In the first, TR2 recruitment to HDAC3 is atRA-dependent and ERK2- sensitive, whereas the second pathway (in which HDAC3 recruitment to PML is slightly enhanced) is responsive to atRA but is independent colocalization with PML was enhanced in both the presence and absence of atRA (60% of the cells). The enhanced, atRA- independent, increase in TR2 recruitment to PML might have been caused by saturation of the endogenous components. In contrast, in a loss-of-function system where endogenous HDAC3 was knocked down, atRA-stimulated association of TR2 with PML NBs was almost completely abolished. These results further support a functional role for HDAC3 in atRA-stimulated recruitment of TR2 to PML in this experimental system. PLoS ONE \| www.plosone.org Effect of ERK and atRA on complex formation of TR2/ HDAC3/PML Because HDAC3 binds both TR2 and PML, it might act as a chaperone for TR2 to PML. atRA activates ERK2, which phosphorylates TR2 at Thr-210. Phosphorylated TR2 then strongly PLoS ONE \| www.plosone.org February 2009 \| Volume 4 \| Issue 2 \| e4363 February 2009 \| Volume 4 \| Issue 2 \| e4363 4 HDAC3 for Shuttling TR2 Figure 3. Effect of atRA and ERK on endogenous complexes with HDAC3. (A) ERK2-dependent association of HDAC3 to TR2 but not PML. ERK2 was inactivated pharmacologically and the atRA-triggered complex formation of HDAC3 with TR2 and PML was monitored. (B) Effects of atRA and ERK2 on HDAC-PML complex formation and HDAC3 subcellular distribution. Nuclear (Nuc)/cytoplasmic (Cyt) distribution of HDAC3 and its association with the import (Importin b; Karyopherin ß 1) or the export (exportin 1, CRM1) machinery were monitored in coimmunoprecipitation (IP) experiments. Immuno blot (IB) panels show all input and protein controls. Numbers above the panels indicate quantified relative values. (C) atRA- triggered nuclear enrichment of HDAC3 in P19 cells, monitored by immunocytochemistry. (D) A kinetic study of endogenous components in P19 cells after atRA treatment at 0, 2, 8, 16, 24, and 48 h (panels 1–5). doi:10.1371/journal.pone.0004363.g003 Figure 3. Effect of atRA and ERK on endogenous complexes with HDAC3. (A) ERK2-dependent association of HDAC3 to TR2 but not PML. ERK2 was inactivated pharmacologically and the atRA-triggered complex formation of HDAC3 with TR2 and PML was monitored. (B) Effects of atRA and ERK2 on HDAC-PML complex formation and HDAC3 subcellular distribution. Nuclear (Nuc)/cytoplasmic (Cyt) distribution of HDAC3 and its association with the import (Importin b; Karyopherin ß 1) or the export (exportin 1, CRM1) machinery were monitored in coimmunoprecipitation (IP) experiments. Immuno blot (IB) panels show all input and protein controls. Numbers above the panels indicate quantified relative values. (C) atRA- triggered nuclear enrichment of HDAC3 in P19 cells, monitored by immunocytochemistry. (D) A kinetic study of endogenous components in P19 cells after atRA treatment at 0, 2, 8, 16, 24, and 48 h (panels 1–5). doi:10.1371/journal.pone.0004363.g003 [40] and this complex formation is not dependent upon TR2 (shown above). We therefore suspected a possibility that atRA could modulate subcellular distribution of HDAC3, thereby enhancing its nuclear distribution. PLoS ONE \| www.plosone.org Effect of ERK and atRA on complex formation of TR2/ HDAC3/PML To examine this possibility and to determine if this particular effect of atRA (stimulation of HDAC3-PML complex formation in the nucleus) was dependent upon the ERK2 pathway, we conducted siRNA-mediated knockdown of ERK2 and monitored subcellular distribution of of ERK2 activation (see the current working model shown in Fig. 4). It is possible that TR2–HDAC3 and HDAC3–PML could exist as two separate complexes because HDAC3–PML was formed even in the absence of TR2–HDAC3 (i.e., when the culture was stimulated with atRA but inhibited by an ERK2 inhibitor). It is known that endogenous HDAC3 is generally distributed in equilibrium between the cytoplasm and the nucleus [26,30]. Further, HDAC3 and PML can directly interact with each other PLoS ONE \| www.plosone.org February 2009 \| Volume 4 \| Issue 2 \| e4363 5 HDAC3 for Shuttling TR2 HDAC3 for Shuttling TR2 HDAC3 for Shuttling TR2 Figure 4. Schematic of events leading to TR2 association with PML NBs for SUMOylation. atRA activates ERK2 nongenomically, which in turns phosphorylates TR2 at Thr-210. This facilitates its recruitment to HDAC3 that shuttles TR2 to PML NBs for subsequent SUMOylation. Two major outstanding questions in this signaling pathway are pointed out with question marks. doi:10.1371/journal.pone.0004363.g004 atRA-stimulated nuclear enrichment of HDAC3 provides some explanation for the increased nuclear HDAC3-PML complex formation. However, how atRA stimulates nuclear retention, or enrichment in the nucleus, of HDAC3 remains to be investigated. This is indicated with a question mark in our current working model shown in Fig. 4. atRA-stimulated nuclear enrichment of HDAC3 provides some explanation for the increased nuclear HDAC3-PML complex formation. However, how atRA stimulates nuclear retention, or enrichment in the nucleus, of HDAC3 remains to be investigated. This is indicated with a question mark in our current working model shown in Fig. 4. We also examined the kinetics of endogenous complex formation at different time points following atRA treatment (Fig. 3D). The phosphorylation of TR2 at Thr-210 was rapidly stimulated, but was diminished by 16 h (panel 2). TR2 association with HDAC3 was consistently apparent at 2 h and subsided after 16 h (panel 1). The kinetics of TR2 recruitment to PML (panel 3) and its subsequent SUMOylation ([33]; panel 4) followed closely that of atRA- stimulated TR2 phosphorylation and HDAC3 association. Among the HDACs examined (HDACs 1–6), only HDAC3 showed detectable levels of expression in the P19 culture system (panel 5). Discussion Interestingly, this particular effect of atRA on HDAC3 nuclear enrichment was not blocked by the addition of siRNA to ERK2 (RNAi). This result suggests that the equilibrium of HDAC3 has shifted in favor of its nuclear localization, and that this phenomenon is ERK2 independent. Thus, the effect of atRA on HDAC3 nuclear enrichment is clearly different from its effect on TR2 phsophorylation that is ERK2- dependent as shown in our previous study. g HDAC3 interacts directly with the DNA-binding domain of TR2 that encompasses Thr-210 [27], and this interaction is enhanced by specific phosphorylation on Thr-210 (Fig. 1D). The exact mechanism of this enhancement remains unclear. Presum- ably, phosphorylation could induce conformational changes in TR2 such that it is more likely to interact with the binding domain of HDAC3. The presumed direct interaction between HDAC3 and PML [40] is increased slightly by atRA treatment, but this is ERK-independent. This remains an interesting point for further investigation. Another important finding of this study is that HDAC3 function in this pathway is independent of its deacetylase activity. Although this observation was made using the TR2 system, HDAC3 can also interact with other proteins, including many nuclear receptors that all harbor a very similar DNA- binding domain. Whether HDAC3 can have a similar role in recruiting other nuclear receptors remains to be determined. HDAC3 interacts directly with the DNA-binding domain of TR2 that encompasses Thr-210 [27], and this interaction is enhanced by specific phosphorylation on Thr-210 (Fig. 1D). The exact mechanism of this enhancement remains unclear. Presum- ably, phosphorylation could induce conformational changes in TR2 such that it is more likely to interact with the binding domain of HDAC3. The presumed direct interaction between HDAC3 and PML [40] is increased slightly by atRA treatment, but this is ERK-independent. This remains an interesting point for further investigation. Another important finding of this study is that HDAC3 function in this pathway is independent of its deacetylase activity. Although this observation was made using the TR2 system, HDAC3 can also interact with other proteins, including many nuclear receptors that all harbor a very similar DNA- binding domain. Whether HDAC3 can have a similar role in recruiting other nuclear receptors remains to be determined. To gain insights into the molecular mechanism of atRA-triggered HDAC3 nuclear enrichment, we monitored in vivo interaction of HDAC3 with exportin (the principal export machinery, CRM1) and importin-b1 (karyopherin b1) (Fig. 3B). Effect of ERK and atRA on complex formation of TR2/ HDAC3/PML The results obtained with this system that contains no foreign constructs validate our proposed model (Fig. 4): atRA stimulates TR2 localization to PML, where SUMOylation of TR2 converts it into an effective repressor of the Oct4 gene [32]. The present study has identified HDAC3 as an important carrier for the transport of TR2 phosphorylated specifically in its DNA-binding domain to PML NBs, a key step in the conversion of TR2’s activity. Discussion In the P19 stem cell differentiation model, atRA reduces Oct4 expression. Our previous studies reported a non-genomic mecha- nism by which atRA stimulates TR2 SUMOylation, a key step in the conversion of TR2 into a repressor of the Oct4 gene. In these earlier studies, we confirmed that atRA rapidly activates ERK2, leading to TR2 phosphorylation at Thr-210 and subsequent recruitment to PML NBs for SUMOylation [32,33]. However, we failed to detect a direct interaction of TR2 with PML ([33]; Fig. 1D), leaving unanswered the means by which phosphorylated TR2 was recruited to PML. In this current study, we are able to identify this missing link: HDAC3 interacts with TR2 in an atRA-enhanced manner. More importantly, the carrier/chaperone function of HDAC3 in this pathway appears to be independent of its deacetylase activity. However, in this newly identified signaling pathway, some outstanding questions have to be addressed as pointed out in our current working model (Fig. 4). Apparently, questions remain that how atRA activates ERK2, and how atRA stimulates HDAC3 shuttling to PML at the molecular level. Figure 4. Schematic of events leading to TR2 association with PML NBs for SUMOylation. atRA activates ERK2 nongenomically, which in turns phosphorylates TR2 at Thr-210. This facilitates its recruitment to HDAC3 that shuttles TR2 to PML NBs for subsequent SUMOylation. Two major outstanding questions in this signaling pathway are pointed out with question marks. doi:10.1371/journal.pone.0004363.g004 HDAC3 and formation of HDAC-PML complex (Fig. 3B). It appeared that HDAC3 was indeed detected in both the cytoplasm and the nucleus in normal culture; whereas atRA treatment stimulated significant nuclear enrichment of HDAC3 (comparing fractions of Nuc and Cyt). Interestingly, this particular effect of atRA on HDAC3 nuclear enrichment was not blocked by the addition of siRNA to ERK2 (RNAi). This result suggests that the equilibrium of HDAC3 has shifted in favor of its nuclear localization, and that this phenomenon is ERK2 independent. Thus, the effect of atRA on HDAC3 nuclear enrichment is clearly different from its effect on TR2 phsophorylation that is ERK2- dependent as shown in our previous study. HDAC3 and formation of HDAC-PML complex (Fig. 3B). It appeared that HDAC3 was indeed detected in both the cytoplasm and the nucleus in normal culture; whereas atRA treatment stimulated significant nuclear enrichment of HDAC3 (comparing fractions of Nuc and Cyt). PLoS ONE \| www.plosone.org Chemicals and Treatments All treatments were done in Dulbecco’s modified Eagle’s medium containing DCC serum. atRA (0.1 mM) was added for 6 h before harvesting, unless mentioned other wise. Activators/inhibitors (Calbiochem) of ERK, sphingosine-1-phosphate (MAPK/ERK activator, 1 mM), 3-(2-aminoethyl)5-([4-ethoxyphenyl]methylene)- 2,4-thiazolidinedione, HCl (ERK2 inhibitor, 25 mM), 5-(2 phenyl- All treatments were done in Dulbecco’s modified Eagle’s medium containing DCC serum. atRA (0.1 mM) was added for 6 h before harvesting, unless mentioned other wise. Activators/inhibitors (Calbiochem) of ERK, sphingosine-1-phosphate (MAPK/ERK activator, 1 mM), 3-(2-aminoethyl)5-([4-ethoxyphenyl]methylene)- 2,4-thiazolidinedione, HCl (ERK2 inhibitor, 25 mM), 5-(2 phenyl- Statistics All statistical data were from averages of three or more independent experiments. Two-tailed Student t test was performed to obtain P values. P value,0.05 was considered to be statistically significant. Immunoprecipitation and Western Blot Analysis Mouse complementary DNAs for TR2, HDAC3, and GAL4 tk- luciferase reporter were as described previously [27,33]. Consti- tutive negative/positive, point/sequential mutations involving residues Thr-210, and Lys-238 in WT TR2 (CMV/FLAG/ GAL4) vector as template were made according to QuikChange XL site-directed mutagenesis kit (Stratagene) as described previously [PNAS]. HDAC3 enzymatically defective mutant Ser- 424RAla was as described previously [42]. Scrambled RNA and siRNAs for Nr2c1, encoding TR2 were from Dharmacon and siRNAs for Mapk1, encoding ERK2 were from Qiagen as described previously [33]. siRNA for Hdac3, encoding HDAC3 were from Qiagen 59-AGAAGAUGAUCGUCUUCAA-39 and 59-GAUGAUGAUGUGUAUAAUA-39. RNAs were introduced using DharmaFECT1 (T-2001–01, Dharmacon) or HiPerfect (no. 301704, Qiagen). Silencing was assessed by Western blots at 48– 72 h. Transfection and reporter assay were as described [33,42]. Assays were conducted at 16–36 h. As described previously [32,33]. Antibodies were FLAG-M2 (F3165) from Sigma, phosphothreonine (ab-9337) from Abcam, PML (05–718) and HDAC3 from Upstate Biotechnology (05-813) and Santa Cruz Biotechnology (sc11417). TR2 (sc-9087), and PML (sc-5621) were from Santa Cruz Biotechnology. ERK2 (9108), and phosphor-p42/p44 ERK (9101) were from Cell Signaling. Exportin 1 (CRM1) and importin-b1 (karyopherin b1) were as described previously [46] Author Contributions Conceived and designed the experiments: PG PCH LNW. Performed the experiments: PG PCH SGH YWL. Analyzed the data: PG LNW. Contributed reagents/materials/analysis tools: LNW. Wrote the paper: PG LNW. Financial support: LNW. We thank the efforts of Justin Reed. We thank the efforts of Justin Reed. Discussion Interestingly, in atR treated cultures, no change was detected in the interaction of HDAC3 with importin-b1 but a clear reduction was detected in its interaction with exportin 1. This suggests that atRA changes the equilibrium of HDAC3, in favor of its nuclear retention, probably due to a selective blockage of its export machinery. Other studies using immunocy- tochemistry has shown HDAC3 localization mainly in many fine punctate foci of the cytoplasm and in a diffused pattern in the nucleus [26,30]. We also observed a similar nuclear and cytoplasmic pattern of HDAC3 in the control cultures, but a nucleus-enriched pattern in cultures treated with atRA (Fig. 3C). This validates the biochemical data shown in Fig. 3B. Therefore, the result showing HDAC3 associates with the nuclear dots that encompass PML NBs [40,43,44]. It also associates with the E3 ligase PIAS in the nuclear dots, an association that relieves the transcriptional repression exerted by HDAC3 during TFII-I mediated gene February 2009 \| Volume 4 \| Issue 2 \| e4363 PLoS ONE \| www.plosone.org 6 HDAC3 for Shuttling TR2 activation [44]. It is unclear whether the effect of HDAC3 in this system involves deacetylase activity [45]. Relevant to this debate, our current findings present evidence for a deacetylase-indepen- dent functional role for HDAC3 in mediating molecular interactions. The functional role of HDAC3 in leukemia has been attributed to its association with the PML–RAR fusion protein in a deacetylase-dependent fashion [13,41]. A variety of therapeutic agents are being tested, targeting HDAC’s deacetylase activity. The present study would suggest a new potential target that is independent of such activity. py razolo[1,5-a]pyridin-3yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine (ERK1/2 inhibitor, 1 mM) were added for 6 h. TSA (Calbiochem) [41] a deacetylase activity inhibitor, was added 1–2 h before and alongwith atRA treatment. TR2/PML Immunohistochemistry Subsequent to ectopic expression/knockdown of HDAC3 (48 h), atRA treatment was done for 6 h. Cells were fixed in 4% formaldehyde and a permeation buffer, blocked for 30 mins, and incubated with primary antibodies at 4uC overnight, followed by secondary fluorescence-conjugated antibodies, at room tempera- ture for 3 h . Images were acquired with a fluorview confocal system (Olympus). 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https://openalex.org/W2892247967	https://hal.archives-ouvertes.fr/hal-02333159/document	English	null	Strain rate influence on mechanical behavior of a single wire entangled material	EPJ web of conferences	2,018	cc-by	2,514	To cite this version: Sandra Guerard, Jérémie Girardot, Philippe Viot. Strain rate influence on mechanical behavior of a single wire entangled material. EPJ Web of Conferences, 2018, 183, pp.03001. hal-02333159 * Corresponding author: sandra.guerard@ensam.eu Strain rate influence on mechanical behavior of a single wire entangled material Sandra Guérard1,*, Jérémie Girardot1, Philippe Viot1 1 Arts et Métiers ParisTech, Univ. Bordeaux, Bordeaux INP, I2M, UMR 5295, F-33000 Bordeaux, France Abstract. In a global context of energy saving, the ratio stiffness – mass is a key parameter for design of mechanical structures. To deal with this major concern, sandwich materials are finding an increasing use: the skins are designed to resist tensile and compressive stresses while the core needs to gather lightweight, shear stresses resistance and high mechanical energy absorption capacities. Firstly made of balsa wood, the core is nowadays classically realized using architectured materials (cellular materials, honeycombs, entangled materials, etc.). Entangled materials are architectured materials with tuneable properties, depending of the dedicated application. Several entangled materials already exist such as mineral or metallic wool; some of them are made of a single ductile metallic wire, entangled in all directions so that the final material becomes a porous continuous media. Such materials, which combine lightness and ductile behaviour, seem to be perfect candidates to dissipate energy during an impact. Compared to conventional materials such as balsa wood or honeycomb, a large amount of energy is indeed dissipated by friction coming from the numerous contacts due to the entanglement. The global aim of this work is focused on the study of energy dissipation mechanisms involved during impact as well as the correlation between architectural parameters of the material (wire diameter and material, volume fraction, etc.) and macroscopic behaviour. The first step that is presented here consists of an experimental investigation using dynamic compression tests to study macroscopic parameters (wire diameter, volume fraction, etc.) on absorbed energy. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). HAL Id: hal-02333159 https://hal.science/hal-02333159v1 Submitted on 25 Oct 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. https://doi.org/10.1051/epjconf/201818303001 EPJ Web of Conferences 183, 03001 (2018) DYMAT 2018 1 Introduction impact or high speed compression has already been performed on this kind of material. In the way of looking for new material alternatives for engineering applications, the single wire entangled material (SWEM) (see Fig 1), also so- called “metal rubber” [1] has been widely studied during the last decades [2]. Applications can be various and be found in vibration absorbers, biocompatible parts or fluid filtering. Its porous geometry and specific intern architecture produce interesting specific mechanisms under compression loading. Concerning the shock absorption application, foam-like material such as metallic or polymer foams, syntactic foams, ceramics, aerogels or honeycomb structures are mainly based on one irreversible mechanism: the hyperelasticity inducing high strains [10]. This irreversible deformation comes from buckling mechanisms at the microstructure level. The present SWEM material has also an architecture where plastic strains occur especially due to its metallic ductile wire. An additional dissipative friction mechanisms coming from the numerous contacts inside the material are also a source of absorption. The main purpose of the subject is to evaluate the part of friction in shock absorption of SWEM material. Static compression [6-8], hysteresis [3], Poisson’s function [4] and damping capacity [5] are mechanisms analysed regarding the manufacturing process of the SWEM which used a multiple coil geometry and confined compression to obtain a cylindrical sample [6, 9]. Wires are mostly metallic with steel, aluminium or nickel-titanium alloys. In [4], a polyamide was used to obtain reversible hysteresis thanks to the low stiffness of the wire. In this work, it is proposed to observe the compressive mechanical response of a single entangled wire sample with two different densities. A ductile copper wire is used. A Hopkinson bar device is used to perform a high speed loading and results are comparing to static compression to evaluate strain rate effects. As it is a preliminary work, a discussion gives some proposals for further investigation. Although the material behaviour is already analysed under static and dynamic loadings, the dynamic part is only dealing with vibrations. In general, results show that this material has a strong interest in terms of damping characteristics. No https://doi.org/10.1051/epjconf/201818303001 EPJ Web of Conferences 183, 03001 (2018) DYMAT 2018 EPJ Web of Conferences 183, 03001 (2018) Fig. 1. Picture of single copper wire entangled sample. initially encircled into a 3 mm-diameter rod by distorting and twisting the wire (Fig 2b). 1 Introduction The spring- like segment has then been stretched so that the distance between two adjacent spirals is equal to the value of the diameter of the rod (here 3 mm) (Fig 2c). The spring-like wire has then been entangled to obtain a rough porous base material (Fig 2d). Those porous samples have finally been placed into a hollow Plexiglas 24-mm inner diameter tube and shaped into final form by applying a compressive force using upper and bottom pistons (Fig 2e). Fig. 2. Schematic diagram of the preparation process (Tan et al. [6]). Fig. 1. Picture of single copper wire entangled sample. Table 1. Characteristics of specimens (dimensions are given as mean value ± 2 std). High strain rate tests were carried out using compression Split Hopkinson Pressure Viscoelastic 38-mm diameter Bars. Dispersion and attenuation were taken into account using Bacon procedure [11]. Classically, pressure is applied to a striker which hits a viscoelastic input bar, where strains and strain rate are measured from strain gauges. Specimen is positioned between input and output bars, where strains gauges also allow to measure strain levels. Strain rate is then determined at the end of the test. QS tests SHPB tests Volume Fraction 17 % 25 % 17 % 25 % Diameter (mm) 24.3 ± 0.3 24.3 ± 0.4 24.3 ± 0.3 24.3 ± 0.5 Height (mm) 31.5 ± 0.6 30.9 ± 0.3 7.7 ± 0.2 7.7 ± 0.02 Initial wire length (mm) 12350 18155 2750 4040 Finally, to assess repeatability of the results, 6 specimens for each configuration were prepared. From all tests, nominal stress, engineering strain and energy were calculated and used as post- treatment parameters. 2.1 Characteristics of the specimens A copper bare wire with a diameter of 500 m was used to prepare the entangled wire preforms. Firstly, volume fraction and dimension of specimens have been chosen. In order to evaluate the influence of volume fraction on the dynamical response of entangled structures, 2 volume fractions were chosen: Vf = 17% and Vf = 25%. Cylindrical shape specimens were chosen to facilitate manufacturing process. Outer diameter was fixed to 24 mm and height to either 31.50 mm for quasi-static (QS) tests or 7.00 mm for dynamic (SHPB) tests. All characteristics of specimen are summarized in Table 1. Fig. 2. Schematic diagram of the preparation process (Tan et al. [6]). 2.3 Mechanical tests Quasi-static or dynamic tests have been carried out on the manufactured specimens. Monotonic quasi-static test were carried out on an electromechanical testing machine (Zwick-Roell 250 kN) equipped with a 10 kN load sensor. A displacement was applied to the specimen placed between two platens so that the strain rate reached 0.005 s-1. Tests were stopped when load was equal to 8 kN (which was reached in the densification region of the stress-strain curve). Table 1. Characteristics of specimens (dimensions are given as mean value ± 2 std). Table 1. Characteristics of specimens (dimensions are given as mean value ± 2 std). 2.2 Manufacturing process Manufacturing of the samples was made following Tan et al. [6] protocol, as presented in Fig. 2 and can be summarized as follows. The copper wire, which length was firstly calculated from chosen volume fraction and diameter and height (Fig 2a), has been 2 2 EPJ Web of Conferences 183, 03001 (2018) DYMAT 2018 EPJ Web of Conferences 183, 03001 (2018) https://doi.org/10.1051/epjconf/201818303001 Fig. 4. Absorbed energy versus engineering strain: (top) Vf = 17 %, (bottom) Vf = 25%. 0 1 2 3 4 5 6 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Energy [J/mm3] Engineering strain Dyn - 25% QS - 25% 3 Results From mechanical tests, Fig. 3 presents nominal stress as a function of engineering strain material response for the two chosen volume fractions and both strain rates. As expected, the mechanical response of the specimen presents typical response of cellular material, i.e., a first linear response, followed by a plastic stress plateau where strain values are increasing while small changes of stress occur, and finally a densification region. Strain rate plays an important role in the mechanical response of the specimen: the stress level of the beginning of the stress plateau strongly depends on both the volume fraction and the strain rate. Considering that a possible application of the material is energy dissipation, absorbed energy per volume unit is plotted as a function of strain on Fig. 4. Fig. 4. Absorbed energy versus engineering strain: (top) Vf = 17 %, (bottom) Vf = 25%. 4 Discussion Fig. 5 makes a comparison between the two densities regarding the absorbed energy at 40% of strain. As expected, the SWEM is more absorbent with a higher density. The strain rate effect is clearly observed as mentioned in all the previous works of literature and confirms the good suitability of SWEM for shock absorption. Fig. 3. Stress-Strain curves for the two volume fractions: (top) Vf = 17 %, (bottom) Vf = 25 %. Fig. 5. Absorbed energy at 40% strain versus strain rate for the two densities. Fig. 5. Absorbed energy at 40% strain versus strain rate for the two densities. Regarding the results of this preliminary experimental campaign, several comments can be pointed out: Fig. 3. Stress-Strain curves for the two volume fractions: (top) Vf = 17 %, (bottom) Vf = 25 %. • The sample slenderness used in this work is valid for dynamic tests. No shear strain was observed during the static or dynamic tests. It is noticed that two different sample heights were used such that further analyses on the size effect in the SWEM must be done; 0 1 2 3 4 5 6 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 Energy [J/mm3] Engineering strain Dyn - 17% QS - 17% 0 1 2 3 4 5 6 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 Energy [J/mm3] Engineering strain Dyn - 17% QS - 17% • The good repeatability for all strain rates and densities validates the manufacturing process that is “handmade”. A recent proposal [8] about using weaving techniques could be very useful to decrease time production of a sample (currently equal to one hour for one sample); • More tests at different strain rates are required to clearly identify the strain rate influence of SWEM. A specific flywheel device [12] could be 3 EPJ Web of Conferences 183, 03001 (2018) DYMAT 2018 https://doi.org/10.1051/epjconf/201818303001 very useful to perform constant strain rate tests and to obtain results at intermediate strain rate. very useful to perform constant strain rate tests and to obtain results at intermediate strain rate. 5. J.F. Hou, H.B. Bai, D.W. Li, Damping capacity measurement of elastic porous wire- mesh material in wide temperature range. Journal of Materials Processing Technology 206, pp. 5 Conclusions The SWEM was investigated under dynamic compression loading with two densities. Although results confirm the good performance of this material for dynamic applications, the present work only presents preliminary results. A more detailed experimental campaign is currently under process using standard Hopkinson bar device but also a specific flywheel compression test, in order to bring more information on the global strain rate influence. Such data will be used to validate a modelling of this material at the wire scale using the Discrete Element Method. Then, this model will be very helpful to analyse the proportion of absorbed energy coming from friction, such that an optimization for dynamic application can be considered like sandwich-core material or even momentum trap device application. 9. L. Courtois, E. Maire, M. Perez, D. Rodney, O. Bouaziz, Y.Brechet, Mechanical properties of monofilament entangled materials, Optical Measurements, Modeling, and Metrology, 5. ( Springer, New York, NY, 2011) 10. P. Qiao, M. Yang, F. Bobaru, Impact Mechanics and High-Energy Absorbing Materials: Review, Journal of Aerospace Engineering, 21, pp. 235– 248 (2008) 11. C. Bacon, An experimental method for considering dispersion and attenuation in a viscoelastic Hopkinson bar, Experimental Mechanics, 38, pp. 242-249 (1998) 12. C. Froustey, M. Lambert, J.L. Charles, J.L. Lataillade, Design of an impact loading machine based on a flywheel device: Application to the fatigue resistance of the high rate pre-straining sensitivity of aluminium alloys, Experimental Mechanics, 47, pp. 709–721 (2007) 4 Discussion 412–418 (2008) Finally, the main question being the part of the friction in the shock absorption, it is needed to perform a simulation at the structure level. This simulation needs to take into account all contacts in the material and to handle high strains (up to 60%) during a dynamic compression loading. Discrete approach was already used to model SWEM [13] by modelling the wire with a coarse “pearl necklace” meshing. Recent work for woven material and dry fabrics using discrete methods [14] could be used to improve representation of the material entanglement. 6. Q. Tan, G. He, Stretching behaviors of entangled materials with spiral wire structure. Materials & Design 46, pp.61-65 (2013) 7. B. Gadot, O. Riu Martinez, S. Rolland Du Roscoat, D. Bouvard, D. Rodney, L. Orgéas, Entangled single-wire NiTi material: A porous metal with tunable superelastic and shape memory properties, Acta Materialia 96, pp. 311- 323 (2015) 8. J. Hu, Q. Du, J. Gao, J. Kang, B. Guo, Compressive mechanical behavior of multiple wire metal rubber, Materials and Design 140, pp. 231–240 (2018) References 1. D.E. Chegodaev, Design of Metal Rubber Components, Press of National Defense Industry, pp. 2–12 (translated by Li Z.Y., 2000) 13. D. Rodney, M. Fivel, R. Dendievel, Discrete modeling of the mechanics of entangled materials, Physical Review Letters, 95 (2005) 2. A.O. Hong-Rui, H.Y. Jiang, H. Yan, et al., Research of a metal rubber isolation system based on complex stiffness, Journal of Harbin Institute of Technology 37, pp. 1615–1629 (in Chinese, 2005) 14. J. Girardot, F. Dau, A mesoscopic model using the discrete element method for impacts on dry fabrics, Matériaux & Techniques 104,408 (2016) 3. E. Piollet, D. Poquillon, G. Michon, Dynamic hysteresis modelling of entangled cross-linked fibres in shear, Journal of Sound and Vibration 383, pp. 248-264 (2016) 4. D. Rodney, B. Gadot, O.R. Martinez, S. Rolland Du Roscoat, L. Orgéas, Reversible dilatancy in entangled single-wire materials. Nature Materials 15, pp. 72–77 (2015) 4
https://openalex.org/W2954633521	https://pn.bmj.com/content/practneurol/20/2/180.full.pdf	English	null	I Am, I Am, I Am: Seventeen Brushes With Death, by Maggie O’Farrell	Practical neurology	2,019	cc-by	932	Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https:// creativecommons.org/licenses/by/4.0/. O’Farrell’s memoirs provided an intriguing substrate for discussion: her neurological illness and her descrip- tion of its effect on all aspects of her life; her depiction of doctors and their key role in her most formative experiences (childhood and mother- hood) and finally, her familiarity in the face of death, and how this shaped her. In the run-up to this book club, there was a healthy mix of admiration for O’Farrell’s literary splendour (a refreshing respite from bland scien- tific text!), and cynicism about some of her more tenuous ‘brushes’ with death. However, with the author present, critical discussion—particu- larly interrogation of the finer details © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ. guest. Protected by copyright. To cite Moullaali TJ, Scott S. Pract Neurol 2020;20:180–181. As we allowed our vulnerabilities to surface, the conversation came full circle with renewed insight and empathy, and we closed by asking O’Farrell what she valued most in a doctor. Together we concluded Book club I Am, I Am, I Am: Seventeen Brushes With Death, by Maggie O’Farrell Contributors TJM wrote the first draft of the report. SS chaired the discussion, and edited and approved the report. In our early exchanges, O’Farrell was an observer. The conversation started where we are conditioned to be comfortable: discussion of medicalised experiences through the distorted and dispassionate doctor’s lens. We shared professional expe- riences with the typical detachment that serves as a coping mechanism to ensure day-to-day maintenance of our sanity: challenging consultations in the clinic and attending cardiac arrest calls where our best efforts were in vain. Our readiness to question our own approaches, our explanations, and the parts we play in the real-life emotional dramas unfolding in every day practice was varied. But O’Far- rell’s presence, and her willingness and ability to describe vividly her personal experiences—many of which she associated with deep emotional pain—encouraged a rare openness and the discussion slowly softened. We began to share our own painful personal stories with O’Farrell, who admitted her book’s impact on others continued to surprise her. p g Patient consent for publication Not required. Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed by Tom Hughes, Cardiff, UK. Book club I Am, I Am, I Am: Seventeen Brushes With Death, by Maggie O’Farrell Book club I Am, I Am, I Am: Seventeen Brushes With Death, by Maggie O’Farrell on October 23, 2024 by guest. Protected by copyright. http://pn.bmj.com/ Pract Neurol: first published as 10.1136/practneurol-2019-002310 on 4 July 2019. Downloaded from of her neurological diagnosis—was tempered. Instead, we drew inspira- tion from O’Farrell’s willingness to share her personal stories with us, both through her writing, and now in person. that our patients require us first to make the correct diagnosis and to deliver appropriate interventions, but by retaining even a glimmer of insight into the human story of ill health and death, we cling to the shared ground that allows us to connect with each other during times of extreme vulnerability. The Edinburgh Neurology Book Club had a unique opportunity to explore Maggie O’Farrell’s1 memoirs ‘I Am, I Am, I Am: Seventeen Brushes With Death’ with the author present, which provided an intimate forum for reflection on the common ground we shared: an unusual familiarity with death. O’Farrell suffered a mysterious neurological illness in childhood— we settled on acute cerebellitis, but the case wasn’t closed—which was a focal point and catalyst for her unsettlingly frequent encounters with death in childhood and early adult- hood. Chapters purposefully meander through time and take their titles from the body part at risk, opening with ‘Neck (1990)’, a chilling recount of a near miss with a murderous pred- ator who claimed the life of another young female traveller only days later. In ‘Lungs (1988)’, her impulsive younger-self jumps into deep water and becomes increasingly disoriented and panicked when the route to the surface evades her vulnerable proprio- ceptive powers. She concludes with ‘Daughter (The present day)’, which O’Farrell explained was the hardest for her to tell—her child’s extreme nut allergy resulted in countless episodes of anaphylaxis in her early life, and as a result, frequent and varied experi- ence of emergency medical care. ‍ ‍ Tom J Moullaali ‍ ‍ ,1 Shona Scott2 1Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK 2Department of Clinical Neurology, Western General Hospital, Edinburgh, UK Correspondence to Dr Tom J Moullaali, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK; tom.moullaali@ed.ac.uk Acknowledgements We extend a huge thank you to Maggie O’Farrell for joining us for the discussion, to Rustam Al-Shahi Salman for organising this edition of Edinburgh neurology book club, and to Richard Davenport for being a fantastic host. ORCID iD T J M ORCID iD Tom J Moullaali http://orcid.org/0000- 180 Moullaali TJ, Scott S. Pract Neurol 2020;20:180–181. doi:10.1136/practneurol-2019-002310 Moullaali TJ, Scott S. Pract Neurol 2020;20:180–181. doi:10.1136/practneurol-2019-002310 Book club Reference 1 O’Farrell M. I am, I am, I am: seventeen 0002-6786-3623 brushes with death. Headline Publishing brushes with death. Headline Publishing Group, 2017. on October 23, 2024 by gue http://pn.bmj.com/ Pract Neurol: first published as 10.1136/practneurol-2019-002310 on 4 July 2019. Downloaded from 181 Moullaali TJ, Scott S. Pract Neurol 2020;20:180–181. doi:10.1136/practneurol-2019-002310
https://openalex.org/W2058349512	https://europepmc.org/articles/pmc4068638?pdf=render	English	null	A liberal strategy of red blood cell transfusion reduces cardiovascular complications in older patients undergoing cardiac surgery	Critical care	2,014	cc-by	245,479	References References 1. Vincent et al.: Adverse events in British hospitals. BMJ 2001, 322:517-519. 1. Vincent et al.: Adverse events in British hospitals. BMJ 2001, 322:517-519. ld f f d f p 2. Building a Safer NHS for Patients: Improving Medication Safety [http://webarchive.nationalarchives.gov.uk/+/www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_4071443] p p Results We assessed 129 prescriptions in the fi rst round, and 111 after intervention, demonstrating a 70% improvement in safe prescribing. Only 24% of prescriptions initially fulfi lled best practice criteria, improving to 94% afterwards. We also reduced the number of infusions running without prescription, 7 (6%) versus 24 (19%). See Figures 1 and 2. Conclusion Our audit supports the need for standardised prescribing practices within critical care, especially when dealing with potentially harmful vasoactive/sedative drugs. With a small, cost-eff ective intervention (£20 for 6,200 stickers), we improved prescribing accuracy, and thus patient safety in intensive care. 3. Safe Prescribing [http://www.medicalprotection.org/uk/england-factsheets/ safe-prescribing] 3. Safe Prescribing [http://www.medicalprotection.org/uk/england-factsheets/ safe-prescribing] P2 The Limpet controlled drug cabinet alarm and camera M Mariyaselvam, D Pearson, P Moondi, P Young Queen Elizabeth Hospital NHS Trust, Kings Lynn, UK Critical Care 2014, 18(Suppl 1):P2 (doi: 10.1186/cc13192) P2 The Limpet controlled drug cabinet alarm and camera M Mariyaselvam, D Pearson, P Moondi, P Young Queen Elizabeth Hospital NHS Trust, Kings Lynn, UK Critical Care 2014, 18(Suppl 1):P2 (doi: 10.1186/cc13192) © 2010 BioMed Central Ltd Figure 1 (abstract P1). Accuracy of prescriptions before intervention. © 2014 BioMed Central Ltd Figure 1 (abstract P1). Accuracy of prescriptions before intervention. Introduction The theft and tampering of controlled drugs (CDs) remains a prevalent patient safety issue. Sadly there are numerous reports of critical care staff stealing CDs for personal use or fi nancial gain and notably there have been some cases where CDs have been substituted for other medications in order to delay detection of the theft. This creates both the hazard of medication errors and potentially exposes patients to opioid intoxicated healthcare workers. As most critical care staff have access to CDs, when drugs are found to be missing it can be diffi cult to identify the perpetrators. Therefore the implementation of a deterrent which also improves the methods of detection is warranted. Methods The Limpet, a device which incorporates a proximity sensor and a camera unit, was installed within the CD cupboard of the critical care unit. MEETING ABSTRACTS MEETING ABSTRACTS P1 Minimising prescribing errors in the ICU DJ M li S S h DJ Melia, S Saha Introduction We aimed to audit the prescribing practice on a busy 14-bedd general ICU, and develop standardised practices and tools to improve safety. Prescribing errors occur as commonly as in 10% of UK hospital admissions, costing 8.5 extra bed days per admission, and costing the National Health Service an estimated £1 billion per annum [1]. The majority of these mistakes are avoidable [2]. Methods We audited the daily infusion charts of all patients in three separate spot checks, over 1 week. We assessed all aspects of prescriptions that make them legal and valid, in accordance with national guidance [3]. New procedures were introduced, which included a standardised prescription sticker, with common, pre- printed, infusion prescriptions on (noradrenaline, propofol, and so forth), and education on using the new prescription stickers. A month later the audit process was repeated.i 34th International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 18-21 March 2014 Publication of this supplement has been supported by ISICEM. Figure 2 (abstract P1). Accuracy of prescriptions after intervention. P1 Minimising prescribing errors in the ICU DJ Melia, S Saha Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P1 (doi: 10.1186/cc13191) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle p Conclusion When CDs are missing it can be extremely stressful for the staff involved. Those who have access to CDs may feel unfairly scrutinised and the potential for false accusation exists. Investigating the theft of CDs is costly and usually involves pharmacists, managers and the police. Until the issue is resolved, potential suspects are usually suspended from work, leading to disruptions in patient care. The utility of the Limpet in modifying staff behaviour by reducing the number of occasions and the duration of time that open drug cupboards are left unattended, has previously been demonstrated [1]. By providing the facility to determine exactly who accessed each CD cupboard at which time, this initial study has shown the benefi ts of the Limpet as a tool for detecting theft. Therefore the installation of the Limpet mitigates the diffi culties of investigating CD theft and is likely to prove an eff ective deterrent. f Introduction The purpose of this study was to assess our practice in identifying and managing inadvertent hypothermia and whether this could be improved by education and introduction of a protocol. Hypothermia is associated with multiple physiological abnormalities including reduced myocardial contractility, peripheral vasoconstriction, increased infection risk and impaired coagulation [1]. Inadvertent hypothermia may therefore be an avoidable risk factor in the critically ill. The UK National Institute of Clinical Excellence has issued guidance for avoidance of inadvertent hypothermia (temperature <36°C) during the perioperative period. We audited our practice against three standards from these guidelines: all patients should have at least 4-hourly temperature observations; no patient should become inadvertently hypothermic; and all inadvertently hypothermic patients should be rewarmed. Reference 1. The Limpet drug cabinet alarm: technology for safer drug stewardship [abstract and poster presentation]. Presented at International Forum on Quality and Patient Safety; April 16-19 2013; London. 1. The Limpet drug cabinet alarm: technology for safer drug stewardship [abstract and poster presentation]. Presented at International Forum on Quality and Patient Safety; April 16-19 2013; London. Methods Data were collected prospectively. Body temperature was recorded routinely by nursing staff using a tympanic thermometer. We noted any occasion where the body temperature dropped below 36°C along with any associated interventions – such as the use of additional bed sheets or a forced air warming device. Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle After the fi rst audit period a simple education programme was delivered. We also introduced a departmental protocol for the prevention and management of inadvertent hypothermia. Six months later we re-audited our practice. Results Data were collected from 130 patients (2,931 patient-hours) in the fi rst audit period and from 87 patients (2,070 patient-hours) in the second audit period. In the fi rst period 29% of patients had at least 4-hourly temperature measurements compared with 40% in the second period (P <0.01). The average number of overdue temperature observations per day was 1.4 in the fi rst period and 0.9 in the second (P <0.01). Twenty-four per cent of patients became hypothermic in the fi rst period compared with 22% in the second (P = 0.07); however, the time these patients remained hypothermic reduced from an average of 7.9 hours to 6.1 hours (P <0.01). An intervention was made and documented in 15% of cases in the fi rst period and 46% in the second (P <0.001). P3 Role of pharmacist in multidisciplinary pediatric intensive care rounds: a retrospective descriptive study S Tripathi, K Graner, K Fryer, G Arteaga Mayo Clinic, Rochester, MN, USA Critical Care 2014, 18(Suppl 1):P3 (doi: 10.1186/cc13193) p p S Tripathi, K Graner, K Fryer, G Arteaga Introduction Multidisciplinary rounding practices in the ICU have now become the standard of care in most institutions. Few objective data are available, however, on the value each individual member of the team brings to the patient care. In our institution, pediatric pharmacists have been an integral part of the PICU rounds since 2002, although their role has evolved over the course of years. This study was undertaken with the primary aim of identifying the impact of pharmacist involvement in PICU rounds, with respect to changes in therapy. Conclusion We saw some improvement following an education programme and introduction of a clinical protocol although there remains room for further improvement. Methods Since January 2003, pharmacists have recorded their clinical interventions and outcomes of those interventions in an institutionally developed Pharmaceutical Care database (P-Care). An intervention is defi ned as any recommendation the pharmacist makes to the patient care team regarding a change in the patient management or medication therapy. P-Care is designed to assist the pharmacist to optimize medication therapy, identify medication-related problems, decrease medication costs, improve pharmacist effi ciency and document pharmacist workload. P5 P5 Incidence of adverse events in a Brazilian coronary ICU VF Paula1, CE Bosso1, OA Souza2, GE Valerio2, SV Ferreira2 1Instituto do Coração de Presidente Prudente, Brazil; 2UNOESTE – Universidade do Oeste Paulista, Presidente Prudente, Brazil Critical Care 2014, 18(Suppl 1):P5 (doi: 10.1186/cc13195) Incidence of adverse events in a Brazilian coronary ICU VF Paula1, CE Bosso1, OA Souza2, GE Valerio2, SV Ferreira2 1Instituto do Coração de Presidente Prudente, Brazil; 2UNOESTE – Universidade do Oeste Paulista, Presidente Prudente, Brazil Critical Care 2014, 18(Suppl 1):P5 (doi: 10.1186/cc13195) p y y Results From 1 January 2003 through 31 December 2012 pharmacists made 24,207 clinical interventions in the PICU and 19,252 of those interventions resulted in changes in medication therapy or therapy monitoring. Interventions that were accepted by the team involved 10,361 (53.8%) drug dosing regimen changes, 292 (1.5%) drug interactions or incompatibility, 969 (5%) drug monitoring suggestions, 1,665 (8.7%) drug routes/methods of administration, 3,895 (20.2%) drug selections, and 2,070 (10.8%) medication profi le/order clarifi cations. As the pharmacist role on the PICU multidisciplinary rounds has evolved, the number of interventions has increased from 1,643 in 2003 to 3,799 in 2012. Of the 19,252 interventions that were implemented, 304 were deemed to be of potentially life-threatening consequences, 10,767 had a moderate impact, and 8,181 had minimal impact on patient outcome. Conclusion To our knowledge this is the largest reported data on pharmacist’s involvement in pediatric intensive care. This can serve as background knowledge on implementing measures on novel methodologies to integrate pharmacists in intensive care practice. Critical Care 2014, 18(Suppl 1):P5 (doi: 10.1186/cc13195) Introduction Security policies for the patient are an interest to any health professional. The rates of adverse events in hospitals reach values ranging between 3.7 and 16.6%, being the highest range (40 to 70%) considered preventable or avoidable [1]. The objective of this study is to analyze the incidence of adverse events and their severity in a Brazilian coronary ICU (CICU). Methods Research conducted from database analysis regarding hospitalizations from 1 May 2012 until 31 October 2013 in a coronary care unit of the city of Presidente Prudente, Brazil. Statistical analysis was performed using EPI INFO, version 3.5.2 software, which was considered signifi cant at P <0.05 two-tailed and CIs at 95% (CI) were used for the odds ratios (OR) estimated in the sample. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Mock thefts were successfully accomplished on six occasions over a 4-week period. On each occasion the Limpet photographed the ‘thief’ and recorded the date and time of access. Therefore, in the event of a real theft it would be possible to quickly and easily indentify the culprit. References Whenever the cupboard was accessed the date and time were recorded and a photograph was taken to identify the staff member. Mock thefts were subsequently undertaken by a designated staff member at random times. This allowed testing of the product to determine the number of times the ‘thief’ was correctly identifi ed. © 2010 BioMed Central Ltd © 2014 BioMed Central Ltd © 2014 BioMed Central Ltd S2 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle Data concerning pharmacists’ interventions were extracted from the P-Care database in yearly increments via a reporting functionality that is available in the P-Care system. This study was exempted for review by the Mayo Clinic IRB. 1. Andrzejowski et al.: Br J Anaesth 2008, 101:627-631. 1. Andrzejowski et al.: Br J Anaesth 2008, 101:627-631. P5 1. Marra AR, et al.: Am J Infect Control 2009, 37:619-625. Organisational changes in service provision outside critical care impact on referral patterns A Tridente1, L Cecchini2, S Lobaz3, A Chick3, S Keep3, S Furmanova4, D Bryden3fi A Tridente1, L Cecchini2, S Lobaz3, A Chick3, S Keep3, S Furmanova4, D Bryden3fi Conclusion Post intervention, a statistically signifi cant improvement in overall bundle compliance was found. Thereby highlighting that through engaging all members of the multidisciplinary team in identifying barriers to noncompliance and delivering education, it is possible to improve compliance. While total compliance was suboptimal as the target was 95%, the bundle redesign has given a tool that records compliance with greater clarity due to the presence of clearly defi ned exclusion criteria. It has also been a signifi cant step in the right direction to improving the reliability of care delivered to patient and reinforces the concept that quality improvement is a continuous cycle. 1St Helens and Knowsley, Liverpool, UK; 2SMH, Manchester, UK; 3STH, Sheffi eld, UK; 4UHW, Cardiff , UK 1St Helens and Knowsley, Liverpool, UK; 2SMH, Manchester, UK; 3STH, Sheffi eld, UK; 4UHW, Cardiff , UK f Critical Care 2014, 18(Suppl 1):P8 (doi: 10.1186/cc13198) f Critical Care 2014, 18(Suppl 1):P8 (doi: 10.1186/cc13198) Introduction Demand for critical care (CC) resources is constantly increasing in the face of limited availability. Guidance for triage exists but may no longer refl ect current practice [1,2]. We previously identifi ed nonmedical and medical factors (comorbidities, physiological derangement and functional status) as predicting likelihood of admission of referred patients to CC [3-5]. Reduction in UK doctors’ working hours and numbers has resulted in new ways of multidisciplinary teams working the hospital at night (H@N), which may have an impact on CC referral. We aimed to establish any eff ect of Introduction Demand for critical care (CC) resources is constantly increasing in the face of limited availability. Guidance for triage exists but may no longer refl ect current practice [1,2]. We previously identifi ed nonmedical and medical factors (comorbidities, physiological derangement and functional status) as predicting likelihood of admission of referred patients to CC [3-5]. Reduction in UK doctors’ working hours and numbers has resulted in new ways of multidisciplinary teams working the hospital at night (H@N), which may have an impact on CC referral. We aimed to establish any eff ect of 1. Foster AJ, et al.: Adverse events among medical patients after discharge from hospital. CMAJ 2004, 170:345-349. Compliance of a ventilator-associated pneumonia care bundle in an adult intensive care setting G Wigmore, R Sethuraman The Princess Alexandra Hospital NHS Trust, Harlow, UK Critical Care 2014, 18(Suppl 1):P6 (doi: 10.1186/cc13196) Compliance of a ventilator-associated pneumonia care bundle in an adult intensive care setting G Wigmore, R Sethuraman The Princess Alexandra Hospital NHS Trust, Harlow, UK Critical Care 2014, 18(Suppl 1):P6 (doi: 10.1186/cc13196) g The Princess Alexandra Hospital NHS Trust, Harlow, UK Introduction Ventilator-associated pneumonia (VAP) is the leading cause of death amongst hospital-acquired infections and is also linked to an increased length of stay and cost of care. The Institute for Healthcare Improvement ventilator bundle is comprised of a series of interventions, which, when implemented together, have been shown to decrease the incidence of VAP. The aim of this study was to determine the compliance of the bundle and if <95% [1] devise strategies to improve compliance. Conclusion Acutely unwell patients require the expertise of the most senior clinicians regarding further management, including planning for end-of-life care. Our audit demonstrated poor adherence to NCEPOD [1] and Department of Health [2] recommendations. The majority of referrals to critical care were made by nonconsultants and for patients who had not been reviewed by a team consultant, prior to referral. The workload in critical care demonstrated that almost one- half of the referrals happen out of hours. These fi ndings have resulted in signifi cant changes to working practice, including the presence of an onsite consultant intensivist for a minimum of 13 hours daily. References p p Methods A retrospective review of the compliance of an existing VAP bundle was conducted for all adult patients ventilated in the ICU of a large district general hospital in 2011. The bundle comprised four elements: head up 30°, peptic ulcer prophylaxis, deep vein thrombosis (DVT) prophylaxis and sedation hold. The bundle was considered compliant if all four were performed. The fi ndings of the audit were presented to the department and, through subsequent discussions, barriers to noncompliance were identifi ed. Following a period of education, a revised and updated bundle was implemented. A repeat audit covering 3 months was subsequently conducted. References 1. An Acute Problem? Report of the National Confi dential Enquiry into Patient Outcome and Death. London: National Confi dential Enquiry into Patient Outcome and Death; 2005. [http://www.ncepod.org.uk/2005report/] 1. An Acute Problem? Report of the National Confi dential Enquiry into Patient Outcome and Death. London: National Confi dential Enquiry into Patient Outcome and Death; 2005. [http://www.ncepod.org.uk/2005report/] g 2. Guidelines on Admission to and Discharge from Intensive Care and High Dependency Units. London: Department of Health; 1996. 2. Guidelines on Admission to and Discharge from Intensive Care and High Dependency Units. London: Department of Health; 1996. Results Pre-intervention, overall compliance of the bundle stood at 32% and subsequently increased to 63% post intervention (P <0.05). Compliance at the level of individual elements varied: head up 30°, 94%; ulcer prophylaxis, 91%; DVT prophylaxis, 85%; sedation hold, 37%. Post intervention, a statistically signifi cant increase in compliance with regard to sedation hold was observed; 72% (P <0.05). The other individual elements did not show a statistical change. However, the new elements that were introduced demonstrated high levels of compliance; cuff pressure 20 to 30 cm H2O 83% and oral hygiene with chlorhexidine 90%. P5 Demographics (gender and average age) and aspects related to adverse events were analyzed, the latter being under the impact and probability of death. S3 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results A total of 1,067 admissions were analyzed in the period, with 57.38% male and 42.63% female. The average age was 67.7 ± 13.2 years. The average CICU length of stay was 3.74 ± 5.51 days. A total of 211 adverse events occurring in 140 diff erent admissions were recorded. The most frequent were drug administration errors (24.3%), pressure ulcer (24.3%), phlebitis (22.1%), loss of enteric tube (13.6%) and central venous cannulation accident (7.1%). The statistical analysis shows that hospitalization time longer than 2 days are related to the occurrence of the events (OR: 8.08 CI: 4.95 to 13.2) and that the presence of the fi rst occurrence is signifi cant to increase the probability of death in the unit (OR = 5.33, CI: 3.49 to 8.12). Pressure ulcer (OR = 16.73, CI: 8.04 to 34.81), enteral tube loss (OR 3.64, CI: 1.36 to 9.75) and drug administration errors (OR = 2.87, CI: 1.31 to 6.31) were also related to higher mortality. Conclusion The research draws attention to a signifi cant incidence of adverse events and shows that their occurrence implies an increase of death in the unit. Therefore, security measures should be employed for the reduction, and thus enhance the quality of service provided by health professionals to patients admitted to a coronary care unit. Reference Referrals to a critical care unit: compliance with the NCEPOD recommendations G Wigmore1, M Campbell2, P Walker2, K Raveendran2 1The Princess Alexandra Hospital NHS Trust, Harlow, UK; 2Barking Havering and Redbridge University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P7 (doi: 10.1186/cc13197) G Wigmore1, M Campbell2, P Walker2, K Raveendran2 1The Princess Alexandra Hospital NHS Trust, Harlow, UK; 2Barking Havering and Redbridge University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P7 (doi: 10.1186/cc13197) Introduction The report of the National Confi dential Enquiry into Patient Outcome and Death (NCEPOD) 2005 [1] provides recommendations to reduce morbidity and mortality among acutely ill patients. It highlights the importance for involvement of consultants in the referral process, clear physiological monitoring plans for each patient, and review by a consultant intensivist within 12 hours of admission to critical care. The objective of the audit was to assess compliance with these recommendations, ensuring safe management of acutely unwell patients and appropriate utilisation of scarce critical care resources. Methods Prospective data on referrals to adult critical care in a 20- bed unit in a teaching hospital were collected over 8 weeks. Collected data included: source, time, seniority of doctor referring and receiving referral, outcome of referral, involvement of team consultant prior to referral, documented management plan and review by a critical care consultant within 12 hours of referral. 1. Foster AJ, et al.: Adverse events among medical patients after discharge from hospital. CMAJ 2004, 170:345-349. P6 Results Seventy-three referrals were analysed, the majority of which were medical. One-half of referrals came out of hours; 24% of referrals were made by a consultant; 51% were seen by a consultant prior to referral; 73% of referrals were admitted; likelihood of admission increased from 63% to 83% if the patient was reviewed by home consultant prior to referral. In 56% of cases the referral was received by a foundation doctor (all referrals were discussed with a consultant intensivist). Among the rejected referrals (maximal ward therapy not reached), 54% were from a trainee below registrar grade. Twenty-fi ve per cent of patients were not seen by a consultant intensivist within 12 hours of referral. Demands on a continuing education online-study program for physicians 1. ACCCM Guidelines for ICU admission, discharge, and triage. Crit Care Med 1999, 27:633-638. y g y Critical Care 2014, 18(Suppl 1):P10 (doi: 10.1186/cc13200) 2. ATS. Fair allocation of intensive care unit resources. Am J Respir Crit Care Med 1997, 156:1282-1301. Introduction Physicians have an intense and irregular workday life. Thus, they need to use their time for continuing education in the most effi cient way. This presents a major challenge to suppliers of continuing study programs. Our online master program Physico-Technical Medicine (PTM) addresses working physicians who want to acquire knowledge in the fi elds of biomedical engineering and medical physics [1]. We attach great importance to the special needs of our participants. Therefore, we investigated which general conditions are most important for the working physician regarding qualifying continuing education. 3. Tridente A, Chick A, Keep S, Furmanova S, Webber S, Bryden DC: Factors aff ecting critical care admission to a UK University Hospital [Poster P508]. Presented at 32nd International Symposium on Intensive Care and Emergency Medicine; March 19-23 2012; Brussels. 4. Tridente A, Chick A, Keep S, Furmanova S, Webber S, Bryden DC: Functional status as a predictor of admission to critical care in acutely unwell patients [Abstract 0424]. Presented at 25th Annual Congress of the European Society of Intensive Care Medicine; October 13-17 2012; Lisbon. 5. Tridente A, Chick A, Keep S, Furmanova S, Webber S, Bryden DC: Non medical factors infl uence likelihood of admission to critical care of acutely unwell patients [Abstract A-570-0025-00363]. Presented at 26th Annual Congress of the European Society of Intensive Care Medicine; October 5-7 2013; Paris. y y Methods The general conditions of our continuing education program (PTM) were examined on the basis of evaluation questionnaires. Additionally, students were asked in interviews about their demands, and they could give anonymous feedback on feedback cards.l y y Results Flexibility: students appreciated fl exibility regarding time and environment of their learning activities. This fl exibility enables the students to use even small time frames or traveling times for learning. Students can choose their preferred time and place for their learning activities. Security in planning: students have to organize their time frames for learning besides their daily work, their family life, and possibly their academic career. Especially phases of attendance should be planned and communicated as early as possible. 1. Guttmann J, et al.: Physikalisch-Technische Medizin (PTM) – ein neuer Master-Online Studiengang für Mediziner. GMS Z Med Ausbild 2011, 28(1):Doc01. Demands on a continuing education online-study program for physicians Transparency of structure: participants ask for transparency in terms of the expected workload and expenditure of time to plan their studies and get focused. Relation to previous knowledge: learning success is supported by relating new contents to previous knowledge from the student’s fi rst medical degree. Mainly clinical examples illustrate abstract topics, which also helps students to recognize the relevance of the content. Individual support: students need contact persons in terms of learning organization, technical support, and for questions regarding the course material. Demand versus supply in intensive care: an ever-growing problem M Tanaka Gutiez, R Ramaiah East Kent Hospitals University NHS Foundation Trust, Ashford, UK Critical Care 2014, 18(Suppl 1):P9 (doi: 10.1186/cc13199) Introduction The demand for intensive care has reached new heights, where medical advancement and aging populations have increased the proportion of patients with multi comorbidities. Ideally, bed occupancy on the ICU should be around 70% whereas beyond 80% has been associated with increased mortality. William Harvey Hospital is a district general hospital with nine ICU beds where the ICNARC national database suggested a consistently high occupancy. The purpose of this study was to assess bed occupancy and its impact on service delivery. Methods Data were collected between 2005 and 2013 to analyse trends in ICU bed occupancy, overnight discharges, surgical cancellations, and nonclinical transfers. Data were gathered from April 2012 to March 2013 using quality indicators (Intensive Care Society): readmissions; premature discharges; discharges at night; cancelled planned surgery; and nonclinical transfers. Regression analysis was conducted to assess: correlation between mortality and eff ects of excess occupancy; and causality of increasing demand for ICU beds. Conclusion We could show that an extra-occupational continuing study program for physicians should be adjusted to their special situation and therefore has to comply with particular requirements. As such we could identify fi ve main requirements which are continuously fulfi lled by the online master program PTM and regularly surveyed by evaluations. P10 0 Demands on a continuing education online-study progra physicians J Seifried, V Titschen, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P10 (doi: 10.1186/cc13200) p y y References 1. Intensive Care Society: Standards for Intensive Care Units. London: ICS; 1997. 2. Iapichino G, Gattinoni L, Radrizzani D, et al.: Volume of activity and occupancy rate in intensive care units. Association with mortality. Intensive Care Med 2004, 30:290-297. 3. Intensive Care Society: Standards, Safety and Quality Committee. Quality Indicators. London: Intensive Care Society; 2010. 3. Intensive Care Society: Standards, Safety and Quality Committee. Quality Indicators. London: Intensive Care Society; 2010. 4. Department of Health: Comprehensive Critical Care. A Review of Adult Critical Care Services. London: Department of Health; 2000. 4. Department of Health: Comprehensive Critical Care. A Review of Adult Critical Care Services. London: Department of Health; 2000. Conclusion Decision-making about admission to CC is based mainly on the assessment of patients’ functional status. Organisational changes in the provision of healthcare services outside CC, such as H@N, have had a signifi cant infl uence on CC workload and patterns of referrals. References Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (29%), surgical cancellations (90%), and nonclinical transfers (88%). Between 2012 and 2013, the calculated demand for beds was 174. There were 1.8 ICU to 100 hospital beds and 4.5 ICU beds per 100,000 population, which dropped signifi cantly when including regional specialty services. A persistent gap between ICU bed supply and demand was associated with increased unfavourable outcomes in all quality indicators. hospital changes in service provision outside CC on the referral pattern and admissions. hospital changes in service provision outside CC on the referral pattern and admissions. Methods Data from prospectively enrolled urgent patient referrals were analysed comparing two periods: before (period 1: 2011/12) and after (period 2: 2013) the introduction of H@N. We collected data on acute physiological parameters, hospital length of stay, demographic and functional status, dependency and comorbidities. STATA was used for these preliminary analyses. Conclusion A steady increase in occupancy over the last 8 years was due to multiple factors, including an increase in clinical services such as in the coronary care centre, head and neck unit, and trauma centre. Presentation of our results to managerial level facilitated increased bed capacity by 22%. f y y Results Comparing the two periods we found no signifi cant diff erences in age, gender distribution, degree of acute physiological derangement, comorbidities, specialty of origin, time spent in hospital prior to referral and grade of referrer. By contrast, the proportion of out- of-hours referrals greatly increased (from 49.3% to 69.7%) along with the proportion of referrals deemed inappropriate for CC (from 37.3% to 54.8%); the proportion of patients accepted increased only marginally (from 46.7 to 50%) in the second period, compared with the fi rst. Neither the number of beds available within the critical care department (P = 0.59) nor receiving the referral out of hours (P = 0.8) infl uenced the likelihood of admission. The factors predicting admission to CC (functional status, acute physiological derangement) did not diff er signifi cantly between the two periods examined. Housebound status was consistently found to be an independent predictor of admission refusal (OR 0.11, 95% CI 0.05 to 0.23, P <0.001). Reference S4 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Convalescence via critical care collaboration C Shute, A Saaymanf y p Critical Care 2014, 18(Suppl 1):P12 (doi: 10.1186/cc13202) y p ,f , Critical Care 2014, 18(Suppl 1):P13 (doi: 10.1186/cc13203) Introduction The aim was to explore the HDU booking process, organ support requirements and fi nancial implications as the current approach to booking surgical cases for HDU has somewhat been based on a ‘just in case’ principle. Introduction A collaborative approach to patient management has been shown to improve patient outcomes. In a resource-limited NHS, critical care beds are at a premium, therefore preventing unnecessary admissions by optimising ward-based management is essential. The objective of this service quality improvement project was to improve collaboration between the critical care directorate and neurosurgical high care unit at a tertiary university teaching hospital. We proposed that the use of a simple ward round tool on collaborative rounds would facilitate systematic patient review, prompt early recognition of those critically unwell and improve patient outcomes. j p p Methods The booking and admission details from October 2012 to July 2013 were gathered from the HDU log-book and Ward Watcher software. The cost of HDU beds and staff were provided by the fi nance department. Data were analysed using MS Excel and SPSS software. Results There were 105 handwritten bookings over 10 months, several missing essential data. Only 89 admissions actually took place; 61 were elective, 66 colorectal, six urgent, 19 scheduled and three unspecifi ed (Figure 1). Most patients required basic cardiovascular and respiratory support (Figure 2). Data revealed 16 cancellations. Each booking requires allocated nursing staff which bears a basic cost of £23/hour (estimated annual loss >£10.000). If we add the daily cost of basic HDU care (approximately £1,000 for single organ support), the losses rise rapidly. We also registered 38 days in delayed discharges. This was predominantly due to ward bed shortages. In fi nancial terms there is at j Methods The booking and admission details from October 2012 to July 2013 were gathered from the HDU log-book and Ward Watcher software. The cost of HDU beds and staff were provided by the fi nance department. Data were analysed using MS Excel and SPSS software. Results There were 105 handwritten bookings over 10 months, several missing essential data. Only 89 admissions actually took place; 61 were elective, 66 colorectal, six urgent, 19 scheduled and three unspecifi ed (Figure 1). Most patients required basic cardiovascular and respiratory support (Figure 2). Data revealed 16 cancellations. Reference Results Bed occupancy remained high between 2005 and 2013 with a 15% increase. This was associated with increased overnight discharges S5 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P12). Admissions per speciality. Figure 2 (abstract P12). Organ support requirements. Figure 1 (abstract P12). Admissions per speciality. P11 Do generic measures fully capture health-related quality of life in adult, general critical care survivors? W Lim1, N Black1, K Rowan2, N Mays1 1London School of Hygiene & Tropical Medicine, London, UK; 2Intensive Care National Audit & Research Centre, London, UK Critical Care 2014, 18(Suppl 1):P11 (doi: 10.1186/cc13201) Introduction We examined the extent to which the two generic health- related quality of life (HRQL) measures recommended for use in adult, general critical care [1] – the SF-36 and EQ-5D – captured survivors’ HRQL, which is important in assessing the eff ectiveness of critical care. Unlike other fi elds of healthcare that employ both generic and specifi c HRQL measures, most recent studies in critical care have used only generic measures, despite uncertainty as to their appropriateness. Methods A patient-based conceptual framework for survivors’ HRQL was built using: a systematic review of the literature; secondary analysis of 40 in-depth interviews with adult critical care survivors; and primary analysis of in-depth interviews with a maximum variation sample of 25 white critical care survivors in England. Two methods were then used to assess the extent to which the SF-36 and EQ-5D captured their HRQL, as detailed in the framework: the content of both instruments was examined, alongside data collected from the in-depth interviews; and the opinions that survivors expressed about how accurately the SF-36 and EQ-5D refl ected their ideas on health and HRQL were analysed and taken into account.i Introduction We examined the extent to which the two generic health- related quality of life (HRQL) measures recommended for use in adult, general critical care [1] – the SF-36 and EQ-5D – captured survivors’ HRQL, which is important in assessing the eff ectiveness of critical care. Unlike other fi elds of healthcare that employ both generic and specifi c HRQL measures, most recent studies in critical care have used only generic measures, despite uncertainty as to their appropriateness. Reference Methods A patient-based conceptual framework for survivors’ HRQL was built using: a systematic review of the literature; secondary analysis of 40 in-depth interviews with adult critical care survivors; and primary analysis of in-depth interviews with a maximum variation sample of 25 white critical care survivors in England. Two methods were then used to assess the extent to which the SF-36 and EQ-5D captured their HRQL, as detailed in the framework: the content of both instruments was examined, alongside data collected from the in-depth interviews; and the opinions that survivors expressed about how accurately the SF-36 and EQ-5D refl ected their ideas on health and HRQL were analysed and taken into account. Figure 1 (abstract P12). Admissions per speciality. Figure 2 (abstract P12). Organ support requirements. Figure 2 (abstract P12). Organ support requirements. Results The patient-based framework was in two parts: the fi rst covered survivors’ personal status which consisted of their physical status, emotional/psychological status and cognitive status; and the second comprised nine subdomains aff ected by their personal status. The latter were: activities and behaviours; physical zone of comfort and/or activity; interactions and relationships with others; perception of, interpretation of, and responses to life; personality; external appearance; suitability and availability of clothes; place of residence; and fi nances. The current generic measures recommended on the basis of expert consensus for use with survivors of adult, general critical care have substantial gaps in their coverage of this conceptual framework for survivors’ HRQL, particularly in relation to their cognitive status and its subsequent impact. Figure 2 (abstract P12). Organ support requirements. least £500/day diff erence between surgical and HDU (potential annual loss >£22,000). In addition there were 13 patients who spent a total of 37 days on the HDU but did not require an HDU level of care. Conclusion The currently limited resource is not being utilised eff ectively with implications on both staff deployment and fi nances. A more holistic approach is needed where all requests are reviewed by a consultant anaesthetist in conjunction with preadmission data. This could better identify patients for HDU care and potentially decrease cancellations. We have developed an improved booking form for this purpose. least £500/day diff erence between surgical and HDU (potential annual loss >£22,000). In addition there were 13 patients who spent a total of 37 days on the HDU but did not require an HDU level of care. Reference q p Conclusion The two most commonly used generic HRQL measures in adult, general critical care have signifi cant gaps in their coverage of survivors’ HRQL. Any (new) critical care-specifi c HRQL measure should be designed specifi cally to capture the impact of critical illness on survivors’ cognitive status and its subsequent eff ects. Reference 1. Angus DC, et al.: Intensive Care Med 2003, 29:368-377. 1. Angus DC, et al.: Intensive Care Med 2003, 29:368-377. P12 Surgical HDU admissions: utilisation, organ support and fi nance G Brakmane, A Molokhia University Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P12 (doi: 10.1186/cc13202) Surgical HDU admissions: utilisation, organ support and fi nance G Brakmane, A Molokhia University Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P12 (doi: 10.1186/cc13202) Convalescence via critical care collaboration C Shute, A Saayman University Hospital Wales, Cardiff , UK Critical Care 2014, 18(Suppl 1):P13 (doi: 10.1186/cc13203) P15 time further data were collected to assess whether our interventions resulted in modifi ed behaviours and to document the number of changes made to patient management as a consequence of the collaborative approach to care. time further data were collected to assess whether our interventions resulted in modifi ed behaviours and to document the number of changes made to patient management as a consequence of the collaborative approach to care. Targeting blood tests in the ICU may lead to a signifi cant cost reduction Targeting blood tests in the ICU may lead to a signifi cant cost reduction R Gray, F Baldwin R Gray, F Baldwin Results Our results showed that improvements were made in all assessed domains. Consultant-led ward rounds increased, attendance by members of the multidisciplinary team (MDT) dramatically improved and as a result MDT discussion was enhanced. In addition, documentation of structured plans improved and review of prescription charts signifi cantly increased. As a consequence of collaborative rounds, a total of 343 changes were made to patient management under the domains of the ward round tool. This was an average of 21.4 changes per collaborative round, with most changes being made to medication charts or decisions regarding thromboprophylaxis. BSUH, Brighton, UK BSUH, Brighton, UK g Critical Care 2014, 18(Suppl 1):P15 (doi: 10.1186/cc13205) Introduction Daily blood tests are an essential diagnostic tool and routine practice in the management of ICU patients, but are associated with cost and a risk of anaemia. There is no evidence for the frequency or breadth of blood testing but there is evidence that by rationalising blood tests, signifi cant cost saving can be made without a negative eff ect on mortality or length of ICU stay [1]. Blood tests in our ICU are requested by nurses, this is a unique practice to the ICU compared with other hospital departments. Conclusion The use of a simple ward round tool combined with a collaborative approach to ward rounds improved MDT involvement and discussion, promoted structured patient review and resulted in positive changes to multiple areas of patient management. In conclusion, structured collaborative rounds result in signifi cant changes to patient management that may prevent admission, or readmission, to critical care which has the potential to reduce healthcare costs and morbidity. Methods We had previously carried out a survey to assess blood requesting practise in UK ICUs. This demonstrated that routine tests were, as in our ICU, nurse led and the majority of staff underestimated the costs of tests. Using the results of this survey and a literature review we developed routine blood test guidelines for our general mixed 26- bed ICU/HDU. Following a period of staff education, we audited our blood requesting practice over 28 days. Results Our blood testing did not comply with our guidelines. Over the 28-day period, €12,849.96 was spent on all blood tests. Table 1 (abstract P15) Table 1 (abstract P15) Number of 28-day cost Annual cost Test inappropriate requests (€) (€) Full blood count 4 21.82 284.44 Urea and electrolytes 6 29.44 383.77 Liver function tests 95 583.18 7,602.1 Coagulation screen 117 1,276.86 16,644.78 C-reactive protein 45 245.55 3,200.91 Bone profi le 112 274.75 3,581.56 Magnesium 88 483.35 6,300.81 p J Jennings1, TD Thomsen2, JW Larsen2, J Markvardt3 1Sheffi eld Teaching Hospital, Sheffi eld, UK; 2Kolding Hospital, part of Lillebaelt Hospital, Kolding, Denmark; 3Danish Building Research Institute/Aalborg University, Copenhagen, Denmark Critical Care 2014, 18(Suppl 1):P14 (doi: 10.1186/cc13204) Introduction Hospital lighting may cause disruption in the circadian rhythm, partly due to suppression of melatonin production. This may create sleep diffi culties and delirium for ICU patients and health issues such as fatigue, poor sleep quality and chronic diseases for ICU staff . Dynamic RGB coloured light which changes colour and intensity in correlation with the time of day has been installed in a Danish ICU, aiming to create lighting conditions being close to daylight variations and supporting patient and staff rhythms. As the eff ect of dynamic light on patients may be biased by illness, organ failure, and so forth, the aim was to examine the infl uence of dynamic light on ICU nurses’ melatonin production, quality of sleep, and well-being. Conclusion In our ICU, unnecessary blood tests have a signifi cant cost burden of approximately €38,000 per year. To limit unnecessary costs, consultants now lead requesting by completing a blood test pro forma on the evening ICU ward round indicating which blood tests are clinically needed the following day. Reference Methods An intervention study examining the impact of dynamic light regarding the impact on the circadian rhythm (measured by melatonin profi les from saliva samples), quality of sleep (sleep effi ciency, number of awakenings and subjective assessment of sleep; measured by sleep monitors and sleep diary), and subjective experiences of well-being, health, and sleep quality (measured by a questionnaire survey). Results from the intervention group were compared with a control group of nurses from a similar ICU without dynamic light. Light conditions were documented by measurements. Data collection was from February to May 2013. 1. Seguin P, et al.: Intensive Care Med 2002, 28:332-335. P14 Can dynamic light improve melatonin production and quality of sleep? Can dynamic light improve melatonin production and quality of sleep? Table 1 (abstract P15) Table 1 (abstract P15) Convalescence via critical care collaboration C Shute, A Saaymanf Each booking requires allocated nursing staff which bears a basic cost of £23/hour (estimated annual loss >£10.000). If we add the daily cost of basic HDU care (approximately £1,000 for single organ support), the losses rise rapidly. We also registered 38 days in delayed discharges. This was predominantly due to ward bed shortages. In fi nancial terms there is at y Methods An initial observation period of behaviours and practice on the neurosurgical unit was conducted with qualitative and quantitative data collection. Following analysis of these outcome measures a simple ward round tool was constructed, with the mnemonic ‘DON’T FORGET’, and we devised a programme for collaborative ward rounds to take place three times per week over a 1-month period. During this S6 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Targeting blood tests in the ICU may lead to a signifi cant cost reduction According to our guidelines, €2,914.96 was spent on inappropriate tests (€37,998.63 per year) (Table 1). Over 40% of this cost was spent on the coagulation screen. P14 Can dynamic light improve melatonin production and quality of sleep? J Jennings1, TD Thomsen2, JW Larsen2, J Markvardt3 1Sheffi eld Teaching Hospital, Sheffi eld, UK; 2Kolding Hospital, part of Lillebaelt Hospital, Kolding, Denmark; 3Danish Building Research Institute/Aalborg University, Copenhagen, Denmark Critical Care 2014, 18(Suppl 1):P14 (doi: 10.1186/cc13204) P18 ICU nursing connectivity and the quality of care in an academic medical center: a network analysis Results Data were collected for 3 months for 106 patients. The 24-hour trends of acoustic parameters corresponded well to the daily routine events in the ICU. The analyses for the fi rst 4 days of the patients’ ICU stay showed that the average SPL varied depending on the day (P = 0.023) and on the time of the day (day/night) (P <0.001). The average noise level decreased from day 1 to day 2 with a signifi cant reduction at night (P = 0.008), but it was found to rebound from day 2 where the increase of the daytime noise level from day 2 to day 4 was signifi cant (P = 0.005). The results of the multiple linear regression showed that various patient conditions infl uenced diff erent types of noise-level parameters. For example, the location of the patient room, the usage of mechanical ventilators and the mortality rate were found to correlate to the 10th-percentile SPL (L90) in the fi rst 48 hours (adjusted R2 = 0.58; P <0.001). Also, the room location, gender and the usage of mechanical ventilators were found to be related to the 50th-percentile SPL (L50) in the same period (adjusted R2 = 0.54; P <0.001). y DM Kelly1, DC Angus1, D Krackhardt2, JM Kahn1 1University of Pittsburgh, PA USA; 2Carnegie Mellon University, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P18 (doi: 10.1186/cc13208) Introduction A collaborative nurse work environment is associated with ICU quality, yet collaborative interaction is diffi cult to measure. Network analysis may be an innovative tool to measure interactions. We sought to determine the feasibility of network analysis to measure ICU nurse connectivity and test whether key network measures were associated with the ICU quality of care. Methods We performed a network analysis in eight ICUs within an urban academic medical center in the United States during 2011. Using scheduling data, we defi ned network ties as instances when two ICU nurses worked together in the same ICU for 4 hours or more. We examined each ICU’s network by visualizing sociograms and by measuring two network properties: density and clustering. Density measures the cohesion within a network on a scale from 0 to 100, with a higher score indicating more cohesion. Clustering assesses the local neighborhoods on a scale from 0 to 100, with a higher score indicating a more decentralized network. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Use of the TM Program for the implementation of the Surviving Sepsis Campaign Protocol at a community Brazilian hospital improved compliance with recommended care bundles and signifi cantly decreased the hospital mortality of septic patients. out to investigate the eff ect of relevant factors (for example, the time of day). Furthermore, multiple linear regression models were established to relate the diff erent types of patient-related data (as independent variables) to each type of the acoustic data (as dependent variable), where independent variables were selected based on an exhaustive search method. Results of the Telemedicine Program for implementation of the Surviving Sepsis Campaign Protocol in a community Brazilian hospital Conclusion We found substantial variation in ICU nursing networks across eight ICUs in one academic medical center. These patterns may have implications for evidence-based practice implementation. More work is needed to understand the role of network analysis as a reliable tool for improving and understanding ICU organization. P18 ICU nursing connectivity and the quality of care in an academic medical center: a network analysis We examined variation in network measures across ICUs and tested the correlation between network measures and the proportion of patients receiving daily interruption of sedation (DIS). Conclusion Noise-level parameters were found to vary depending on the day of ICU stay, the time of day, and other indicators of the patient’s status. For a rigorous analysis of the infl uence of noise on patients’ outcome, the eff ects of these factors must fi rst be controlled or corrected. f Reference 1. Van Rompaey et al.: Crit Care 2012, 16:R73. Results of the Telemedicine Program for implementation of the Surviving Sepsis Campaign Protocol in a community Brazilian hospital Results There was wide variation in the networks, with density ranging from 79 to 96 and clustering ranging from 88 to 97. Two sample sociograms are shown in Figure 1: ICU A had a very high density (96) and clustering coeffi cient (97) suggesting a cohesive and decentralized network, contrasting with ICU H that had the lowest density (79) and clustering coeffi cient (88). Greater density and clustering was positively associated with DIS (B = 0.02 (–0.10, 0.14); B = 0.003 (–0.07, 0.07)) but did not achieve statistical signifi cance in our small sample. C Abreu Filho1, M Steinman1, A Andrade2, F Piza1, A Caluza1, J Teixeira2, M Bracco2, R Abaladejo1, E Silva1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P17 (doi: 10.1186/cc13207) Introduction Sepsis has high incidence around the world, approximately 400,000 new cases occur annually in Brazil. In developing countries, it is still an important cause of death due to poor adherence to best- practice medicine protocols. The Brazilian mortality rate of septic shock is around 60%. The aim of this study is to describe the initial results for the implementation of the Brazilian initiative of a Telemedicine (TM) Project for therapeutic support of septic patients in a community hospital in São Paulo, Brazil. Figure 1 (abstract P18). Sociograms of ICU A and ICU H. Figure 1 (abstract P18). Sociograms of ICU A and ICU H. p Methods Since May 2012, a TM Program has been implemented at two hospitals in São Paulo – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity – due to a partnership with Brazilian Health Ministry. A TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and Medigraf Gowireless® technology. Mobile Intern MXP ISDN/IP Cisco® and Medigraf Gowireless® for the remote hospital (HMMD) via a dedicated connection. At HMMD, the sepsis protocol, based on the Surviving Sepsis Campaign, has been started for every recruited patient admitted to the emergency department (ED) or the ICU, and assessed by the Central Command through TM with an experienced consultant of HIAE. We compared the results of this group of patients with the group of patients with diagnosis of severe sepsis and septic shock, admitted to HMMD during 2011, who were not assisted with TM resources. P19 P19 Compassion fatigue and burnout among healthcare professionals in the ICU M Van Mol1, E Kompanje1, J Bakker1, M Nijkamp2 1Erasmus MC, Rotterdam, the Netherlands; 2Open University, Heerlen, the Netherlands Critical Care 2014, 18(Suppl 1):P19 (doi: 10.1186/cc13209) Analysis of the acoustic environment in an ICU using patient information as a covariate y Results A total of 55 nurses (89%) from the intervention ICU (ICU1) and 58 nurses (88%) from the control ICU (ICU2) participated. No signifi cant diff erences were found between the two groups regarding personal characteristics. The nurses from ICU1 described their work light as comfortable, relaxing and natural compared with artifi cial, institutional and gloomy in ICU2. Preliminary analyses did not shown any signifi cant diff erences in melatonin level. During a 10-day period, the nurses from ICU2 assessed their actual sleep as less eff ective (OR 2.17; P = 0.03) and felt less rested (OR 1.89; P = 0.006) compared with nurses from ICU1. The nurses in ICU2 had 16% more awakenings (P = 0.05) during sleep, but there were no signifi cant diff erences in duration of awakenings or in total sleep effi ciency between the two groups. M Park1, P Vos2, A Kohlrausch1, B Vlaskamp1, AW Oldenbeuving2 1Philips Research Laboratories, Eindhoven, the Netherlands; 2St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P16 (doi: 10.1186/cc13206) M Park1, P Vos2, A Kohlrausch1, B Vlaskamp1, AW Oldenbeuving2 1Philips Research Laboratories, Eindhoven, the Netherlands; 2St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P16 (doi: 10.1186/cc13206) M Park1, P Vos2, A Kohlrausch1, B Vlaskamp1, AW Oldenbeuving2 1Philips Research Laboratories, Eindhoven, the Netherlands; 2St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P16 (doi: 10.1186/cc13206) Introduction Noise levels in ICUs are often very high, potentially aff ecting patient health outcome, which are also considered to be among the risk factors contributing to ICU delirium [1]. In the current study, multivariate linear regression models were established to relate the acoustic data to the indicators of patient status. Methods Acoustic measurements were taken in eight single-bed patient rooms in a level 3 ICU, while patient information was also collected, including APACHE IV score and mortality rate together with other potentially relevant variables; for example, the usage of mechanical ventilators, and so on. The hourly and daily trends of the acoustic data were obtained, and an analysis of variance was carried fi Conclusion Most participants from the intervention ICU found the dynamic light agreeable and assessed their sleep more positively than participants from the control ICU. No signifi cant diff erences were found between monitored sleep effi ciency and melatonin level. S7 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Prevalence, risk factors and consequences of intra-team confl icts in the ICU G Burghi1, F Verga1, M Acevedo2, V Martinez1, V Carrasco3, C Quiroga4, G Pittini5, G Fariña6, M Godino1, S Mareque7, P Alzugaray3, H Bagnulo1, E Azoulay8 Results All references, retrieved from electronically database search (n = 1,432) and manual search (n = 3), resulted in 100 relevant publications for full-text screening. Subsequently, articles with only an abstract available (n = 28), a language barrier (n = 27), or prevalence not shown in percentages (n = 23) were removed. Finally, a sample of 22 eligible articles were appraised as methodologically sound and systematically analysed for this review. The prevalence of BO in the ICU varied from 1.2 to 49% and diff ered in defi ning a severe BO. Two studies reported the prevalence of CF, 7.3% and 40%, and fi ve studies described the prevalence of STS in a range from 21 to 44%. 1Hospital Maciel, Montevideo, Uruguay; 2COMERO, Rocha, Uruguay; 3Sanatorio Americano, Montevideo, Uruguay; 4Hospital Español, Montevideo, Uruguay; 5CAAMEPA, Pando, Uruguay; 6Casa de Galicia, Montevideo, Uruguay; 7CAMS, Mercedes, Uruguay; 8Hopital Saint Louis, Paris, France Critical Care 2014, 18(Suppl 1):P21 (doi: 10.1186/cc13211) Introduction The ICU is a stressful place with several sources of confl icts. Among ICU workers, confl icts lead to burnout, loss of psychological well-being, and deterioration of the work performance. Intra-team confl icts (confl icts that occur among members of the intensive care team) are one of the most frequent types of confl icts in the ICU. The objectives of this study were to determine the prevalence, risk factors and consequences associated with intra-team confl icts in the ICU. Introduction The ICU is a stressful place with several sources of confl icts. Among ICU workers, confl icts lead to burnout, loss of psychological well-being, and deterioration of the work performance. Intra-team confl icts (confl icts that occur among members of the intensive care team) are one of the most frequent types of confl icts in the ICU. The objectives of this study were to determine the prevalence, risk factors and consequences associated with intra-team confl icts in the ICU. Methods A survey was conducted in 12 Uruguayan adult ICUs in 2009. ICUs and clinician’s characteristics were assessed for their association with the prevalence of confl icts. All factors associated with confl icts were introduced into a multivariable model. References 1. Multz AS, et al.: Am J Respir Crit Care Med 1998, 157(5 Pt 1):1468-1473. 2. Pronovost PJ, et al.: JAMA 2002 288:2151-2162. f g p g Methods A review of the literature between 1998 and December 2013 was conducted using eight databases, and included the keywords CF, STS, BO, ICU, nurses and physicians. The references were screened for relevancy at title/abstract using predefi ned inclusion and exclusion criteria. Studies were limited to original and review articles in the English language. Two independent researchers assessed the methodological soundness of each article. P19 Compassion fatigue and burnout among healthcare professionals in the ICU Results From January to December 2011, 283 septic patients were treated at HMMD, 161 (56.8%) patients were male, mean age was 53.7 years and the hospital mortality was 65.3%. After the implementation of the TM Program, 189 septic patients were admitted to the hospital and evaluated by skilled doctors of HIAE via TM, with the institution of the Surviving Sepsis Campaign protocol. In total, 68.9% of the consultations originated from the ICU and 31.1% from the ED; 111 patients were male (58.4%), mean age was 54.1 years and the hospital mortality was 32.9% (P <0.05). M Van Mol1, E Kompanje1, J Bakker1, M Nijkamp2 1Erasmus MC, Rotterdam, the Netherlands; 2Open University, Heerlen, the Netherlands Critical Care 2014, 18(Suppl 1):P19 (doi: 10.1186/cc13209) Critical Care 2014, 18(Suppl 1):P19 (doi: 10.1186/cc13209) Introduction This study comprises a systematic review of studies focusing on the prevalence of compassion fatigue (CF) and burnout Introduction This study comprises a systematic review of studies focusing on the prevalence of compassion fatigue (CF) and burnout S8 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (BO) among professionals in the ICU, to indicate the size of this problem. Both CF and BO have an important impact on daily quality of life of professionals and may threaten patient care. A growing body of research suggests that BO appears to be common among ICU nurses [1] and physicians [2] due to a highly stressful work environment. However, BO might be overestimated and seen as a fashionable diagnosis [3]. The overlap of BO with CF, secondary traumatic stress (STS) and vicarious traumatisation has recently been explored [4]; CF seems to apply more to ICU staff and might be a lead in future preventive strategies. compared with NDG. Discordance was associated with higher ICU and hospital mortality (35.5 vs. 11%; OR = 4.09; P <0.001 and 45.2 vs. 20.1%; OR = 2.77; P = 0.02 respectively).i Conclusion The occurrence of divergences, even during the fi rst days of ICU admission, about medical plans of care for critically ill patients is frequent and is associated with higher ICU and hospital mortality and more use of medical resources. Prevalence, risk factors and consequences of intra-team confl icts in the ICU Conclusion The emotional price of working at the ICU can become a burden in personal life, but the size of the problem in ICU staff remains unclear. Because the decreased well-being of the professionals might negatively infl uence the quality of care, preventive strategies should be developed in order to alleviate the distressed. Further exploration of CF might provide suffi cient starting points. ql Methods A survey was conducted in 12 Uruguayan adult ICUs in 2009. ICUs and clinician’s characteristics were assessed for their association with the prevalence of confl icts. All factors associated with confl icts were introduced into a multivariable model. g p References References 1. Mealer et al.: Am J Respir Crit Care Med 2007, 175:693-697. 2. Embriaco et al.: Am J Respir Crit Care Med 2007, 175:686-682. 3. Kaschka et al.: Deutsch Arztebl Int 2011, 108: 781-787. 4. Meadors et al.: J Pediatr Health Care 2008, 22: 24-34. Results A total of 384 questionnaires were collected, 74.5% (n = 286) were nurses and 23.2% (n = 89) were intensivists. From the 384 forms, 45.8% perceived at least one confl ict in the past year. Confl icts were perceived more frequently by intensivists (61.8%) than nurses (40.9%) (P = 0.001). A worse relationship with intensivists (OR 1.3; 95% CI 1.09 to 1.59; P = 0.004) is independently associated with confl icts. However, lower grade of irritability (OR 0.8, 95% CI 0.73 to 0.97; P = 0.01) and a higher position in the ICU (OR 0.57 95% CI 0.36 to 0.92; P = 0.02) are factors independently associated with a lower incidence of intra-team confl icts. Interestingly, this study confi rms that workers with at least one confl ict had more frequently: libido disturbances (56% vs. 40%; P = 0.002), eating problems (52% vs. 40%; P = 0.02), as well as the wish to leave the ICU (53% vs. 39%; P = 0.005). P20 Eff ect of divergences about patient’s care plan on the outcome of critically ill patients EL Queiroz, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P20 (doi: 10.1186/cc13210) P20 Eff ect of divergences about patient’s care plan on the outcome of critically ill patients EL Queiroz, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P20 (doi: 10.1186/cc13210) P20 Eff ect of divergences about patient’s care plan on the outcome of critically ill patients EL Queiroz, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P20 (doi: 10.1186/cc13210) Critical Care 2014, 18(Suppl 1):P20 (doi: 10.1186/cc13210) Introduction There is an actual discussion about the best way to provide medical care for critically ill patients, particularly between the classical opened versus closed ICU models [1,2]. In Brazil, all ICUs have to have a full-time and dedicated physician at the unit but the primary physician also can visit his patient every day and, most of the time, participates in the patient’s care plan. This hybrid model creates opportunity for patient’s care plan divergences between the ICU staff and the primary physician. The objective of this study is to evaluate the eff ect of divergences about patient’s care plan on the outcome of critically ill patients. Conclusion The prevalence of confl icts is very high among ICU workers in Uruguay. We have identifi ed diff erent risk factors associated with the development of confl icts. These results confi rm previous fi ndings and highlights that strategies to decrease intra-team confl icts in the ICU are urgently warranted. References g Methods A literature review was conducted. PubMed was searched. The following combinations of Mesh terms were used: [(decision- making) OR (communication)] AND (intensive care) OR (intensive care units)] AND [(cultural diversity) OR (multiculturalism) OR (migrants) OR (culture) OR (ethnicity)]. A total of 111 studies were retrieved. We excluded nonempirical studies, studies not written in English and articles published before 2003. After screening the titles and abstracts 12 articles were selected. The full text of these articles was analyzed and synthesized with a thematic-synthesis approach. During the process we performed additional searches based on the bibliographies of all the relevant articles.i 1. Cullen DJ, et al.: Therapeutic intervention scoring system: a method for quantitative comparison of patient care. Crit Care Med 1974, 2:57-60. 1. Cullen DJ, et al.: Therapeutic intervention scoring system: a method for quantitative comparison of patient care. Crit Care Med 1974, 2:57-60. 2. Hamel MB, et al.: Seriously ill hospitalized adults: do we spend less on older patients? Support investigators. Study to understand prognoses and preference for outcomes and risks of treatments. J Am Geriatr Soc 1996, 44:1043-1048. New policy for ICU visits: prospective study New policy for ICU visits: prospective study A Moughabghab, S Gemayel, N Azar, R Bousaba A Moughabghab, S Gemayel, N Azar, R Bousaba y Lebanese Canadian Hospital, Beyrouth, Lebanon Lebanese Canadian Hospital, Beyrouth, Lebanon C i i l C 2014 18(S l 1) P23 (d i 10 1186/ 13213) p y Critical Care 2014, 18(Suppl 1):P23 (doi: 10.1186/cc13213) Results Culturally specifi c expectations about communication and decision-making, diff erent views on gender roles, organization of the care and expression and resolving emotional and social crises may cause extra stress. The communication becomes more complex when the language of the care providers diff ers from the language of the patient and family. Intercultural communication can be improved by specifi c actions such as ethnic matching, using an interpreter and the formation of cultural-competent healthcare professionals. Introduction ICU visits are generally limited to 2 hours per day selected at any time during the 24 hours. At our institution we abide to the international regulations which were recently modifi ed allowing family members and signifi cant others to be present throughout the day accompanying their ICU-admitted relatives, participating in an uninterrupted timely fashion and no time limits with their daily activities. References Conclusion There is a lack of knowledge regarding the specifi c experiences of patients, families and care providers with communi- cation and especially decision-making in multicultural ICUs in Western Europe. Methods From January 2012 through April 2012, 200 questionnaires were fi lled by families of ICU-admitted patients; each questionnaire includes 13 questions concerning their overall degree of satisfaction at our unit and evaluating the quality. Accommodating spaces were off ered for families of ICU patients, off ering them a state of freedom within the ICU premises surrounding their beloved sick relatives; additionally they were constantly advised by a designated secretary to share their experience and any possible insight to improve the overall polity of our service. Symptoms of anxiety, depression and post-traumatic stress in pairs of patients and their family members during and following ICU stay: who suff ers most? R Fumis1, O Ranzani2, P Martins1, G Schettino1 1Hospital Sírio Libabês, São Paulo, Brazil; 2Hospital das Clínicas, Universidade de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P25 (doi: 10.1186/cc13215) Results Two hundred patients were included in the study, the average age of patients was 68.55 years and the average length of ICU stay was 5.2 days. Most family (85%) desired their presence accompanying their related patients within the ICU premises when meals were served. Those families expressed satisfaction while off ering psychological support to their beloved ill ones. Fifteen per cent of the families argued about the permitted number of visitors (one person) per visit and 12% addressed lack of communication between the medical staff and patient’s families. Introduction To compare the incidence of anxiety, depression and post-traumatic stress symptoms in pairs of patients and respective family members during the ICU stay and at 30 and 90 days post ICU discharge. According to the literature, both patients and family members suff er from psychological distress during and following an ICU stay [1]. Although these issues have been discussed, to date few studies have addressed the pairs at three times. p Conclusion Compared with the limited visit status within the ICU ward in the past, the majority of families expressed general acceptance and satisfaction toward the newly adopted policy. Additionally and by this new policy, patients displayed marked improvement at the psychological level, being more cooperative and experiencing less episodes of agitation. Dealing with cultural diversity during the process of communication and decision-making in the ICU: a literature review Dealing with cultural diversity during the process of communication and decision-making in the ICU: a literature review Dealing with cultural diversity during the process of communication and decision-making in the ICU: a literature review RV Van Keer, RD Deschepper, LH Huyghens, WD Distelmans, JB Bilsen Vrije Universiteit Brussel, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P24 (doi: 10.1186/cc13214) RV Van Keer, RD Deschepper, LH Huyghens, WD Distelmans, JB Bilsen Vrije Universiteit Brussel, Brussels, Belgium RV Van Keer, RD Deschepper, LH Huyghens, WD Distelmans, JB Bilsen Vrije Universiteit Brussel, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P24 (doi: 10.1186/cc13214) i Results TISS-28 scores at 24 and 72 hours of ICU admission were analyzed for 4,128 patients. Mean patient age was 68.3 (SD ± 17.6), 46.8% were female and 37% were surgically ill. The mean APACHE II score was 16 (SD ± 8) and 40% were submitted to mechanical ventilation at any time of the stay. Overall mortality was 14.4%. Neither APACHE II score adjusted for age nor TISS-28 in 24 and 72 hours of admission diff ers among age groups. However, mortality was signifi cantly higher in patients aged 70 years or older (P <0.001). Introduction Studies have shown that communication in the ICU is related to the cultural background of all involved actors. As a consequence, hospitals in multiethnic areas in western countries are currently forced to rethink their models of communication as they are increasingly transformed into spaces of cultural encounter. During this process one needs to be aware of the cultural diff erences in attitudes and experiences towards communication in critical medical situations. However, the growing academic interest in intercultural relations in the critical care has not yet led to the construction of cumulative knowledge. The aim of this study is to review the experiences of the involved actors, namely the care providers, the patients and their family members, with cultural diversity during the process of communication and decision-making in the ICU. Conclusion Mortality remained higher in older patients despite an absence of age-related diff erences in resource use at ICU. Comparing our results to a previous prospective cohort study [2], we emphasize the lower overall mortality of our population (14.4% vs. 50%). Limitations of our analysis include our unicenter design and lack of data for a closed model ICU. P24 P24 Methods During 7 years (2007 to 2013), TISS-28 was prospectively applied to all consecutive adult critically ill patients at 24 and 72 hours of ICU admission in a private hospital in Brazil. Demographic data, diagnoses on admission, comorbidities, ICU length of stay and mortality were recorded. Patients were stratifi ed according to their ages. 2. Bisaillon S, Li-james S : Family partnership in care: integrating families into the coronary intensive care unit. Can J Cardiovasc Nurs 1997, 8:43-46. 1. Derwick D, Kotagal M: Restricted visiting hours in ICU. J Am Med Assoc 2004, 292:736-737. P22 Do we spend less on older critically ill patients? Relationship among intensity of care, severity of illness and mortality MD Rosa, AC Pecanha Antonio, M Mattioni, L Tagliari, F Schaich, J Maccari, R Oliveira, C Teixeira, TF Tonietto, N Brandão da Silva Hospital Moinhos de Vento, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P22 (doi: 10.1186/cc13212) Methods A prospective court study was conducted (from January to May 2013) to point out the patient’s care divergences (blood transfusion, diuretics, antibiotics, vasopressors, mechanical ventilation, and so forth) that happened in the fi rst 72 hours of ICU admission in a 30-bed adult Brazilian ICU. We enrolled only patients that stayed more than 48 hours in the ICU. Introduction The Therapeutic Intervention Scoring System (TISS-28) quantifi es the type and number of intensive care treatments; therefore, it indicates the workload of ICU and may be used for calculating costs [1]. A previous cohort study [2] demonstrated that seriously ill older patients receive fewer invasive procedures and less resource-intensive hospital care, showing a preferential allocation of hospital services to younger patients regardless of severity of illness. The objective of this study is also to compare resource utilization across diff erent ages of critically ill patients in an open model, mixed ICU. Results In a court of 357 patients at least one divergence between the ICU medical staff and the primary(s) physician(s) were identifi ed in 31 cases (8.6% – divergence group (DG)). The age (67.9 years), gender (55.2% of male), SAPS3 score (45.7) and reasons for ICU admission (emergency surgery 7.6%, nonemergency surgery 30%, clinical 62.4%) were similar in DG and nondivergence (NDG); however, the ICU length of stay (6.2 vs. 3.9 days, P = 0.023), use of mechanical ventilation (48.4% vs. 27%, P = 0.012), vasopressors (77.4% vs. 46%, P = 0.001) and blood transfusion (41.9% vs. 27.6%, P = 0.073) were higher in the DG S9 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P27 was 51.79 ± 13.47 and 64% were spouses. Patients had symptoms of anxiety (26.1%), depression (12%) or both (8.7%) during the ICU stay. Regarding family members, the incidence of anxiety, depression or both during the ICU was 33.2%, 9.8% and 8.2% respectively. Symptoms of PTSD within patients were found in 6.9% at 30 days and disappeared at 90 days. Diff erently, these symptoms in family members were found in 6.2% at 30 days and 9.0% at 90 days. There was a positive correlation between HADS at ICU and IES at 30 days for family members (r = 0.527, P <0.001). The HADS score over time between patients, family members of patients who deceased and who survived were diff erent (P = 0.002). In post-hoc analysis, group A presented less symptoms than group C at 30 days (P = 0.001) and less than groups B and C at 90 days (P <0.001 for both). At 90 days, group B showed less symptoms than group C (P = 0.039).f P27 Family satisfaction in the ICU: a 6-month experience M Zouka1, A Myrou2, I Soultati2, T Aslanidis2, A Euthymiou2, E Geka2, E Anastasiou2, V Ourailoglou2, M Giannakou2 1Papageorgiou General Hospital, Thessaloniki, Greece; 2AHEPA University Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P27 (doi: 10.1186/cc13217) Introduction The aim of the study was to prospectively assess family satisfaction in the intensive care setting and identify fi elds of possible improvement. Introduction The aim of the study was to prospectively assess family satisfaction in the intensive care setting and identify fi elds of possible improvement. Methods The study was performed over a 6-month period in a 10-bed ICU. A properly translated and validated Family Satisfaction in the ICU (FS-ICU 24) questionnaire was used. A total of 126 questionnaires were handed out to family members upon patient discharge. The number of questionnaires returned for evaluation was 120 (91.5% participation). Five-point Likert scale responses were transformed to percentage scores, higher values representing better satisfaction. Conclusion Family members of ICU patients suff er more than the patients principally when their loved one died. Patients’ symptoms of anxiety, depression and PTSD decrease with time but in family members these symptoms continue along the 90 days. R f Results Overall satisfaction with care was reported as very good or excellent by 81% of family members (32% and 49% respectively) both for ICU staff concern and caring as well as symptom management. Heart-focused anxiety in critically ill patients’ relatives Z Konstanti, M Gouva, G Nakos, V Koulouras University of Ioannina, Greece Critical Care 2014, 18(Suppl 1):P26 (doi: 10.1186/cc13216) Introduction A series of studies have shown an association between the ICU environment and symptoms of psychological distress in critically ill patients’ relatives [1-3]. The aim of this study was to investigate the risk of cardiophobia and panic attack crisis on ICU patients’ family members. Methods In the period from March 2012 to July 2013, we studied 223 family members (81 men and 142 women, mean age 41.5 ± 11.0 years) of critically ill patients. These patients were admitted to the intensive care department with various medical and surgical conditions (147 patients, 103 men and 44 women, mean age 58.3 ± 18.6 years). We used a questionnaire in which were included: social–demographic characteristics of the patients’ family environment, and the Cardiac Anxiety Questionnaire (CAQ). The results from the CAQ [4] were compared with those of the general population of Greece (GGP). Conclusion Most family members were highly satisfi ed with ICU care their patients received. Nevertheless, our study showed that the fi elds of communication with the nurses as well as the waiting room atmosphere defi nitely need further improvement. Concerning emotional support and decision-making, the study revealed that currently used measures in our ICU still do not apply to all families and probably require a few changes. P26 Heart-focused anxiety in critically ill patients’ relatives Z Konstanti, M Gouva, G Nakos, V Koulouras University of Ioannina, Greece Critical Care 2014, 18(Suppl 1):P26 (doi: 10.1186/cc13216) Qualitative analysis of a family satisfaction in an adult ICU C Ebm1, D Dawson2, M Cecconi2, A Rhodes2 1Wiener Privatklinik, Vienna, Austria; 2St George’s NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P28 (doi: 10.1186/cc13218) 1Wiener Privatklinik, Vienna, Austria; 2St George’s NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P28 (doi: 10.1186/cc13218) Introduction Patient satisfaction is a key determinant of the quality of care within the hospital environment. Critically ill patients often lack capacity. Under such circumstances, family members are often main decision-makers and their satisfaction about the ICU experience resembles an important surrogate in evaluating the quality of care. We aimed to measure family satisfaction with diff erent aspects of medical care. Methods In February 2013, surveys, including 24 qualitative questions, were sent out to relatives of 50 patients 1 month after their discharge from ICU St George’s, UK. Responses were graded 1 to 5, with higher values representing a greater degree of satisfaction.i Conclusion The hospitalization of a patient in the ICU is considered to be a factor that is aff ecting the mental health of patients’ relatives. Our results highlight a risk for psychologically induced symptoms on ICU relatives and underline the need for a psychosocial support system for them. The cardiophobia and self-monitoring of heart activity which infl icts the patients’ relatives appears to be more intense in the siblings, parents and partners of patients. Results Most respondents were satisfi ed with the overall performance and the respect received (mean ± SD score 4.7 ± 0.68). Skills and competency of doctors (4.38 ± 0.67) and nurses (4.57 ± 0.68) was also perceived very positive. However, family satisfaction with communication with doctors (3.52 ± 1.47) and nurses (3.71 ± 0.96), as well as inclusion in decision-making (3.39 ± 1.24), resulted in somewhat lower scores. In particular, ease, clarity, consistency, honesty, and completeness of information given by doctors resulted in inhomogeneous perceptions between relatives (Figure 1). Additionally, the majority of responders felt only moderately satisfi ed with time and support to make a decision. P28 Qualitative analysis of a family satisfaction in an adult ICU C Ebm1, D Dawson2, M Cecconi2, A Rhodes2 1Wiener Privatklinik, Vienna, Austria; 2St George’s NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P28 (doi: 10.1186/cc13218) p y q References y Referencesi 1. Wall RJ et al.: Refi nement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit Care Med 2007, 35:271-279. 2. Heyland DK et al.: Family satisfaction with care in the intensive care unit: results of a multiple center study. Crit Care Med 2002, 30:1413-1418. p g p p Results In comparison with the general Greek population, the relatives of critically ill patients demonstrate statistically signifi cant higher scores (P <0.001) on the scales of fear and anxiety in regards of thoracic and heart sensations (1.1 ± 0.9 vs. 0.9 ± 0.9 in GGP), avoidance of activities considered to reproduce cardiac symptoms (1.3 ± 1.0 vs. 1.1 ± 0.9 in GGP), heart-focused attention and self-monitoring of cardiac activity (0.9 ± 0.7 vs. 0.7 ± 0.6 in GGP), and at total CAQ score (1.1 ± 0.6 vs. 0.9 ± 0.6 in GGP). Analysis of variance among family members on heart- focused attention and self-monitoring of cardiac activity showed that hospitalization provoked heart-focused attention (that is, the main factor of perceived panic crisis risk). In accordance with the Bonferroni criterion, we found that patients’ siblings demonstrated statistically signifi cant diff erence compared with patients’ children (P = 0.015), with the latter showing lower levels of heart-focused attention and self- monitoring of cardiac activity. Further MANCOVA analysis showed a statistically signifi cant correlation between age and cardiophobia, and also between education level and heart-focused anxiety (P = 0.041 and P = 0.044 respectively). References Even the medical team elaborated their interest towards this policy as being a more profound and comprehensive way of supporting the ICU patients. Further investigations are currently being employed to strengthen the outcome of the study. References Methods A prospective study conducted in a 22-bed adult general ICU including patients staying >48 hours. The Hospital Anxiety and Depression Scale (HADS) was completed by pairs (patients/respective family members). They were interviewed by telephone at 30 and 90 days after ICU discharge with the Impact of Event Scale (IES) and the HADS. We separated them into three diff erent groups (patients – group A, family member of patient who survived – group B and family member of patient who deceased – group C). 1. Derwick D, Kotagal M: Restricted visiting hours in ICU. J Am Med Assoc 2004, 292:736-737. p g p Results A total of 184 pairs were interviewed at the ICU. Mean patient age was 59.33 ± 15.5 years. The admission SAPS III was 47.6 ± 15.7 points. Median ICU LOS was 4 days (2 to 47). Family’s age 2. Bisaillon S, Li-james S : Family partnership in care: integrating families into the coronary intensive care unit. Can J Cardiovasc Nurs 1997, 8:43-46. 2. Bisaillon S, Li-james S : Family partnership in care: integrating families into the coronary intensive care unit. Can J Cardiovasc Nurs 1997, 8:43-46. S10 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P27 Favorable scores were also noted in terms of decision-making, 89% of relatives reporting very good and excellent rates as regards information needs. On the other hand, most of the family members (73%) felt neither included nor excluded from the decision-making process. Moreover, a small number of them (6%) answered that they received poor emotional support. Finally, the frequency of communication with ICU nurses and the atmosphere in the waiting room were noted as fair by 28% and 47% of subjects respectively.i Reference 1. Myhren H, et al.: Patients’ memory and psychological distress after ICU stay compared with expectations of the relatives. Intensive Care Med 2009, 35:2078-2086. 1. Myhren H, et al.: Patients’ memory and psychological distress after ICU stay compared with expectations of the relatives. Intensive Care Med 2009, 35:2078-2086. References 1. Azoulay E, et al.: Intensive Care Med 2003, 29:1498-1504. 2. Garrouste-Orgeas M, et al.: Crit Care Med 2008, 36:30-35. 3. Stayt CL: J Adv Nursing 2007, 57:623-630. 4. Dragioti E, et al.: Psychol Rep 2011, 109:77-92. P30 Advance care planning in critically ill haematology patients V Metaxa1, J Lambert2, A Barrow3, J De Vos4 1King’s College Hospital, London, UK; 2Royal London Hospital, London, UK; 3John Radcliff e Hospital, Oxford, UK; 4Royal Surrey County Hospital, Guildford, UK Critical Care 2014, 18(Suppl 1):P30 (doi: 10.1186/cc13220) Introduction Signifi cant improvements in chemotherapy, haemato- poietic stem cell transplantation and general intensive care mean that more patients are now living longer and often present to the ICU at various stages of their disease [1,2]. Encouraging patients with haematological malignancy (HM) to express their wishes and values on end of life (EoL) prior to ICU admission would ensure that their autonomy is respected. The aim of our study was to determine whether patients with HM that were admitted and died in the ICU had any form of advance care planning (ACP) documented prior to their admission. Methods Data were collected on all adult patients with HM that were admitted and died in the ICU of a tertiary haematology referral centre in London during the period of 1 year. g p y Results Information was collected for 34 patients and their charac- teristics are shown in Table 1. In 62% of the patients EoL decisions were made, and do-not-attempt-resuscitation documentation was found in 65% of the cases. Documentation on information exchange, and participation in the deliberation or the decision-making process was found in only 30% of the patients, even though more than 75% of the haematology physicians estimated the prognosis of these patients as moderate (17%) or poor (59%). In the vast majority of cases (31/34) the deaths occurred after withholding or withdrawal of treatment in ICU. Conclusion In general, relatives felt very satisfi ed with the ICU, especially with the care of the patients and the professional workforce. The complete decision-making process was rated moderately good, which highlights some areas of improvement in involving relatives in the care of their beloved by providing regular, clear, easy to understand and consistent information. Conclusion In general, relatives felt very satisfi ed with the ICU, especially with the care of the patients and the professional workforce. The complete decision-making process was rated moderately good, which highlights some areas of improvement in involving relatives in the care of their beloved by providing regular, clear, easy to understand and consistent information. Table 1 (abstract P30). P29 Outcomes of ventilated surgical and medical ICU patients: do patients die from ARDS or with ARDS? Conclusion Only a small number of patients with HM that die in the ICU have documented ACP prior to admission. Since the majority of ICU patients lack personal decision-making capacity, ACP would ensure that care is consistent with patients’ wishes, EoL actions are congruent with their values, the burden on family and healthcare providers is alleviated and cost is decreased. L Fuchs1, D Talmor2 1Soroka University Medical Center, Beer Sheba, Israel; 2Beth Israel Deaconess Medical Center, Boston, USA Critical Care 2014, 18(Suppl 1):P29 (doi: 10.1186/cc13219) Introduction Patients with acute respiratory distress syndrome (ARDS) have a high mortality rate. Whether this excess mortality is contributed by ARDS or is a consequence of prolonged mechanical ventilation is unclear. References 1. Azoulay E, et al.: Intensive Care Med 2003, 29:1498-1504. 2. Garrouste-Orgeas M, et al.: Crit Care Med 2008, 36:30-35. 3. Stayt CL: J Adv Nursing 2007, 57:623-630. 4. Dragioti E, et al.: Psychol Rep 2011, 109:77-92. S11 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion In general, relatives felt very satisfi ed with the ICU, especially with the care of the patients and the professional workforce. The complete decision-making process was rated moderately good, which highlights some areas of improvement in involving relatives in the care of their beloved by providing regular, clear, easy to understand and consistent information. Figure 1 (abstract P28). Team performance. Figure 1 (abstract P28). Team performance. References 1. Bird GT, et al.: Br J Anaesth 2012, 108:452-459. 2. Howell DA, et al.: Palliat Med 2011, 25:630-641. Methods A longitudinal retrospective study focusing on noncardiac ICU patients who required mechanical ventilation. Patients were classifi ed as having ARDS on admission, late-onset ARDS or no ARDS. The analysis included patients ventilated for longer than 48 hours. Primary outcomes were mortality at 28 days, 1 year and 2 years from ICU admission. P30 Patient characteristics Mean age 30 Median APACHE II score 26 >3 organ support 60% Leukaemias >50% Table 1 (abstract P30). Patient characteristics Death rate of patients admitted to a Brazilian ICU on weekends and holidays y Methods We analyzed the records of 11,230 ICU admissions. ASA and elective/nonelective nature of the admission were combined into eight categories. We used the chi-square test and accepted the null hypothesis if P >0.05. Areas under the ROC curve using ASA and ASA/ admission categories were constructed. y P Batista, R Passos, R Oliveira, M Ribeiro, F Alves H i l S R f l S l d B hi B il P Batista, R Passos, R Oliveira, M Ribeiro, F Alves Hospital Sao Rafael, Salvador Bahia, Brazil Critical Care 2014, 18(Suppl 1):P34 (doi: 10.1186/cc13224) p Critical Care 2014, 18(Suppl 1):P34 (doi: 10.1186/cc1322 g Results We included 5,998 patients admitted to the ICU immediately after surgery. Forty-one percent were older than 65 years and 51% were female. Length of stay previous to ICU admission was 2.5 ± 6.1 days and duration of ICU stay was 1.9 ± 2.4 days. Elective hospital admission occurred in 65% of the patients. Neurosurgery with 21% of the procedures was followed by abdominal surgery (20%), and orthopedic surgery (15%). The death rate at the hospital was 6.9% (2.8% in ASA I patients and 31% in ASA IV/E). Higher ASA classifi cation was signifi cantly associated with the death rate both in the ICU and at the hospital (P <0.00001). The nonelective nature of the hospital admission was also associated with higher risk of death at the ICU (P <0.0001) and in the hospital (P <0.0001). Gender and hour of admission was not associated with the death rate. The combination of ASA classifi cation with the nonelective nature of the hospital admission produced an eight- category index signifi cantly associated with mortality (P <0.00001). Analyzing hospital mortality, the area under the ROC curve was 0.77 (95% CI 0.74 to 0.79) for this index and was 0.72 (95% CI 0.69 to 0.75) for ASA. When analyzed for death at the ICU the AUROC was 0.71 (95% CI 0.69 to 0.75, P <0.0001) for ASA and 0.75 (95% CI 0.71 to 0.78, P <0.0001) for ASA nonelective admission index. Introduction Several studies conducted in a number of diff erent populations indicate that patients admitted to hospitals on weekends have a higher mortality rate. Many factors have been proposed to explain these observations and in most studies, however, this eff ect disappeared after controlling for illness severity. P33 Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P33 (doi: 10.1186/cc13223) P33 Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P33 (doi: 10.1186/cc13223) qi y p g Results A total of 220 nurses and 55 physicians participated. Scales showed good reliability with all Cronbach’s alpha ≥0.8 and signifi cant between unit variability (all P ≤0.011). On a scale from 0 (‘never’) to 5 (‘very often’), ICU staff rated frequency of futile care as median 3.6 (IQR: 3 to 4). On a seven-point Likert scale with 7 denoting maximal values, intention to quit was rated as 2.3 (1 to 4). Nurses gave signifi cantly lower ratings of job satisfaction than physicians (4 (3 to 5) vs. 5 (4 to 6), P ≤0.001), perceived futility more often than physicians (P ≤0.001), and rated all aspects of nurse–physician collaboration worse than physicians, including attendings’ inclusive leadership, collaboration, decision-making and psychological safety (all P ≤0.001). Futility and intention to quit were each predicted by high workload and low psychological safety (all P ≤0.009). Among nurses, inclusive leadership by the head nurse prevented intention to quit (P = 0.001) and a composite score of good nurse–physician interactions predicted less perception of futile care (P ≤0.001). Introduction The aim of the ICU is to give the best life care to the admitted patients in order to preserve and restore the patients’ quality of life. In some critical patients, life-supporting therapies become actions to support the end stage of their life [1]. This is the paradox of the ICU. Patients aff ected by amyotrophic lateral sclerosis (ALS) belong to this category: for these patients, all medical actions do not improve the quality of life but just prolong it. ALS or Charcot’s disease is the most common motor neuron disease which causes severe motor disability and evolves in a few years to death [2]. We want to present our experience in patients aff ected by ALS in the ICU.f p pf y Methods We collected data about patients aff ected by ALS admitted to the ICU from 1 January 2010 to 31 October 2013. We considered entry diagnosis, mean age, need to perform tracheostomy and/or percutaneous endoscopic gastrostomy (PEG), and mortality in the ICU. P33 decision-making, psychological safety, workload, intention to quit and a newly developed scale for perceived futile care. English questions were translated into German by state-of-the art forward and backward translations. Reliability – that is, internal consistency and inter-rater reliability of scales – was evaluated. Predictors of perceived futile care and intention to quit were identifi ed by multiple regressions. P33 Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P33 (doi: 10.1186/cc13223) P31 A new questionnaire to determine the eff ect of team interaction in the ICU on perceived futility and intention to quit: results of a pilot study in two German hospitals Results A total of 1,396 patients were enrolled between 2001 and 2008: 485 (34.7%) had ARDS on admission (early-onset ARDS), 219 (15.6%) developed ARDS during their ICU stay (late-onset ARDS) and 692 (49.5%) did not meet ARDS criteria prior to ICU discharge or death. Twenty-eight-day mortality rates were 23.7%, 25.6% and 25.7% for early, late and non-ARDS respectively. After adjusting for age, weight on admission, unit of admission (MICU vs. SICU), severity of disease score, comorbidity score, and primary diagnosis on admission, and mortality risk at 28 days, 1 year and 2 years were not signifi cantly diff erent between the three study groups. Neither severity of ARDS or timing of ARDS (early versus late) was associated with mortality. Sensitivity analysis, analyzing all ventilated patients, including those who were ventilated less than 48 hours, showed the same results. y p D Schwarzkopf1, F Bloos1, A Meier-Hellmann2, C Icke2, N Riedemann1, CS Hartog1 g 1Jena University Hospital, Jena, Germany; 2Helios Hospital Erfurt, Germany Critical Care 2014, 18(Suppl 1):P31 (doi: 10.1186/cc13221) 1Jena University Hospital, Jena, Germany; 2Helios Hospital Erfurt, Germany Critical Care 2014, 18(Suppl 1):P31 (doi: 10.1186/cc13221) Introduction Perceived futility of care may jeopardize patient quality of care and increase ICU staff turnover. It is related to workload and interdisciplinary collaboration. Our aim was to evaluate concepts from team science, namely inclusive leadership (a style that invites and appreciates others’ contributions) and psychological safety (a key antecedent of speaking up and learning behavior), and to determine their eff ect on perceived futility and intention to quit.f Conclusion Neither the presence of ARDS or the severity or timing contribute independently to the short and long mortality risk after adjustment for age, severity of disease, comorbidity score and diagnosis on presentation in patients hospitalized in a noncardiac ICU with acute respiratory failure. f Methods A staff survey in four interdisciplinary ICUs and two intermediate care units of two tertiary care hospitals. The questionnaire contained validated scales to assess inclusive leadership of head nurses and attending physicians, nurse–physician collaboration, collaborative S12 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P33 Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P33 (doi: 10.1186/cc13223) Results Sixty patients were admitted for neuromuscular respiratory failure complicated by aspiration pneumonia. Mean age was 43 years. The male to female ratio was 3:1. All patients underwent percutaneous tracheostomy and PEG. One of them died in the ICU because of septic shock. In every case we honestly communicated the worsening of the disease and we perceived the awareness of it by the patients and/or their family. In no case did patients ask us to withdraw the necessary cures such as percutaneous tracheostomy and PEG. In no case did they ask us to die. In Europe the ‘end of life care’ law is very dyshomogeneous because of diff erent cultures, traditions, religions, and beliefs. In Italy euthanasia is not allowed by law. It is the physician often by himself that has to make the fi nal decision according to the patient’s mind.f Conclusion The scales of the new questionnaire showed good reliability and diff erentiated between nurses and physicians. Psychological safety and inclusive leadership proved to be important concepts. P32 ASA helps prediction of the death rate in surgical ICU patients P Batista, R Passos, C Gomes, A Oliveira Hospital Sao Rafael, Salvador Bahia, Brazil Critical Care 2014, 18(Suppl 1):P32 (doi: 10.1186/cc13222) i g p Conclusion Our experience shows that patients aff ected by severe motor disability are not always willing to die. It is essential that decisions should be patient-centered and taken multidisciplinarily, based on open and emphatic communication, involving family and caregivers. References Introduction Many scoring systems have been designed to predict mortality in surgical patients; however, these systems require the collection of several parameters that may not be available at ICU admission. As a result, these classifi cation systems are not used as a routine part of clinical practice. The aim of this study was to evaluate the prognostic value of ASA classifi cation predicting ICU and hospital mortality. 1. Servillo G, Vargas M: Transl Med UniSa 2011, 1:237-242 2. Gordon PH: Aging Dis 2013, 4:295-310. 1. Servillo G, Vargas M: Transl Med UniSa 2011, 1:237-242 2. Gordon PH: Aging Dis 2013, 4:295-310. Autopsy-detected diagnostic errors in critically ill patients with cirrhosis N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) Table 1 (abstract P35). Cluster 1 Cluster 2 Cluster 3 Cluster 4 (n = 353) (n = 755) (n = 667) (n = 272) ICU LOS, mean ± SD (days) 0.5 ± 0.2 2.0 ± 0.8 7.2 ± 2.8 25.6 ± 15.2 Mechanical ventilation 282 (79.9%) 631 (83.6%) 604 (90.6%) 269 (98.9%) Tracheostomy 3 (0.9%) 13 (1.7%) 14 (2.1%) 127 (46.7%) CPR 20 (5.7%) 19 (2.5%) 16 (2.4%) 8 (2.9%) Conclusion Cluster analysis can identify unique typologies of death within ICUs. This approach may be a novel method to more specifically Table 1 (abstract P35). Cluster 1 Cluster 2 Cluster 3 Cluster 4 (n = 353) (n = 755) (n = 667) (n = 272) ICU LOS, mean ± SD (days) 0.5 ± 0.2 2.0 ± 0.8 7.2 ± 2.8 25.6 ± 15.2 Mechanical ventilation 282 (79.9%) 631 (83.6%) 604 (90.6%) 269 (98.9%) Tracheostomy 3 (0.9%) 13 (1.7%) 14 (2.1%) 127 (46.7%) CPR 20 (5.7%) 19 (2.5%) 16 (2.4%) 8 (2.9%) Conclusion Cluster analysis can identify unique typologies of death within ICUs. This approach may be a novel method to more specifi cally target ICU eff orts to reduce mortality and improve the quality of death. P36 Independent risk factors associated with the decision to withhold therapeutic intervention in patients admitted to the emergency room L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P36 (doi: 10.1186/cc13226) Introduction The decision to withhold therapeutic intervention in a Introduction Even with the availability of new and more eff ective diagnostic procedures over the past decades, autopsy remains one of the most reliable methods to validate clinical diagnoses. The aim of this review was to determine whether autopsy plays a role in extending knowledge about the cause of death in patients with cirrhosis who died in the ICU. Methods We conducted a retrospective review of medical records and postmortem fi ndings in critically ill patients with cirrhosis who were admitted to a major university teaching medical ICU (MICU) between August 2007 and August 2013. Agreement between diagnoses before death and postmortem fi ndings were compared using the Goldman system [1]. Review was independently performed by a fellow in critical care medicine and a board-certifi ed attending. Autopsy diagnoses included histologic and microbiological fi ndings. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 role of the physician is to cure, treat and alleviate suff ering. When the fi rst two goals are not possible the medical role should be dedicated to end-of-life treatments adjusted to the patient’s benefi t [1,2]. patients admitted on weekends and on weekdays were found in the group admitted after surgery (respectively 10% and 30%, P <0.0001), originating from the emergency unit (respectively 30% and 16%; P <0.0001), originating from step-down units (respectively 31% and 17%; P <0.0001), patients from clinical teams compared with surgical teams (respectively 28% and 13%; P <0.0001), previous hospital length of stay lower versus higher than 2 days (respectively 15% and 25%; P <0.0001) and age ≥65 years (respectively 21% and 18%; P <0.0001). The mortality ratios were signifi cantly diff erent between these groups. Multiple logistic regression showed that the inclusion of other variables reduces the odds ratio associated with admission on weekends and holidays to 1.22 (95% CI 1.08 to 1.39, P <0.002) for ICU mortality and to 1.23 (95% CI 1.09 to 1.38, P <0.001). i Methods A retrospective cohort study including all adult patients with sepsis admitted to the emergency room (ER) at a tertiary care, university hospital between 1 July 2011 and 30 June 2012. u e s ty osp ta bet ee Ju y 0 a d 30 Ju e 0 . Results During the study period 162 patients with sepsis were admitted to the ER, of which 40 (25%) had withheld therapeutic decisions. Comparing this group with the group without therapeutic limitations, patients in the fi rst group were older (81 ± 13 vs. 68 ± 14, P <0.001), with more comorbidities (90% vs. 66%, P = 0.004) and a higher proportion needing help in daily activities (Karnofsky performance status (KPS) <70% = 55% vs. 8%, P <0.001). The hospital mortality in patients with a decision to limit the therapeutic intervention was signifi cantly higher (83% vs. 43%, P <0.001). Variables independently associated with the withholding therapy decision were age (adjusted OR per year = 1.078, P <0.001), presence of comorbidities (adjusted OR = 4.632, P = 0.030), chronic wounds (adjusted OR = 5.965, P = 0.005) and patient’s needed of help in daily activities (KPS <70%, adjusted OR = 5.391, P = 0.012). In the fi rst group a lower proportion received antibiotics (70% vs. References 1. Damghi et al.: BMC Emergency Med 2011, 11:12. 2. Conte et al.: Intensive Care Med 2010, 36:765-772. 1. Damghi et al.: BMC Emergency Med 2011, 11:12. 2. Conte et al.: Intensive Care Med 2010, 36:765-772. y Results Four clusters were identifi ed. Short ICU LOS and low life- sustaining therapy utilization but relatively frequent CPR characterized Cluster 1. Intermediate ICU LOS and moderate life-sustaining therapy utilization characterized Clusters 2 and 3. Prolonged ICU LOS and high life-sustaining therapy utilization (except CPR) characterized Cluster 4. Age and severity of illness decreased across clusters with the oldest, sickest patients in Cluster 1 and the youngest, least sick patients in Cluster 4. See Table 1. Death rate of patients admitted to a Brazilian ICU on weekends and holidays We undertook the present study to explore the eff ect that day of ICU admission may have on death rate. Methods We analyzed 11,230 electronic records from patients admitted from 1 January 2006 to 30 June 2013. The ICU admission dates were categorized as normal weekdays and as weekends and holidays. The dependent variables were ICU mortality and hospital mortality. The chi- square test and Student t test were used as appropriate and signifi cant association was accepted when P <0.05. Multiple logistic regression (backward conditional) was used accepting a variable when P <0.05 and rejecting a variable with P >0.1. SPSS version 19.0 was used. Methods We analyzed 11,230 electronic records from patients admitted from 1 January 2006 to 30 June 2013. The ICU admission dates were categorized as normal weekdays and as weekends and holidays. The dependent variables were ICU mortality and hospital mortality. The chi- square test and Student t test were used as appropriate and signifi cant association was accepted when P <0.05. Multiple logistic regression (backward conditional) was used accepting a variable when P <0.05 and rejecting a variable with P >0.1. SPSS version 19.0 was used. Results Forty-nine percent of these patients were female, 53% was admitted immediately after surgery, 56% had <2 days of previous hospital admission, and 19.2% were admitted during weekends and holidays. Mortality in the ICU for admissions on weekends and holidays was 24% and was 13% for those admitted on weekdays (OR = 2.23, 95% CI 2.02 to 2.47; P <0.0001). The hospital mortality was respectively 35% and 20% (OR = 2.23, 95% CI 2.02 to 2.47; P <0.0001). Diff erences between Conclusion These results show that the ASA index can be used, preferably in combination with other data from electronic records, to predict mortality for surgical ICU patients. S13 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 How many ways are there to die? Identifi cation of ICU death typologies using cluster analysis L Reineck, A Barnato, R Arnold, D Angus, J Kahn University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P35 (doi: 10.1186/cc13225) How many ways are there to die? Identifi cation of ICU death typologies using cluster analysis L Reineck, A Barnato, R Arnold, D Angus, J Kahn University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P35 (doi: 10.1186/cc13225) Introduction Although avoidance of death is a key goal of critical care, so too is provision of high-quality end-of-life care when life-prolonging therapy is not desired. One often-used measure of ICU performance is risk-adjusted mortality, yet this measure treats all deaths equally and thus is fl awed. In this work, we identify diff erent death typologies using cluster analysis, with the ultimate goal of more narrowly targeted ICU quality improvement measures and eff orts. Conclusion In this study the decision to withhold therapy was independently associated with increasing age, the presence of comorbidities and loss of functional autonomy. For the same level of intervention such as antibiotic administration, the decision to withhold therapy did not infl uence the effi cacy of therapeutic attitudes. References Methods We performed k-means cluster analysis in 2,047 ICU decedents admitted to a university medical center in 2011/12. Variables for the analysis included ICU length of stay (LOS), mechanical ventilation, tracheostomy, gastrostomy tube insertion, dialysis, enteral or parenteral nutrition, and cardiopulmonary resuscitation (CPR). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 99%, P <0.001) and when those were considered inadequate for the agent responsible for the sepsis episode it was less frequently changed (15% vs. 50%, P = 0.028). However, no diff erences were found regarding the elapsed time from admission to the ER until the fi rst medical contact or the time since the recognition of sepsis and antibiotic administration, although the group with withholding decisions had less specimens collected for microbiology: blood cultures (68% vs. 91%, P <0.001) or other specimens (58% vs. 96%, P <0.001). Conclusion The higher death rate on weekends and holidays may be related to case mix. The interplay of other variables possibly related either to admission on weekends or higher death rate should be sought. Autopsy-detected diagnostic errors in critically ill patients with cirrhosis N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) The records were also reviewed for demographics, APACHE II score and all performed diagnostic procedures. P36 Independent risk factors associated with the decision to withhold therapeutic intervention in patients admitted to the emergency room L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P36 (doi: 10.1186/cc13226) Results Of 641 patients admitted with diagnosis of cirrhosis, 86 (13%) died in the MICU. Forty-fi ve (52%) patients underwent an autopsy. Forty-two autopsy reports were available for review, three histologic and microbiological reports were missing. Major missed diagnoses (principal underlying disease related to death and primary cause of death itself) were present in seven patients (17%), four in class I (10%) and three in class II (7%) (Table 1). Minor missed diagnoses were present in 13 patients (31%) (class III and IV). Postmortem fi ndings were in complete agreement (class V) with clinical cause of death in almost one-half of the patients (n = 19, 45%). P36 Independent risk factors associated with the decision to withhold therapeutic intervention in patients admitted to the emergency room Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. Conclusion In KSA, abdomino-pelvic traumas are serious injuries aff ecting mainly young male victims; however, with a lower mortality than predicted. P39 Radiation exposure in trauma patients is aff ected by age M Wagner1, JV Vonk2, CW Wichman1, AH Hegde3, JO Oliveto3 1Creighton University, Omaha, NE, USA; 2University of Iowa, Iowa City, IA, USA; 3UNMC, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P39 (doi: 10.1186/cc13229) Critical Care 2014, 18(Suppl 1):P39 (doi: 10.1186/cc13229) Introduction Trauma patients are subjected to higher radiation exposure (RE) as a function of Injury Severity Score (ISS) [1]. Analysis of this dataset was done to ascertain if RE varied with patient age. Class I = missed major diagnosis that would have changed management and might have resulted in cure or prolonged survival. Class II = missed major diagnosis that would not have modifi ed ongoing patient care. p g Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). RE as a function of the ISS and age was explored for 7,661 trauma patients. The ISS groupings were as follows: ISS 1 to 8, ISS 9 to 15, ISS 16 to 25, and ISS >25. Age groupings were 19 to 39, 40 to 59, 60 to 79, and >80 years. Conclusion Despite declining rates worldwide, autopsy remains an important tool for quality and safety assurance. In this retrospective study, autopsy showed that knowledge of the correct premortem diagnosis would have altered therapy in 10% of critically ill cirrhotic patients. y Results The data were analyzed in a hierarchical fashion: the mean exposure by age group was compared by one-sided, two-sample t tests. Diff erences were set up to test if mean exposure decreased with increasing age. Bonferroni adjustment was used for all multiple comparisons to maintain the overall error rate at 0.05. P36 Independent risk factors associated with the decision to withhold therapeutic intervention in patients admitted to the emergency room Introduction The decision to withhold therapeutic intervention in a patient is a complex decision, wrapped in profound ethical debates. The S14 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P37). Major missed diagnosis Class I (n = 11) Class II (n = 3) 1. Metastatic linitis plastica 1. Acute pancreatitis 2. Oesophageal variceal bleeding 2. Necrotizing mucor mycosis pneumonia 3. Disseminated mucor mycosis 3. Focal aspergillosis (myocardial) 4,5,6. Invasive aspergillosis (n = 3) 7. Invasive candidiasis 8. Herpes simplex pneumonia 9. Pneumocystis pneumonia 10. Gastric rupture 11. Disseminated HCC, vs. porta thrombosis Class I = missed major diagnosis that would have changed management and might have resulted in cure or prolonged survival. Class II = missed major diagnosis that would not have modifi ed ongoing patient care. Table 1 (abstract P37). Major missed diagnosis Class I (n = 11) Class II (n = 3) 1. Metastatic linitis plastica 1. Acute pancreatitis 2. Oesophageal variceal bleeding 2. Necrotizing mucor mycosis pneumonia 3. Disseminated mucor mycosis 3. Focal aspergillosis (myocardial) 4,5,6. Invasive aspergillosis (n = 3) 7. Invasive candidiasis 8. Herpes simplex pneumonia 9. Pneumocystis pneumonia 10. Gastric rupture 11. Disseminated HCC, vs. porta thrombosis Class I = missed major diagnosis that would have changed management and might have resulted in cure or prolonged survival. Class II = missed major diagnosis that would not have modifi ed ongoing patient care. Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. Conclusion In KSA, abdomino-pelvic traumas are serious injuries aff ecting mainly young male victims; however, with a lower mortality than predicted. Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. Conclusion In KSA, abdomino-pelvic traumas are serious injuries aff ecting mainly young male victims; however, with a lower mortality than predicted. Reference 1. Goldman et al.: The value of autopsy in three medical eras. N Engl J Med 1983, 308:1000-1005. P36 Independent risk factors associated with the decision to withhold therapeutic intervention in patients admitted to the emergency room Of the six independent diff erence tests conducted, fi ve were signifi cant: 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; 40 to 59 versus >79; and 60 to 79 versus >79 (P <0.01). Analysis was done for ISS categories: ISS 1 to 8, ISS 9 to 15, ISS 16 to 25, and ISS >25. For ISS 1 to 8, fi ve diff erences were signifi cant: 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; 40 to 59 versus >79; and 60 to 79 versus >79 (P <0.01). For ISS 9 to 15, all diff erences were signifi cant: 19 to 39 versus 40 to 59; 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; 40 to 59 versus >79; and 60 to 79 versus >79 (P <0.01). For ISS 16 to 25, four diff erences were signifi cant: 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; and 40 to 59 versus >79 (P <0.01). For ISS > 25, none of the diff erences were signifi cant. Reference 1. Wagner M, et al.: 258 Increased Injury Severity Score is associated with increased radiation exposure in trauma patients. Crit Care Med 2013, 41:A59. P38i Profi le, outcomes, and predictors of mortality of abdomino-pelvic trauma patients in a tertiary ICU in Saudi Arabia S Haddad, Z Youssef, S Azzam, A Aldawood, A Al-Zahrani, H Al-Zamel, H Tamim, A Deeb, Y Arabi King Abdulaziz Medical City, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P38 (doi: 10.1186/cc13228) Introduction Saudi Arabia (KSA) has the world’s highest number of deaths from motor vehicle accidents (MVAs). Numerous trauma victims sustain abdomino-pelvic injuries which are associated with considerable morbidity and mortality. The purpose of this study is to describe the profi le, outcomes and predictors of mortality of patients with abdomino-pelvic trauma admitted to the ICU in a tertiary care trauma center in Riyadh, KSA. gi Conclusion Previous studies show signifi cant RE in trauma patients [1]. We demonstrated the signifi cance of RE to trauma patients, that the amount of RE has gone up chronologically over time, and that patients with an increasing ISS have a higher RE. We sought to determine whether age played a factor in RE. Based on our statistical analyses, older patients receive less RE for a given ISS. Although this is a large assessment of RE and trauma patients broken down by ISS and patient age, data analysis by year was limited by the small number of patients in high ISS groups at higher ages. y Methods This is a retrospective analysis of a prospectively collected ICU database. All consecutive patients older than 14 years with abdomino-pelvic trauma from March 1999 to June 2013 were included. The followings were extracted: demographics, injury severity, mechanism and type of injury, associated injuries, use of vasopressors and mechanical ventilation, and worse laboratory results in the fi rst 24 hours. The primary outcome was hospital mortality. Secondary outcomes were ICU mortality, mechanical ventilation duration, need for tracheotomy, and ICU and hospital length of stay. We compared the profi le, outcomes, and the predictors of mortality between survivors and nonsurvivors. Reference Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) p Results We included a total of 469 patients with oncological and hematological malignancies, of whom 8.9% (n = 42) were admitted to the ICU and 18.7% (n = 88) did not survive until the 30-day follow- up. There was a strong association of initial proADM levels and 30-day mortality risk (odds ratio (OR) per 10-fold increase 9.9, 95% CI 4.3, 22.9) with an AUC of 0.67 (95% CI 0.60, 0.74). This association remained signifi cant after multivariate adjustment for initial vital signs (blood pressure, pulse, temperature) and comorbidities (chronic heart failure, chronic obstructive pulmonary disease, diabetes, coronary heart disease) with an adjusted OR of 9.0 (95% CI 3.1, 26.4). There was also a signifi cant association of proADM and LOS (adjusted regression coeffi cient per 10-fold increase: 6.6, 95% CI 2.0, 11.2). Introduction Critical care (CC) admission has traditionally been viewed as likely to result in a poor outcome for immune-compromised haemato-oncological (HO) patients [1,2]. Recent studies have challenged such views [3,4]. We recently reported results from a cohort of HO patients admitted to CC, showing the pAO2/FiO2 (P/F) ratio to be the only independent predictor of mortality [5]. We report analyses of a larger cohort of HO patients admitted to CC. g p Methods We assessed outcome for HO patients at CC (primary outcome) and hospital discharge, and at 6-month and 1-year follow- up. Single variable logistic regression analyses, adjusted by age, gender and haematological diagnosis, and multivariate analyses were performed to identify independent predictors of outcome using STATA. Results A total of 225 HO patients were admitted to CC. Median age was 59 (interquartile range (IQR) 46 to 66) years. The most common haematological diagnoses on admission were acute myeloid leukaemia in 57 (25.3%) cases, non-Hodgkin lymphoma in 54 (24%) cases and multiple myeloma in 42 (18.7%) patients. Median APACHE II score was 21 (IQR 17 to 26). A total of 164 patients (72.9%) had at least one organ supported. Unit and hospital mortality rates were 34.7% (78 patients) and 49.3% (111 patients), respectively. At 6-month and 1-year follow-up, mortality increased to 63.1% (142 patients) and 70.5% (153 patients), respectively. P40 Survival rate and predictors of outcome in intubated patients with haematological malignancies in a Greek ICU V Tsolaki, M Karapetsa, G Ganeli, A Mpouzia, E Zakynthinos University Hospital of Larissa, Greece Critical Care 2014, 18(Suppl 1):P40 (doi: 10.1186/cc13230) Results Of 9,974 trauma patients during the study period, 702 patients with abdomino-pelvic trauma were admitted to the ICU. The average age was 30.7 ± 14.4 years and the majority was male (89.5%). MVA was the most common cause of abdomino-pelvic trauma (86%). Pelvis (46.2%), liver (25.8%), and spleen (23.1%) were the most frequent injuries; and chest (55.6%), head (41.9%), and lower extremities (27.5%) were the most commonly associated injuries. Mechanical ventilation was required in 89.6%, emergency surgery was performed in 45% and vasopressors were used in 46.6% of patients. The most commonly performed interventions during the ICU stay were surgery (39.5%) and chest tube insertion (33.3%). Of the 702 patients with abdomino-pelvic trauma, 115 (16.4%) patients did not survive. Associated head trauma and retroperitoneal hematoma, higher level of lactic acid on admission and ISS, and advanced age were independent risk factors for fatality. Introduction The admission of patients with haematological malignancies to the ICU is associated with high mortality, especially when mechanical ventilation is required [1,2]. The purpose of the present study was to explore survival rates and the predictors of survival in this group of patients. Methods A retrospective study of intubated patients with haemato- logical malignancies, admitted to a general ICU in central Greece, due to any cause. S15 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results During a 10-year period (2003 to 2013), 16 patients with haematological malignancies (nine with acute myelogenous leukaemia, four with non-Hodgkin lymphoma, one with Hodgkin lymphoma and two with multiple myeloma, mean age 50.75 ± 16.59) (male/ female 3/13, mean APACHE II score 23.18 ± 6.67, mean SOFA score 12.50 ± 2.82, CRP 16.00 ± 10.13, WBC 2.055 ± 30.053) were admitted to the ICU. The majority of patients were admitted due to ARDS (PO2/ FiO2 164 ± 109), one patient was admitted due to intestinal rupture and peritonitis and the other one due to intracerebral haemorrhage. P40 In the majority of the patients (13/16) diagnosis of the malignancy was made during the present admission and only three had the malignancy for a longer period (5 months to 3 years). The mean ICU length of stay was 10.56 ± 16.19 days. A total 68.75% (11/16) of the patients were intubated upon admission, whereas the mean time to intubation for the rest of the patients was 6.37 ± 4.16 hours. Neither intubation upon admission nor time to intubation was correlated with survival. Type of haematological malignancy, duration of immunosuppression and preceding length of stay in the general ward did not correlate with survival either. The survival rate was 18.7% and in linear regression analysis, duration of treatment with NIV in the general ward, increased SOFA score and the number of platelets (<50.000) upon admission to the ICU were independent predictors of survival (R2 = 0.77, P = 0.017). mortality, in agreement with previously published data [6]. The CC mortality rate was 34.7%; at 1 year, mortality had risen to 70.5%. Acknowledgement AT and KB are joint fi rst authors. References 1. Ewig: Eur Respir J 1998, 12:116-122. 2. Rubenfeld: Ann Int Med 1996, 125:625-633. 3. Kroschinsky: Intensive Care Med 2002, 28:1294-1300. 4. Cuthbertson: JICS 2008, 9:135-140. 5. Browett: Br J Aanaesth 2013, 110:860-885. 6. Bird: Br J Aanaesth 2012, 108:452-459. 1. Ewig: Eur Respir J 1998, 12:116-122. 2. Rubenfeld: Ann Int Med 1996, 125:625-633. 3. Kroschinsky: Intensive Care Med 2002, 28:1294-1300. 4. Cuthbertson: JICS 2008, 9:135-140. 5. Browett: Br J Aanaesth 2013, 110:860-885. 6. Bird: Br J Aanaesth 2012, 108:452-459. P42 Early risk stratifi cation in patients with oncological and hematological malignancies in the emergency department A Rast, D Steiner, A Kutz, M Bargetzi, B Mueller, P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P42 (doi: 10.1186/cc13232) Introduction There are no well-validated risk scores for patients with oncological and hematological malignancies presenting to the emergency department (ED). Previous research found the prognostic blood biomarker pro-adrenomedullin (proADM) to be associated with infection-related complications and mortality and thus may be helpful in managing febrile patients with malignancies. Yet the prognostic value of proADM in general oncological patients presenting to the ED remains unclear. Herein, the objective of this study is to evaluate the prognostic potential of proADM and clinical parameters in a consecutive cohort of patients with oncological and hematological malignancies with regard to ICU admission and 30-day mortality. P41 Predictors of outcome in patients with haematological malignancies admitted to critical care A Tridente1, K Browett2, J Hall 2, Y Sorour3, J Snowden2, S Webber2 1St Helens and Knowsley, Liverpool, UK; 2STH, Sheffi eld, UK; 3DBH, Doncaster, UK Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) P41 Predictors of outcome in patients with haematological malignancies admitted to critical care A Tridente1, K Browett2, J Hall 2, Y Sorour3, J Snowden2, S Webber2 1St Helens and Knowsley, Liverpool, UK; 2STH, Sheffi eld, UK; 3DBH, Doncaster, UK Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) P40 Conclusion The present retrospective study indicates that patients with haematological malignancies have poor survival when they are admitted to the ICU. Longstanding treatment with NIV before ICU admission, high SOFA scores and low platelet levels upon admission negatively aff ect survival. References 1. Cherif H, et al.: Support Care Cancer 2007, 15:1393-1398. 2. Comet AD, et al.: Eur J Haematol 2005, 74:511-516. 1. Cherif H, et al.: Support Care Cancer 2007, 15:1393-1398. 1. Cherif H, et al.: Support Care Cancer 2007, 15:1393 1398. 2. Comet AD, et al.: Eur J Haematol 2005, 74:511-516. Methods We enrolled all consecutive patients with oncological and hematological malignancies seeking ED care at a tertiary care hospital from February 2013 to October 2013. We prospectively collected various clinical features, and measured blood parameters including proADM upon admission. To assess outcomes, data from electronic medical records – that is, ICU admission, length of stay (LOS), and post- acute care location – were used and we contacted all patients 30 days after hospital admission. Logistic regression models with area under the receiver operating curve (AUC) were used to assess association of baseline parameters and outcomes. Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) The APACHE II score (OR = 0.93, 95% CI = 0.89 to 0.97, P <0.001), number of organs supported (OR = 0.34, 95% CI = 0.23 to 0.48, P <0.001), P/F ratio (OR = 1.06, 95% CI = 1.03 to 1.09, P <0.001), inotropic requirement (OR = 0.27, 95% CI = 0.15 to 0.49, P <0.001), and IMV status (OR = 0.06, 95% CI = 0.03 to 0.14, P <0.001) infl uenced unit survival at single variable analyses. At multivariate analysis, the P/F ratio (OR = 1.05, 95% CI = 1.02 to 1.09, P = 0.002) and IMV status (OR = 0.12, 95% CI = 0.04 to 0.35, P <0.001) independently predicted outcome. Conclusion Organ failures and need for organ support correlated with outcome P/F ratio and need for IMV were independent predictors of fi Conclusion This study including consecutive patients with oncological and hematological malignancies found a moderate association of proADM with 30-day mortality and LOS. proADM in combination with clinical parameters may help to improve site-of-care decisions for these patients in the future. Predictors of outcome in patients with haematological malignancies admitted to critical care A Tridente1, K Browett2, J Hall 2, Y Sorour3, J Snowden2, S Webber2 1St Helens and Knowsley, Liverpool, UK; 2STH, Sheffi eld, UK; 3DBH, Doncaster, UK l l d PP Dobesh, TR McGuire, DG Klepser, AL Himmelberg, DA Roberts, KM Olsen PP Dobesh, TR McGuire, DG Klepser, AL Himmelberg, DA Roberts, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P46 (doi: 10.1186/cc13236) University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P46 (doi: 10.1186/cc13236) Introduction The purpose of this study was to evaluate the impact of obesity on outcomes in patients with severe sepsis. Since obesity is considered an infl ammatory disease and is associated with elevations in several infl ammatory mediators important in the outcome of sepsis, the relationship between obesity and outcome in septic patients was studied. Methods This retrospective cohort study included all patients over the age of 40 with a confi rmed diagnosis of severe sepsis and an ICU stay at our academic medical center from 1 January 2005 to 31 March 2011. Obesity was defi ned as a body mass index of 30 or greater. Data on other patient demographics and APACHE II score at the time of sepsis were collected from patient charts. Outcomes measured included in- hospital mortality, development of acute respiratory distress syndrome (ARDS), days on mechanical ventilation, hospital cost, and length of stay.i Conclusion Regardless of the EWS, critical care teams are heavily involved in the management of patients outside critical care units. Fifty per cent of patients reviewed by the critical care team subsequently required admission to a critical care unit. The trigger threshold (7 and above) for referral to a critical care team currently recommended by the EWS escalation protocol is more likely to predict need for critical care admission. However, one in four patients referred below the threshold also required admission to a critical care environment. This study questions the safety of introducing such a protocol into acute hospitals. Will noncritical care staff be forced to wait until patients deteriorate further and reach the trigger threshold for referral or will the role of the critical care team expand further to look after all patients with abnormal EWS in hospital? Results We identifi ed 824 patients who met the inclusion criteria for this study. Of these patients, 257 (31.2%) were classifi ed as obese. The mean APACHE II score was similar between obese and nonobese patients (23.3 vs. 22.4; P = 0.068). Obese patients had a similar rate of in-hospital mortality (31.9% vs. Factors aff ecting the clinical response to National Early Warning score triggers Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). Group analysis was done for both ISS and NISS using the following severity score ranges (SSR) 1 to 8, 9 to 15, 16 to 25, and >25. I Kolic1, S McCartney1, S Crane1, Z Perkins2, A Taylor1 1South London Healthcare NHS Trust, London, UK; 2East Kent Hospitals University NHS Trust, Ashford, UK Critical Care 2014, 18(Suppl 1):P45 (doi: 10.1186/cc13235) Results The analysis conducted in previous research [1] was repeated for the data recategorized by NISS. The conclusions were identical; increased NISS is associated with increased radiation exposure. A total of 465 patients fell into diff erent SSR when classifi ed by NISS. The distribution of exposure for each SSR, NISS versus ISS, was compared using Wilcoxon’s test. For the range 1 to 8, there was no detectable diff erence. There was a signifi cant shift in the distribution of exposures for the remaining three ranges (P <0.02). The exposure shift was downward for NISS compared with ISS. Introduction We aimed to assess actions taken in response to variations in the National Early Warning (NEW) score and to identify factors associated with a poor response. The NEW score is a physiological score, which prescribes an appropriate response for the deteriorating patient in need of urgent medical care. This allows enhanced observation and clinical review of patients, identifying patients at risk of acute mortality. Methods We performed a prospective observational study of adult patients admitted to an acute medical ward in a London district general hospital over a 2-week period. Patient characteristics, NEW score, time of day, day of week and clinical response data were collected for the fi rst 24 hours of admission. Patients with less than a 12-hour hospital stay were excluded. The primary outcome measure was the quality of clinical response. Data were analysed with univariate and multivariate logistic regression. Conclusion In this analysis, with the exception of patients in groups 1 to 8 there was a signifi cant decrease in RE in the NISS groups when compared with the compared ISS group. Further analysis is needed to determine the cause for this change and whether this diff erence will be clinically signifi cant. P45 determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). Group analysis was done for both ISS and NISS using the following severity score ranges (SSR) 1 to 8, 9 to 15, 16 to 25, and >25. determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). Group analysis was done for both ISS and NISS using the following severity score ranges (SSR) 1 to 8, 9 to 15, 16 to 25, and >25. determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. Factors aff ecting the clinical response to National Early Warning score triggers I Kolic1, S McCartney1, S Crane1, Z Perkins2, A Taylor1 1South London Healthcare NHS Trust, London, UK; 2East Kent Hospitals University NHS Trust, Ashford, UK Critical Care 2014, 18(Suppl 1):P45 (doi: 10.1186/cc13235) Early warning scores: breaking or building barriers to critical care E Dunne1, RO Leary1, K Srinivasan2, B Ahmed3, D Galvin4, R Ni Mhuircheartaigh1, B Marsh1 j g Conclusion Clinical response to NEW score triggers is signifi cantly worse at weekends, highlighting an important patient safety concern. Reference Introduction A prospective multicentre observational study was carried out to assess the extent to which critical care teams manage patients in hospital who are cared for outside the critical care unit. The Health Service Executive (HSE) in Ireland is in the process of implementing a national Early Warning Score (EWS) and, at an EWS of 7 or above, a referral to critical care is recommended. This study recorded the EWS of patients referred to the critical care team and describes the subsequent interventions made by the critical care team and patient outcomes.fi 1. Royal College of Physicians: National Early Warning Score (NEWS): Standardising the Assessment of Acute Illness Severity in the NHS. Report of a Working Party. London: RCP; 2012. 1. Royal College of Physicians: National Early Warning Score (NEWS): Standardising the Assessment of Acute Illness Severity in the NHS. Report of a Working Party. London: RCP; 2012. P43 Calculated radiation exposure for trauma patients is lower when using the New Injury Severity Score versus the Injury Severity Score to calculate injury severity M Wagner1, J Vonk2, C Wichman1, A Hegde3, J Oliveto3 1Creighton University, Omaha, NE, USA; 2University of Iowa, Iowa City, IA, USA; 3UNMC, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P43 (doi: 10.1186/cc13233) Introduction We studied radiation exposure (RE) in trauma patients [1]. To adjust for injury severity the New Injury Severity Score (NISS) or the Injury Severity Score (ISS) can be used to group patients. We sought to Conclusion Organ failures and need for organ support correlated with outcome. P/F ratio and need for IMV were independent predictors of S16 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Reference 1. Wagner M, et al.: 258 Increased Injury Severity Score is associated with increased radiation exposure in trauma patients. Crit Care Med 2013, 41:A59. Results During the study period 200 patients were included with a median age of 70 (20 to 102) years. NEW scores were evenly distributed between day and night (52% vs. 48%) with a greater proportion on weekdays compared with weekend days (82% vs. 18%). The majority of patients scored <5 (93% vs. 7%). Forty-seven (27%) patients received an inadequate clinical response. Univariate analysis showed no association with time of day (night 34% vs. day 38%, OR 0.83 (0.47 to 1.49), P = 0.556). However, day of the week (weekend 56% vs. weekday 32%, OR 2.8 (1.30 to 5.84), P = 0.01) and increasing score (NEWS ≥5 100% vs. NEWS <5 31%, OR 65 (3.8 to 1100), P < 0.0001) were signifi cantly associated with an inadequate response. Day of the week was independently associated with an inadequate response after adjusting for confounders (OR 3.08 (1.27 to 7.46), P = 0.013). Impact of obesity on outcomes in patients with sepsis Methods Six critical care departments in university-affi liated hospitals across Ireland collected data on all referrals to the critical care team over a 6-week period. Data were anonymised, coded and analysed centrally. Results A cumulative total of 399 calls were made to the critical care teams in the six hospitals. The most common reason for referral was to request a critical care review of a patient (n = 319, 79.9%). Other reasons for referral included cardiac arrest, request to transfer patients from other hospitals and requests for vascular access. The average duration spent by the critical care team reviewing patients on the wards was 57 minutes. This increased up to 67 minutes for cardiac arrest calls. Of the 319 critical care reviews, 160 (50.2%) patients were subsequently admitted to critical care. A total 118 of this 160 had EWS of 7 or above, while 42 scored less than 7 but were still deemed to require admission to critical care. P44 Early warning scores: breaking or building barriers to critical care E Dunne1, RO Leary1, K Srinivasan2, B Ahmed3, D Galvin4, R Ni Mhuircheartaigh1, B Marsh1 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Tallaght Hospital, Dublin, Ireland; 3St Vincent’s University Hospital, Dublin, Ireland; 4University College Hospital, Galway, Ireland Critical Care 2014, 18(Suppl 1):P44 (doi: 10.1186/cc13234) Reference 1. Schuetz P, et al.: Eff ect of procalcitonin-based guidelines vs standard guidelines on antibiotic use in lower respiratory tract infections: the ProHOSP randomized controlled trial. JAMA 2009, 302:1059-1066. Methods We used a platform of a negative randomized control trial in subjects (n = 51) with a diagnosis of severe sepsis with ≥1 organ failure. The cohort of severe septic subjects was stratifi ed by obesity status based on the body mass index (BMI >30). Primary outcomes: 30-day and 180-day mortality; secondary outcome: diff erence in median (IQR) of fi ve infl ammatory cytokines including tumor necrosis factor alpha (TNFα), TNFα-receptor 2, interleukin (IL)-6, IL-1-receptor-antagonist (IL- 1ra) and IL-10. The measurement of median baseline cytokine levels was done in serum by Luminex technology. Statistical signifi cance was defi ned as P <0.05. Obesity is not associated with poor outcomes in older patients with sepsis Conclusion Clinical parameters and discharge levels of proADM allow accurate long-term prognostication in COPD patients independent of initial type of exacerbation. The focus on the best use of long-term prognostic information to improve patient care and clinical outcomes seems promising/rational. MI Restrepo1, P Faverio2, LF Reyes3, A Anzueto1 1University of Texas Health Science Center, San Antonio, TX, USA; 2University of Milan-Bicocca, Monza, Italy; 3Universidad de La Sabana, Bogota, Colombia Critical Care 2014, 18(Suppl 1):P47 (doi: 10.1186/cc13237) Introduction Studies suggest that obesity may infl uence mortality in patients who develop sepsis. However, the mechanisms linked to improved outcomes are unclear. Our aim was to assess the impact of obesity on mortality at 30 and 180 days and cytokine expression. Introduction Studies suggest that obesity may infl uence mortality in patients who develop sepsis. However, the mechanisms linked to improved outcomes are unclear. Our aim was to assess the impact of obesity on mortality at 30 and 180 days and cytokine expression. Methods We used a platform of a negative randomized control trial in subjects (n = 51) with a diagnosis of severe sepsis with ≥1 organ failure. The cohort of severe septic subjects was stratifi ed by obesity status based on the body mass index (BMI >30). Primary outcomes: 30-day and 180-day mortality; secondary outcome: diff erence in median (IQR) of fi ve infl ammatory cytokines including tumor necrosis factor alpha (TNFα), TNFα-receptor 2, interleukin (IL)-6, IL-1-receptor-antagonist (IL- 1ra) and IL-10. The measurement of median baseline cytokine levels was done in serum by Luminex technology. Statistical signifi cance was defi ned as P <0.05. PP Dobesh, TR McGuire, DG Klepser, AL Himmelberg, DA Roberts, KM Olsen 33.7%; P = 0.810) compared with nonobese patients, but a signifi cantly higher rate of development of ARDS (49.4% vs. 34.4%; P <0.001). Obese patients also had signifi cantly S17 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Frailty predicts need for medical review but not degree of organ support after complex orthopaedic surgery N Singatoullina1, A Panickar1, A Dennis1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Univeristy of Leicester Hospitals, Leicester, UK N Singatoullina1, A Panickar1, A Dennis1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Univeristy of Leicester Hospitals, Leicester, UK Critical Care 2014, 18(Suppl 1):P49 (doi: 10.1186/cc13239) Critical Care 2014, 18(Suppl 1):P49 (doi: 10.1186/cc13239) Introduction Based on expert opinion and case note review, the UK National Confi dential Enquiry into Peri-operative Outcome has recommended provision of perioperative level 2 and 3 care to support major surgery in older people, and particularly those with comorbidity [1]. We wished to identify whether we could predict if the need was uniform and whether any factors could predict the degree of organ supports needed. i Results Fifty-one subjects with severe sepsis were included in the study; 37% of the patients were obese (BMI >30). Paradoxically, obese severe septic patients had lower 30-day mortality (n = 1 (5%) vs. n = 9 (28%), P = 0.069) and 180-day mortality (n = 1 (5%) vs. n = 13 (41%), P = 0.008), when compared with nonobese. The expression of TNFα, TNFα-receptor 2, IL-6, IL-1ra and IL-10 was not statistically signifi cant diff erent among obese versus nonobese severe septic patients. f Conclusion Obesity is associated with lower mortality rates at 30 and 180 days in patients diagnosed with severe sepsis. This survival benefi t was not associated with lower cytokine production among obese patients. Further studies are needed to assess the mechanisms associated with the survival benefi t related to obesity in patients with severe sepsis. Methods A retrospective note review of all patients admitted to a level 2 critical care unit in the 12-month period from 1 January 2012 to 31 December 2012 undergoing revision hip surgery either as a two-stage or single-stage process. Surgery was undertaken at a national referral unit and chosen to represent an appropriate group of older, comorbid patients. Predefi ned preoperative and perioperative data were collected from chart review, along with postoperative physiological data whilst the patient was in critical care. This included frailty, comorbidities, operative blood loss, anaesthetic technique and level and duration or organ supports including the need for additional medical review whilst on the unit. Frailty was assessed preoperatively Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 more days on mechanical ventilation (6.2 days vs. 5.0 days; P = 0.005). There was no relationship between mortality in obese patients on mechanical ventilation (34.4% vs. 39.5%; P = 0.26) or ARDS (33.9% vs. 42.6%; P = 0.13) compared with nonobese patients. Hospital costs and length of stay did not diff er between the groups. Results Overall mortality in the 469 included COPD patients was 55% (95% CI 0.5 to 0.6) with a 14% (95% CI 0.1 to 0.2) mortality incidence rate per year. Patients with pneumonic COPD exacerbation had a more pronounced infl ammatory response compared with patients with nonpneumonic exacerbation with regard to levels of initial C-reactive protein levels (median 158 mg/dl vs. 39 mg/dl, P <0.0001), procalctionin (median 0.4 μg/l vs. 0.1 μg/l, P <0.0001) and proADM (median 1.3 nmol/l vs. 0.9 nmol/l, P <0.0001), but long-term survival was similar (HR 1.0, 95% CI 0.8 to 1.2). In univariate regression models, proADM was signifi cantly associated with mortality after 1, 3 and 6 years (HR 16.1 (95% CI 6.9 to 37.7), 10.5 (95% CI 5.7 to 19.6) and 10.4 (95% CI 6.2 to 17.7), respectively). There was no eff ect modifi cation by type of exacerbation. A model including clinical parameters (age, coronary heart disease, heart failure, diabetes mellitus, chronic renal failure, neoplastic disease, pneumonia, smokers) and proADM showed good discrimination of long-term survivors from nonsurvivors with AUC of 0.74 (95% CI 0.6 to 0.7). g yf g Conclusion Obesity signifi cantly increased the incidence of ARDS and days on mechanical ventilation in patients with sepsis. Previous work has reported that obesity is associated with elevations in infl ammatory cytokines and adipokines, particularly IL-6, which is a known risk factor for ARDS. The higher rate of ARDS in obese patients with sepsis identifi es a high-risk group where new therapies may be most benefi cial and where new methods of preventing ARDS can be targeted. P48 Frail patients were signifi cantly more likely to need additional medical input in the postoperative period whilst on critical care (Figure 1, P = 0.002) but this was not signifi cantly linked to need for vasopressors, evidence of sepsis or choice of anaesthetic technique. Methods This study was conducted in a 20-bed mixed ICU of a teaching hospital. The study sample was extracted from a dataset of all ICU patients treated for more than 72 hours between 1 January 2007 and 1 October 2012. Demographic characteristics and clinical characteristics at admission and during the ICU stay were collected. Characteristics of patients alive 1 year after ICU discharge were compared with patients who died within the fi rst year after ICU discharge. Descriptive statistics were calculated. Multivariate analysis of 1-year mortality was performed using a logistic regression model with backward elimination. Survival was analysed by the Kaplan–Meier method using the time interval from day of ICU discharge until death. Conclusion In complex revision orthopaedic surgery, the need for postoperative level 2/3 support cannot be predicted from any preoperative or intraoperative factors but patient frailty does indicate the need for medical input in the postoperative period. References 1. Wilkinson K, et al.: An Age Old Problem. London: NCEPOD; 2010. 2. Rockwood K, et al.: CMAJ 2005, 173:489-495. 1. Wilkinson K, et al.: An Age Old Problem. London: NCEPOD; 2010. 2. Rockwood K, et al.: CMAJ 2005, 173:489-495. 1. Wilkinson K, et al.: An Age Old Problem. London: NCEPOD; 2010. 2. Rockwood K, et al.: CMAJ 2005, 173:489-495. y g Results During the study period, 740 patients were treated for more than 72 hours in the ICU. The ICU mortality was 106/740 (14%). The data of 617 ICU survivors were further analysed (17 patients were lost to follow up). One-year mortality was 175/617 (28%), of which 85/175 (49%) patients died during hospital stay after ICU discharge. Frailty measures in the critically ill: are we approaching a critical age? A systematic review g y R Pugh1, DL John1, C Thorpe2, C Subbe2 g , , p , 1Glan Clwyd Hospital, Bodelwyddan, UK; 2Ysbyty Gwynedd, Bangor, UK Critical Care 2014, 18(Suppl 1):P50 (doi: 10.1186/cc13240) g p 1Glan Clwyd Hospital, Bodelwyddan, UK; 2Ysbyty Gwynedd, Bang 1Glan Clwyd Hospital, Bodelwyddan, UK; 2Ysbyty Gwynedd, Bangor, UK Critical Care 2014, 18(Suppl 1):P50 (doi: 10.1186/cc13240) y p y y y y g Critical Care 2014, 18(Suppl 1):P50 (doi: 10.1186/cc13240) Introduction As the general population ages, the proportion of critically ill patients in whom frailty may adversely aff ect outcome is likely to rise. The aim of this systematic review was to evaluate performance of frailty measures in predicting ICU, hospital and long-term outcomes following intensive care admission. Conclusion Of patients being treated for more than 72 hours in the ICU, 28% died within 1 year after ICU discharge. One-half of them within the hospital stay after ICU discharge. High age at ICU admission, high APACHE IV predicted mortality score, high number of comorbidities, readmission and an admission diagnosis within the categories ‘cardiovascular’ and ‘sepsis’ are associated with an increased 1-year mortality after ICU discharge in this population. The burden of patients dying after ICU discharge underlines the necessity for clear ICU discharge criteria and post-ICU care. Methods We performed a literature search for original studies in: EMBASE, MEDLINE, Web of Knowledge, Cochrane database of systematic reviews, and Database of Abstracts of Reviews of Eff ects, using the terms ‘frailty’, ‘frail elderly’, ‘critical care’, ‘critically ill’, ‘critical illness’ and ‘intensive care’. Our study inclusion criteria were that the study: included patients cared for in intensive care, captured data relating to premorbid frailty, and reported ICU, hospital and/or long- term outcome data. Results Initial searches identifi ed 606 reports, of which, following review of abstracts and removal of duplicates, 66 full-text papers were evaluated. Of these, 11 met inclusion criteria. A further 19 papers that met inclusion criteria were identifi ed from relevant review articles and reference lists. There was huge variation in populations studied, methodology, frailty measures utilised and reported outcome measures. Of the 30 included studies, 11 studies evaluated patients undergoing major (including cardiothoracic) surgery, two studies specifi cally assessed patients with pneumonia, one study investigated patients in a burns ICU and one study restricted its investigation to former nursing home residents. Long-term physical functioning and health-related outcomes in survivors of intensive care K Solverson, C Doig K Solverson, C Doig University of Calgary, Canada Critical Care 2014, 18(Suppl 1):P52 (doi: 10.1186/cc13242) Introduction The long-term impact of critical illness on survivors’ physical and mental health remains unknown. Our aim was to create a follow-up clinic for survivors of critical illness in order to quantitatively examine muscle weakness, physical functioning and mental health and relate these fi ndings to health-related quality of life (HRQL) and ICU risk factors. Methods Study patients were selected from a 24-bed multidisciplinary ICU. Fifty-six patients who were ≥18 years old, stayed longer than 4 days in the ICU and did not have an acute brain injury were followed-up at 2 and 4 months post hospital discharge. Peripheral muscle strength (grip, triceps, biceps, hamstrings, quadriceps and dorsifl exors) and physical functioning were objectively assessed using hand-held dynamometry and the six-minute walk test (6MWT); both were compared with age/ sex normative data. HRQL and mental health were assessed using the Short Form-36 (SF-36), EuroQol-5D (EQ-5D) and Hospital Anxiety and Depression Scale. p y p y Conclusion Measures of frailty appear to predict mortality and functional dependence following critical admission across a wide range of clinical conditions. However, comparative data regarding the relative accuracy and reliability of frailty measures in the intensive care population are currently defi cient. P48 Long-term outcome in COPD patients with pneumonic and nonpneumonic exacerbation: a 6-year prospective follow-up study EG Grolimund, AK Kutz, MA Alan, BM Mueller, PS Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P48 (doi: 10.1186/cc13238) Figure 1 (abstract P49). Proportion of patients requiring additional medical review on critical care by degree of frailty. Introduction Predicting long-term outcome in patients surviving a pneumonic or nonpneumonic COPD exacerbation remains challenging. This study investigates the association of clinical parameters and the prognostic blood marker pro-adrenomedullin (proADM) measured upon hospital discharge with 6-year mortality in well-defi ned cohort of COPD patients. Methods We prospectively followed consecutive COPD patients from a previous Swiss multicenter trial (2006 to 2008) [1] over a 6-year follow- up and investigated all-cause mortality following hospital discharge. Patients and/or treating general practitioners were contacted by telephone interview to assess the vital status of patients. We used Cox regression models and the area under the receiver operating characteristics curve (AUC) to investigate associations of baseline predictors and mortality. Figure 1 (abstract P49). Proportion of patients requiring additional medical review on critical care by degree of frailty. S18 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 determine the 1-year mortality after discharge from the ICU in patients who were treated in the ICU for more than 72 hours and to identify predictors for 1-year mortality. using the Rockwood assessment tool by trained staff [2]. Data were analysed using Microsoft Excel for Mac 2011 and Stata/IC 11.2 for Mac. Results A total of 182 patients with a mean age of 69.8 years (range 29 to 92) were identifi ed. Frail patients were signifi cantly more likely to need additional medical input in the postoperative period whilst on critical care (Figure 1, P = 0.002) but this was not signifi cantly linked to need for vasopressors, evidence of sepsis or choice of anaesthetic technique. using the Rockwood assessment tool by trained staff [2]. Data were analysed using Microsoft Excel for Mac 2011 and Stata/IC 11.2 for Mac. Results A total of 182 patients with a mean age of 69.8 years (range 29 to 92) were identifi ed. p p References Referencesi 1. Rockwood et al.: A global clinical measure of fi tness and frailty in elderly people. CMAJ 2005, 173:489-495. 2. Knaus et al.: APACHE-acute physiology and chronic health evaluation: a physiologically based classifi cation system. Crit Care Med 1981, 9:591-597. Results The median age of patients was 60 years, 68% were admitted for respiratory illnesses, they were severely ill (median APACHE II, 19) and had long ICU lengths of stay (median, 11 days). Muscle strength was signifi cantly reduced when compared with normative data in all muscle groups at the 2-month and 4-month visits (2-month average indexed overall strength, 72%, P <0.05). The median distance walked during the 6MWT was 382 m at 2 months (median percent predicted, 72%) and it did not signifi cantly change at the 4-month visit. Reduced peripheral muscle strength was signifi cantly correlated with lower distance walked during the 6MWT. Reported HRQL by the SF-36 was below national averages at both 2-month and 4-month visits (2-month P48 Independent predicting factors of 1-year mortality were: age at ICU admission (OR: 1.03; 95% CI: 1.01 to 1.05), APACHE IV predicted mortality score (OR: 1.02; 95% CI: 1.02 to 1.03), number of comorbidities (one or two co morbidities OR: 2.14; 95% CI: 1.42 to 3.23) (>3 comorbidities OR: 2.56; 95% CI: 1.16 to 5.62), readmission after ICU discharge within the same period of hospital stay (OR: 1.98; 95% CI: 1.13 to 3.46) and the diagnosis at admission (cardiovascular OR: 4.31; 95% CI: 1.73 to 10.76) (sepsis OR: 2.67; 95% CI: 1.50 to 4.77). Frailty measures in the critically ill: are we approaching a critical age? A systematic review The most commonly used measures of frailty were: measures of Activities of Daily Living (n = 9), which predicted need for long-term institutional care, 30-day, 90-day, 6-month and 12-month mortality; Clinical Frailty Score [1] (n = 5), which predicted ICU mortality, hospital mortality, 12-month mortality, quality of life and functional dependence; and Knaus score [2] (n = 4), which predicted ICU mortality, hospital and 12-month mortality. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 physical composite score (PCS) 36.2, mental composite score 48.1; 50 = national average). Reduced muscle strength was associated with low scores on the SF-36 physical function and general health domains. Performance on the 6MWT correlated with the SF-36 including the PCS (P = 0.001). Screening positive for anxiety was associated with both poor 6MWT performance and reporting dysfunction on the EQ- 5D domains. ICU/hospital length of stay, number of days ventilated, severity of illness and organ dysfunction were not found to be predictive of muscle strength or physical functioning. had a signifi cantly higher logistic EuroSCORE (20.3 vs. 10.6; P <0.001). The logistic EuroSCORE was a reasonable predictor of prolonged ICU/ in-hospital mortality (OR 1.04, 95% CI 1.04 to 1.05, P <0.001) with a receiver operating characteristic (ROC) curve of 0.72. The relationship between a patient’s logistic EuroSCORE and predicted risk of prolonged ICU is shown in the fi gure; including low-risk, medium-risk and high risk groups. Around 50% of the entire cohort of patients had a logistic EuroSCORE of 10 or less and an associated risk of prolonged ICU stay of 5% or less. See Figure 1. physical composite score (PCS) 36.2, mental composite score 48.1; 50 = national average). Reduced muscle strength was associated with low scores on the SF-36 physical function and general health domains. Performance on the 6MWT correlated with the SF-36 including the PCS (P = 0.001). Screening positive for anxiety was associated with both poor 6MWT performance and reporting dysfunction on the EQ- 5D domains. ICU/hospital length of stay, number of days ventilated, severity of illness and organ dysfunction were not found to be predictive of muscle strength or physical functioning. had a signifi cantly higher logistic EuroSCORE (20.3 vs. 10.6; P <0.001). The logistic EuroSCORE was a reasonable predictor of prolonged ICU/ in-hospital mortality (OR 1.04, 95% CI 1.04 to 1.05, P <0.001) with a receiver operating characteristic (ROC) curve of 0.72. The relationship between a patient’s logistic EuroSCORE and predicted risk of prolonged ICU is shown in the fi gure; including low-risk, medium-risk and high risk groups. Around 50% of the entire cohort of patients had a logistic EuroSCORE of 10 or less and an associated risk of prolonged ICU stay of 5% or less. See Figure 1. y Conclusion Our study gives qualitative evidence that survivors of critical illness have reduced muscle strength, physical functioning and HRQL after hospital discharge. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Also, we have shown muscle weakness is predictive of overall physical functioning, which in turn impacted HRQL and mental health. No ICU risk factors were identifi ed that predicted defi cits in muscle strength or physical functioning. Conclusion Using an existing risk prediction model, a patient’s risk of prolonged ICU stay can be calculated using contemporaneous data. This information could be relevant for aiding in providing informed consent for cardiac surgery patients. P54 Six-month outcomes in lung cancer patients surviving ICU admission: results from a multinational multicenter study M Soares1, JF Timsit2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, BF Almeida7, UV Silva8, LC Azevedo9, JI Salluh1, E Azoulay10 1DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil; 2Hôpital A. Michallon Chu de Grenoble, France; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton NHS Foundation Trust, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Fundação Pio XII – Hospital do Câncer de Barretos, Barretos, Brazil; 9Hospital Sírio Libanês, São Paulo, Brazil; 10Hôpital Saint Louis, Paris, France Critical Care 2014, 18(Suppl 1):P54 (doi: 10.1186/cc13244) Six-month outcomes in lung cancer patients surviving ICU admission: results from a multinational multicenter study Six-month outcomes in lung cancer patients surviving ICU admission: results from a multinational multicenter study M Soares1, JF Timsit2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, BF Almeida7, UV Silva8, LC Azevedo9, JI Salluh1, E Azoulay10 1DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil; 2Hôpital A. Michallon Chu de Grenoble, France; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton NHS Foundation Trust, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Fundação Pio XII – Hospital do Câncer de Barretos, Barretos, Brazil; 9Hospital Sírio Libanês, São Paulo, Brazil; 10Hôpital Saint Louis, Paris, France Critical Care 2014, 18(Suppl 1):P54 (doi: 10.1186/cc13244) Prediction of 1-year mortality of patients treated for more than 72 hours in an ICU S Steenbergen, S Rijkenberg, H Endeman S Steenbergen, S Rijkenberg, H Endeman S Steenbergen, S Rijkenberg, H Endeman Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands Critical Care 2014 18(Suppl 1):P51 (doi: 10 1186/cc13241) S Steenbergen, S Rijkenberg, H Endeman Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands g j g Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P51 (doi: 10.1186/cc13241) Introduction ICU or hospital mortality rates have been reported as the endpoint of ICU therapy for many years. The aim of this study was to S19 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Survival and quality of life in patients acquiring acute kidney injury in the fi rst 24 hours of ICU admission Survival and quality of life in patients acquiring acute kidney injury in the fi rst 24 hours of ICU admission I Soliman, L Peelen, D De Lange, D Van Dijk University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P55 (doi: 10.1186/cc13245) Introduction The population of the UK is ageing, with the fastest increase in those ≥85 years. Increased age has been repeatedly associated with adverse outcome and it is uncertain to what extent this relates to the changes of ageing in themselves, or due to other considerations. Age is a key variable in the majority of scoring systems that relate patient characteristics to adverse outcome. We aimed to assess change in age distribution of patients admitted to our ICU over 20 years and examine the relationship between age of patient, mortality and length of stay (LOS). i Soliman, L Peelen, D De Lange, D Van Dijk i I Soliman, L Peelen, D De Lange, D Van Dijk , , g , j University Medical Center, Utrecht, the Netherlands University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P55 (doi: 10.1186/cc13245) y , , Critical Care 2014, 18(Suppl 1):P55 (doi: 10.1186/cc13245) Introduction Survival and quality of life (QoL) in ICU patients with acute kidney injury (AKI) have been repeatedly reported as poor [1-3]. It is unknown whether early AKI, occurring during the fi rst 24 hours of ICU treatment, is also a strong predictor of poor long-term outcome. Our aim was to describe the long-term outcomes of this specifi c group of ICU patients. Methods Data were extracted from electronic records (WardWatcher) and analysed using SPSS 20, GraphPad Prism 5.0 and Excel 2007. Methods Data were extracted from electronic records (WardWatcher) and analysed using SPSS 20, GraphPad Prism 5.0 and Excel 2007. Results ICU patients have become older by 4.4 months/year. By 2013 the median age was 66 and 15% of all patients are now ≥80 years – a 36% increase since 1993. Compared with the reference group (61 to 70 years), those in the older deciles have increased risk of ICU and hospital mortality (P <0.01). Fifteen per cent of all those 81 to 90 years old and 20% of those >91 years old who do not die on the ICU go on to die on the ward. It is unknown what proportion of these post-ICU deaths was unexpected. References 1. Morgera S et al.: Crit Care Med 2008, 36(4 Suppl):S193-S197. 2. Nisula S et al.: Crit Care 2013, 17:R250. 3. Brinkman S et al.: Crit Care Med 2013, 41:1229-1236. Conclusion Post-hospital mortality in critically ill lung cancer patients is relatively high and many patients require anticancer treatments after discharge. PS before ICU admission is a major determinant of both mortality and ability to receive optimal anticancer treatment in these patients. P53 A Grayson, N Coulson, N Scawn Introduction The aim of this study was to determine an appropriate risk model to identify patients at high risk of prolonged ICU stay and to aid patient consent prior to cardiac surgery. Introduction The aim of this study was to determine an appropriate risk model to identify patients at high risk of prolonged ICU stay and to aid patient consent prior to cardiac surgery. Methods Data were prospectively collected on 5,440 consecutive cardiac surgery cases between April 2009 and March 2012. The primary outcome measure was the combined outcome of prolonged ICU stay (length of stay greater than 20 days) and/or in-hospital mortality. Logistic regression was performed to assess the predictability of logistic EuroSCORE against the primary outcome. Low-risk, medium- risk and high-risk groups were identifi ed and subsequent risk of 1-year mortality assessed. Survival status was determined at 1 year. Introduction Information about lung cancer patients surviving critical illnesses is very scarce. Our aim was to evaluate the outcomes and continuing of anticancer treatments in lung cancer patients surviving ICU admission. Methods Secondary analysis of a prospective multicenter study including patients admitted for >24 hours to 22 ICUs in six countries from Europe and South America during 2011. Readmissions and patients in cancer remission >5 years were excluded. Logistic regression was used to identify predictors for hospital mortality. Results A total of 192 (3.5%) patients had a prolonged ICU stay and 187 (3.4%) in-hospital deaths occurred, resulting in a combined primary outcome of 349 (6.4%). At 1 year, 371 (6.8%) deaths occurred. The risk of death in-hospital and at 1 year was signifi cantly higher in patients with prolonged ICU stay (in-hospital mortality, 15.6% vs. 3.0%; P <0.001/1 year, 27.6% vs. 6.1%; P <0.001). The mean logistic EuroSCORE for all patients was 10.9. Patients with prolonged ICU stay Results A total of 449 patients (small-cell (SCLC) = 55; non-SCLC = 394)) were admitted to ICUs, and out of them 275 (SCLC = 29; NSCLC = 246) were discharged alive from the hospital. Among them, 200 (73%) patients were alive and 72 (26%) had died at 6 months; three Figure 1 (abstract P53). Risk of long stay versus 1-year mortality. Figure 1 (abstract P53). Risk of long stay versus 1-year mortality. Increasing age of patients admitted to intensive care, and association between increased age and greater risk of post-ICU death Increasing age of patients admitted to intensive care, and association between increased age and greater risk of post-ICU death B Creagh-Brown, S Green B Creagh-Brown, S Green Royal Surrey County Hospital, Guildford, UK Critical Care 2014, 18(Suppl 1):P56 (doi: 10.1186/cc13246) Critical Care 2014, 18(Suppl 1):P56 (doi: 10.1186/cc13246) P53 S20 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (1%) patients were lost to follow-up. Mortality rates were far lower in the patient subset with nonrecurrent/progressive cancer and a good performance status (PS), even those with sepsis, multiple organ dysfunctions, and need for ventilatory support. Cancer recurrence or progression occurred in 53 (26%) hospital survivors. Anticancer treatments were recommended for 108 (39%) hospital survivors and administered to 102. Treatments used were variable combinations of surgical resection (7%), radiation therapy (34%), and chemotherapy (80%). The initial treatment plan required reduction or modifi cation in 35 (34%) patients. Post-hospital mortality was nonsignifi cantly lower in the patients given the initial treatment plan than in the other patients (17% vs. 32%, P = 0.065). Poor PS was the only factor associated with a lower probability of receiving the initial treatment plan (OR = 0.20; 95% CI, 0.05 to 0.87; P = 0.032). At 6 months, 71% patients were at home, 15% were hospitalized, and 7% were in hospice care; the location was unknown for 6% patients. PS at 6 months was 3 to 4 in 19 (9.5%) survivors. Results Out of 5,934 admissions, 269 patients were identifi ed with early AKI. After ICU discharge a large and signifi cant diff erence in survival between included patients and the Dutch population, matched for age and gender, was seen (P <0.001). The median QoL index in surviving patients was 0.65 (interquartile range (IQR) 0.45 to 1.00), versus 0.86 (IQR 0.84 to 0.89) in the Dutch population (P <0.001). Low QoL was found in 11/59 (18.6%) survivors. In total, poor ICU outcome was seen in 171/269 (63.6%) patients. See Figure 1. p g Conclusion Patients developing AKI in the fi rst 24 hours of ICU stay are prone to poor outcome. Future research into prognostic factors for ICU patients should include early AKI in their models. R f Outcomes of military patients treated at the UK Royal Centre for Defence Medicine 2007 to 2013 AM Johnston1, J Henning2, D Harrison3 1Royal Centre for Defence Medicine, Birmingham, UK; 2James Cook University Hospital, Middlesbrough, UK; 3ICNARC, London, UK Critical Care 2014, 18(Suppl 1):P57 (doi: 10.1186/cc13247) AM Johnston1, J Henning2, D Harrison3 1Royal Centre for Defence Medicine, Birmingham, UK; 2James Cook University Hospital, Middlesbrough, UK; 3ICNARC, London, UK Critical Care 2014, 18(Suppl 1):P57 (doi: 10.1186/cc13247) Introduction UK military personnel injured overseas are repatriated to the Royal Centre for Defence Medicine (RCDM) based at the Queen Elizabeth Hospital Birmingham (QEHB) in Birmingham UK. We report the demographics and outcomes of military patients treated on the ICU at RCDM using data from the Intensive Care National Audit and Research Centre over a 6.5-year period. Conclusion Very old patients with cancer present acceptable rates of survival after ICU admission. Similarly to younger patients, organ function evaluation in the fi rst hours of ICU can predict outcomes in this specifi c population. y p Methods Data on 570 admissions of 527 patients to the ICU at RCDM/ QEHB were analysed by ICNARC using standard methodology. y y g gy Results Some physiology and CCMDS data were missing for 175 patients. Age, sex and mortality are described in Table 1. A total of 90.9% of patients had traumatic injuries, 2.1% received CPR prior to ICU admission, 1.5% prehospital. A total of 20.6% had head, neck or spinal trauma. A total of 85.7% were transferred directly to the ICU from a military hospital overseas, others coming to the ICU following surgery at RCDM. Of the 382 patients with APACHE II score data the mean score was 11.0 (SD 4.9), probably refl ecting stabilisation in military hospitals overseas or during aeromedical critical care transfer. The mean number of ICU days was Level 3: 7.6 (SD 11.6); Level 2: 2.0 (SD 2.8). A total of 70.4% of patients required advanced respiratory support for a mean of 7.5 days, and 33% required advanced cardiovascular support for a mean of 3.7 days. Reference 1. Bagshaw SM, Webb SA, Delaney A, et al.: Very old patients admitted to intensive care in Australia and New Zealand: a multi-centre cohort analysis. Crit Care 2009, 13:R45. 1. Bagshaw SM, Webb SA, Delaney A, et al.: Very old patients admitted to intensive care in Australia and New Zealand: a multi-centre cohort analysis. Crit Care 2009, 13:R45. Very old patients with cancer admitted to the ICU: outcome and predictive factors of mortality Introduction The rate of very old patients (≥80 years old) admitted to intensive care has increased in the last years. Older age is associated with a higher prevalence of chronic diseases, including cancer [1]. The aim of the present study was to describe characteristics, outcomes and predictive factors of mortality in very old patients admitted to the ICU. Methods We performed a retrospective analysis of all cancer patients who were 80 years or older admitted to the ICU between January 2009 and December 2012 in a tertiary reference cancer center. Data were collected from medical records. Results A total of 597 patients with cancer were included in the analysis. Hospital mortality was 28.5%. These patients were more likely to have a solid tumor, a localized disease and underwent surgery, chemotherapy or radiotherapy recently. Variables associated with hospital mortality in these patients were: lung cancer, metastatic cancer and at ICU admission vasopressor requirements, acute respiratory failure, low hemoglobin levels, elevated creatinine levels, elevated bilirubin levels, acidosis and hyperlactatemia. By the multivariate analysis, the following factors were independent factors associated with hospital mortality: lung cancer (odds ratio (OR) = 6.3, 95% CI = 2.6 to 15.0, P <0.001), lactate levels on ICU admission (OR = 1.03, 95% CI = 1.02 to 1.04), P <0.001), bilirubin levels on ICU admission (OR = 1.16, 95% CI = 1.04 to 1.30, P = 0.007) and creatinine levels on ICU admission (OR = 1.58, 95% CI = 1.35 to 1.85, P <0.001). P58 Very old patients with cancer admitted to the ICU: outcome and predictive factors of mortality L Hajjar1, M Franca1, J Almeida1, J Fukushima1, F Bergamin1, C Zambolim1, P Camargo1, C Park1, E Osawa1, M Sundin2, R Nakamura1, F Galas1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Heart Institute, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P58 (doi: 10.1186/cc13248) Survival and quality of life in patients acquiring acute kidney injury in the fi rst 24 hours of ICU admission Older patients had prolonged hospital LOS (P <0.01) but not ICU LOS. See Figures 1 and 2. p Methods All patients admitted to our mixed ICU from July 2009 to May 2012 with early AKI were included. All survivors received the EuroQoL EQ-6D-3L questionnaire. Early AKI was defi ned as creatinine >1.5 mg/ dl and urine output <150 ml/8 hours. Poor ICU outcome was defi ned as either death or EuroQoL index <0.4 at 1-year follow-up. Figure 1 (abstract P55). Survival. Figure 1 (abstract P55). Survival. Conclusion There are increasing numbers of older patients on ICUs in the UK. In analyses uncorrected for severity of illness or comorbidities, older patients are more likely to die on the ICU, and on the ward after ICU. They also spend longer in hospital prior to discharge. the UK. In analyses uncorrected for severity of illness or comorbidities, older patients are more likely to die on the ICU, and on the ward after ICU. They also spend longer in hospital prior to discharge. Figure 1 (abstract P55). Survival. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S21 Figure 2 (abstract P56). Post-ICU in-hospital mortality, by age. Conclusion The data on resource utilisation for this group of patients may inform planning of critical care support for military operations overseas. Endpoint resuscitation-based prediction model for early mortality of severe sepsis and septic shock Endpoint resuscitation-based prediction model for early mortality of severe sepsis and septic shock R Sinto, S Suwarto, R Sedono, K Harimurti, A Sejati Faculty of Medicine, University of Indonesia, Jakarta, Indonesia Critical Care 2014, 18(Suppl 1):P62 (doi: 10.1186/cc13252) Introduction The fi rst description of SAPS II dates back to 1993 [1], but little is known about the scoring accuracy in daily practice and factors possibly aff ecting it. The purpose of this study was to evaluate accuracy of SAPS II scoring by means of a nationwide survey. Introduction There is an unknown role for macrocirculation and microcirculation endpoint resuscitation, which are combined as the component of a prediction model for early mortality of patients with severe sepsis and septic shock [1,2]. The aim of this study is to develop a prediction model for early mortality (fi rst 120 hours after onset [3]) of patients with severe sepsis and septic shock based on macrocirculation and microcirculation endpoint resuscitation. Methods Twenty clinical scenarios, covering a broad range of illness severity, were randomly assigned to a convenience sample of clinicians or nurses of Swiss adult ICUs. They were asked to assign a SAPS II score (including details for each item of the score) for one scenario. Results were compared with a reference, as defi ned by fi ve experienced researchers using a Delphi method, and cross-matched with data related to training and quality control for scoring, information on daily practice of scoring, and structural and organizational properties of each participating ICU. p Methods A retrospective cohort study was conducted in adult patients with severe sepsis and septic shock hospitalized in the ICU, Cipto Mangunkusumo Hospital, Indonesia. Patients’ outcome and time to outcome were observed during the fi rst 120 hours after severe sepsis and/or septic shock onset. Independent predictors for early mortality were identifi ed by Cox’s proportional hazard regression analysis and each was quantifi ed to develop an early mortality prediction model. The calibration and discrimination abilities of the model were determined. Results Subjects consisted of 268 patients. Early mortality developed in 70 patients. Two independent predictors for early mortality were: lactate clearance <10% (adjusted hazard ratio (HR) 11.81 (95% CI 6.50 to 21.46)) and number of organ dysfunctions (two organs, adjusted HR 1.47 (95% CI 0.58 to 3.72); >2 organs, adjusted HR 3.79 (95% CI 1.65 to 8.69)). 1. Moreno RP, et al.: Intensive Care Med 2010, 36:1207-1212. References 1. National Oesophago-cancer Audit Annual Report 2013. The NHS Information Centre [https://catalogue.ic.nhs.uk/publications/clinical/ oesophago-gastric/nati-clin-audi-supp-prog-oeso-gast-canc-2013/clin-audi- supp-prog-oeso-gast-2013-rep.pdf] Results Forty-six patients were identifi ed in our hospital at the time of the snapshot as being high risk according to NEWS. After recategorising this cohort of patients using ViEWS, 36 were classifi ed as high risk (in this instance meaning a score of 5 or more). Subjectively the authors did not have any clinical concerns created by moving 10 patients out of the high-risk classifi cation. 2. Michelet P, et al.: Br J Surg 2009, 96:54-60. Conclusion ViEWS is more specifi c without being less sensitive. We have replaced NEWS with ViEWS and feel that this is clinically safe. References Endpoint resuscitation-based prediction model for early mortality of severe sepsis and septic shock A scoring system as the predictive model was performed by assigning 1 point for two organ dysfunctions, 6.5 points for >2 organ dysfunctions, and 12 points for lactate clearance <10%. This scoring system was stratifi ed into two levels: low-risk (score <12, probability for early mortality 7.8%) and high-risk (score ≥12, probability for early mortality 72.3%) groups. The Hosmer–Lemeshow test revealed good precision (P = 0.745) and the area under the receiver operating characteristic curve showed very good discrimination ability (0.91 (95% CI 0.87 to 0.97)). Results Sixty-three (81%) of 78 adult ICUs participated in this survey. A perfect match with each single reference item was found in 27 (7.8%) of 345 scorings. The participants’ mean SAPS II scoring was 42.6 ± 23.4, with a bias of +5.7 (95% CI 2.0 to 9.5) as compared with the reference score. There was no evidence of variation of the bias according to case severity, number of beds in the unit, number of residents during workshifts, linguistic area, profession (physician vs. nurse), experience, initial SAPS II training, and presence of a quality control system. The items with highest scoring accuracy were bilirubin, temperature and chronic disease (93%, 93% and 91% respectively), whereas the lowest agreement was found for urinary output and for the Glasgow Coma Scale (63% and 64%). Conclusion This nationwide survey suggests a wide variability of SAPS II scoring results. On average, SAPS II was overestimated by more than 10%, irrespective of the profession or experience of the scorer or the structural characteristics of the ICUs. At least one person per unit involved in the scoring should be trained by the national society and should be responsible for the scoring quality. Reference Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 and nine patients had died by 1 year (12.5%). The median length of ICU and hospital stay was 4 days and 14 days respectively. Six patients had an anastomotic leak (of which two were chyle leaks). Use of non- invasive ventilation (in 23.1% of patients) was not associated with an anastomotic leak (chi-square P = 0.53), nor was the amount of fl uid given intraoperatively (Mann–Whitney U P = 0.410). Six patients had to be reintubated and this was associated with a signifi cantly increased length of both ICU and hospital stay (Mann–Whitney U P = 0.01 and 0.03 respectively). Lower P/F ratios were also associated with a signifi cant increase in length of both ICU and hospital stay (P = 0.007 and 0.043). P60 1. National Early Warning Score (NEWS). Standardising the Assessment of Acute- illness Severity in the NHS. London: Royal College of Physicians; July 2012. [http //wwwrcplondon ac uk/resources/national early warning score news] 1. National Early Warning Score (NEWS). Standardising the Assessment of Acute- illness Severity in the NHS London: Royal College of Physicians; July 2012 illness Severity in the NHS. London: Royal College of Physicians; July 2012. [http://wwwrcplondon ac uk/resources/national-early-warning-score-news] SwissScoring: a nationwide survey about SAPS II assessing accuracy M Previsdomini1, B Cerutti2, P Merlani3, HU Rothen4, M Kaufmann5, A Perren1 1Ospedale Regionale Bellinzona e Valli, Bellinzona, Switzerland; 2Unit of Development and Research in Medical Education, Geneva, Switzerland; 3Ospedale Regionale, Lugano, Switzerland; 4Bern University Hospital, Bern, Switzerland; 5University Hospital Basel, Switzerland Critical Care 2014, 18(Suppl 1):P60 (doi: 10.1186/cc13250) SwissScoring: a nationwide survey about SAPS II assessing accuracy M Previsdomini1, B Cerutti2, P Merlani3, HU Rothen4, M Kaufmann5, A Perren1 1 d l l ll ll ll l d 2 f [http://www.rcplondon.ac.uk/resources/national-early-warning-score-news] 2. Prytherch D, et al.: Resuscitation 2010, 81:932-937. p p y g 2. Prytherch D, et al.: Resuscitation 2010, 81:932-937. 2. Prytherch D, et al.: Resuscitation 2010, 81:9 1Ospedale Regionale Bellinzona e Valli, Bellinzona, Switzerland; 2Unit of Development and Research in Medical Education, Geneva, Switzerland; 3Ospedale Regionale, Lugano, Switzerland; 4Bern University Hospital, Bern, Switzerland; 5University Hospital Basel, Switzerland Critical Care 2014, 18(Suppl 1):P60 (doi: 10.1186/cc13250) Abandoning the National Early Warning Score in our district general hospital B Mylrea Lowndes, M Mercer, H Robinson Torbay Hospital, Torquay, UK Critical Care 2014, 18(Suppl 1):P61 (doi: 10.1186/cc13251) Introduction Early Warning Scores (EWS) are used in UK hospitals to identify patients who are acutely unwell or needing urgent review. The National EWS (NEWS) [1] was implemented in our institution and led to a noticeable increase in the numbers of patients being triggered for escalation of medical care, although clinically this was thought unwarranted. This led to a potentially dangerous lack of faith in the NEWS by clinical staff . We assessed whether it was safe to move to VitalPAC™ EWS (ViEWS), a commercially available electronic EWS [2]. y Conclusion The overall mortality and morbidity rate was comparable with that seen nationally. Our data suggest that the use of non-invasive ventilation was not associated with anastomotic breakdown. A lower P/F ratio in the postoperative period was associated with prolonged ICU and hospital stay. y Methods All patients scored as ‘high risk’ by NEWS (with a score of 6 or more) in a snapshot audit of patients in our 500-bed acute district general hospital were identifi ed and reviewed clinically. All of these patients were then recategorised using ViEWS. The clinical safety of this recategorisation was then assessed. P59 A retrospective review of mortality and complications following oesophagectomy in a large UK teaching hospital A retrospective review of mortality and complications following oesophagectomy in a large UK teaching hospital N Pawley, CM Ball, K Wickenden, B Riley, M Clapham, B Eltayeb, A Glossop, A Raithatha Sheffi eld Teaching Hospital, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P59 (doi: 10.1186/cc13249) g y g g N Pawley, CM Ball, K Wickenden, B Riley, M Clapham, B Eltayeb, A Glossop, A Raithathafifi Table 1 (abstract P57). Case mix, demographics and outcome Table 1 (abstract P57). Case mix, demographics and outcome Introduction Just over 1,200 curative oesophagectomies are carried out in the UK annually. Although in-hospital mortality rates have fallen (12 to 13% in 1998 to 2.5% 2013), complication rates remain high [1] with anastomotic failure and respiratory failure common postoperatively [2]. The aim of this retrospective review was to examine the outcomes in patients who underwent oesophagectomies in our unit between January 2010 and October 2012. Table 1 (abstract P57). Case mix, demographics and outcome n 570 Male (%) 97.9 Age 25.7 Trauma (%) 90.9 Died (%) 6.8 LOS 8.8 MV (%) 70.4 CRRT (%) 10.9 Neuro (%) 24.8 CV (%) 33.2 y Methods We examined demographic data, survival (30 day and 1 year) and length of ICU and hospital stay. Case notes were reviewed to identify postoperative complications including anastomotic breakdown, reintubation and respiratory failure. Data were analysed to examine the relationship between the use of postoperative non- invasive ventilation and intraoperative fl uid volume and the incidence of postoperative complications.i p p p Results Seventy-two patients were identifi ed as having undergone an oesophagectomy between January 2010 and October 2012. Median age was 65 and 82% were male. One patient died within 30 days (1.39%) S22 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P63 Is the Golden hour important? Looking at disability and health-related quality of life in a Portuguese trauma registry J Estilita1,2, CC Dias3, A Costa-Pereira3,4, C Granja1,2,3,4, I Aragão4, L Orwelius3,4,5 1DEICM, Algarve Hospital Centre, Portugal; 2University of Algarve, Portugal; 3CINTESIS, FMUP, Porto, Portugal; 4FMUP, Porto, Portugal; 5Linköping University, Linköping, Sweden Critical Care 2014, 18(Suppl 1):P63 (doi: 10.1186/cc13253) P63 Is the Golden hour important? Looking at disability and health-related quality of life in a Portuguese trauma registry J Estilita1,2, CC Dias3, A Costa-Pereira3,4, C Granja1,2,3,4, I Aragão4, L Orwelius3,4,5 1DEICM, Algarve Hospital Centre, Portugal; 2University of Algarve, Portugal; 3CINTESIS, FMUP, Porto, Portugal; 4FMUP, Porto, Portugal; 5Linköping University, Linköping, Sweden Critical Care 2014, 18(Suppl 1):P63 (doi: 10.1186/cc13253) g Results Signifi cant predictors of the development of complications were age, number of rib fractures, chronic lung disease, use of pre- injury anticoagulants and oxygen saturation levels. The fi nal model demonstrated an excellent c-index of 0.97. Introduction The Golden hour (GH) complies the concept that defi nitive trauma care must be initiated within 60 minutes in order to improve outcome, but evidence is confl icting [1,2]. A variety of injury and health loss scores are used in post-impact care for assessing the injury severity, probability of survival and long-term loss of health [3]. The aim of this study is to establish a relationship between the concept of GH and disability or health-related quality of life (HRQoL) in trauma patients and to compare injury scores and health loss scores in this population. Conclusion In our two-phase study, we have developed and validated a prognostic model that can be used to assist in the management of blunt chest wall trauma patients. The fi nal risk score provides the clinician with the probability of the development of complications for each individual patient. References 1. Demirhan R, Onan B, Oz K, Halezeroglu S: Comprehensive analysis of 4205 patients with chest trauma: a 10-year experience. Interact Cardiovasc Thorac Surg 2009, 9:450-459. Methods We analyzed patients from a trauma registry of a 700-bed university hospital between January 2003 and December 2007 who were alive 6 months after injury. A follow-up interview with the EQ-5D questionnaire was conducted. Data included patient demographics, type of injury, ISS, RTS and TRISS scores, ICU and hospital length of stay (LOS), mortality and EQ-5D domains. g 2. Battle CE, Hutchings H, Evans PA: Risk factors that predict mortality in patients with blunt chest wall trauma: a systematic review and meta- analysis. Injury 2012, 43:8-17. 2. Battle CE, Hutchings H, Evans PA: Risk factors that predict mortality in patients with blunt chest wall trauma: a systematic review and meta- analysis. Injury 2012, 43:8-17. g References 1. Orwelius L, et al.: J Trauma Acute Care Surg 2012, 72:504-512. 2. Aitken LM, et al.: J Trauma Acute Care Surg 2012, 72:1702-1708. 3. European Commission Mobility and Transport Road Safety [http:// ec.europa.eu/transport/road_safety/specialist/knowledge/postimpact/ data_and_information_systems/impairment_disability_and_loss_of_ function_scales_and_scores.htm] 3. European Commission Mobility and Transport Road Safety [http:// ec.europa.eu/transport/road_safety/specialist/knowledge/postimpact/ data_and_information_systems/impairment_disability_and_loss_of_ function_scales_and_scores.htm] j y Methods To develop crush syndrome, both hind limbs of rats were compressed for 6 hours under weights (3.0 kg each). Rats in the treated group were intravenously administrated BMMNCs (1×107 cells in 1 ml phosphate-buff ered saline (PBS)) immediately after weight removal (BMMNC group) and PBS alone was administered in the control group (CR group). The sham group underwent the same procedure without compression of hind limbs (SH group). The rats were observed over 7 days after the injury to evaluate survival. To estimate anti- infl ammatory eff ects of BMMNCs, sera were collected 3 hours, 6 hours and 24 hours after the injury. The levels of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) were measured by ELISA and statistically analyzed. Reference S23 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion A prediction model for early mortality of patients with severe sepsis and septic shock can be developed based on two parameters, lactate clearance and number of organ dysfunctions. A model has been developed to predict and classify mortality risk. References of this patient group is challenging as a result of the delayed onset of complications. The aim of this study was to develop and validate a prognostic model that can be used to assist in the management of blunt chest wall trauma. Methods There were two distinct phases to the overall study; the development and the validation phases. In the fi rst study phase, the prognostic model was developed through the retrospective analysis of all blunt chest wall trauma patients (n = 274) presenting to the emergency department of a regional trauma centre in Wales (2009 to 2011). Multivariable logistic regression was used to develop the model and identify the signifi cant predictors for the development of complications. The model’s accuracy and predictive capabilities were assessed. In the second study phase, external validation of the model was completed in a multicentre prospective study (n = 237) in 2012. The model’s accuracy and predictive capabilities were re-assessed for the validation sample. A risk score was developed for use in the clinical setting. 1. Trzeciak S, et al.: Acad Emerg Med 2008, 15:399-413. 2. Rivers EP, et al.: Minerva Anestesiol 2012, 78:712-724. 3. Macias WL, et al.: Crit Care Med 2004, 32(5 Suppl):S223-S228. 1. Trzeciak S, et al.: Acad Emerg Med 2008, 15:399-413. 2. Rivers EP, et al.: Minerva Anestesiol 2012, 78:712-724. 3. Macias WL, et al.: Crit Care Med 2004, 32(5 Suppl):S223-S228. Transplantation of bone marrow-derived mononuclear cells can improve the survival rate and suppress the infl ammatory response in a rat crush injury model Transplantation of bone marrow-derived mononuclear cells can improve the survival rate and suppress the infl ammatory response in a rat crush injury model J Nakagawa, N Matsumoto, K Yamakawa, T Yamada, H Matsumoto, T Muroya, H Ogura, T Shimazu Osaka University Graduate School of Medicine, Suita-shi, Osaka, Japan Critical Care 2014, 18(Suppl 1):P65 (doi: 10.1186/cc13255) Introduction Crush syndrome is often encountered in natural disasters. It is a critical condition leading to multiple organ failure. However, the mechanisms by which the local traumatic injuries aff ect distant organs remain unknown. We paid attention to bone marrow-derived mononuclear cells (BMMNCs) as therapeutic strategy against crush injury. Transplantation of BMMNCs can elicit protection and regeneration against damaged organs through their paracrine properties and its clinical application has been realized to treat ischemia reperfusion-related various diseases. We have previously reported that multiple organ damage based on systemic infl ammatory response is induced in crush injury and the pathogenesis is largely dependent on massive ischemia–reperfusion. We investigated whether BMMNCs could suppress systemic infl ammation and improve mortality in a rat model of crush injury. Conclusion The GH may be important as a survival outcome but not as a HRQoL outcome. Patients with severe problems in EQ-5D physical health domains showed lower probability of survival, were older and had a longer ICU LOS. R f P65 y Results There were 78% (n = 589) males and the average age was 44 ± 20 years. Most trauma cases originated from metropolitan areas and 91% (n = 688) of patients were managed within the GH. There was an association of mortality with older age and penetrating trauma. Mortality was higher for those with shorter length of stay. Those treated within the GH were less likely to die. In the HRQoL analysis, over one-half of the patients had moderate to severe problems in the Usual Activities domain. The median EQ-5D Index was 0.66 (0.47 to 0.98). There was no association of GH with HRQoL domains 6 months after discharge from the ICU. For the TRISS there was a lower probability of survival for the patients with severe problems in the physical health domains. The same results were obtained for association with age and LOS. Impact of a dedicated trauma desk in ambulance control on the identifi cation of major trauma in Scotland Impact of a dedicated trauma desk in ambulance control on the identifi cation of major trauma in Scotland i j J Bruce1, A Parker2, M Donald3, M Esposito2, L Curatolo2, A Kennedy2, K Simpson2, C Morton2, J Cormack2, M Austin2 1St Johns Hospital, Livingston, UK; 2Scottish Ambulance Service, Edinburgh, UK; 3Ninewells Hospital, Dundee, UK Critical Care 2014, 18(Suppl 1):P66 (doi: 10.1186/cc13256) J Bruce1, A Parker2, M Donald3, M Esposito2, L Cu J Bruce1, A Parker2, M Donald3, M Esposito2, L Curatolo2, A Kennedy2, K Simpson2, C Morton2, J Cormack2, M Austin2 1St Johns Hospital, Livingston, UK; 2Scottish Ambulance Service, Edinburgh, UK; 3Ninewells Hospital, Dundee, UK Critical Care 2014, 18(Suppl 1):P66 (doi: 10.1186/cc13256) Results We included a total of 1,452 patients (58% males, mean age 66.6 years). A total of 20.1% (n = 292) were classifi ed as high treatment priority, 5.4% (n = 79) were admitted to the ICU and 4.4% (n = 64) died within 30 days. The initial MTS showed a good prognostic accuracy to predict treatment priority (AUC 0.75) and ICU admission (AUC 0.76), but not for mortality prediction (AUC 0.58). Initial ProADM levels were independent predictors for all three outcomes and signifi cantly improved the MTS score to AUCs of 0.78 for treatment priority, 0.80 for ICU admission and 0.84 for mortality. Introduction In this study we determine the impact of a dedicated trauma desk on the identifi cation of patients suff ering from major trauma and on time to allocate critical care resources. Trauma is increasingly recognised as a serious global health problem and is the leading cause of death in those under the age of 45, accounting for 1,300 deaths in Scotland each year [1]. In addition, trauma causes considerable short-term and long-term morbidity and has personal, social and fi nancial impact on the population [2]. Evidence and expert opinion strongly support the presence of appropriately trained, clinically active, personnel integrated and fundamental to the command and control structure in order to optimise the right resource being dispatched to the right patient at the right time.f y Conclusion Within this large cohort of consecutive unselected medical patients seeking ED care, the MTS instrument in combination with a prognostic biomarker (ProADM) allowed accurate initial risk assessment in regard to treatment priority, ICU admission and mortality. Predicting outcomes after blunt chest wall trauma: development and external validation of a new prognostic model l 1 1 h 2 1 C Battle1, S Lovett1, H Hutchings2, PA Evans1 C Battle1, S Lovett1, H Hutchings2, PA Evans1 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2014, 18(Suppl 1):P64 (doi: 10.1186/cc13254) Critical Care 2014, 18(Suppl 1):P64 (doi: 10.1186/cc13254) Introduction Blunt chest wall trauma accounts for over 15% of all trauma admissions to emergency departments worldwide [1]. Reported mortality rates vary between 4 and 60% [2]. Management y Results The survival rate at day 7 in the BMMNC group was 80.0%, and that in the CR group was 47.6%, revealing that the 7-day survival S24 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 System (MTS), have not been well validated in unselected medical patients. Herein, we performed a prospective cohort study to assess the prognostic potential of the MTS and the prognostic biomarker pro- adrenomedullin (ProADM) to identify patients at high initial treatment priority, patients with admission to the ICU, and patients who die within a 30-day follow-up. was signifi cantly improved by the BMMNC injection (P <0.05). Crush injury-induced upregulation of serum IL-6 was signifi cantly reduced by the BMMNC treatment at all time points (P <0.05). The level of TNFα decreased signifi cantly in the BMMNC group compared with that in the CR group 24 hours after the compression release (P <0.05). These fi ndings suggest that transplantation of BMMNCs has an ability to evade the devastating condition following crush injury by suppressing systemic infl ammation. y p Methods This is a prospective, observational cohort study including all consecutive medical patients seeking ED care between June 2013 and October 2013, except nonadult and nonmedical patients. We collected detailed clinical information including the initial MTS and measured ProADM levels on admission in all patients. Initial treatment priority was adjudicated by two independent, blinded physicians based on all available results at the time of ED discharge to the medical ward. To assess outcomes, data from electronic medical records were used and all patients were contacted by telephone 30 days after hospital admission. The prognostic performance of MTS and ProADM was assessed in multivariate regression models with area under the receiver operating curve (AUC) as an overall measure of discrimination. P68 Since September 2013, we newly developed k-support to the Kainan Fire Department Mugi branch offi ce, Kainan Fire Department Hiwasa branch offi ce, Kaifu Fire-fi ghting Union Kainan fi re department, and Muroto Fire-Fighting Headquarters Touyou branch offi ce, and reviewed our experience of this approach in the use of k-support during the fi rst 3 months of the introduction. Results k-support was used for 62 patients, and 13 (21.0%) of them were categorized as neurosurgical diseases. The detail of neurosurgical diseases consisted of six patients (46.1%) with head injury, followed by fi ve patients (38.5%) with cerebral infarction, one patient (7.7%) with cerebral hemorrhage and one patient (7.7%) with miscellaneous diseases. We shared the information by ambulance services to tweet the patient information or transmit vital signs and electrocardiograms. We experienced a case that was diagnosed with cardiogenic embolism and tried the ‘drip and ship’ method of rt-PA. The outcomes were as follows: hospitalization, 28 (45.2%); discharge, 18 (29.0%); and transfer, 16 (25.8%). Conclusion Introduction of k-support is the fi rst trial in Japan. For emergency diseases such as ischemic heart diseases and stroke, we could perform responses earlier than ever by providing this system. Conclusion A clinician focusing on major trauma in the ambulance control room improves activation times of prehospital critical care teams. The number of patients receiving life-saving and limb-saving interventions has substantially increased. The stand-down rate of teams following activation by the trauma desk is considerably reduced. References 1. Major Trauma in Scotland. Edinburgh: Royal College of Surgeons; 2012. 2. Scottish Trauma Audit Group – Trauma Report, Edinburgh: NHS National Services Scotland, Information Services Division Publications; 2012. 1. Major Trauma in Scotland. Edinburgh: Royal College of Surgeons; 2012. 2. Scottish Trauma Audit Group – Trauma Report, Edinburgh: NHS National Services Scotland, Information Services Division Publications; 2012. pp gi Results k-support was used for 62 patients, and 13 (21.0%) of them were categorized as neurosurgical diseases. The detail of neurosurgical diseases consisted of six patients (46.1%) with head injury, followed by fi ve patients (38.5%) with cerebral infarction, one patient (7.7%) with cerebral hemorrhage and one patient (7.7%) with miscellaneous diseases. We shared the information by ambulance services to tweet the patient information or transmit vital signs and electrocardiograms. We experienced a case that was diagnosed with cardiogenic embolism and tried the ‘drip and ship’ method of rt-PA. Predicting outcomes after blunt chest wall trauma: development and external validation of a new prognostic model l 1 1 h 2 1 yl Conclusion The administration of BMMNCs reduced production of infl ammatory cytokines and improved survival rate in a rat model of crush injury. Cell therapy using BMMNCs might become a novel therapy against crush injury. P68 The outcomes were as follows: hospitalization, 28 (45.2%); discharge, 18 (29.0%); and transfer, 16 (25.8%). Conclusion Introduction of k-support is the fi rst trial in Japan. For emergency diseases such as ischemic heart diseases and stroke, we could perform responses earlier than ever by providing this system. P68 P68 Introduction of the Kaifu telemedicine system for emergency medicine to ambulance services with improvement of the survival rates F Obata1, R Tabata2, K Mori1, T Kageji3, K Tani2, H Bando1 1Tokushima Prefectural Kaifu Hospital, Tokushima, Japan; 2Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; 3Tokushima University Hospital, Tokushima, Japan Critical Care 2014, 18(Suppl 1):P68 (doi: 10.1186/cc13258) Results There were 190 activations of critical care teams during the study period compared with 73 from the historical data from the same time frame in the previous year, representing a 160% increase in activations. The mean time from emergency call to allocation of critical care resource was 6 minutes compared with 19 minutes from historical data. The stand-down rate for prehospital critical care resource from emergency calls identifi ed by trauma desk clinicians was 31.5% compared with 56% for those by nontrauma desk clinicians or dispatchers. Introduction Our hospital has introduced the telemedicine system for emergency medicine (k-support) using a smart device to rectify health disparity and reduce the burden to general physicians since February 2013. We have expanded to the ambulance services’ k-support toward further survival rate improvement. The mean time from emergency call to allocation of critical care resource was 6 minutes compared with 19 minutes from historical data. The stand-down rate for prehospital critical care resource from emergency calls identifi ed by trauma desk clinicians was 31.5% compared with 56% for those by nontrauma desk clinicians or dispatchers. p Methods The registration device, patient information and image information such as magnetic resonance imaging and computed tomography taken in the hospital were transferred in real time to k-support using the smart device. Since September 2013, we newly developed k-support to the Kainan Fire Department Mugi branch offi ce, Kainan Fire Department Hiwasa branch offi ce, Kaifu Fire-fi ghting Union Kainan fi re department, and Muroto Fire-Fighting Headquarters Touyou branch offi ce, and reviewed our experience of this approach in the use of k-support during the fi rst 3 months of the introduction. Methods The registration device, patient information and image information such as magnetic resonance imaging and computed tomography taken in the hospital were transferred in real time to k-support using the smart device. Impact of a dedicated trauma desk in ambulance control on the identifi cation of major trauma in Scotland A combined score has the potential to signifi cantly improve initial risk assessment in patients, which may translate into faster and more targeted care and better clinical patient outcomes. g g g Methods From October 2012 to April 2013 a trauma desk, staff ed by experienced paramedics, was created in ambulance control with access to all emergency calls across Scotland. The operational hours of the desk were 08:00 to 18:00, 7 days a week. A customised database provided a data entry portal. Retrospective data were collated for comparison. Primary outcome measures were the number of emergency calls identifi ed as potential major trauma and the time to allocation of critical care resources. Secondary outcome measures were the eff ects on stand-down rates of prehospital critical care teams. Evaluation and prevention of violence in the emergency department in Lebanon Evaluation and prevention of violence in the emergency department in Lebanon p Z Fadel, N Hachem, C El Ramy, G Abi Nader, D Haykal Sainte Famille University, Batroun, Lebanon Critical Care 2014, 18(Suppl 1):P71 (doi: 10.1186/cc13261) T Yamashita1, K Yamazumi2, H Takayama3, Y Sakamoto1 1Saga University, Saga, Japan; 2Ureshino Medical Center, Ureshino, Saga, Japan; 3Nagasaki Medical Center, Omura, Nagasaki, Japan Critical Care 2014, 18(Suppl 1):P69 (doi: 10.1186/cc13259) Introduction At the hospital we are faced with situations of violence, whether verbal or physical, especially in the emergency department (ED) [1]. The objective of this study is to evaluate the phenomenon of violence at hospitals in Lebanon, especially in the ED, and to recommend techniques to prevent it. Introduction Japanese emergency medical technicians are not allowed to perform some advanced medical practices such as a tracheal intubation, establishment of an intravenous line, administration of a drug to a patient who retains their own circulation. So medical staff have to be dispatched to the accident scene to give a patient such medical practices. In Japan, we have developed and dispersed the educational course of the Disaster Medical Assistance Team since 2005. But the role of medical teams at the scene is not widely known among rescue workers. It is also distant for medical staff to coordinate rescue teams, because most of them do not work with rescue workers in their daily work. We have felt a need for practical training to achieve the coordination of rescue and medical teams. Methods A questionnaire consisting of 18 questions was sent to the caregivers in the ED of three randomly selected hospitals in Beirut, Lebanon in 2012. A total of 111 people (nurses, aides, doctors, interns, residents, social workers and security guards) responded to the survey questionnaire. Results The majority of the surveyed people are young women (62%) aged between 20 and 40 years (78%) with a nursing degree (74%) and professional experience <5 years (48%). In total, 59% of respondents have experienced violence in the ED during the night (58%) from the patients (31%) or their companions (68%). The caregivers most aff ected by the violence are nurses (54%) and employees of reception (46%). Violence can be verbal (threats 47%, insults 36%, criticism 18%) or physical (hitting 43%, slapping 40%, stabbing 17%). P72 P70 Complementary cooperation of an ambulance helicopter and car with medical doctors: meaning of simultaneous dispatch K Ishii, K Kurosawa, S Tanabe, T Noguchi Oita University Hospital, Oita, Japan Critical Care 2014, 18(Suppl 1):P70 (doi: 10.1186/cc13260) P72 Epidemiology and critical care management of patients admitted after intentional self-poisoning GP Misselbrook, N Sudhan Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P72 (doi: 10.1186/cc13262) Introduction Recently, the dispatch system with a medical doctor in pre-hospital care has made rapid progress in Japan. We usually use an ambulance helicopter or an ambulance car/rapid response car for dispatch. In this report, we analyzed the cases in which the medical doctors were dispatched using both means at the same time. Introduction Intentional self-poisoning is one of the most common presentations to acute medical units across the UK [1]. To our knowledge no studies have been published on incidence of admissions to critical care in England after overdose. Our aim was to investigate the epidemiology, clinical features and outcomes of patients admitted to critical care after intentional self-poisoning to establish patterns in our community. Methods We analyzed 29 cases with simultaneous dispatch with the medical doctors using ambulance helicopter and car during October 2012 to September 2013. Methods We performed a retrospective data collection using our critical care database ‘Metavision’ to select all patients admitted with a diagnosis of ‘overdose’. Records were scrutinised to collect information on patient demographics, clinical features and medical management. Results Thirty-eight patients (male:female ratio 1:1.53) were admitted to critical care over a 1-year period (September 2011 to 2012). This represented 2.45% of the total admissions to critical care during the same period. The sample had a signifi cantly younger median age (45 years) than the standard patient population in critical care (68 years) during the same period (P <0.0001). Despite the young age and paucity of comorbidities, there was no diff erence in length of stay between overdose patients (2.0 days) and all other patients on critical care (1.58 days, P = 0.3). The median number of agents ingested was three Methods We performed a retrospective data collection using our critical care database ‘Metavision’ to select all patients admitted with a diagnosis of ‘overdose’. Records were scrutinised to collect information on patient demographics, clinical features and medical management. Results The average distance of dispatch was 25.6 km. The Manchester Triage System in optimizing triage in adult general medical emergency patients: the Triage Projectf g y g j D Steiner, A Kutz, AC Rast, S Haubitz, L Faessler, M Batschwaroff , U Buergi, B Mueller, P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P67 (doi: 10.1186/cc13257) Introduction Some patients presenting to the emergency department (ED) currently face inacceptable delays in initial treatment due to suboptimal initial triage. Triage scores, such as the Manchester Triage S25 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P69 Training to achieve coordination of rescue and ambulance and medical teams T Yamashita1, K Yamazumi2, H Takayama3, Y Sakamoto1 1Saga University, Saga, Japan; 2Ureshino Medical Center, Ureshino, Saga, Japan; 3Nagasaki Medical Center, Omura, Nagasaki, Japan Critical Care 2014, 18(Suppl 1):P69 (doi: 10.1186/cc13259) P71 Evaluation and prevention of violence in the emergency department in Lebanon The dissatisfaction of the patient in his care (42%) and his anxiety (33%) are the most important factors in the generation of violence that may have repercussions on the care workers and their psychological status. Methods We held a disaster-relief training workshop from 2010 to 2013. Prior to starting the drill, we gave two lectures about the role of a medical team in a rescue site and the basic knowledge of rescue skills. The drill was organized for 1 to 1.5 hours. The scenario was secret for participants. Before and after the workshop, we had a questionnaire survey for attitude changes about a collaborative work between a rescue team and a medical team. Results A total of 160 people participated in the workshop (63 rescue workers, 27 ambulance workers, 33 paramedics, 37 medical staff ). At fi rst, rescue workers tended to downplay the importance about the division of roles or the establishment of command and control system between rescue teams and medical teams, but their attitude changed after the drill (P <0.05). They also understood the need to know about basic trauma survey skills and some medical terms. Conclusion Violence in the ED may be due to the heavy workload of the caregivers causing a delay in care. Secondly, patients in the ED may feel insuffi ciently informed and heard by the nursing staff . The priority given to emergencies depending on the severity and not the order of arrival can be misunderstood [2]. Therefore, we recommend the following actions: encourage caregivers to improve their knowledge and training on the management of patients in emergency situations; train emergency caregivers to mediation, nonviolent communication and managing stressful situations; and increase the number of nurses and security guards in the ED and motivate them to ensure a better quality of care and minimize the delay in their care. y Conclusion To achieve mutual understanding, it is important to have drills in which both rescue teams and medical teams participate. 1. Prudhomme C: L’infi rmière et les urgences. Paris: Maloine; 2000:422. 2. Goddet B: La violence à l’hôpital, in Soins, Pratique et savoir infi rmier. Masson 1998, 624:3-23. P70 P72 The corres- pondence to the plural patients was three trauma cases. In nine cases, the ambulance car was fi rst selected for the dispatch vehicle and the ambulance helicopter was added. In three cases, both devices were canceled on the dispatch way to the scene. In seven cases, the ambulance car was canceled. In 10 cases, the patients were transferred by the ambulance helicopter. Results Thirty-eight patients (male:female ratio 1:1.53) were admitted to critical care over a 1-year period (September 2011 to 2012). This represented 2.45% of the total admissions to critical care during the same period. The sample had a signifi cantly younger median age (45 years) than the standard patient population in critical care (68 years) during the same period (P <0.0001). Despite the young age and paucity of comorbidities, there was no diff erence in length of stay between overdose patients (2.0 days) and all other patients on critical care (1.58 days, P = 0.3). The median number of agents ingested was three Conclusion In the selection of the dispatch means, the time to contact with the patient is the most important factor. However, the decision of the suitable means is occasionally quite diffi cult at the moment of fi rst information. The ambulance helicopter and car were complementary to each other in the dispatch mean of the medical doctors. S26 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (1 to 7) with 84.2% ingesting ≥2 agents. Hypnotics and antidepressants made up 45% of the agents ingested. A total 92.1% of the sample was admitted out-of-hours or at weekends. Fifty per cent had a past history of overdose, 25% had a history of alcohol misuse. A total of 79% of patients were referred to critical care due to a low conscious level but only 50% required IPPV and 20% received vasopressors/inotropes. The mortality rate was 2.6%, with one further death 6 months after discharge due to alcoholic liver disease. Estimated fi nancial cost was £80,555 or £2,119 per patient (57 level 3 bed-days, 35 level 2 bed-days). Conclusion Intentional self-poisoning mortality rates are low in spite of the number of patients admitted. P73 Price per unit: the cost of alcohol-related admissions to a regional ICU R Matthews, A Revill Introduction The objective was to analyze the clinical features of multiple organ dysfunction syndrome (MODS) induced by severe heat stroke, and to investigate the pathogenesis, diagnosis and prevention strategies in patients with MODS caused by severe heat stroke. Introduction The objective was to analyze the clinical features of multiple organ dysfunction syndrome (MODS) induced by severe heat stroke, and to investigate the pathogenesis, diagnosis and prevention strategies in patients with MODS caused by severe heat stroke. Methods We retrospectively studied nine cases of MODS caused by severe heat stroke, and systemically reviewed the relevant literature. Results (1) Nine patients with MODS caused by severe heat stroke were all exposed in hot and humid environments. All nine patients are severe heat stroke, including seven cases of classic heat stroke (77.8%) and two cases of exertional heat stroke (22.2%). (2) Among nine cases of MODS caused by severe heat stroke, eight patients met the diagnostic criteria for SIRS. In addition, there was a signifi cant increase in blood PMN proportion and serum CRP of all nine patients. Of note, there was also a marked increase in serum IL-6 (average (36 ± 19) pg/ml; reference range (0 to 5.9 pg/ml)); and TNFα level (average (21 ± 10) pg/ml; reference range (0 to 8.1 pg/ml)), while IL-8 was normal (average (22 ± 25) pg/ ml; reference range (0 to 62 pg/ml)). (3) There were 34 organ damages involved in all nine patients with MODS induced by severe heat stroke. The kidney, circulatory and liver accounted for 58.8% of all these organ dysfunctions. The incidence of severe heatstroke-induced organ dysfunction in turn was the kidney, circulation, liver, blood coagulation, metabolism, brain, lungs, and gastrointestinal tract. (4) During hospitalization, the common complication was pulmonary infection in patients with MODS caused by heat stroke. (5) After early intensive care of organ supportive treatment, there was a quick improvement and recovery in most cases in several days. Seven patients survived, and the average length of stay in hospital was 9.5 days. Introduction A number of studies have demonstrated the signifi cant and increasing morbidity and mortality associated with alcohol-related disease in the UK, and the corresponding impact this has on critical care services [1]. Reference 1. Clark D, et al.: Epidemiology and outcomes of patients admitted to critical care after self-poisoning. JICS 2011, 12:268-273. J Liu1, G Zou1, Y Wu1, W Li2 J Liu , G Zou , Y Wu , W Li 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Critical Care 2014, 18(Suppl 1):P74 (doi: 10.1186/cc13264) P73 Price per unit: the cost of alcohol-related admissions to a regional ICU R Matthews, A Revill Plymouth Hospitals NHS Trust, Plymouth, UK Critical Care 2014, 18(Suppl 1):P73 (doi: 10.1186/cc13263) P72 Despite the young age of patients and lack of comorbidities, their length of stay is similar to the average length of stay for all patients admitted to the unit, representing a signifi cant fi nancial cost. Self-poisoning requiring critical care support is more common out-of-hours when less senior expertise is available. Education of junior doctors into management of overdose is therefore vital to ensure the early identifi cation and appropriate treatment of these patients. number of admissions per month varied from zero in May to a peak of 14 in November, with the majority (40%) of admissions occurring over the autumn months (Figure 1). Eighty-nine per cent of patients with alcohol-related conditions required support for at least two organ failures, which subsequently equated to an overall cost to the unit of £725,308 and 12% of an approximate £6 million annual budget. Conclusion The results of this study support the hypothesis that alcohol-related disease contributes considerably to admissions to this ICU. Furthermore, they have shown that the fi nancial impact was proportionally greater than the percentage number of admissions attributable to alcohol consumption, refl ecting the high frequency of multiorgan failure in this patient cohort. References number of admissions per month varied from zero in May to a peak of 14 in November, with the majority (40%) of admissions occurring over the autumn months (Figure 1). Eighty-nine per cent of patients with alcohol-related conditions required support for at least two organ failures, which subsequently equated to an overall cost to the unit of £725,308 and 12% of an approximate £6 million annual budget. pp g Conclusion The results of this study support the hypothesis that alcohol-related disease contributes considerably to admissions to this ICU. Furthermore, they have shown that the fi nancial impact was proportionally greater than the percentage number of admissions attributable to alcohol consumption, refl ecting the high frequency of multiorgan failure in this patient cohort. R f References 1. Geary T, et al.: Anaesthesia 2012, 67:1132-1137. 2. Jones L, et al.: Alcohol-attributable Fractions for England. Liverpool: Centre for Public Health, Liverpool John Moores University; June 2008. P73 Price per unit: the cost of alcohol-related admissions to a regional ICU This study was designed to similarly evaluate the current burden of alcohol-related disease on admission rates to a regional ICU, but also to calculate the fi nancial costs of such admissions.i i Methods Data-entry fi elds derived from recommendations in a public heath publication [2] were introduced to the local electronic records system to enable prospective data collection for all admissions that were either wholly or partially attributable to alcohol consumption. Using locally defi ned values for the cost per day of an admission to the ICU, depending on the maximum level of organ support required during the admission, it was possible to calculate the total expense incurred by the unit for each alcohol-related admission. y Results In 1 year from December 2012 to November 2013 inclusive, the ICU recorded 84 alcohol-related admissions, accounting for approximately 9% of annual unplanned admissions. With an average length of stay (5.8 days) similar to that of all other unplanned admissions, this totalled 534 ICU bed-days. A total of 86% of patients with alcohol-related conditions were male with an average age of 46.4 years (range 15 to 83 years), and the majority (42%) presented with chronic conditions partially attributable to alcohol consumption. The Figure 1 (abstract P73). Conclusion Severe heat stroke results in signifi cant abnormal changes in infl ammatory markers in patients with MODS induced by heat stroke. The types of organ dysfunction in heat stroke-induced MODS are usually distinct from those of infection and trauma. After active cooling and intensive organ function supportive treatment, most patients recovered in a relatively short period. Acknowledgements Supported by the Research Projects CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. Eff ect of low-dose hydrocortisone on gene expression profi les after severe burn injury J Plassais1, MA Cazalis1, F Venet2, G Monneret2, A Pachot1, S Tissot2 1Joint Unit Hospices Civils de Lyon/bioMérieux, Lyon, France; 2Hospices Civils de Lyon, Hôpital Edouard Herriot, Lyon, France Critical Care 2014, 18(Suppl 1):P75 (doi: 10.1186/cc13265) Eff ect of low-dose hydrocortisone on gene expression profi les after severe burn injury J Plassais1, MA Cazalis1, F Venet2, G Monneret2, A Pachot1, S Tissot2 1Joint Unit Hospices Civils de Lyon/bioMérieux, Lyon, France; 2Hospices Civils de Lyon, Hôpital Edouard Herriot, Lyon, France Critical Care 2014, 18(Suppl 1):P75 (doi: 10.1186/cc13265) P77 Low socioeconomic status, ethnicity and geographical location confers high risk of signifi cant accidental burns injuries in London JS Heng, O Clancy, I Jones, J Atkins, J Leon-Villapalos, A Williams, M Hayes, M Vizcaychipi Chelsea & Westminster Hospital, London, UK Critical Care 2014, 18(Suppl 1):P77 (doi: 10.1186/cc13267) Results Severe burn injury induced the deregulation of a considerable number of genes (n >2,200 at S1) in comparison with controls with an increased number of deregulated genes over time. Within burn patients, more than 300 genes were deregulated by hydrocortisone over time. The treatment had a rapid eff ect on gene expression, 339 and 627 genes were diff erentially expressed at S2 and S3 respectively. However, the number of these genes decreased drastically at S4 (only 24 genes signifi cant). The genes identifi ed at S2 were mostly related to the decrease of growth, development and quantity of leukocytes but these biological processes were not found signifi cant at S3, indicating that the action of glucocorticoid in the response to burn injury is short lived and time dependent. Introduction The majority of burns injuries are considered accidental, although previous studies have identifi ed demographic factors associated with higher risk of burns such as socioeconomic deprivation [1] and being from ethnic minority groups [2]. This study aims to identify population subgroups in London at high risk of burns injuries requiring admission to a burns centre through geographic mapping and socioeconomic statistics. Conclusion This study is an informative overview of the genomic responses after burn injuries. More importantly, it is the fi rst study providing information about mechanisms involved in glucocorticoid’s reduced shock duration after burn. Methods Records of all paediatric and adult inpatients admitted to the burns centre at Chelsea and Westminster Hospital were retrospectively reviewed for age, ethnic group and deprivation score of residence, as measured by the English Index of Multiple Deprivation 2010. Corresponding population data for London were obtained. Reference 1. Keck M, et al.: Wien Med Wochenschr 2009, 159:13-14. Results In total, 2,195 patients from London were admitted between January 2009 and August 2013, with 1,963 (89.4%) classifi ed as having accidental injuries. A total 1,725 (87.8%) of accidental burn injuries occurred in the patients’ own homes. Patients from ethnic minorities have the highest rate of burn injury at 7.1 per 100,000 population per annum (P <0.0001). Patients below the median for socioeconomic deprivation in London are more likely to suff er burn injuries (P <0.0001). Patients from the most deprived quartile are more likely to suff er burns injuries of >10% TBSA (P = 0.04), and have a trend towards higher rates of ICU admission (P = 0.144). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P76). Numbers of complex burns in Scotland 2010 to 2012 2010 2011 2012 Adult 304 392 399 Paediatric 195 185 138 Adult >15% 27 28 26 Paediatric >15% 0 2 4 Mortality 5 7 13 Table 1 (abstract P76). Numbers of complex burns in Scotland 2010 to 2012 shock. Patients need vasopressor infusion to maintain adequate delivery of oxygen but it could have deleterious eff ects on skin perfusion and worsen the burn depth. This shock could result from the interplay of the initial hypovolemia and the release of multiple infl ammatory mediators [1]. It has been shown that a low-dose of hydrocortisone could reduce the shock duration but the mechanisms involved remain unclear. We investigated the systemic genomic response after severe burn injuries and determine whether patterns of gene expression could be associated with a low dose of glucocorticoids. shock. Patients need vasopressor infusion to maintain adequate delivery of oxygen but it could have deleterious eff ects on skin perfusion and worsen the burn depth. This shock could result from the interplay of the initial hypovolemia and the release of multiple infl ammatory mediators [1]. It has been shown that a low-dose of hydrocortisone could reduce the shock duration but the mechanisms involved remain unclear. We investigated the systemic genomic response after severe burn injuries and determine whether patterns of gene expression could be associated with a low dose of glucocorticoids. Methods Thirty burn patients with over 30% of total body surface area were enrolled into a randomized double-blind clinical study. Fifteen patients were treated with a low dose of hydrocortisone and 15 patients were treated with placebo. Whole blood samples were collected after shock onset (S1) before any treatment, 1 day after treatment beginning (S2), and 120 hours and 168 hours after the burn injury (S3/S4). Blood samples of 13 healthy volunteers were collected. Pangenomic expression was evaluated with Aff ymetrix HG-U133plus 2.0 microarrays. Moderated t tests and F test were used to compare burn patients with controls and gene expression profi les between the two groups (B–H correction, P <0.05). sources. As data quality improves, detailed analysis of mortality data will allow COBIS to identify contributing issues aff ecting burns patients. Some issues identifi ed already are that patients with burns often die soon after their discharge from hospital of other related and unrelated causes. P77 Low socioeconomic status, ethnicity and geographical location confers high risk of signifi cant accidental burns injuries in London JS Heng, O Clancy, I Jones, J Atkins, J Leon-Villapalos, A Williams, M Hayes, M Vizcaychipi Chelsea & Westminster Hospital, London, UK Critical Care 2014, 18(Suppl 1):P77 (doi: 10.1186/cc13267) Domestic accidental burns were mapped to their respective administrative wards, and the rate of burns per 100,000 was calculated and divided into quintiles. The areas with the top quintile of burn injury rate, of up to 18.8 per 100,000 population per year, were almost four times the national average. Eff ect of low-dose hydrocortisone on gene expression profi les after severe burn injury Introduction The systemic eff ect of infl ammatory mediators released after severe burn is a severe cardiovascular dysfunction called burn Introduction The systemic eff ect of infl ammatory mediators released after severe burn is a severe cardiovascular dysfunction called burn S27 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Subsequent analysis of this will allow COBIS to identify and address issues that may be contributing to these statistics. Care of Burns in Scotland: 3-year data from the Managed Clinical Network National Registry CJ Gilhooly1, J Kinsella2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University of Glasgow, UK Critical Care 2014, 18(Suppl 1):P76 (doi: 10.1186/cc13266) Care of Burns in Scotland: 3-year data from the Managed Clinical Network National Registry CJ Gilhooly1, J Kinsella2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University of Glasgow, UK Critical Care 2014, 18(Suppl 1):P76 (doi: 10.1186/cc13266) g CJ Gilhooly1, J Kinsella2i Introduction The Managed Clinical Network for Care of Burns in Scotland (COBIS) was launched in April 2007. Primary aims included establishing and maintaining a registry of complex burn injury in Scotland and setting mechanisms to regularly audit outcome of burn treatment against nationally agreed standards of care. On behalf of COBIS, we present 3-year incidence and mortality data of Scottish patients admitted with a complex burn injury in this abstract. y Conclusion Ethnicity, socioeconomic deprivation and geographical location appear to be risk factors for burn injuries. Identifying such groups may allow the development of targeted preventative strategies. References Methods From January 2010 onwards, data were prospectively collected for all patients in Scotland with complex burn injury admitted to Scottish burns units. Data collection was initially on a paper pro forma, but subsequently evolved into a web-based audit data capture system to securely link hospital sites involved in the delivery of care of complex burns. Data collected included extent and mechanism of burn, presence of airway burn or smoke inhalational injury, comorbidities, complications, length of stay, interventions and mortality. Quality, completeness and consistency of data collection are audited with feedback to the individual units. 1. Mistry RM, Pasisi L, Chong S, Stewart J, She RB: Socioeconomic deprivation and burns, Burns 2010, 36:403-408. 1. Mistry RM, Pasisi L, Chong S, Stewart J, She RB: Socioeconomic deprivation and burns, Burns 2010, 36:403-408. 1. Mistry RM, Pasisi L, Chong S, Stewart J, She RB: Socioeconomic deprivation and burns, Burns 2010, 36:403-408. 2. Tan KT, Prowse PM, Falder S: Ethnic diff erences in burn mechanism and severity in a UK paediatric population, Burns 2012, 38:551-555. 2. Tan KT, Prowse PM, Falder S: Ethnic diff erences in burn mechanism and severity in a UK paediatric population, Burns 2012, 38:551-555. 2. Tan KT, Prowse PM, Falder S: Ethnic diff erences in burn mechanism and severity in a UK paediatric population, Burns 2012, 38:551-555. P78f Survey of severe sepsis and septic shock management in Thailand: THAI-SHOCK SURVEY 2013 K Chittawatanarat1, B Patjanasoontorn2, S Rungruanghiranya3, Thai SCCM Study Group4 1Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 2Khon Khaen University, Khon Khaen, Thailand; 3Srinakharinwirot University, Nakornnayok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) Central venous pressure (CVP) and fl uid challenge test were more frequently used for preload evaluation than new fl uid responsiveness methods. Less than 15% still used a pulmonary artery catheter in their routine practice. Antipyretics in the emergency department – intravenous paracetamol versus intramuscular diclofenac: a comparative study N Purayil, N Vamanjore, F Paramba, O Mohammad, P Chandra Hamad Medical Corporation, Doha, Qatar Critical Care 2014, 18(Suppl 1):P79 (doi: 10.1186/cc13269) Introduction Fever is a common problem in adults visiting the emergency department (ED) [1]. Although it is important to treat the cause of fever, symptomatic management of fever is also necessary. Multiple studies are available on the effi cacy of various antipyretics in children, but very little has been done in adults [1,2]. Hence we aimed to compare the antipyretic effi cacy of intravenous (i.v.) paracetamol and intramuscular (i.m.) diclofenac in adult patients presenting with fever to the ED. Methods In this parallel-group, open-label trial, participants aged 14 to 75 years who had a temperature more than 38.5°C were enrolled and treated in the ED, Alkhor, Hamad Medical Corporation, during the period June 2008 to December 2011. Patients were randomly assigned to receive either 1,000 mg i.v. paracetamol or 75 mg i.m. diclofenac. The primary outcome was degree of reduction in mean oral temperature at 90 minutes and the secondary outcomes were degree of reduction at 30, 60 and 120 minutes. The effi cacy of drugs was assessed by a superiority comparison. Analysis was done using intention-to-treat (ITT) principles. Conclusion Most physicians manage shock with protocols. Hemodynamic endpoints are preferred to tissue perfusion targets. Early antimicrobial therapy and de-escalation are routine practices without use of infective biomarkers. Crystalloid is preferred rather than colloid at initial resuscitation. CVP and fl uid challenge are still more popular than new fl uid responsiveness methods on preload assessment. Hydrocortisone is the most common steroid prescription in septic shock but the threshold of initiation, frequency and discontinuation are varied. Results In total, 139 patients received i.v. paracetamol and 150 received i.m. diclofenac. The mean age was 35.5 ± 14.24 and 36.4 ± 14.98 years in the i.m. diclofenac and i.v. paracetamol groups respectively. The majority of the patients were males in both groups. After 90 minutes both groups showed a signifi cant reduction in mean temperature, i.m. diclofenac showing a greater reduction (–1.44 ± 0.43(95% CI –1.4, –2.5)) than i.v. Survey of severe sepsis and septic shock management in Thailand: THAI-SHOCK SURVEY 2013 K Chittawatanarat1, B Patjanasoontorn2, S Rungruanghiranya3, Thai SCCM Study Group4 1Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 2Khon Khaen University, Khon Khaen, Thailand; 3Srinakharinwirot University, Nakornnayok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) Introduction A pragmatic survey of shock management in Thai physicians is unavailable. The objective of this study is to identify shock management patterns for severe sepsis and septic shock in Thailand. Methods Two thousand questionnaires were sent to physicians involved in caring for shock patients across Thailand. The frequency scale was defi ned as fi ve levels by patient proportion estimation at routine practice. Introduction A pragmatic survey of shock management in Thai physicians is unavailable. The objective of this study is to identify shock management patterns for severe sepsis and septic shock in Thailand. Methods Two thousand questionnaires were sent to physicians involved in caring for shock patients across Thailand. The frequency scale was defi ned as fi ve levels by patient proportion estimation at routine practice. g p p p Methods Two thousand questionnaires were sent to physicians involved in caring for shock patients across Thailand. The frequency scale was defi ned as fi ve levels by patient proportion estimation at routine practice. Conclusion Performing noncontrast abdominal MDCT to the patient with renal insuffi ciency for evaluating acute abdominal symptoms, severe sepsis or septic shock in the emergency department was feasible and eff ective. Results Between April and August 2013, a total of 533 questionnaires (26.7%) were returned. For severe sepsis and septic shock management, a total of 406 physicians (76.2%) reported their routine usage of quantitative resuscitation protocols. Urine output, mean arterial pressure and central venous pressure are more frequently used than central venous oxygen saturation and lactate as the resuscitation endpoint. Nearly 80% of these had an ‘often and always’ resuscitation goal within 6 hours. Most physicians (65.3%) never used procalcitonin. The antimicrobial empirical treatments were started within 1 hour for 87.7% and these were continued less than 5 days in 67.3% before de- escalation. Crystalloid was the common initial fl uid therapy in 98.9%. The most common used vasopressor was norepinephrine (69.6%). The median of cortisol threshold level for steroid replacement therapy was 15 (interquartile range, 5 to 19) mg/dl. Almost all of the physicians used hydrocortisone (96.4%). The median daily dose of hydrocortisone was 300 mg (interquartile range, 200 to 300). Nearly 50% divided the dose every 8 hours and 31.8% infused continuously. The duration for tapering was varied (33.6% in 2 to 3 days). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ease of administration, i.m. diclofenac can be used as a fi rst-choice antipyretic in febrile adults in the ED. References ease of administration, i.m. diclofenac can be used as a fi rst-choice antipyretic in febrile adults in the ED. References symptoms or searching for an infection focus such as sepsis. But contrast dye can cause renal damage especially in critically ill patients. Noncontrast CT can be alternative options but few studies have shown the eff ectiveness of noncontrast MDCT. We want to evaluate the eff ectiveness of noncontrast abdominal MDCT in the emergency department. symptoms or searching for an infection focus such as sepsis. But contrast dye can cause renal damage especially in critically ill patients. Noncontrast CT can be alternative options but few studies have shown the eff ectiveness of noncontrast MDCT. We want to evaluate the eff ectiveness of noncontrast abdominal MDCT in the emergency department. 1. Karbasi SA, et al.: Comparison of antipyretic eff ectiveness of equal dose of rectal and oral acetamenophen in children. J Pediatr (Rio J) 2010, 86:228-233. Methods This is a retrospective chart review study conducted in a single tertiary academic hospital from January 2011 to April 2012. Patients were enrolled if they visited the emergency department with acute abdominal symptoms or infection signs, had elevated serum creatinine level, and received noncontrast 16-channel MDCT.fi 2. Pernerstofer et al.: Acetaminophen has a greaterantipyretic effi cancy thanaspirin in endotoxemia. A randomised double blind placebo controlled trail. Clin Pharmacol Ther 1999, 66:51-57. Results During study period, 78 patients with renal insuffi ciency received noncontrast abdominal CT. Causes of CT were infection focus (48, 61.5%), abdominal pain (21, 26.9%), GI bleeding (4, 5.2%), abnormal labs (3, 3.9%), ureter stone (1, 1.3%) and abdominal distension evaluation (1, 1.3%). Any abnormal CT fi ndings were detected in 61 (78.2%) patients. For 42 (53.8%) patients, noncontrast CT fi ndings showed diagnostic abnormal fi ndings. For excluding abdominal pathology, 35 (44.9%) were helpful. In one (1.6%) case with hematochezia, noncontrast CT showed no benefi t for patient diagnosis. Additional contrast-enhanced CTs were performed in 32 (41%) and additional fi ndings were found in fi ve cases (6.4%). There were 40 patients with severe sepsis or septic shock. Abnormal CT fi ndings were found in 30 (75%) cases. Additional contrast enhanced CT was done for 13 (32.5%) patients but no additional information was detected. P80 Survey of severe sepsis and septic shock management in Thailand: THAI-SHOCK SURVEY 2013 K Chittawatanarat1, B Patjanasoontorn2, S Rungruanghiranya3, Thai SCCM Study Group4 1Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 2Khon Khaen University, Khon Khaen, Thailand; 3Srinakharinwirot University, Nakornnayok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) Survey of severe sepsis and septic shock management in Thailand: THAI-SHOCK SURVEY 2013 K Chittawatanarat1, B Patjanasoontorn2, S Rungruanghiranya3, Thai SCCM Study Group4 1Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 2Khon Khaen University, Khon Khaen, Thailand; 3Srinakharinwirot University, Nakornnayok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) Eff ectiveness of noncontrast abdominal multidetector CT for evaluating the patient with renal insuffi ciency in the emergency department Results In a population of approximately 5.3 million, the annual incidence of complex burn injury is 499 to 537 (9 to 10 per 100,000). The incidence of a major burn is 5% of burn admissions. The hospital mortality from a burn is 1 to 2.2%. See Table 1. HJ Lee, E Kang, JH Shin d HJ Lee, E Kang, JH Shin SMG-SNU Boramae Medical Center, Seoul, South Korea Conclusion From these data, Scotland now has comprehensive national fi gures for complex burn injury. This allows for benchmarking against other international indices, few of which provide comprehensive data. COBIS data can now also be correlated with other mortality data Introduction Contrast-enhanced abdominal multidetector CT (MDCT) is an important and accurate diagnostic approach for acute abdominal S28 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 shown that only 20 to 35% of patients trigger the clinical EWS prior to cardiac arrest. Jarvis and colleagues proposed that an EWS based on common laboratory fi ndings can predict patient mortality [3]. The aim of this study, as part of a wider review of cardiac arrests in our hospital, was to determine whether the laboratory early warning score (LEWS) might be of use identifying patients at risk of cardiac arrest in our trust. Methods Retrospective data collected identifi ed cardiac arrest calls that lead to CPR or defi brillation over 6 months. The LEWS was calculated according to the formula devised by Jarvis and colleagues [3]. LEWS ≥4 for males and ≥5 for females was taken as being a ‘trigger’ as suggested by Jarvis and colleagues [3]. References 1. Hillman K, et al.; MERIT study investigators. Introduction of the medical emergency team (MET) system: a cluster-randomised controlled trial. Lancet 2005, 365:2091-2097. 1. Hillman K, et al.; MERIT study investigators. Introduction of the medical emergency team (MET) system: a cluster-randomised controlled trial. Lancet 2005, 365:2091-2097. 2. Lee A, et al.: The Medical Emergency Team. Anaesth Intens Care 1995, 23:183-186. 2. Lee A, et al.: The Medical Emergency Team. Anaesth Intens Care 1995, 23:183-186. Results The mean age was 64 ± 19.8 years and 48% (n = 513) were male. There were 513 (48.9%) early calls and 537 (51.1%) late calls. The main reasons for RRT activation were respiratory failure in 18.2% (n = 191) and severe sepsis/septic shock in 13.1% (n = 138). The distribution of RRT activation was uniform over the 24-hour period, with 50.5% (n = 531) of calls during the day (7:00 a.m. through 7:00 p.m.) and 49.5% (n = 520) overnight (7:00 p.m. through 7:00 a.m.). A total of 460 patients (43.7%) were admitted to the ICU. The multivariate analysis showed the following variables as signifi cantly associated with hospital mortality: age (OR 1.03; 95% CI 1.01 to 1.04), late (>48 hours) RRT call (OR 2.73; 95% CI 1.79 to 4.71), acute change in oximetry saturation to <90% (OR 1.94; 95% CI 1.28 to 2.95) and acute change in respiratory rate to <8 or >28 breaths/minute (OR 1.79 95% CI 1.09 to 2.94). Hospital mortality predictive factors following Rapid Response Team activation H Palomba, F Piza, M Jaures, A Capone H Palomba, F Piza, M Jaures, A Capone Hospital Israelita Albert Einstein, São Paulo, Brazil p Critical Care 2014, 18(Suppl 1):P82 (doi: 10.1186/cc13272) Introduction The Rapid Response Team (RRT) represents an important advance in the management of deteriorating ward patients and is recommended as a patient safety measure. Most studies on RRT evaluate the eff ects of its implementation on rate reduction of cardiopulmonary arrest outside the ICU and hospital mortality, with limited information on the criteria for RRT calls and predictive factors associated with hospital mortality [1,2]. Therefore, our objective was to determine what factors are associated with hospital mortality for patients seen by the RRT at the Hospital Israelita Albert Einstein (HIAE). Methods A total of 1,051 patients assessed by RRT between January and December 2012 at the HIAE, a general hospital with 650 beds, were included in this study. Multivariate analysis was used to evaluate what variables were associated with hospital mortality. Early RRT call was defi ned as RRT activation <48 hours from hospital admission and late RRT call if it happened >48 hours from hospital admission. Conclusion ERT system implementation was associated with pro gressive reduction in cardiac arrests over the 3-year period, especially statistically signifi cant in diff erence in the fourth year after implementation. We also found an inverse association between number of ERT use and the risk of occurrence of cardiac arrests, but we found no diff erence in overall hospital mortality rate. P82 Results Of a total 623 ERT cases from June 2009 until December 2012, there were 72 calls in 2009, 196 calls in 2010, 139 calls in 2011 and 245 calls in 2012.The number of ERT calls per 1,000 admissions in year 2009 to 2010 was 7.69, 5.61 in 2011 and 9.38 in 2013. The number of Code Blue calls per 1,000 admissions decreased signifi cantly from 2.28 to 0.99 per 1,000 admissions (P <0.001). The incidence of cardiac arrest decreased progressively from 1.19 to 0.34 per 1,000 admissions and was signifi cant in diff erence in year 2012 (P <0.001). The overall hospital mortality rate decreased by 8% from 15.43 to 14.43 per 1,000 admissions (P = 0.095). P83 Long-term outcome of the Emergency Response Team system in in-hospital cardiac arrest Jarvis et al.: Resuscitation 2013, 84:1494-1499. 1. NCEPOD 2012 [http://www.ncepod.org.uk] Methods To investigate the outcome of the ERT system in in-hospital cardiac arrest and the overall hospital mortality rate, we conducted a prospective, controlled before and after examination of the long- term eff ect of an ERT system on the incidence of cardiac arrest. We performed chi-square analysis to fi nd statistical signifi cance. 2. NICE 2007 [http://www.nice.org.uk] 3. Jarvis et al.: Resuscitation 2013, 84:1494-1499. Survey of severe sepsis and septic shock management in Thailand: THAI-SHOCK SURVEY 2013 K Chittawatanarat1, B Patjanasoontorn2, S Rungruanghiranya3, Thai SCCM Study Group4 1Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 2Khon Khaen University, Khon Khaen, Thailand; 3Srinakharinwirot University, Nakornnayok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) paracetamol (–1.35 ± 0.46 (95% CI –1.3, –3.1) P <0.0001). After 120 minutes, a signifi cant diff erence was observed in the mean change from the baseline temperature between the groups, –1.81 ± 0.46 (95% CI –1.7, –2.9) in i.m. diclofenac versus –1.63 ± 0.55 (95% CI –1.5, –4.1) in the i.v. paracetamol group (P <0.0001). Signifi cant changes in temperature were observed in favor of i.m. diclofenac over i.v. paracetamol at each time point from 60 minutes through 120 minutes inclusive.i Laboratory early warning score versus clinical early warning score as a predictor of imminent cardiac arrest M Keil, S Hutchinson, T Leary Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P81 (doi: 10.1186/cc13271) Introduction NCEPOD reported in 2012 that 75% of patients had warning signs for cardiac arrest present prior to their arrest [1]. NICE recommends a vital sign-based early warning score (EWS) to identify patients at risk of deterioration or death [2]. In our trust, audit has Conclusion Both i.m. diclofenac and i.v. paracetamol showed signifi cant antipyretic activity, with diclofenac having a greater eff ect. In view of S29 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P83 Long-term outcome of the Emergency Response Team system in in-hospital cardiac arrest Long-term outcome of the Emergency Response Team system in in-hospital cardiac arrest J Suriyachaisawat, E Surakarn Bangkok Hospital Group, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P83 (doi: 10.1186/cc13273) y g Results Eighty-nine cardiac arrest calls lead to CPR and/or defi brillation in this time period. Of these, 65 patients had had blood tests within the last 24 hours. Median LEWS was 6 for females and 7 for males (range 1 to 12). Most patients (77%) had a LEWS trigger (≥4 for females and ≥5 for males). Introduction To improve early detection and the mortality rate of in- hospital cardiac arrest, the Emergency Response Team (ERT) system was planned and implemented since June 2009 to detect pre-arrest conditions and for any concerns. The ERT consisted of on-duty physicians and nurses from emergency department. ERT calling criteria [1,2] consisted of acute change of HR <40 or >130 beats per minute, systolic blood pressure <90 mmHg, respiratory rate <8 or >28 breaths per minute, O2 saturation <90%, acute change in conscious state, acute chest pain or worried about the patients. From the data on ERT system implementation in our hospital in the early phase (during June 2009 to 2011), there was no statistical signifi cance in diff erence in in-hospital cardiac arrest incidence and overall hospital mortality rate. Since the introduction of the ERT service in our hospital, we have conducted a continuous educational campaign to improve awareness in an attempt to increase use of the service. Conclusion The collected data suggest that more patients at risk of cardiac arrest in our hospital might be identifi ed using a LEWS rather than a clinical EWS. It is evident that for a clinical EWS regular observations need to be taken and are subject to user error. Clinical EWS could also not be sensitive enough or there is a failure to implement it successfully. LEWS can be generated automatically when bloods are taken. The downside is that it relies on bloods being done. It is beyond the scope of this study to examine the sensitivity/specifi city of LEWS or suggest that LEWS should replace EWS. We suggest that LEWS may compliment EWS by identifying a diff erent group of patients. The ongoing data collection aims to correlate the clinical EWS for each patient directly with their LEWS to confi rm the initial fi ndings. 1. NCEPOD 2012 [http://www.ncepod.org.uk] 2. NICE 2007 [http://www.nice.org.uk] 3. References References 1. Winters BD, et al.: Rapid response systems: going beyond cardiac arrest and mortality. Crit Care Med 2013, 41:911-912. 2. Leach LS, et al.: Rapid response teams: qualitative analysis of their eff ectiveness. Am J Crit Care 2013, 22:198-210. References 1. Winters BD, et al.: Rapid response systems: going beyond cardiac arrest and mortality. Crit Care Med 2013, 41:911-912. 2. Leach LS, et al.: Rapid response teams: qualitative analysis of their eff ectiveness. Am J Crit Care 2013, 22:198-210. References 1. Winters BD, et al.: Rapid response systems: going beyond cardiac arrest and mortality. Crit Care Med 2013, 41:911-912. 2. Leach LS, et al.: Rapid response teams: qualitative analysis of their eff ectiveness. Am J Crit Care 2013, 22:198-210. References 1. Winters BD, et al.: Rapid response systems: going beyond cardiac arrest and mortality. Crit Care Med 2013, 41:911-912. 2 L h LS t l R id t lit ti l i f th i Use of low-dose CT KUB: is it becoming the easy way out? P Shah, D Wilson Homerton University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P85 (doi: 10.1186/cc13275) Use of low-dose CT KUB: is it becoming the easy way out? P Shah, D Wilson Homerton University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P85 (doi: 10.1186/cc13275) Introduction In 1995, Smith and colleagues fi rst proposed the use of low-dose CT KUB for the diagnosis of ureteric colic [1]. Since then, popularity of this imaging modality has increased due to a number of reasons: a sensitivity of 94 to 97%, a specifi city of 96 to 99%, lack of intravenous contrast injection and speed of examination. The UK College of Emergency Medicine considers CT KUB as best practice for radiological investigation of renal colic. A review of the literature suggests that the true positive rate (number of patients diagnosed with an obstructing or symptom causing calculus) should be between 47.5 and 67% and alternative diagnoses should be confi rmed in approximately 10% of patients imaged [2]. Conclusion The results underline that the unit’s guidelines were not being followed. The re-audit does show an improvement in adherence. Patients are being exposed to unnecessary blood tests, which not only is implicated in iatrogenic anaemia, but also places a signifi cant fi nancial burden on the department. Continued staff education and encouragement are required in order to aid the transition from current to recommended practice. Decreasing central-line blood draws by consolidation of phlebotomy timing: results of a quality improvement project G Arteaga, S Tripathi, Y Ouellette, M Nemergut, G Rohlik, K Fryer, C Huskins Mayo Clinic, Rochester, MN, USA Critical Care 2014, 18(Suppl 1):P87 (doi: 10.1186/cc13277) pp y p g Methods All patients over the age of 18 years who had a CT KUB requested from the emergency department of the Homerton University Hospital, London over a period of 3 months, between May 2012 and July 2012, were identifi ed. Individual case notes were examined and data were collected on an Excel spreadsheet. Data were analysed and results were divided into positive for renal colic (true positive), positive for other pathology (other signifi cant diagnoses) and negative results. Results A similar previous audit carried out in 2009 at the same hospital demonstrated a true positive rate of 48.5%. The 2012 audit examined the outcome of 124 consecutive scans and a true positive rate of 29% (P = 0.0006) was identifi ed. Alternative diagnoses were 10% in 2009 compared with 17% in 2012 (P = 0.02). Bled dry? An audit of blood sampling practices on an adult intensive therapy unit Bled dry? An audit of blood sampling practices on an adult intensive therapy unit Methods A prospective observational evaluation of ICU unplanned admission through in-hospital ward referral over a 3-month duration. Anonymised data collection included baseline demographics, timing and grade of referral, Modifi ed Early Warning Score (MEWS) at time of referral, clinical reason for referral, senior review prior to referral, MEWS trend 24 hours prior to referral, if appropriate basic intervention commenced, time delay between referral and assessment by the ICU team, organ failure score, length of stay (LOS) and survival status. RK Vincent, P Temblett RK Vincent, P Temblett ABMU Health Board, Swansea, UK Critical Care 2014, 18(Suppl 1):P86 (doi: 10.1186/cc13276) Introduction The aim of this audit was to evaluate whether guidelines produced in a local intensive therapy unit (ITU) with regard to blood sampling practices were being adhered to. The volume of blood taken and cost was also evaluated. g g y Results So far 22 patients have been enrolled, of which 60% of the unplanned admissions were ‘out of hours’, 60% of admissions were from medical wards and 33% from surgical wards. Pneumonia was the main reason for referral and admissions were due to respiratory failure requiring advanced ventilator care. Twenty per cent of the patients did not have senior medical review for at least 4 hours prior to the ICU referral. Basic interventions such as antibiotics and intravenous fl uids were not started in a few patients (13%) prior to admission. The mean ICU review time from referral was 70 minutes and the mean MEWS at the time of referral was 8. Methods A retrospective audit investigating the number of routine blood tests ordered on an ITU in January 2013 was performed. There were no exclusion criteria. Computer-based data collection systems were used to gather data with regard to patient details and when blood tests were processed. The collection bottles used were examined to see how much blood was needed to fi ll them. Thirty nurses were asked how much ‘dead space’ blood they discarded, and an average was recorded. The cost of the blood tests was also calculated. Following a period of education regarding the contents of the guidelines, this was re-audited in July 2013 retrospectively. Conclusion The study is due to be completed by mid-January 2014. P86 in order to identify the associative trends that led to the admission and hence to mitigate the processes of interventional strategies. in order to identify the associative trends that led to the admission and hence to mitigate the processes of interventional strategies. References 1. Hopkins P, Wolff AH: Intensive care transfers. Crit Care 2002, 6:123 f 2. McQuillan P et al.: Confi dential inquiry into quality of care before admission to intensive care. Br Med J 1998, 316:1853-1858. 1. Smith RC, et al.: Radiology 1995, 194:789-794. Epidemiology of unplanned intensive care admissions through in- hospital referrals at a tertiary referral centre university hospital J Rigby, N Bradshaw, B Carr, R Matsa Epidemiology of unplanned intensive care admissions through in- hospital referrals at a tertiary referral centre university hospital J Rigby, N Bradshaw, B Carr, R Matsa University Hospital of North Staff ordshire, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P84 (doi: 10.1186/cc13274) g y, , , University Hospital of North Staff ordshire, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P84 (doi: 10.1186/cc13274) Introduction The provision of intensive care has lagged behind demand [1]. Intensive care services in the UK have signifi cant resource restriction. Although rationing of beds has been a priority, equally important is to establish patient safety and also identify strategies to prevent admissions [2], which echoes the importance of early recognition of deteriorating patients and prompt intervention. This study evaluates the epidemiology of unplanned admissions to the ICU Conclusion In this study, hospital mortality predictive factors for patients seen by the RRT were: age, acute respiratory failure and late RRT call. S30 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 2. Roth CS, et al.: Ann Emerg Med 1985, 14:311-31515. Use of low-dose CT KUB: is it becoming the easy way out? P Shah, D Wilson Homerton University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P85 (doi: 10.1186/cc13275) Introduction The direct central line entry rate is believed to be a major contributor to the risk of central line infections. At Mayo Clinic there was historically no schedule for obtaining blood for analysis in the pediatric ICU. A policy was implemented in May 2013 to restrict blood draws to three times daily for nonemergent blood draws only. We subsequently conducted this study to determine whether implementation of this policy was associated with a reduction of blood draws as well as central-line unique entries. Conclusion The true positive rate of CT KUB for renal colic has decreased signifi cantly. We are getting more negative scan results and more scans diagnosing other signifi cant pathology. The authors believe clinicians’ thresholds for imaging may have decreased due to the apparent low- dose radiation of CT KUBs. Furthermore, there is a perceived ease of access to CT KUB imaging and hence this modality appears to be being used to identify other signifi cant pathology. Methods Data from the laboratory as well as database for Central Line Unique Entry were analyzed at baseline and after implementation of the policy change for identifi cation of any decrease in line entry/blood draw rate. As per Mayo Clinic policy, IRB approval was not required for a QI project. Results In the pre-implementation phase there were a total of 4,602 blood draws in 5,227 total patient-days, (0.88 blood draws/patient- days). After consolidation, there were 1,095 blood draws in 1,491 patient-days (0.73 blood draws/patient-day; 17% reduction). Of these line entries, 24.7% were arterial line entry, 50.5% central line entry and Bled dry? An audit of blood sampling practices on an adult intensive therapy unit With the limitation of incompleteness, the fi ndings so far have alluded that there is a necessitation for change and education for early identifi cation and intervention in deteriorating patients. The completed study with outcomes (that is, survival rates and ICU LOS) will help us identify whether delay or defi ciency in the early management had a causal relationship. f Results The initial audit examined 901 patient-days. Urea and electrolytes (U&Es) and full blood counts (FBC) were requested in line with the guidelines. Liver function tests (LFTs), bone profi le, magnesium and a clotting screen were ordered approximately four times more than advocated. It was shown that many bone profi les and magnesium tests were probably inappropriate requests. Moreover, twice as much blood was taken from patients compared with that recommended by guidelines (almost 16 litres in total in January). The cost of the routine blood tests in January was €11,019. If guidelines had been followed, the estimated yearly saving would be €65,588. During the repeat audit, 731 patient-days were examined. The amount of times a U&E or FBC were requested was largely unchanged, but the amount of times a LFT, bone profi le, magnesium and clotting screen were ordered reduced by approximately 50%. Almost one-third more blood was taken from patients when compared with the suggested volume in the guidelines. The cost of the blood tests done in July was €5,423. Despite an improvement in the frequency of blood testing, an estimated €21,907 per year could still be saved. Reference 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. Results In the simulation study 10/15 clinicians (67%) injected atropine directly into arterial cannula via a three-way tap. Five of 15 doctors (34%) injected safely. In the laboratory study, swabbing of the arterial hubs showed bacterial contamination of all of the samples in the standard group (20/20) and no contamination in the NIC group (0/20), P <0.0001. Eighty-fi ve per cent (17/20) of samples in the standard arterial group showed onward transmission of pathogens to the patient circulation, and none of the patient samples taken from the NIC group were contaminated (0/20), P <0.0001. In the survey, 80% of the nurses found manual handling of a new device equally simple and 20% even simpler. Sampling of the blood was equally challenging for 87% participants. Ninety-four per cent found similar durability of the new device and better protection against accidental intra-arterial injection. Sixty-six per cent of the nursing staff would replace the standard system with a NIC. P90 Should we avoid invasive treatment in cancer patients with pericardial tamponade? F Galas, J Fukushima, E Osawa, C Park, J Almeida, D Nagaoka, L Hajjar Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P90 (doi: 10.1186/cc13280) Introduction Patients with cancer now live longer due to advances in oncologic treatment. ICU admission is progressively more frequent in this population due to complications of both disease and therapy. Pericardial eff usion leading to tamponade and hemodynamic compromise is a common fi nding in the advanced stages of disease and results in death if not treated on an urgent basis. However, we do not know midterm outcomes of these patients after pericardial drainage. The aim of this study was to evaluate prospectively cancer patients with pericardial tamponade regarding mortality in 6 months. Conclusion Most of the doctors would inject the drug intra-arterially. Currently, no other device is available to eliminate accidental intra- arterial injection or bacterial transmission. The nursing staff found the management of the NIC equally challenging. References S31 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 bleeding remains a challenge in tangential excision of the burn wound. Although various techniques to reduce intraoperative blood loss have been described, there is an absence of uniformity and consistency in their application. In the literature, the blood loss in burn surgery is estimated to be at least 123 ± 106 ml per percentage body surface area excised [1]. Recently we developed a novel hemostatic technique using a silicone gel dressing (SI-AID®; ALCARE Co., Ltd, Tokyo, Japan) to stop intraoperative bleeding. Briefl y, soon after tangential excision with the Humby knife, the wounds were sprayed with thrombin and with 1:100,000 adrenalin solution and wrapped tightly with SI-AID® for a full 10 minutes. Burn wounds on limbs were tangentially excised under tourniquet control and wrapped with SI-AID® before defl ation of the tourniquet. After defl ation of the tourniquets, we waited for a full 10 minutes. When the SI-AID® was removed, any major bleeders were cauterized, and the grafts were applied after rinsing the wounds with warm saline. 12.5% were by peripheral venipuncture. After policy implementation, these numbers were 10.9%, 49.7%, and 23.8%, respectively. The average central line unique entry after blood draw consolidation decreased from 10 to 6 line entries/central line-day. Consolidation of blood draws was associated with a cost saving of $7,200/year. Conclusion Consolidating time frames for blood draws in the PICU was associated with decreased central line entries, decreased utilization of vascular access teams, and decreased phlebotomy cost. We hypothesize that this policy will be associated with a decreased incidence of CLABSI when more patients are included for analysis. P89 Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery A Osuka, Y Kuroki, H Kojima, M Sekido, S Okuma, S Onishi, M Ueyama Social Insurance Chukyo Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) P89 Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery A Osuka, Y Kuroki, H Kojima, M Sekido, S Okuma, S Onishi, M Ueyama Social Insurance Chukyo Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) g Reference 1. Sen S, et al.: Complications after unintentional intraarterial injection of drugs: risks, outcomes, and management strategies. Mayo Clin Proc 2005, 80:783-795. Results Fifty-three patients (50%) were female. Most patients had a previous diagnosis of lung neoplasia (46%), followed by breast neoplasia (15%) and hematological neoplasia (15%). The mean Karnofsky performance status of patients was 70. All these patients underwent surgical drainage in a mean time of 1.5 hours since ICU admission until surgery. Length of ICU stay was 8 days (2 to 15) and of hospital stay was 16 days (8 to 31). ICU mortality was 75.2%, hospital mortality was 83% and at 3 months 92% of patients were dead. Reference Methods We evaluated consecutively 105 patients with cardiac tamponade consecutively admitted to the ICU of the Cancer Institute, a reference cancer hospital, during a 3-year period. Baseline characteristics and clinical data were collected prospectively. Patients were followed during a 6-month period. Introducing an arterial non-injectable connector into clinical practice Methods This is a prospective observational study. From 1 January to 31 October 2013 we collected preoperative and 24-hour postoperative hemoglobin levels from the patients who underwent tangential excision for burn injury, and calculated blood loss in the perioperative period. The data for amounts of blood transfusion, excised area, and harvest area were also collected. The Queen Elizabeth Hospital, Kings Lynn, UK Critical Care 2014, 18(Suppl 1):P88 (doi: 10.1186/cc13278) p g y Critical Care 2014, 18(Suppl 1):P88 (doi: 10.1186/cc13278) Introduction The standard arterial system for blood withdrawal provides no impediment to intra-arterial injection [1] and bacterial contamination. We assessed the probability of inadvertent intra-arterial injection, transmission of bacterial contamination and surveyed the nursing staff following introduction of a non-injectable connector (NIC). Results Nine patients, 13 operations were included. The mean excised area was 8.3 ± 4.6% body surface area. Estimated blood loss was 35.3 ± 38.3 ml per percentage body surface area excised. Intraoperative transfusion requirements were 86.2 ± 134.5 ml per case. The mean skin graft take rate was 4.5 ± 2.2%. Methods The simulation study data were descriptive. Fifteen junior doctors managed a case of bradycardia. A simulated patient had a peripheral intravenous cannula, central venous catheter and brachial arterial cannula. Atropine was available on request. A laboratory controlled trial compared a transmission of bacteria through standard arterial ports against the NIC when attached to an arterial sampling hub. The colonization rates were compared using a two-tailed Fisher’s exact test. A closed-circuit arterial sampling system was designed. A contaminated syringe tip was inserted into the conventional arterial hub or the NIC to take an arterial sample. Transducer fl ush fl uid fl owing to the patient bloodstream was cultured. In a survey the nursing staff were asked questions regarding the NIC system, including its manual handling, sampling of the blood and its durability. Conclusion The application of our new technique during burn excision and grafting resulted in a remarkable reduction in blood loss and transfusion requirements. Reference ROTEM: Multiplate monitoring in the ICU and outcome scores U Schött, A Larsson Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P93 (doi: 10.1186/cc13283) Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P93 (doi: 10.1186/cc13283) q p pp p Results Preoperatively, the HB group (n = 31) was characterized by lower blood fi brinogen and decreased clot amplitude at ROTEM compared with the LB group (n = 40). Intraoperatively, larger amounts of fi brinogen, fresh frozen plasma and platelets were deemed necessary to normalize the coagulation parameters in the HB group. Postoperatively, the incidence of major thromboembolic and ischemic events did not diff er between the two groups (<10%) and the observed in-hospital mortality was signifi cantly less than expected by the POSSUM score (22% vs. 35% in HB group and 5% vs. 13% in LB group). Conclusion ROTEM-derived information is helpful to detect early coagulation abnormalities and to monitor the response to hemostatic therapy. Early goal-directed management of coagulopathy may contribute to improve outcome after cardiovascular surgery. R f Introduction Hemostatic disorders are common in intensive care patients and can be used as prognostic markers. The aim of this prospective clinical study was to study platelet function in intensive care patients using point-of-care viscoelastic and platelet aggregometry instruments and platelet count (PLC). Our hypothesis was that measurement of platelet function would give more information about disseminated intravascular coagulation (DIC), morbidity and mortality than PLC alone. Methods Patients admitted to the ICU were monitored with ROTEM viscoelasticity and a new Multiplate platelet aggregometer; routine coagulation analyses; International Society of Thrombosis and Haemostasis and Japanese Association for Acute Medicine DIC calculated scores; Sequential Organ Failure Assessment scores, Simplifi ed Acute Physiology Score III – Expected Mortality Rate; and real in-hospital mortality. Nonparametric tests were chosen for all statistical evaluation. P <0.05 was considered signifi cant. 1. Ranucci M, et al.: The deadly triad of cardiac surgery. Ann Thorac Surg 2013, 96:478-485. Results A total of 128 patients with diff erent diagnoses were studied, with 330 sampling events. Multiplate analyses correlated signifi cantly with PLC and ROTEM. However, there were more test results below normal limits for Multiplate analyses than for ROTEM in patients with thrombocytopenia. Multiplate, ROTEM and PLC results were low in patients with high SOFA scores and in patients with overt DIC scores. These test results at admission to the ICU did not diff er between survivors and nonsurvivors, and did not correlate with the length of stay in the ICU. Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery y A Osuka, Y Kuroki, H Kojima, M Sekido, S Okuma, S Onishi, M Ueyama Social Insurance Chukyo Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) A Osuka, Y Kuroki, H Kojima, M Sekido, S Okuma, S Onishi, M Ueyama Social Insurance Chukyo Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) Conclusion Cardiac tamponade is a serious complication in cancer patients. Despite adequate treatment, high rates of mortality are observed. Prospective studies are needed to better defi ne whether in these patients end-of-life discussion should be implemented early in the diagnosis, avoiding futility. y p , g y , p Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) Introduction The purpose of this study is to demonstrate the effi cacy of our novel hemostatic technique for burn surgery. Signifi cant Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S32 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P91 Goal-directed hemostatic therapy using rotational thromboelastometry in patients requiring emergent cardiovascular surgery D Sartorius, JL Waeber, G Pavlovic, M Aldenkortt, MJ Licker Hôpital Cantonal Universitaire de Genève, Geneva, Switzerland Critical Care 2014, 18(Suppl 1):P91 (doi: 10.1186/cc13281) 1.2. The average measured value of INR in men was 1.50, and in women was 1.35. APTT normal values were 0.8 to 1.2. The average measured value of APTT in men was 1.09, and in women was 1.14. Platelet count was 150×109/l to 300×109/l level of platelets; in men was 252.57, in women was 226.78. ExTEM CT normal values at 41 to 74 seconds: measured value of CT in men was 62.58 and in women was 62.07. MCF normal values were 50 to 72 mm; the measured value of MCF in men was 69.88, and in women was 73.28. Considered administration of blood derivatives: fresh frozen plasma, 123 transfusion units (TU); platelets, 4 TU. In the examined cases no blood products were administered to avoid the risk of bleeding before the elective procedure. No periprocedural bleeding was observed. Introduction Massive bleeding remains a leading cause of potentially preventable death after cardiovascular surgery [1]. Conventional coagulation tests (CCT) fail to characterize the multiple hemostatic abnormalities observed in surgical patients and are further limited by their slow results and poor correlation with transfusion requirements. We assessed the clinical impact of goal-directed coagulation manage- ment based on rotational thromboelastometry (ROTEM) in patients undergoing an emergent cardiovascular surgical procedure. ROTEM: Multiplate monitoring in the ICU and outcome scores U Schött, A Larsson Only EMR diff ered between survivors and nonsurvivors and correlated with ICU length of stay. P92 Thromboelastometric examination on the ICU before elective procedures P Lukas University Hospital Motol, Prague, Czech Republic Critical Care 2014, 18(Suppl 1):P92 (doi: 10.1186/cc13282) Thromboelastometric examination on the ICU before elective procedures P Lukas University Hospital Motol, Prague, Czech Republic Critical Care 2014, 18(Suppl 1):P92 (doi: 10.1186/cc13282) Introduction In critically ill patients a number of elective procedures with a potential risk of bleeding are performed. In this population, coagulation disorder is frequently observed according to the commonly used laboratory parameters of APTT (activated partial thromboplastin time), INR (international normalized ratio) or platelet count. Thromboelastometric examination (ROTEM) evaluates coagulation of whole blood and thus allows the testing of all components of secondary hemostasis. Conclusion In this study, Multiplate and ROTEM did not provide any more information than PLC about DIC, morbidity and mortality. Multiplate showed lower platelet function in more patients with low platelets than ROTEM. In patients without thrombocytopenia, all patients had normal ROTEM results, but 36% of Multiplate measurements were low. This higher sensitivity of the Multiplate to measure lowered platelet function needs to be linked to real bleeding in larger patient series to defi ne its clinical signifi cance. Methods Identifi cation of patients with pathological values of INR, APTT and platelet count before the planned intervention. Through a questionnaire we found a potential correction of coagulopathy carried out by the physician. Performed ROTEM methodology: ExTEM (external pathway thromboelastometry). Inclusion criteria: normal curves and values, CT (clotting time), MCF (maximum clot fi rmness). In those patients with normal values according to ExTEM examination, no blood products were administered. Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery p p p g Conclusion Examination EXTEM in these patients proved to be eff ective and effi cient in predicting bleeding complications in relation with interventions routinely performed on the ICU. Also, these specifi c cases proved not indicated and thus ineff ective administration of blood products. Methods Over a 2-year period, data from 71 patients were collected prospectively and blood samples were obtained for coagulation testing. Administration of packed red blood cells (PRBC) and hemostatic products was guided by an algorithm using ROTEM- derived information and hemoglobin level. Based on the amount of PRBC transfused, two groups were considered: high bleeders (≥5 PRBC; HB) and low bleeders (<5 PRBC; LB). Data were analyzed using the chi- square test, unpaired t test and ANOVA as appropriate. P92 P92 Thromboelastometric examination on the ICU before elective procedures P Lukas University Hospital Motol, Prague, Czech Republic Critical Care 2014, 18(Suppl 1):P92 (doi: 10.1186/cc13282) References References 1. Chee YL, et al.: Br J Haematol 2008, 140:496-504. 2. BCSH Blood Transfusion Task Force: Br J Haematol 2003, 122:10-23. References 1. Chee YL, et al.: Br J Haematol 2008, 140:496-504. 2. BCSH Blood Transfusion Task Force: Br J Haematol 2003, 122:10-23. Methods We prepared both types of bag: parenteral nutrition bags whose composition was defi ned in the unit, including sodium heparin (77 UI/ml); and bags containing only sodium heparin diluted in 50% dextrose (193 UI/ml). These bags (n = 6 per type) were infused over a period of 24 hours with and without in-line fi ltration. Heparin activity was measured using a chromogenic anti-Xa method in bags just being prepared (references for other measures) and after 24-hour infusion and in effl uents at the end of infusion line after 24 hours. Heparin stability in parenteral nutrition bags prepared in a neonatal ICU y Conclusion This study demonstrates inconsistent use of FFP, with no signifi cant diff erence in PT between patients who were transfused and those that were not. The lack of eff ect of FFP transfusion on PT and APTT creates additional confusion for its prophylactic usage. There is a need for further clarifi cation around coagulopathy and interventional radiology in the critical care setting. The low absolute incidence of bleeding complications and risk of complications from transfusion lends further support to the view that FFP should be used therapeutically rather than as prophylactic ‘cover’ [1]. 1Faculté de Pharmacie, Lille, France; 2Institut de Pharmacie, CHRU, Lille, France; 3Fédération de Biologie Pathologie du CHRU, Lille, France; 4Hôpital Jeanne de Flandre, CHRU, Lille, France Critical Care 2014, 18(Suppl 1):P96 (doi: 10.1186/cc13286) Introduction Heparin is commonly given in our neonatal ICU (NICU) by continuous intravenous infusion. Heparin is diluted in parenteral nutrition bags and administered over a period of 24 hours with in-line fi ltration. However, there are no data on heparin stability in parenteral nutrition bags, especially on its compatibility with 50% dextrose mainly present in bags. The aim of our in vitro study was to determine heparin stability in parenteral nutrition bags prepared in a NICU after 24-hour infusion and to assess its interaction or not with 50% dextrose. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Clot initiation was prolonged with both drugs and detected by both ROTEM and ReoRox. Clot formation was more decreased with tinzaparin than enozaparin and only detected by ReoRox. Results Patients who received FFP had a mean PT of 18.5 seconds while those who did not receive FFP had a mean PT of 16.7 seconds (unpaired t test, P = 0.275). In the nine patients given FFP, the pre- transfusion mean PT was 18.5 seconds whereas the post-transfusion mean PT was 17.1 seconds (paired t test, P = 0.235). The pre-transfusion mean APTT was 41.6 seconds compared with a post-transfusion mean APTT of 38.1 seconds (paired t test, P = 0.127). No patient had platelet levels below the recommended transfusion threshold [2], but one patient nevertheless received a double-dose platelet transfusion. One patient had a recorded immediate bleeding complication. Their PT was 15.6 seconds and APTT was 40.9 seconds and they did not receive FFP. One patient had an anaphylactic reaction whilst receiving FFP. P94 Retrospective observational study of interventional radiology and critical care coagulopathy SP Hibbs, S McKechnie, M Little, M Desborough John Radcliff e Hospital, Oxford, UK Critical Care 2014, 18(Suppl 1):P94 (doi: 10.1186/cc13284) Results During March 2013 to November 2013, 40 patients were identifi ed as relevant, 26 men of average age 53 years and 14 women of average age 59 years. Central venous cannulation, 12 cases; surgical revision, nine cases; thoracic drainage, six cases; percutaneous endoscopic gastrostomy, fi ve cases; nephrostomy, two cases; epidural catheter, two cases; tracheostomy, two cases; permanent pacemaker, one case; and bronchoscopy, one case. INR normal values were 0.8 to Introduction Estimation of bleeding risk in critical care patients undergoing interventional radiological procedures is often made on the basis of coagulation tests. If these tests are abnormal, fresh frozen plasma (FFP) is often given to reduce the risk of bleeding, despite Introduction Estimation of bleeding risk in critical care patients undergoing interventional radiological procedures is often made on the basis of coagulation tests. If these tests are abnormal, fresh frozen plasma (FFP) is often given to reduce the risk of bleeding, despite S33 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods Enoxaparin (Klexane) and tinzaparin (Innohep) were added to 2 ml citrated blood from 10 intensive care patients to obtain plasma concentrations of 0, 0.5, 1.0 and 1.5 IU/ml enoxaparin and tinzaparin, respectively. The study was approved by the local ethics committee and with written consent (relatives). Clot formation and clot retraction was studied using ROTEM and ReoRox. a poor evidence base for this practice [1]. There is a relatively better evidence base for prophylactic platelet transfusion [2] but clinical practice is inconsistent. Through a retrospective study we aimed to establish the thresholds triggering use of FFP and platelet transfusion prior to percutaneous drain insertion in critical care patients.i Methods Enoxaparin (Klexane) and tinzaparin (Innohep) were added to 2 ml citrated blood from 10 intensive care patients to obtain plasma concentrations of 0, 0.5, 1.0 and 1.5 IU/ml enoxaparin and tinzaparin, respectively. The study was approved by the local ethics committee and with written consent (relatives). Clot formation and clot retraction was studied using ROTEM and ReoRox. Results ROTEM analysis showed prolonged clot formation (CT) with increasing concentrations of enoxaparin and tinzaparin (more so). ReoRox analysis showed that the initiation of clot formation (COT1) increased with increasing doses of enoxaparin and tinzaparin (more so), as did the progression of clot formation (COT2 – COT1), thus resulting in a prolongation until complete clot formation (COT2). See Table 1. Conclusion Clot initiation was prolonged with both drugs and detected by both ROTEM and ReoRox. Clot formation was more decreased with tinzaparin than enozaparin and only detected by ReoRox. Methods We identifi ed 68 consecutive chest, abdominal or pelvic drain insertions in 54 critical care patients between 1 January 2008 and 11 October 2012 at the John Radcliff e Hospital, Oxford. The prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet counts prior to each procedure were recorded to demonstrate triggers used for FFP and platelet transfusion. In patients who underwent transfusion, the next PT, APTT and platelet count post transfusion were recorded. Results ROTEM analysis showed prolonged clot formation (CT) with increasing concentrations of enoxaparin and tinzaparin (more so). ReoRox analysis showed that the initiation of clot formation (COT1) increased with increasing doses of enoxaparin and tinzaparin (more so), as did the progression of clot formation (COT2 – COT1), thus resulting in a prolongation until complete clot formation (COT2). See Table 1. Data presented as median (SD). P <0.05 compared with 0 IU/ml LMWH. P96 P96 Heparin stability in parenteral nutrition bags prepared in a neonatal ICU A Foinard1, M Perez1, B Décaudin2, C Barthélémy1, A Tournoys3, L Storme4, P Odou2 1Faculté de Pharmacie, Lille, France; 2Institut de Pharmacie, CHRU, Lille, France; 3Fédération de Biologie Pathologie du CHRU, Lille, France; 4Hôpital Jeanne de Flandre, CHRU, Lille, France Critical Care 2014, 18(Suppl 1):P96 (doi: 10.1186/cc13286) P95 Conclusion We conclude that there is no loss of heparin activity when drug is infused over 24 hours for both types of bag prepared, with and without in-line fi ltration, showing heparin activity remains stable during this period and there is no interaction of drug with other nutrient components of bags, especially 50% dextrose. y j y Methods Rabbits were treated with a high intravenous bolus dose of edoxaban (1,200 μg/kg) followed by the administration of Beriplex® (25 to 75 IU/kg). Bleeding was assessed based on the time to hemostasis and the total blood loss after induction of a standardized kidney injury. In parallel, the following biomarkers of hemostasis were determined: factor Xa inhibition, prothrombin time (PT), activated partial thrombo- plastin time (aPTT), whole blood clotting time (WBCT), and thrombin generation (TGA). Results The results confi rmed increased and prolonged bleeding of edoxaban-treated animals following standardized kidney injury compared with vehicle administration. Parallel monitoring of biomarkers of hemostasis showed a prolongation of PT, aPTT, WBCT, and changes in thrombin generation parameters. Subsequent administration of Beriplex® resulted in a dose-dependent reversal of edoxaban-induced bleeding as indicated by reduced time to hemostasis and total blood loss. Both parameters achieved statistical signifi cance compared with placebo at the Beriplex® dose of 50 IU/kg under fully blinded study conditions. The biomarkers correlating best with Beriplex®-mediated edoxaban anticoagulation reversal included PT, WBCT and endogenous thrombin potential.f P97 Bivalirudin or heparin: which anticoagulation strategy for critically ill cardiac surgery patients? F Pappalardo1, B Arnaez1, M Celinska-Spodar1, M Pieri1, F Isella1, S Silvetti1, A Montisci1, O Saleh1, A Koster2 1San Raff aele Scientifi c Institute, Milan, Italy; 2Heart and Diabetes Centre, North Rhine-Westphalia Ruhr-University Bochum, Bad Oeyenhausen, Germany Critical Care 2014, 18(Suppl 1):P97 (doi: 10.1186/cc13287) Introduction Anticoagulation with unfractionated heparin in cardiac surgery patients has several limitations, and above all the risk of heparin-induced thrombocytopenia. Bivalirudin is a direct thrombin inhibitor, use of which in cardiac surgery patients has expanded in recent years. The aim of the study was to analyze two strategies for introducing bivalirudin in this setting (as secondary drug switching from heparin or as primary anticoagulant) and to evaluate clinical outcomes. Conclusion In summary, Beriplex® treatment eff ectively reversed edoxaban-induced anticoagulation in an animal model of acute bleeding at clinically relevant dose levels. Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs g p g M Honickel1, O Grottke1, J Van Ryn2, H Ten Cate3, R Rossaint1, H Spronk3 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany; 3Maastricht University, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P99 (doi: 10.1186/cc13289) g g M Honickel1, O Grottke1, J Van Ryn2, H Ten Cate3, R Rossaint1, H Spronk3 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany; 3Maastricht University, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P99 (doi: 10.1186/cc13289) y p p Results Bivalirudin treatment was associated with a reduction of major bleeding (P = 0.05) compared with the control group. Interestingly, in an intention-to-treat analysis, patients receiving primary bivalirudin showed a signifi cant reduction in minor bleeding (P = 0.04), and mortality (P = 0.01) compared with the secondary bivalirudin group, and similarly if compared with UFH and secondary bivalirudin patients (P = 0.01 and P = 0.05 respectively). Predictors of hospital mortality at multivariate analysis included urgent admission (OR = 2.7; 95% CI, 1.03 to 7.2; P = 0.04), septic shock (OR = 8.0; 95% CI, 2.26 to 28.7; P <0.005) and primary therapy with UFH (OR = 19.2; 95% CI, 2.2 to 163.9; P = 0.007). Conclusion Novel anticoagulant strategies might play a crucial role in critically ill cardiac surgery patients. In a propensity-matched population, our study showed that primary bivalirudin anticoagulation may reduce bleeding complications and mortality. Further studies are therefore warranted. Results Bivalirudin treatment was associated with a reduction of major bleeding (P = 0.05) compared with the control group. Interestingly, in an intention-to-treat analysis, patients receiving primary bivalirudin showed a signifi cant reduction in minor bleeding (P = 0.04), and mortality (P = 0.01) compared with the secondary bivalirudin group, and similarly if compared with UFH and secondary bivalirudin patients (P = 0.01 and P = 0.05 respectively). Predictors of hospital mortality at multivariate analysis included urgent admission (OR = 2.7; 95% CI, 1.03 to 7.2; P = 0.04), septic shock (OR = 8.0; 95% CI, 2.26 to 28.7; P <0.005) and primary therapy with UFH (OR = 19.2; 95% CI, 2.2 to 163.9; P = 0.007). Introduction The reversal of the anticoagulant eff ects of the new oral anticoagulants in severely bleeding patients requires new therapeutic strategies. This study investigated the eff ectiveness of a specifi c antidote for idarucizumab to reverse bleeding in a dabigatran anticoagulated pig trauma model. P95 P95 Monitoring of treatment with low molecular weight heparins using viscoelastic devices U Schött1, A Larsson1, O Thomas1, N Tynngård2 1Lund University, Lund, Sweden; 2Linköping University, Linköping, Sweden Critical Care 2014, 18(Suppl 1):P95 (doi: 10.1186/cc13285) Introduction Few studies have investigated the use of viscoelastic devices for monitoring of treatment with LMWHs and to our knowledge there are no studies comparing diff erent LMWHs or diff erent visocoelastic methods. fl Results Our results show values of heparin activity measured in bags and effl uents with and without in-line fi ltration after 24-hour infusion for both types of bag assessed (Tables 1 and 2). Results are expressed as median values (minimum to maximum) in percent. Table 1 (abstract P95). ROTEM and FOR variables for enoxaparin and tinzaparin Enoxaparin Tinzaparin 0 IU/ml 0.5 IU/ml 1.0 IU/ml 1.5 IU/ml 0.5 IU/ml 1.0 IU/ml 1.5 IU/ml ROTEM CT (seconds) 175 ± 38 191 ± 89 214 ± 109 249 ± 94* 223 ± 53* 289 ± 84* 326 ± 125* CFT (seconds) 77 ± 23 87 ± 21 85 ± 16 83 ± 22 74 ± 27 84 ± 45 80 ± 21 Angle (°) 75 ± 4 74 ± 4 75 ± 3 74 ± 4 75 ± 5 73 ± 5 73 ± 4 MCF (mm) 62 ± 5 61 ± 7 63 ± 5 63 ± 6 68 ± 7* 64 ± 8 65 ± 6* MCL (%) 8 ± 4 6 ± 3+ 5 ± 4 2 ± 5* 5 ± 4* 6 ± 4 2 ± 4* ReoRox COT1 (seconds) 30 ± 5 35 ± 7* 39 ± 8* 38 ± 11* 36 ± 4* 45 ± 9* 48 ± 15* COT2 (seconds) 65 ± 11 76 ± 13* 82 ± 14* 85 ± 20* 74 ± 5* 91 ± 16* 108 ± 29* COT2 – COT1 (seconds) 34 ± 10 41 ± 8* 43 ± 9* 46 ± 10* 37 ± 4 46 ± 11* 51 ± 15* Data presented as median (SD). P <0.05 compared with 0 IU/ml LMWH. Table 1 (abstract P95). ROTEM and FOR variables for enoxaparin and tinzaparin S34 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P96). P95 Methods Data from 100 propensity matched patients who received heparin (Group H, n = 50) or bivalirudin (Group B, n = 50), from January 2009 to January 2012, in a cardiac surgery ICU of a university hospital were analyzed. Bivalirudin was administered as either fi rst-line or second-line drug after heparin discontinuation if heparin-induced thrombocytopenia was presumed. P99 Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs M Honickel1, O Grottke1, J Van Ryn2, H Ten Cate3, R Rossaint1, H Spronk3 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany; 3Maastricht University, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P99 (doi: 10.1186/cc13289) Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs P95 Results of heparin activity in parenteral nutrition bags Bags at T0 + Effl uent at T0 + Bags at T0 24 hours 24 hours With fi ltration 100 95.45 97.73 (84.09 to 109.09) (79.55 to 102.27) (75.00 to 104.55) Without fi ltration 100 97.62 97.62 (90.48 to 114.29) (83.33 to 107.14) (83.33 to 114.29) Table 2 (abstract P96). Results of heparin activity in bags with sodium heparin in 50% dextrose Bags at T0 + Effl uent at T0 + Bags at T0 24 hours 24 hours With fi ltration 100 95.10 98.04 (77.45 to 108.82) (90.20 to 109.80) (95.10 to 100.98) Without fi ltration 100 92.23 94.17 (85.44 to 107.77) (88.35 to 100.00) (86.41 to 108.74) Conclusion We conclude that there is no loss of heparin activity when drug is infused over 24 hours for both types of bag prepared, with and without in-line fi ltration, showing heparin activity remains stable during this period and there is no interaction of drug with other nutrient components of bags, especially 50% dextrose. Table 1 (abstract P96). Results of heparin activity in parenteral nutrition bags P98 Reversal of edoxaban-induced anticoagulation by the four-factor prothrombin complex concentrate Beriplex® in a rabbit model E Herzog1, F Kaspereit1, W Krege1, B Doerr1, J Mueller-Cohrs1, I Pragst1, Y Morishima2, G Dickneite1 1CSL Behring GmbH, Marburg, Germany; 2Daiichi Sankyo Co., Ltd, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P98 (doi: 10.1186/cc13288) Introduction The oral direct and selective factor Xa inhibitor edoxaban (Daiichi Sankyo) is currently available in Japan for the prophylaxis of venous thromboembolism (VTE) in patients undergoing major orthopedic surgery and is undergoing investigation in phase III trials for the prevention of stroke in patients with atrial fi brillation and the treatment and secondary prevention of VTE. The primary complication of any available anticoagulant therapy is the risk of bleeding. Rapid reversal of anticoagulation may be necessary in patients requiring emergency treatment due to uncontrolled bleeding. Prothrombin complex concentrates (PCC) are frequently used to reverse the eff ect of vitamin K antagonists such as warfarin and have also been suggested to be potentially eff ective in reversing the eff ects of the new oral anticoagulants. The present study was therefore designed to determine whether the four-factor PCC Beriplex® can eff ectively reverse bleeding and normalize coagulation following edoxaban administration in a rabbit kidney injury model. Plasma-free hemoglobin and microvascular response to fresh or old blood transfusion in septic patients Plasma free hemoglobin and microvascular response to fresh or old blood transfusion in septic patients A Donati1, E Damiani1, R Domizi1, AT Colesnicenco1, E Montesi1, S Ciucani1, P Pelaia1, C Ince2 1Università Politecnica delle Marche, Ancona, Italy; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P101 (doi: 10.1186/cc13291) A Donati1, E Damiani1, R Domizi1, AT Colesnicenco1, E Montesi1, S Ciucani1, P Pelaia1, C Ince2 P Pelaia1, C Ince2 1Università Politecnica delle Marche, Ancona, Italy; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P101 (doi: 10.1186/cc13291) Results Dabigatran levels were 1,147 ± 370 ng/ml with no diff erences between groups prior to injury. BL in sham animals was 409 ± 53 ml 10 minutes after injury and 700 ± 107 ml after 4 hours (survival rate 100%). Anticoagulation with dabigatran (control animals) resulted in signifi cantly higher BL 10 minutes after injury (801 ± 66 ml, P <0.05). Mortality in these animals was 100%, with a mean survival time of 121 minutes (range: 90 to 153 minutes; P <0.05 vs. sham and idarucizumab-treated animals). Total BL in dabigatran-treated animals was 2,977 ± 316 ml. In contrast, treatment with idarucizumab was associated with a dose-dependent reduction in BL. The lowest dose of 30 mg/kg resulted in 17% mortality (1/6 animals) and BL was reduced by 50% (1,586 ± 619 ml). BL was further reduced in animals receiving 60 (1,077 ± 103 ml) or 120 mg/kg idarucizumab (1,137 ± 121 ml; both 100% survival). Hemodynamic parameters and markers of shock were similar to pre-trauma levels in latter groups receiving idarucizumab.i Introduction Free hemoglobin (fHb) can scavenge nitric oxide and induce vasoconstriction [1]. The fHb content may be higher in older blood bags. We studied whether old red blood cell (RBC) transfusion increases plasma fHb in septic patients and if this aff ects the microvascular response. Methods Twenty septic patients randomly received either fresh (<10 days storage) or old (>15 days) RBC transfusion. Plasma fHb was measured before and 1 hour after transfusion; the sublingual microcirculation was assessed with sidestream dark-fi eld imaging. The perfused boundary region (PBR) was measured as an index of glycocalyx damage [2]. The thenar Tissue Hb index (THI) was measured (near-infrared spectroscopy). g g Conclusion This study demonstrates for the fi rst time that anticoagulation with dabigatran can be reversed eff ectively and safely by idarucizumab. P101 dabigatran-treated animals were randomized (n = 6/group) to a single injection of idarucizumab at 30, 60 or 120 mg/kg i.v. or vehicle (control animals). Blood loss and hemodynamic variables were monitored over 4 hours or until time of death. Data were analyzed by ANOVA (± SD) and by the log-rank test. Plasma-free hemoglobin and microvascular response to fresh or old blood transfusion in septic patients Even under supra-therapeutic dabigatran concentrations, the antidote decreased blood loss and mortality in this lethal pig animal model. Thus, data from clinical studies are warranted to confi rm the fi ndings of this study. p py Results fHb increased in the old RBC group (Figure 1). THI increased in both groups, while SDF parameters were unaltered. Negative correlations were found between ΔfHb and changes in total vessel density (r = –0.57, P <0.01; Figure 2) and THI (r = –0.71, P <0.001). These relations were lacking in patients with PBR <2.68 μm. Figure 1 (abstract P101). Changes in fHb. Figure 2 (abstract P101). Relation between changes in fHb and TVD. Figure 1 (abstract P101). Changes in fHb. Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs Methods After ethical approval, male pigs (n = 30) were given dabigatran etexilate for 3 days (30 mg/kg bid p.o.), and the sham group (n = 6) received placebo. To achieve supra-therapeutic anticoagulation, dabigatran was infused prior to injury on day 4 in anesthetized pigs, and the sham group received placebo. A standardized blunt liver injury was infl icted and blood loss (BL) was recorded 10 minutes post trauma. The Conclusion Novel anticoagulant strategies might play a crucial role in critically ill cardiac surgery patients. In a propensity-matched population, our study showed that primary bivalirudin anticoagulation may reduce bleeding complications and mortality. Further studies are therefore warranted. S35 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Fatty acid composition of blood plasma in multiple organ dysfunction syndrome AN Osipenko1, AV Marochkov2 Introduction The aim of study was to assess the fatty acid (FA) composition of blood plasma in multiple organ dysfunction syndrome (MODS). Introduction The aim of study was to assess the fatty acid (FA) composition of blood plasma in multiple organ dysfunction syndrome (MODS). Table 1 (abstract P103). Univariate analysis of logistic regression for in-hospital mortality Table 1 (abstract P103). Univariate analysis of logistic regression for in-hospital mortality HR 95% CI P PAI-1 day 2 1.02 1.0 to 1.06 <0.01 TAT day 2 1.49 1.02 to 19 <0.05 PMG day 3 0.64 0.1 to 0.95 <0.01 SOFA day 1 1.2 0.9 to 1.69 0.21 IL-6 day 1 0.99 0.9 to 1.00 0.21 Methods The objects of the study were 18 people with multiple organ failure (35.6 ± 8.7 years) of various etiologies. The blood of 16 healthy volunteers aged 37.7 ± 3.2 years served as control. There were also analyzed blood plasma and fragments of artery luminal part from patients who died from diff erent causes. Analysis of FA was conducted using capillary gas–liquid chromatography. Quantitative evaluation of individual FA content was made as a mass percentage of their total. Statistical analysis was performed using the Mann–Whitney U test (P <0.05). Results Normalized contents of oleic and palmitoleic monounsaturated FA increase in patients with MODS, while the levels of stearic saturated FA and polyunsaturated FA decrease, as compared with the control. Considering these results, we conclude that blood plasma FA composition in MODS biased towards composition of FA characteristic of adipose and muscle tissue triglycerides, which quantitatively predominant monounsaturated FA, and stearic and polyunsaturated FA have a signifi cantly lower level [1,2]. Thus, the composition of the FA in MODS refl ects the degree of lipolysis activation in fat depots. As a result of these processes there should occur an imbalance in vascular endothelial cells between monounsaturated and polyunsaturated FA and, as a consequence, changes in metabolism of eicosanoids and other lipid vasoactive mediators should develop. One should note that in plasma of people who died in a hospital environment due to various reasons, similar changes in FA blood composition are observed. The degree of change of FA composition in postmortem blood samples presumably primarily depends on severity and duration of a previous critical and serious condition. Postmortem changes presumably exert some infl uence on blood plasma FA composition. References References 1. Malcom G, et al.: Am J Clin Nur 1989; 50:288-291. 2. Andersson A, et al.: Am J Clin Nutr 2002; 76:1222-1229. Methods We retrieved data on 2,852 consecutive patients undergoing cardiac surgery at revascularization at Duke between 1 January 2004 and 31 December 2008. We compared patients receiving or not prophylatic ε-aminocaproic acid in coronary artery bypass graft surgery, or valve procedures or combined ones. We evaluated baseline characteristics, intraoperative data and severe clinical endpoints during 30 days. 1. Malcom G, et al.: Am J Clin Nur 1989; 50:288 291. 2. Andersson A, et al.: Am J Clin Nutr 2002; 76:1222-1229. P100 S36 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Old RBC transfusion increased plasma fHb in septic patients. Increasing plasma fHb levels after transfusion were associated with decreased microvascular density and lower increase in tissue Hb content. This relation might be blunted when the glycocalyx is preserved. References Methods A prospective observational study of adult patients with severe sepsis was conducted in a single academic hospital. Plasma was analyzed for coagulation and fi brinolysis markers on days 1, 2, and 3. Patients were stratifi ed according to the age 67 years, which was the point of the Youden index (maximum sensitivity + specifi city – 1) in a receiver operation characteristics plot for a logistic regression model of in-hospital mortality. 1. Donadee C, et al.: Circulation 2011, 124:465-476. 2. Donati A, et al.: Microvasc Res 2013, 90:86-89. 1. Donadee C, et al.: Circulation 2011, 124:465-476. 1. Donadee C, et al.: Circulation 2011, 124:465-476. 2. Donati A, et al.: Microvasc Res 2013, 90:86-89. Results For the in-hospital survival rate, that of older sepsis patients was signifi cantly lower than younger patients, 9/15 (60.0%) versus 27/28 (96.4%), P <0.05. Older patients had markedly higher total plasmin activator inhibitor-1 (TPAI-1) on day 1, thrombin–antithrombin complex (TAT) on days 2 and 3, and fi brin monomer complex on day 2, and markedly lower plasminogen (PMG) on day 3 and alpha-plasmin inhibitor (αPI) on days 2 and 3 compared with younger patients (all P <0.05). Age was an independent predictor of high TAT on days 2 and 3, high fi brin monomer complex on day 2, low PMG on day 3, and low αPI on days 2 and 3 after adjusting for some cofactors and covariables. TPAI-1 on day 2, TAT on day 2, and PMG on day 3 were risk factors of in- hospital mortality in older sepsis patients (Table 1). 2. Donati A, et al.: Microvasc Res 2013, 90:86-89. Fatty acid composition of blood plasma in multiple organ dysfunction syndrome Moreover, the composition of FA in blood plasma in the case of MODS is similar to that of FA in the luminal part of the artery wall. This suggests a decrease in the infl uence of intertissue diff erences in lipid composition on metabolic processes. Conclusion In severe sepsis, older patients displayed a biomarker profi le suggestive of enhanced response of coagulation and fi brinolysis system compared with younger patients. Change of some markers depended on age and may contribute to the poor outcome in older patients. ε-Aminocaproic acid does not increase adverse eff ects in cardiac surgery: an analysis of 2,852 casesf Critical Care 2014, 18(Suppl 1):P104 (doi: 10.1186/cc13294 p Conclusion Given that the monounsaturated FAs in the case of MODS enter the bloodstream primarily from adipose and muscle tissues, one can assume that their blood plasma level refl ects the degree of hypermetabolism processes in the organism. Introduction Antifi brinolytic drugs recently have been associated with adverse outcomes in patients undergoing cardiac surgery. We reviewed our experience with prophylatic ε-aminocaproic acid in patients undergoing cardiac surgery at InCor – Heart Institute. p References 1. Martin GS, et al.: N Engl J Med 2003, 348:1546-1554. 2. Gando S, et al.: Crit Care 2013, 17:R111. P100 Primary bivalirudin anticoagulation for patients with an implantable ventricular assist device M Pieri, B Arnaez, AL Di Prima, M Celinska-Spodar, S Ajello, O Saleh, F Isella, A Montisci, F Pappalardo San Raff aele Scientifi c Institute, Milan, Italy Critical Care 2014, 18(Suppl 1):P100 (doi: 10.1186/cc13290) Introduction Bivalirudin is a direct thrombin inhibitor that is increasingly used in patients undergoing mechanical circulatory support as it presents many advantages compared with heparin. The aim of this study was to describe our experience with bivalirudin as primary anticoagulant in patients undergoing ventricular assist device (VAD) implantation. p Methods An observational study on the 12 consecutive patients undergoing VAD implantation from October 2011 at out institution. Five patients were implanted the Heart Mate II LVAD (Thoratec Corp., Pleasanton, CA, USA), six patients the Heartware HVAD (HeartWare Inc., Miramar, FL, USA), and one patient a Cardiowest Total Artifi cial Heart (Syncardia Systems Inc., Tucson, AZ, USA). Patients received a continuous infusion of bivalirudin, with a starting dose of 0.025 μg/ kg/hour. The target activated partial thromboplastin time (aPTT) was between 45 and 60 seconds. Figure 1 (abstract P101). Changes in fHb. Results Patients never received heparin during hospitalization nor had a prior diagnosis of HIT. Preoperative platelets count was 134,000 ± 64,000 platelets/mm3. The bivalirudin dose was 0.040 ± 0.026 mg/kg/hour, and the duration of therapy was 5 (5 to 12) days. The lowest platelet count during treatment was 73,000 ± 23,000 platelets/mm3. No thromboembolic complications occurred. Two episodes of minor bleeding from chest tubes which subsided after reduction or temporary suspension of bivalirudin infusion were observed. Six patients required blood red cell transfusions, and one patient had one fresh frozen plasma transfusion. No platelet transfusions were performed during treatment. The ICU stay was 8 (7 to 17) days, and the hospital stay 25 (21 to 33) days. y y y y Conclusion Bivalirudin is a valuable option for anticoagulation in patients with VAD, and can be easily monitored with aPTT. The use of a bivalirudin-based anticoagulation strategy in the early postoperative period may overcome many limitations of heparin, and above all the risk of HIT which is higher in patients undergoing cardiac surgery. Bivalirudin should no longer be regarded as a second-line therapy for anticoagulation in patients with VAD. Figure 2 (abstract P101). Relation between changes in fHb and TVD. P104 P104 ε-Aminocaproic acid does not increase adverse eff ects in cardiac surgery: an analysis of 2,852 cases S Zeff erino1, L Camara1, J Almeida2, F Galas1, L Camara1, J Auler Jr1, J Fukushima1, A Leme1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P104 (doi: 10.1186/cc13294) Eculizumab treatment of atypical haemolytic uraemic syndrome: results from the largest prospective clinical trial to date LE weekers1, JM Campistol2, M Espinosa3, AO Gaber4, J Menne5, E Minetti6, F Provot7, E Rondeau8, M Ogawa9, CL Bedrosian9, M hourmant10 1University of Liège Hospital, Liège, Belgium; 2Servicio de Nefrología, Hospital Clínic, Barcelona, Spain; 3Hospital Universitario Reina Sofía, Córdoba, Spain; 4Methodist Hospital, Houston, TX, USA; 5Hannover Medical School, Hannover, Germany; 6Careggi University Hospital, Florence, Italy; 7CHU Lille, France; 8Hôpital Tenon, Paris, France; 9Alexion Pharmaceuticals, Inc., Cheshire, CT, USA; 10Institut d’ImmunoTransplantation Urologie et Nephrologie, Nantes, France Critical Care 2014, 18(Suppl 1):P105 (doi: 10.1186/cc13295) Results As of March 2013, there were 1,288 patients randomized in the ABLE study. The median patient age was 63 years (IQR 50 to 74). The majority of patients were emergent admissions (97%) with nonoperative diagnoses (85%), including respiratory illness (27%) and sepsis (18%). Bleeding was infrequent (0.4%). At least one comorbidity was common (43%), frequently signifi cant cardiac disease (14%) and diabetes (12%). There is signifi cant variability in the median pre- transfusion hemoglobin across diff erent sites (P <0.05). The median pre-transfusion hemoglobin by site was 75 g/l (IQR 71 to 77). Six of 24 sites had a signifi cantly higher, and four had a signifi cantly lower, median transfusion threshold compared with the median. Signifi cant variability in the median pre-transfusion hemoglobin remained in multivariate analysis, after adjustment for age and type of ICU.f Introduction Atypical haemolytic uraemic syndrome (aHUS) is a rare, life-threatening disease in which patients experience un- controlled complement activation leading to systemic thrombotic microangiopathy (TMA). We report on the eff ect of eculizumab (Ecu), a terminal complement inhibitor approved for the treatment of aHUS, in the largest prospective clinical trial in aHUS. Introduction Atypical haemolytic uraemic syndrome (aHUS) is a rare, life-threatening disease in which patients experience un- controlled complement activation leading to systemic thrombotic microangiopathy (TMA). We report on the eff ect of eculizumab (Ecu), a terminal complement inhibitor approved for the treatment of aHUS, in the largest prospective clinical trial in aHUS. Methods Adult aHUS patients (≥18 years) with platelets <150×109/l and LDH ≥1.5 ULN were recruited into a 26-week single-arm, phase 2 study evaluating Ecu treatment. Patients with STEC-HUS (Shiga toxin and E. coli) and severe ADAMTS13 defi ciency (activity <5%) were excluded. Identifi cation of complement mutation was not required for enrolment. The primary endpoint was complete TMA response (platelet and LDH normalisation (>150×109/l and <ULN, respectively) and <25% increase in serum creatinine from baseline (BL)). Eculizumab treatment of atypical haemolytic uraemic syndrome: results from the largest prospective clinical trial to date Other effi cacy evaluations measured platelet counts and eGFR improvement (by MDRD). Conclusion There was a 16 g/l diff erence in median hemoglobin between the lowest and highest site and wide inter-site variation. These diff erences may be due to transfusion education, monitoring and blood-bank enforcement practices. Site, not country, remains a signifi cant predictor of transfusion. Future directions in transfusion best practices should focus on local transfusion culture. Signifi cant and nonevidence-based variability persists in transfusion thresholds for critically ill patients. y Results Forty-one patients were enrolled (mean (SD) age 40.3 (15.3) years; 28 (68%) females), 30 (73%) of whom with fi rst presentation of aHUS (median 2 weeks before Ecu). BL mean (SD) platelets and eGFR were 119 (66.1) ×109/l and 17.3 (12.1) ml/minute/1.73 m2, respectively. Thirty-eight (93%) patients received Ecu for 26 weeks. Table 1 shows improvements in outcomes. Dialysis was discontinued in 20 (83%) among the 24 patients requiring dialysis at inclusion. Ecu was generally well tolerated, with no deaths or unexpected safety concerns. Meningococcal infections occurred in two patients, one of whom continued treatment. A liberal strategy of red blood cell transfusion reduces cardiovascular complications in older patients undergoing cardiac surgery Table 1 (abstract P105). Outcomes at 26 weeks in adult aHUS patients receiving Ecu Complete TMA response, n (%) patients 30 (73); 95% CI 57 to 86 Platelet count normalisation, n (%) patients 40 (98); 95% CI 87 to 100 Mean (SD) platelet count increase from baseline, ×109/l 135 (114); P <0.0001 Mean (SD) eGFR increase from baseline ml/minute/1.73 m2 29.3 (23.6); P <0.0001 Table 1 (abstract P105). Outcomes at 26 weeks in adult aHUS patients receiving Ecu Introduction Owing to their high risk in cardiac surgery, it is essential to defi ne which transfusion strategy results in a lower rate of cardio- vascular complications in older patients [1]. The aim of this study was to compare clinical outcomes after the implementation of either a restrictive or a liberal transfusion strategy in patients aged 60 years and above. Conclusion Results from this large prospective clinical trial in adult patients with aHUS confi rm prior trials with Ecu to inhibit complement- mediated TMA, improve renal and haematological outcomes and show the benefi t of early diagnosis and treatment. There were no unexpected safety concerns. The trial is ongoing. Methods This study was a substudy of the Transfusion Requirements After Cardiac Surgery study. In this subgroup analysis we included all patients aged 60 years and above randomized to a restrictive or a liberal strategy of RBC transfusion. A composite endpoint for cardiovascular complications was used and defi ned as a combination of 30-day all- cause mortality and severe cardiovascular morbidity. P106 Results The primary composite endpoint – all-cause 30-day mortality, cardiogenic shock, or myocardial infarction – occurred in 9.6% of patients in the liberal strategy group and in 18.4% in the restrictive strategy group (P = 0.041). The incidence of cardiogenic shock was 5.2% in the liberal group and 12.8% in the restrictive group (P = 0.031) and of myocardial infarction was 2.2% in the liberal group and 5.6% in the restrictive group (P = 0.203). There was no signifi cant diff erence between transfusion strategies in 30-day mortality rates (4.4% vs. 8%, respectively; P = 0.23). Response of coagulation and fi brinolysis system was diff erent between older and nonolder patients with severe sepsis Response of coagulation and fi brinolysis system was diff erent between older and nonolder patients with severe sepsis D Kudo, S Yamanouchi, T Sato, R Nomura, T Omura, N Miyagawa, S Kushimoto Tohoku University Hospital, Sendai, Japan Critical Care 2014, 18(Suppl 1):P103 (doi: 10.1186/cc13293) Response of coagulation and fi brinolysis system was diff erent between older and nonolder patients with severe sepsis D Kudo, S Yamanouchi, T Sato, R Nomura, T Omura, N Miyagawa, S Kushimoto Tohoku University Hospital, Sendai, Japan Critical Care 2014, 18(Suppl 1):P103 (doi: 10.1186/cc13293) y Results A total of 2,852 patients were included in the analysis and 1,389 (48.7%) received prophylactic ε-aminocaproic acid. The others did not receive any antifi brinolytic. In the risk-adjusted model, survival was similar among patients treated with aminocaproic acid or not treated (1.9 vs. 1.6%, P = 0.48). Bleeding in 24 hours was reduced in the treated group as compared with the nontreated (391 vs. 472 ml, P = 0.012). There were no diff erences regarding acute renal failure, stroke and infection in 30 days. y p p Critical Care 2014, 18(Suppl 1):P103 (doi: 10.1186/cc13293) Introduction The present study investigated the relationship between age [1] and biomarkers of coagulation and fi brinolysis [2] and their impact on outcome in severe sepsis patients. S37 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion In cardiac surgery, prophylactic use of aminocaproic acid reduces bleeding and does not result in higher incidence of acute complications. (RBC) transfusion practice remains variable and is infl uenced by patient characteristics and institutional transfusion culture. Methods We performed a multicenter retrospective analysis within the ongoing prospective randomized, Age of Blood Evaluation (ABLE) trial. The patient population included patients admitted to the ICU with an anticipated length of invasive or non-invasive mechanical ventilation >48 hours and who required a fi rst RBC transfusion during the fi rst 7 days of ICU admission. As of March 2013, completed and verifi ed data from 45 sites in Canada, France, and the UK were included. Sites with at least 12 patients were included in site analysis. The primary outcome is the association of enrolling centers on the median hemoglobin prior to transfusion. P107 A lib P107 A liberal strategy of red blood cell transfusion reduces cardiovascular complications in older patients undergoing cardiac surgery R Nakamura1, JL Vincent2, J Fukushima1, J Almeida1, F Bergamin1, C Park1, E Osawa1, M Sundin1, A Muller1, F Galas1, L Hajjar1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P107 (doi: 10.1186/cc13297) y References 1. Carson, et al.: Cochrane Database 2012, 4:CD002042. 2. Barr PJ, et al.: Transfusion 2011, 51:1684-1694. Evaluation of the implementation of a massive transfusion protocol K Balvers, M Coppens, JH Klinkspoor, SS Zeerleder, JC Goslings, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P110 (doi: 10.1186/cc13300) Introduction A massive transfusion protocol (MTP) aims to provide standardized and early delivery of blood products and prohemostatic agents by keeping pre-thawed fresh frozen plasma (FFP) available. Implementation of MTP is assumed to result in transfusion with higher ratios of FFPs and platelets to red blood cells (RBCs). Pre-thawing may also result in waste of FFPs. These MTP benefi ts or disadvantages have not yet been demonstrated. The aim of this study was to evaluate effi cacy of a MTP 1 year after implementation in our level I trauma center in an academic hospital. y j g Results A total of 4,952 patients were enrolled with an in-hospital mortality rate of 4% and a mean age of 58 ± 21 years. In total, 4,683 (95%) patients had their hematocrit measured: 3,857 (82%) patients had a normal hematocrit (30 to 44%), 413 (8.8%) patients had 25 to 29% hematocrit, and 66 (1.4%) patients ≤24% hematocrit. A total of 248 (5.3%) had a high hematocrit (≥45%). After adjusting for age, present or prior cancer, diabetes I and II, end-stage renal disease, AIDS and liver disease, anemia remained an independent predictor of in-hospital mortality with odds ratios of respectively 1.7 and 2.5 with worsening anemia as well as high hematocrit, with odds ratio 2.3 (Table 1). The AUC for the model was 0.78. Methods A retrospective analysis of an electronic blood bank transfusion database comparing massive transfusion before (January to December 2011) and after (January to December 2012) implementation of MTP. Activation of MTP consists of delivery of packages of 6 units of RBC, 6 units of pre-thawed FFP and 2 units of platelets collected from fi ve donors. Massive bleeding was defi ned as transfusion ≥10 units of RBCs. Statistics by t test and Mann–Whitney U test. Table 1 (abstract P108). Eff ect of severity of anemia or high hematocrit on in-hospital mortality Hematocrit Mortality OR P value ≥45% 2.3(1.3 to 4.1) 0.007 30 to 44%, ref. 1.0 25 to 29% 1.7(1.1 to 2.6) 0.019 < 20% 2.5(1.1 to 5.8) 0.032 Table 1 (abstract P108). Eff ect of severity of anemia or high hematocrit on in-hospital mortality Results In 2012, a total of 101 MTP activations was registered. Accurate prediction of massively bleeding patients (n = 30) was 29.7% of MTP activations. Of all massively bleeding patients in 2012, MTP was not activated in 55.2%. P109 P109 New simplifi ed criteria for predicting massive transfusion in trauma T Yumoto, A Iida, E Knaup, N Nosaka, S Morisada, T Hirayama, N Shiba, K Tsukahara, H Nosaka, Y Kinami, M Terado, H Yamanouchi, K Sato, T Ugawa, S Ichiba, Y Ujike Okayama University Hospital, Okayama, Japan Critical Care 2014, 18(Suppl 1):P109 (doi: 10.1186/cc13299) P109 New simplifi ed criteria for predicting massive transfusion in trauma T Yumoto, A Iida, E Knaup, N Nosaka, S Morisada, T Hirayama, N Shiba, K Tsukahara, H Nosaka, Y Kinami, M Terado, H Yamanouchi, K Sato, T Ugawa, S Ichiba, Y Ujike Okayama University Hospital, Okayama, Japan Critical Care 2014, 18(Suppl 1):P109 (doi: 10.1186/cc13299) , , p, , , y , ukahara, H Nosaka, Y Kinami, M Terado, H Yamanouchi, K Sato, Variation in red blood cell transfusion thresholds in critically ill patients K Wilton, R Fowler, T Walsh, J Lacroix, J Callum Sunnybrook Hospital, Toronto, Canada Sunnybrook Hospital, Toronto, Canada Critical Care 2014, 18(Suppl 1):P106 (doi: 10.1186/cc13296) Critical Care 2014, 18(Suppl 1):P106 (doi: 10.1186/cc13296) Introduction Restrictive transfusion practice in recent randomized trials and systematic reviews continues to have favorable outcomes [1]. Despite this, substantial variability in transfusion practice persists [2]. It is important to identify contemporary variability, and which patient, provider, and institutional factors drive this variability. Therefore, we performed a retrospective analysis hypothesizing that red blood cell Conclusion In this prospective, randomized clinical trial, older patients submitted to a restrictive strategy of RBC transfusion had a rate of cardiovascular complications in 30 days after cardiac surgery twice as high than a liberal strategy. In this group of patients, probably Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S38 Methods We retrospectively analyzed trauma patients transported to our center for the recent 2 years. Patients transferred from other hospitals with minor injuries or confi rmed cardiac arrest at the scene were excluded. untreated anemia would be more harmful than in a younger or healthier population undergoing cardiac surgery. Reference untreated anemia would be more harmful than in a younger or healthier population undergoing cardiac surgery. Reference 1. Hajjar LA, Vincent JL, Galas FR, Nakamura RE, Silva CM, Santos MH, et al.: Transfusion requirements after cardiac surgery: the TRACS randomized controlled trial. JAMA 2010, 304:1559-1567. Results A total of 297 trauma patients were included in this study. Thirty-one (10.4%) patients required MT. Sensitivity and specifi city for the Assessment of Blood Consumption (ABC) score were 48% and 99%, respectively. Because blunt injuries account for most trauma patients in Japan, we established new simple criteria using signifi cant factors that were derived from the examination on arrival. If trauma patients met any of the following conditions – that is, shock index (SI) >1, base excess (BE) <–3 mmol/l, and positive focused assessment of sonography for trauma (FAST) – sensitivity and specifi city was 97% and 80%, respectively. The area under the receiver operating characteristic curve of ABC and the new criteria was 0.889 (95% CI, 0.815 to 0.963) and 0.927 (95% CI, 0.881 to 0.974), respectively. Anemia and high hematocrit are associated with in-hospital mortality in emergency department patients with suspected infection Anemia and high hematocrit are associated with in-hospital mortality in emergency department patients with suspected infection M Jessen1, S Skibsted1, N Shapiro2 1Aarhus University Hospital, Aarhus, Denmark; 2Beth Israel Deaconess Medical Center, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P108 (doi: 10.1186/cc13298) M Jessen1, S Skibsted1, N Shapiro2 1Aarhus University Hospital, Aarhus, Denmark; 2Beth Israel Deaconess Medical Center, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P108 (doi: 10.1186/cc13298) Conclusion We suggest new criteria for early predicting MT in trauma using SI, BE, and FAST. This method may be valid especially in such areas because blunt injuries are the major cause of trauma in Japan. Reference Introduction Anemia and its association with mortality among emergency department (ED) patients with suspected infection has to our knowledge never been investigated. We hypothesize that anemia as well as high hematocrit increases the risk of in-hospital death among these patients. Nunez TC, et al.: J Trauma 2009, 66:346-352. P110 p Methods A prospective observational study of adult ED patients with suspected infection presenting to an urban, academic medical center ED at Beth Israel Deaconess Medical Center, Boston, MA, USA. Inclusion criterion was clinically suspected infection at ED presentation. Patients were enrolled over a 1-year period. Laboratory and clinical data were collected at enrollment. Primary outcome was in-hospital mortality. Logistic regression was performed determining the independent mortality odds adjusting for confounders. P110 Evaluation of the implementation of a massive transfusion protocol K Balvers, M Coppens, JH Klinkspoor, SS Zeerleder, JC Goslings, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P110 (doi: 10.1186/cc13300) P111 P111 Blood product transfusions in septic patients are associated with mortality, ARDS, and more days on mechanical ventilation PP Dobesh, DG Klepser, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P111 (doi: 10.1186/cc13301) Blood product transfusions in septic patients are associated with mortality, ARDS, and more days on mechanical ventilation PP Dobesh, DG Klepser, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P111 (doi: 10.1186/cc13301) Blood product transfusions in septic patients are associated with mortality, ARDS, and more days on mechanical ventilation Evaluation of the implementation of a massive transfusion protocol K Balvers, M Coppens, JH Klinkspoor, SS Zeerleder, JC Goslings, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P110 (doi: 10.1186/cc13300) In patients for whom MTP was activated, the RBC:FFP ratio was 1:0.9. In patients for whom MTP was activated and who were massively bleeding, the RBC:FFP ratio was 1:0.9, which was signifi cantly higher compared with 1:0.6 in massive bleeders in 2011 (n = 70) (P = 0.001). The median of blood products administered was 12.5 (6 to 21) in massive bleeding after MTP implementation, compared with 8 (3 to 13) in 2011 (P <0.001). In patients for whom MTP was activated, 9.7% of thawed FFPs were not transfused and wasted. When massive transfusion was accurately predicted, the waste of FFPs was 4.8% versus a waste of 16.2% in the group of unjustifi ed MTP activation. Conclusion Implementation of MTP is associated with an increase of blood products transfused and a signifi cant shift of RBC:FFP ratio to 1:1. However, MTP is also associated with a 9.7% waste of FFP. Improving prediction for massive bleeding patients may result in a decrease of wasted, costly blood products. Conclusion Anemia and elevated hematocrit seem to be associated with in-hospital mortality among patients with suspected infection. Treatment of high or low hematocrit as a part of the ED resuscitation could be a subject for further investigation. P113 Infl ammatory properties of microparticles in stored red blood cell transfusion productsf Infl ammatory properties of microparticles in stored red blood cell transfusion products M Straat1, R Nieuwland1, R Van Bruggen2, N Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Sanquin, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P113 (doi: 10.1186/cc13303) Results We identifi ed 824 patients who met the inclusion criteria for this study. Of those patients, 543 (66%) received at least 1 unit of blood products during hospitalization. Patients receiving blood products had signifi cantly higher in-hospital mortality (36.1% vs. 26.9%; P = 0.003) and a higher rate of development of ARDS (45.3% vs. 27.1%; P <0.001) compared with patients not receiving blood products. Patients receiving packed red blood cells (PRBCs) (60%) did not demonstrate a signifi cant increase in mortality (35.1% vs. 28.8%; P = 0.058), while patients receiving platelets (20%) did have higher mortality (45.1% vs. 29.5%; P <0.001). Transfusions of PRBCs or platelets were both associated with a higher development of ARDS (P <0.001 for both). There was a signifi cant increase in days on mechanical ventilation (7.0 days vs. 2.3 days; P <0.001), hospital cost ($88,331 vs. $35,047; P <0.001), and length of stay (17.3 days vs. 9.7 days; P <0.001) for patients receiving blood products, regardless of the type. These diff erences were seen despite the mean APACHE II score being similar (22.8 vs. 22.5; P = 0.645). M Straat1, R Nieuwland1, R Van Bruggen2, N Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Sanquin, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P113 (doi: 10.1186/cc13303) Introduction Blood transfusion is associated with increased morbidity and mortality in the critically ill [1]. Adverse eff ects of transfusion may be mediated by changes in the blood product that accumulate with storage time [2]. The mechanisms, however, are largely unknown. Erythrocyte-derived microparticles (MPs) have been found in transfusion bags [3,4] and their concentration increases with storage duration [5]. We hypothesize that accumulation of MPs during storage induces a proinfl ammatory state in the recipient. l Methods Microparticles were isolated by high-speed centrifugation from red blood cell transfusion bags stored for 41 or 42 days. Whole blood from healthy volunteers was incubated for 24 hours with supernatant from the bags either containing MPs or depleted from MPs. Controls were incubated with medium or LPS (10 ng/ml). TNFα, IL- 10 and IL-6 were measured in supernatant by ELISA. Data are expressed as median and range. References References 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. 2. Koch CG, et al.: N Engl J Med 2008, 358:1229-1239. 3. Rubin O, et al.: Transfusion 2012, 53:1744-1754. 4. Van Der Meijden PE, et al.: J Thromb Haemost 2012, 10:1355-1362. 5. Almizraq R, et al.: Transfusion 2013, 53:2258-2267. References 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. 2. Koch CG, et al.: N Engl J Med 2008, 358:1229-1239. 3. Rubin O, et al.: Transfusion 2012, 53:1744-1754. 4. Van Der Meijden PE, et al.: J Thromb Haemost 2012, 10:1355-1362. 5. Almizraq R, et al.: Transfusion 2013, 53:2258-2267. 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. 2. Koch CG, et al.: N Engl J Med 2008, 358:1229-1239. 3. Rubin O, et al.: Transfusion 2012, 53:1744-1754. 4. Van Der Meijden PE, et al.: J Thromb Haemost 2012, 10:1355-1362. 5. Almizraq R, et al.: Transfusion 2013, 53:2258-2267. 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. Methods The Transfusion Requirements After Cardiac Surgery (TRACS) study is a prospective, randomized, controlled clinical noninferiority trial conducted between February 2009 and February 2010 in an ICU at a university hospital cardiac surgery referral center in Brazil. Consecutive adult patients (n = 502) who underwent cardiac surgery with cardiopulmonary bypass were eligible; analysis was by intention to treat. P113 Infl ammatory properties of microparticles in stored red blood cell transfusion productsf Conclusion Patients with sepsis receiving blood products, particularly platelets, were signifi cantly more likely to develop ARDS, had more days on mechanical ventilation, and had higher mortality. The lack of an increase in mortality associated with PRBC transfusion may be due to the benefi t in oxygen delivery or sample size. Results MP-depleted supernatant induced a modest increase in median levels of TNFα (9.2 (2.3 to 22.2) pg/ml) and IL-6 (2,140.7 (1,507 to 2,199) pg/ml) compared with the negative controls (2.3 (2.3 to 2.3) pg/ ml and 94.4 (64.7 to 124.1) pg/ml, respectively), while IL-10 levels were not aff ected. Addition of MP-containing supernatant resulted in highly increased levels of TNFα (699 (687 to 742) pg/ml), IL-6 (37,443 (26,493 to 40,967) pg/ml) and IL-10 (1,201 (1,178 to 1,533) pg/ml) compared with negative controls. This MP-induced increase in cytokine production was comparable with the increase observed after the addition of LPS. Transfusion requirements in septic shock patients: a randomized controlled trial Transfusion requirements in septic shock patients: a randomized controlled trial F Bergamin1, J Almeida1, C Park1, E Osawa1, J Silva1, F Galas1, D Nagaoka1, J Fukushima1, S Vieira1, L Candido1, CO Oshiro1, JL Vincent2, L Hajjar1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P112 (doi: 10.1186/cc13302) p Conclusion Erythrocyte-derived MPs from aged red blood cell transfusion bags induce a strong infl ammatory response in vitro, which is largely negated when MPs are removed. Whether MPs mediate adverse eff ects of blood transfusion in the critically ill remains to be determined. Introduction Perioperative red blood cell transfusion is commonly used to address anemia, an independent risk factor for morbidity and mortality in critically ill patients [1]; however, evidence regarding optimal blood transfusion practice in septic shock is lacking. The aim of this study was to defi ne which is the best transfusion strategy in septic shock patients regarding 28-day mortality and clinical outcomes: restrictive or liberal. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (46.3%) were included in the liberal strategy and 73 patients (53.7%) in the restrictive strategy. Occurrence of 28-day mortality was similar between groups (54% in liberal group vs. 56.2% in restrictive group; P = 0.395). outcomes compared with those who do not require transfusions. It is not uncommon for patients with sepsis to require blood product transfusions. The need for transfusions may be indicative of infl ammatory consumption of blood cells, active blood loss, or impaired hematopoiesis. Regardless of the etiology, need for transfusion may be an indicator of a more severely ill patient and a valuable prognostic factor. Conclusion Among cancer patients with septic shock, the use of a restrictive perioperative transfusion strategy compared with a more liberal strategy resulted in similar rates of 28-day-mortality. R f Methods This retrospective cohort study included all patients over the age of 40 years with a confi rmed diagnosis of sepsis and an ICU stay at our academic medical center from 1 January 2005 to 31 March 2011. Use of blood product transfusion, patient demographics, and APACHE II score at the time of sepsis were collected from patient charts. Outcomes of interest were in-hospital mortality, development of acute respiratory distress syndrome (ARDS), days on mechanical ventilation, hospital cost, and length of stay.i 1. Hajjar LA, Vincent JL, Galas FR, Nakamura RE, Silva CM, Santos MH, et al.: JAMA 2010, 304:1559-1567. 1. Hajjar LA, Vincent JL, Galas FR, Nakamura RE, Silva CM, Santos MH, et al.: JAMA 2010, 304:1559-1567. Blood product transfusions in septic patients are associated with mortality, ARDS, and more days on mechanical ventilation y y p y p Critical Care 2014, 18(Suppl 1):P109 (doi: 10.1186/cc13299) PP Dobesh, DG Klepser, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P111 (doi: 10.1186/cc13301) Introduction Several predicting models have been described to identify the necessity for massive transfusion (MT) for trauma patients [1]. The purpose of this study is to validate the simplifi ed scoring systems reported previously and establish new criteria at emergency and in the ICU in Japan. Introduction The objective of this study was to determine whether septic patients requiring blood product transfusions have worse S39 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Application of damage control resuscitation strategies to patients with severe traumatic hemorrhage: review of plasma to packed red blood cell ratios at a single institution Introduction Prothrombin complex concentrate (PCC) has been suggested as a measure to terminate trauma and dabigatran-induced bleeding. Owing to the confl icting data concerning such therapy, we investigated the impact of a four-factor PCC to terminate massive bleeding following the infl iction of multiple trauma in dabigatran anticoagulated pigs. K Jung, K Lee, Y Kim K Jung, K Lee, Y Kim g Ajou University School of Medicine, Suwon, South Korea Ajou University School of Medicine, Suwon, South Korea Critical Care 2014, 18(Suppl 1):P117 (doi: 10.1186/cc133.07) Ajou University School of Medicine, Suwon, South Korea Critical Care 2014, 18(Suppl 1):P117 (doi: 10.1186/cc133.07) Critical Care 2014, 18(Suppl 1):P117 (doi: 10.1186/cc133.07) Introduction When treating trauma patients with severe hemorrhage, massive blood transfusions are often needed. Damage control resuscitation strategies can be used for such patients, but an adequate fresh frozen plasma:packed red blood cell (FFP:PRBC) administration ratio must be established. Introduction When treating trauma patients with severe hemorrhage, massive blood transfusions are often needed. Damage control resuscitation strategies can be used for such patients, but an adequate fresh frozen plasma:packed red blood cell (FFP:PRBC) administration ratio must be established. g p g Methods After ethical approval, 24 male pigs were administered dabigatran etexilate (30 mg/kg bid p.o.) for 3 days. On day 4, dabigatran in anaesthetised animals was infused prior to injury to achieve supra- therapeutic levels. Twelve minutes after infl iction of bilateral femur fractures and standardised blunt liver injury, animals randomly received PCC (25, 50 or 100 IU/kg; n = 6) or placebo (n = 6). Time-adjusted blood loss as primary endpoint (observation period 300 minutes) and a panel of coagulation variables were continually measured. Data were analysed by two-way ANOVA. Data are mean ± SEM.l Methods We retrospectively reviewed the medical records of 100 trauma patients treated with massive transfusions from March 2010 to October 2012. We divided the patients into two groups according to the FFP:PRBC ratio: a high-ratio group (≥0.5) and a low-ratio group (<0.5). The patient demographics, fl uid and transfusion quantities, laboratory values, complications, and outcomes of both groups were analyzed and compared. y y y Results Concentrations of dabigatran prior to infl iction of trauma was comparable between groups (590 ± 40 ng/ml). Anticoagulation with dabigatran and trauma caused severe coagulopathy as shown by prolonged TEM variables (CT, CFT), PT and aPTT. Following PCC application these eff ects were partially reversed. P116 Eff ect of a fi xed dose of fresh frozen plasma on systemic infl ammation and endothelial damage in nonbleeding critically ill patients M Straat, M Muller, J Meijers, M Schultz, N Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P116 (doi: 10.1186/cc13306) Methods We describe a fatal TRALI in a patient with infl uenza A (H1N1), suggesting a relationship between a fi rst-hit lung injury followed by the second lung impairment after blood transfusions. Results We report a 57-year-old female, without a previous medical record. She had an acute onset of fever, cough, muscle pain and progressive dyspnea leading to acute respiratory distress syndrome. The test for infl uenza A H1N1 was positive. She was recovering, but on day 12 of admission, after 1 hour of platelet transfusion, she started with intensive tachycardia, dyspnea and hypoxemia. Her mechanical ventilation parameters increased dramatically. She was in plan for extubation with FiO2 of 30% and positive end-expiratory pressure of 8, which became 100% and 14 respectively. The P:F ratio dropped to 62. Her leukocytes were 10.6×109/l a few hours earlier and went down to 1.5×109/l after the onset. Previous lactate was normal, but jumped to 42 mg/dl. She was free of vasopressors and after the off ending transfusion went through refractory shock and died approximately 24 hours after the blood transfusion. Introduction Fresh frozen plasma (FFP) is associated with onset of acute lung injury [1], the mechanism of which is largely unknown. On the other hand, FFP may be benefi cial, as a higher ratio of FFP to red blood cells decreases mortality in bleeding trauma patients [2] and is associated with an endothelial stabilizing eff ect in vitro [3]. We investigated the eff ect of transfusion with FFP on host response and markers of endothelial damage in nonbleeding critically ill patients. Methods This was a substudy of a multicenter trial in which nonbleeding critically ill patients with an increased International Normalized Ratio (1.5 to 3.0) were randomized to omitting or administering a prophylactic transfusion of FFP (12 ml/kg) prior to an invasive procedure. In 38 patients randomized to receive FFP transfusion, we measured levels of factor VIII, von Willebrand factor, and markers of proinfl ammatory response before and after transfusion. Data are presented as medians. Results FFP transfusion resulted in a signifi cant decrease of TNFα (from 12.3 to 3.1 pg/ml, P = 0.01), von Willebrand factor (from 475 to 424%, P <0.01) and factor VIII (from 246 to 244%, P <0.01). FFP did not alter levels of IL-1β, IL1-RA, IL-8, IL-10, MCP1, MIP1A or sCD40L. References 1. Toy P, et al.: Transfusion-related acute lung injury: defi nition and review. Crit Care Med 2005, 33:721-726. 1. Toy P, et al.: Transfusion-related acute lung injury: defi nition and review. Crit Care Med 2005, 33:721-726. 2. Kleinman S, et al.: Toward an understanding of transfusion-related acute lung injury: statement of a consensus panel. Transfusion 2004, 44:1774-1789. 2. Kleinman S, et al.: Toward an understanding of transfusion-related acute lung injury: statement of a consensus panel. Transfusion 2004, 44:1774-1789. Conclusion FFP transfusion is not associated with a proinfl ammatory response in the critically ill. Rather, FFP seemed to have an endothelial stabilizing eff ect. P116 Eff ect of a fi xed dose of fresh frozen plasma on systemic infl ammation and endothelial damage in nonbleeding critically ill patients M Straat, M Muller, J Meijers, M Schultz, N Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P116 (doi: 10.1186/cc13306) Patients had some degree of lung injury at baseline as refl ected by a lung injury score of 2 (0.8 to 2.5), which did not change following transfusion. None of the patients developed TRALI.l Conclusion For our knowledge this is the fi rst case reported of TRALI in an infl uenza A (H1N1) patient. Although blood transfusion can be life saving, it also can be a life-threatening intervention. Prevention is still the best hit. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion The use of high doses of PCC reversed the anticoagulant eff ects of dabigatran, which was associated with a signifi cant reduction of blood loss. However, the results of this study show that suffi cient concentrations of PCC are necessary to overcome thrombin inhibition. in capillary leakage and subsequent pulmonary edema [2]. TRALI is a clinical diagnosis with the following criteria: acute onset within 6 hours of blood transfusion, PaO2/FIO2 ratio <300 mmHg, or worsening of the P:F ratio, bilateral infi ltrative changes on chest radiograph, no sign of hydrostatic pulmonary edema (pulmonary arterial occlusion pressure ≤18 mmHg or central venous pressure ≤15 mmHg) and no other risk factor for acute lung injury [2].l in capillary leakage and subsequent pulmonary edema [2]. TRALI is a clinical diagnosis with the following criteria: acute onset within 6 hours of blood transfusion, PaO2/FIO2 ratio <300 mmHg, or worsening of the P:F ratio, bilateral infi ltrative changes on chest radiograph, no sign of hydrostatic pulmonary edema (pulmonary arterial occlusion pressure ≤18 mmHg or central venous pressure ≤15 mmHg) and no other risk factor for acute lung injury [2]. Conclusion The use of high doses of PCC reversed the anticoagulant eff ects of dabigatran, which was associated with a signifi cant reduction of blood loss. However, the results of this study show that suffi cient concentrations of PCC are necessary to overcome thrombin inhibition. P116f P116 Eff ect of a fi xed dose of fresh frozen plasma on systemic infl ammation and endothelial damage in nonbleeding critically ill patients M Straat, M Muller, J Meijers, M Schultz, N Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P116 (doi: 10.1186/cc13306) f References 1. Vlaar AP, et al.: Crit Care Med 2010, 38:771-778. 2. Holcomb JB, et al.: Ann Surg 2008, 248:447-458. 3. Peng Z, et al.: Shock 2013, 40:195-202. 1. Vlaar AP, et al.: Crit Care Med 2010, 38:771-778. 1. Vlaar AP, et al.: Crit Care Med 2010, 38:771-778. 2. Holcomb JB, et al.: Ann Surg 2008, 248:447-458. 3. Peng Z, et al.: Shock 2013, 40:195-202. 2. Holcomb JB, et al.: Ann Surg 2008, 248:447-458. 3 Peng Z et al : Shock 2013 40:195 202 RWTH Aachen University Hospital, Aachen, Germany; CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany; 3Maastricht University Medical Center, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P115 (doi: 10.1186/cc13305) Infl uenza A (H1N1): the fi rst hit for transfusion-related acute lung injury? j y F Piza1, F Carvalho1, H Li2, T Crochemore1, R Rodrigues1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital das Clinicas da Faculdade de Medicina da USP, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P114 (doi: 10.1186/cc13304) j y F Piza1, F Carvalho1, H Li2, T Crochemore1, R Rodrigues1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital das Clinicas da Faculdade de Medicina da USP, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P114 (doi: 10.1186/cc13304) This is a randomized controlled parallel-group trial, which included 300 patients admitted to a cancer ICU with diagnosis of septic shock. Patients were randomly assigned to a liberal strategy of blood transfusion (to maintain hemoglobin >9 g/dl) or to a restrictive strategy (hemoglobin >7 g/dl). Mortality in 28 days was the main outcome. Secondary outcomes were clinical complications days free of organ dysfunction, ICU and hospital length of stay, adverse eff ects of transfusion and 60-day mortality.i Introduction Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities [1]. A two-hit hypothesis has been proposed for TRALI. The fi rst hit is underlying patient factors, resulting in adherence of primed neutrophils to the pulmonary endothelium, such as severe pneumonia due to infl uenza A H1N1. The second hit is caused by mediators in the blood transfusion that activate the endothelial cells and pulmonary neutrophils, resulting Results A total of 136 patients were included in the fi rst part of the trial. Mean age was 62 ± 14 years, SAPS 3 at admission was 65 ± 15 and all patients had the diagnosis of solid neoplasm. Sixty-three patients S40 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Validation of infl ationary non-invasive blood pressure monitoring in emergency room patients Validation of infl ationary non-invasive blood pressure monitoring in emergency room patients J Sasaki, S Hori Keio University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P120 (doi: 10.1186/cc13310) y p Results Oral DE prolonged clot formation (CFT: 159 ± 39 seconds) and PT (27 ± 9 seconds). Following the 90-minute infusion of dabigatran, the mean plasma levels of dabigatran increased to 1,423 ± 432 ng/ ml. This supra-therapeutic level was associated with a further prolongation of PT, aPTT, and the EXTEM variables CT and CFT. These changes in coagulation parameters were compounded by blood loss following trauma (total blood loss at 60 minutes: 1,978 ± 265 ml). Sixty minutes after trauma, four out of fi ve animals had no measurable clot formation (EXTEM CFT ≥4,000 seconds) and clot strength (EXTEM MCF) had reduced to 11 ± 7 mm. Both PCCs and aDabi-Fab, but not rFVIIa, reversed the eff ects of dabigatran on thromboelastometry parameters (clotting time and clot formation time) and PT at all time points. In contrast, aPTT was only normalised by idarucizumab. Plasma concentrations of dabigatran remained elevated after PCC therapy, but were not measureable after idarucizumab.f Introduction Currently, most non-invasive blood pressure (NIBP) monitoring is based on the oscillometric method and determines the blood pressure during cuff defl ation [1]. On the other hand, a measurement during cuff infl ation may be advantageous, as cuff infl ation requires lower cuff pressure and shorter duration than defl ation. In surgical patients during anesthesia, the infl ationary NIBP has reasonable accuracy compared with conventional defl ationary NIBP [2]. Few studies have reported NIBP monitoring using the infl ationary NIBP in ER patients with various unstable conditions. A purpose of this study is to verify the usefulness of the infl ationary NIBP monitoring in the emergency department. g y p Methods A total of 2,981 NIBP data were collected from 174 patients (age; 56.5 ± 22.2 (7 to 92) years) who have been accommodated in the resuscitation area of the ER at Keio University Hospital, using alternately two algorithms with a standard monitor (BSM-6000; Nihon Kohden Inc., Tokyo, Japan). One algorithm consists of continuous infl ationary and defl ationary measurement in a single cycle (dual algorithm, 1,502 data) performed in order to verify a success rate and a precision of data. Application of damage control resuscitation strategies to patients with severe traumatic hemorrhage: review of plasma to packed red blood cell ratios at a single institution Due to ongoing blood loss both PT and TEM variables prolonged over time in PCC 25 IU/kg substituted animals. Accordingly, no-PCC (38.5 ± 4.7 ml/minute) and PCC 25 IU/kg (22.6 ± 5.5 ml/minute) animals showed highest blood loss (P <0.05 vs. PCC 50 IU/kg and PCC 100 IU/kg) with a mean survival time of 106 minutes (no-PCC animals) and 204 minutes for PCC 25 IU/kg animals, respectively. All animals of the PCC 50 IU/kg and PCC 100 IU/kg group survived. Blood loss in both groups was comparable (PCC 50 IU/ kg: 5.9 ± 0.2 ml/minute; PCC 100 IU/kg 6.0 ± 0.3 ml/minute). y Results There were 68 patients in the high-ratio group and 32 in the low-ratio group. There were statistically signifi cant diff erences between groups in the quantities of FFP, FFP:PRBC, platelets, and crystalloids administered, as well as the initial diastolic blood pressure. When comparing the incidence of complications, bloodstream infections were noted only in the high-ratio group, and the diff erence was statistically signifi cant (P = 0.028). Kaplan–Meier plots revealed that the 24-hour survival rate was signifi cantly higher in the high-ratio group (71.9% vs. 97.1%, P <0.001). The 30-day survival rate was also higher in the high-ratio group (56.2% vs. 67.6%), but the diff erence was not statistically signifi cant (P = 0.117). S41 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion For treating patients with severe hemorrhagic trauma, raising the FFP:PRBC ratio to 0.5 or higher may increase the chances of survival (especially the 24-hour survival rate). Eff orts to minimize bloodstream infections during the resuscitation process must be increased. References Studies suggest that low doses of both nitrite and carbon monoxide may protect tissues and organs from this reperfusion injury by limiting mitochondrial free radical production. We explored the eff ects of very small doses of nitrite and carbon monoxide on tissue injury in a porcine model of hemorrhagic shock. Studies suggest that low doses of both nitrite and carbon monoxide may protect tissues and organs from this reperfusion injury by limiting mitochondrial free radical production. We explored the eff ects of very small doses of nitrite and carbon monoxide on tissue injury in a porcine model of hemorrhagic shock. g Methods Fluid resuscitation of hemorrhagic shock is frequently associated with reperfusion injury and secondary organ damage. P119 Attenuation of ischemia–reperfusion injury in swine resuscitated for hemorrhagic shock by low-dose inhaled nitrite or carbon monoxide monoxide H Haugaa1, H Gomez2, D Maberry2, A Holder2, O Ogundele2, A Botero2, D Escobar2, L Gordon2, S Shiva2, C Dezfulian2, B Kenney2, TI Tønnessen1, B Zuckerbraun2, MR Pinsky2 1Oslo University Hospital, Oslo, Norway; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P119 (doi: 10.1186/cc13309) monoxide H Haugaa1, H Gomez2, D Maberry2, A Holder2, O Ogundele2, A Botero2, D Escobar2, L Gordon2, S Shiva2, C Dezfulian2, B Kenney2, TI Tønnessen1, B Zuckerbraun2, MR Pinsky2 1Oslo University Hospital, Oslo, Norway; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P119 (doi: 10.1186/cc13309) Conclusion These data suggest that infl ationary NIBP has reasonable accuracy and suffi cient rapidity compared with defl ationary NIBP in emergency room patients. P119 Results The success rate of the infl ationary NIBP (completed only by infl ationary method) was 69.0%. The bias and precision of systolic pressure and diastolic pressure (diff erence of systolic and diastolic pressure between infl ationary and defl ationary NIBP) were –0.6 ± 8.8 and 3.5 ± 7.5 mmHg, respectively (Figure 1). Infl ationary NIBP could also determine NIBP more quickly compared with defl ationary NIBP (16.8 vs. 29.1 seconds, median) (Figure 2).l In a trauma experimental pig model prothrombin complex concentrates and a specifi c antidote (idarucizumab) are eff ective to reverse the anticoagulant eff ects of dabigatran O Grottke1, J Van Ryn2, R Rossaint1 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany Critical Care 2014, 18(Suppl 1):P118 (doi: 10.1186/cc13308) In a trauma experimental pig model prothrombin complex concentrates and a specifi c antidote (idarucizumab) are eff ective to reverse the anticoagulant eff ects of dabigatran O Grottke1, J Van Ryn2, R Rossaint1 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany Critical Care 2014, 18(Suppl 1):P118 (doi: 10.1186/cc13308) Validation of infl ationary non-invasive blood pressure monitoring in emergency room patients The defl ationary algorithm (1,479 data) consists of only conventional defl ationary measurement performed in order to verify the duration of the measurement cycle. Conclusion Both PCC and aPCC are eff ective in reducing coagulopathy in a porcine trauma model with dabigatran anticoagulation. The ex vivo addition of idarucizumab fully corrected all coagulation measures and signifi cantly decreased plasma concentrations of dabigatran. No signifi cant eff ects on haemostasis were observed after the application of rFVIIa. Application of damage control resuscitation strategies to patients with severe traumatic hemorrhage: review of plasma to packed red blood cell ratios at a single institution Studies suggest that low doses of both nitrite and carbon monoxide may protect tissues and organs from this reperfusion injury by limiting mitochondrial free radical production. We explored the eff ects of very small doses of nitrite and carbon monoxide on tissue injury in a porcine model of hemorrhagic shock. 1. Duchesne JC, Hunt JP, Wahl G, et al.: Review of current blood transfusions strategies in a mature level I trauma center: were we wrong for the last 60 years? J Trauma 2008, 65:272-276. 1. Duchesne JC, Hunt JP, Wahl G, et al.: Review of current blood transfusions strategies in a mature level I trauma center: were we wrong for the last 60 years? J Trauma 2008, 65:272-276. 2. Duchesne JC, McSwain NE Jr, Cotton BA, et al.: Damage control resuscitation: the new face of damage control. J Trauma 2010, 69:976-990. 2. Duchesne JC, McSwain NE Jr, Cotton BA, et al.: Damage control resuscitation: the new face of damage control. J Trauma 2010, 69:976-990. 1. van Montfrans GA: Blood Press Monit 2001, 6:287-290. 2. Onodera J, et al.: J Anesth 2011, 25:127-130. In a trauma experimental pig model prothrombin complex concentrates and a specifi c antidote (idarucizumab) are eff ective to reverse the anticoagulant eff ects of dabigatran In a trauma experimental pig model prothrombin complex concentrates and a specifi c antidote (idarucizumab) are eff ective to reverse the anticoagulant eff ects of dabigatran O Grottke1, J Van Ryn2, R Rossaint1 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany Critical Care 2014, 18(Suppl 1):P118 (doi: 10.1186/cc13308) f O Grottke1, J Van Ryn2, R Rossaint1 O Grottke1, J Van Ryn2, R Rossaint1 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany Critical Care 2014, 18(Suppl 1):P118 (doi: 10.1186/cc13308) Introduction Strategies to reverse the anticoagulant eff ects of the new oral anticoagulants in severely bleeding patients remain challenging and confl icting results have been presented regarding the effi cacy of PCCs to alter dabigatran-induced coagulopathy. Thus, this study assessed the ability of PCC, activated PCC (aPCC), recombinant FVII (rFVIIa) and idarucizumab to reverse the anticoagulant eff ects of dabigatran in a porcine model of trauma. g y Conclusion We conclude that at low doses, nebulized sodium nitrite and inhaled carbon monoxide are associated with tissue protection during resuscitation from severe hemorrhagic shock. g g Acknowledgement This work was supported in part by DoD grant W81XWH-11-2-0049, NHLBI HL07820, and 2013121 from The South- Eastern Norwegian Health Authorities. Methods Studies were performed in fi ve pigs. Dabigatran etexilate (DE) was given orally for 3 days (30 mg/kg bid) and on the 4th day dabigatran was infused 90 minutes (30 minutes: 0.77 mg/kg/hour; 60 minutes 0.52 mg/kg/ minute) prior to blunt liver injury. Blood samples were taken before and after dabigatran infusion and 60 minutes post injury. Two doses of PCC (30, 60 IU/kg), aPCC (30, 60 IU/kg), rVIIa (90, 180 μg/kg) and idarucizumab (30, 60 mg/kg) were added to blood samples ex vivo. Coagulation was assessed by thromboelastometry, PT and diluted TT. One-way analysis of variance with the Dunnett post-hoc test for multiple comparisons was used for statistical analysis. Data are presented as mean ± SD. P118 Results Although no increase in blood nitrite concentrations was observed after inhalation, nitrite was associated with signifi cant decreases in blood, muscle, and peritoneal fl uid lactate concentrations (P <0.05), whereas both nitrite and carbon monoxide were associated with signifi cant decreases in glycerol in peritoneal fl uid (P <0.05). Following resuscitation, the muscle mitochondrial respiratory control ratio was preserved in the nitrite and carbon monoxide groups and reduced in the control group. Adjuvant drugs had no eff ects on any gross hemodynamic parameters. P121l Infl uence of the oscillometric calibration method on accuracy and precision of continuous non-invasive arterial pressure measurements using the CNAP™ device JY Wagner1, I Negulescu2, M Schöfthaler2, A Hapfelmaier2, AS Meidert2, W Huber2, RM Schmid2, B Saugel1 1University Medical Center Hamburg–Eppendorf, Hamburg, Germany; 2Klinikum rechts der Isar, Technical University Munich, Germany Critical Care 2014, 18(Suppl 1):P121 (doi: 10.1186/cc13311) Infl uence of the oscillometric calibration method on accuracy and precision of continuous non-invasive arterial pressure measurements using the CNAP™ device JY Wagner1, I Negulescu2, M Schöfthaler2, A Hapfelmaier2, AS Meidert2, W Huber2, RM Schmid2, B Saugel1 1University Medical Center Hamburg–Eppendorf, Hamburg, Germany; 2Klinikum rechts der Isar, Technical University Munich, Germany Critical Care 2014, 18(Suppl 1):P121 (doi: 10.1186/cc13311) Introduction The time profi le of the arterial pulse is known to have features such as the dicrotic notch and subsidiary peaks. Such features may provide useful information about the vascular system and are traditionally explained in terms of aortic valve closure or multiple refl ections from impedance mismatches within the arterial system. However, experimental evidence of such refl ections has been elusive. It has been proposed that arterial dynamics may obey a nonlinear equation [1]. This model predicts the existence of multipeaked solitons which can travel long distances without dissipation. We demonstrate that within the soliton model it is not necessary to model valve closure or wave refl ection: single or multiple notches arise de novo even from featureless theoretical LV pressure pulse profi les. We show that a number of clinically relevant features of the invasive blood pressure are reproduced by the soliton model and examine the role of LV pulse energy on pulse wave shape and progression. Introduction The CNAP™ system (CNSystems Medizintechnik AG, Graz, Austria) provides continuous non-invasive arterial pressure (AP) measurements based on the volume clamp method using a fi nger cuff . Finger AP values are calibrated to oscillometric upper-arm AP measurements. In the present study we investigated the infl uence of the calibration approach based on oscillometric upper-arm cuff measurements on the accuracy and precision of the CNAP™ device in comparison with invasively obtained AP measurements. y Methods The datasets of simultaneously recorded invasive (via arterial catheter) and non-invasive (using the CNAP™ system) AP measurements in 43 patients treated in the medical ICU of a university hospital were analyzed in this study. P122 P122 Arterial pulse waveform as an n-soliton evolution of the left ventricular pressure pulse B Feix, A Ercole Division of Anaesthesia, University of Cambridge, UK Critical Care 2014, 18(Suppl 1):P122 (doi: 10.1186/cc13312) g y References Introduction Fluid resuscitation of hemorrhagic shock is frequently associated with reperfusion injury and secondary organ damage. 1. van Montfrans GA: Blood Press Monit 2001, 6:287-290. 2. Onodera J, et al.: J Anesth 2011, 25:127-130. 1. van Montfrans GA: Blood Press Monit 2001, 6:287-290. 2. Onodera J, et al.: J Anesth 2011, 25:127-130. 1. van Montfrans GA: Blood Press Monit 2001, 6:287-290. 2. Onodera J, et al.: J Anesth 2011, 25:127-130. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S42 Figure 1 (abstract P120). Precision of measurements between infl ationary and defl ationary NIBP. Figure 1 (abstract P120). Precision of measurements between infl ationary and defl ationary NIBP. calibration of the CNAP™ device and the corresponding IAP values resulted in a mean diff erence (± standard deviation) of –0.8 mmHg (±8 mmHg), –5 mmHg (±14 mmHg), and +10 mmHg (±9 mmHg), respectively. (3) CAC of the CNAP™ fi nger AP values to IAP values instead of calibration to oscillometric upper-arm AP measurements resulted in a mean diff erence (± standard deviation) of +4 mmHg (±8 mmHg), +5.5 mmHg (±14 mmHg), and +3 mmHg (±7 mmHg), respectively. The accuracy of CAC-CNAP™-derived systolic and diastolic AP compared with the CNAP™-derived AP calibrated to oscillometric AP was signifi cantly higher (P = 0.004 and P <0.001, respectively). Conclusion When using the CNAP™ system, calibration to oscillometric upper-arm AP values integrated into the CNAP™ system is a relevant source of diff erence between CNAP™-derived continuous non-invasive AP measurements and invasively assessed AP values. calibration of the CNAP™ device and the corresponding IAP values resulted in a mean diff erence (± standard deviation) of –0.8 mmHg (±8 mmHg), –5 mmHg (±14 mmHg), and +10 mmHg (±9 mmHg), respectively. (3) CAC of the CNAP™ fi nger AP values to IAP values instead of calibration to oscillometric upper-arm AP measurements resulted in a mean diff erence (± standard deviation) of +4 mmHg (±8 mmHg), +5.5 mmHg (±14 mmHg), and +3 mmHg (±7 mmHg), respectively. The accuracy of CAC-CNAP™-derived systolic and diastolic AP compared with the CNAP™-derived AP calibrated to oscillometric AP was signifi cantly higher (P = 0.004 and P <0.001, respectively). Figure 2 (abstract P120). Frequency histogram of measurement time. P121l To confi rm this, we use the direct view of the tip by transesophageal echocardiography (TEE), a method which has the most recognized high sensitivity and specifi city [1]. antero-posterior radiograph of the chest (RX). New techniques have been developed to control the seat of the CVC, to avoid complications during the procedure and the RX control post insertion. The aim of this study is to demonstrate the ECG’s P-wave amplitude (PWA) increases as we approach the atrio-caval junction (ACJ) by recording the intra- cavity and show that this method is more sensitive and specifi c compared with RX control. To confi rm this, we use the direct view of the tip by transesophageal echocardiography (TEE), a method which has the most recognized high sensitivity and specifi city [1]. Figure 1 (abstract P122). i Methods In 55 adult patients, hospitalized in the ICU, a CVC was placed. We excluded patients with cardiac arrhythmias or pacemaker wearers. All CVCs were placed with an ultrasound-guided puncture technique of the internal jugular vein (IJV) or subclavian vein (SV). The CVC was introduced by the Seldinger technique. Introducing the CVC along the Seldinger guide links to the same terminal on the cable connection as the intra-cavity derivation ECG (set CVC Certofi x B; Braun), in turn connected to the adapter for ECG (Certodyn Universaladapter B; Braun). Then the detection mode of the adapter is converted by the ECG trace outside (through surface electrodes) to the ECG mode intra-cavity and an increase of PWA confi rmed the CVC in the vicinity of the ACJ. Through the TEE with esophageal average scan at 120° we measured the distance between the tip and the ACJ. The results were expressed as mean with standard deviation.i Results Forty-fi ve CVCs were placed in the IJV while 10 were in the SV. No complications or arrhythmias were detected during the procedure. All CVCs produced an increase in PWA. Where the PWA increased by 25% compared with normal, the TEE scanning tip was 2.5 ± 1.3 cm from the ACJ. Where the PWA increased by 33%, the TEE scanning tip was 1.9 ± 1.1 cm from the ACJ. Where the PWA increased by 50%, the TEE scanning tip was 1.3 ± 0.6 cm from the ACJ. P121l The RX reports have described briefl y the presence of the catheter in the superior vena cava without making explicit the exact position relative to the ACJ. P121l The following comparative analyses between the two AP measurement techniques were performed: (1) comparison of CNAP™-derived AP values with invasive AP (IAP) measurements; (2) comparison of the CNAP™ oscillometric AP values used for the calibration of fi nger AP with IAP measurements; and (3) computer- aided calibration (CAC) of the CNAP™ fi nger AP values to IAP values instead of calibration to oscillometric upper-arm AP measurements with IAP measurements. gy p p p g Methods A model for the arterial pressure is given by solutions to a KdV equation with constants depending on the properties of the artery [1]. This can be solved with the initial condition of a parabolic left ventricular pressure pulse. Results The evolution of the arterial pulse along the arterial tree is shown in Figure 1. We also predict arterial pulses for increasing left ventricular ejection energies. Conclusion Our simple model explains many features of the arterial pulse observed in clinical practice such as the development of the dicrotic notch, the change in shape along the arterial tree and the steepening and acceleration with hypertension. Some phenomena that have traditionally been attributed to arterial wave refl ections or resonance of the invasive arterial pressure measurement can instead be explained by intrinsic properties of the arterial pulse. Results (1) The comparison of CNAP™-derived AP values with IAP measurements revealed a mean diff erence (± standard deviation) for mean AP, systolic AP, and diastolic AP of +0.6 mmHg (±10 mmHg), +11 mmHg (±17 mmHg), and –6 mmHg (±9 mmHg), respectively. (2) The comparison between the oscillometric AP values used for Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S43 Reference 1. Laleg TM, et al.: Biomed Signal Process 2007, 2:163-170. P123 Tackling the burden of postsurgical complications in the USA: would perioperative goal-directed therapy help? G Manecke, A Asemota, F Michard UCSD Medical Center, San Diego, CA, USA Critical Care 2014, 18(Suppl 1):P123 (doi: 10.1186/cc13313) Figure 1 (abstract P122). antero-posterior radiograph of the chest (RX). New techniques have been developed to control the seat of the CVC, to avoid complications during the procedure and the RX control post insertion. The aim of this study is to demonstrate the ECG’s P-wave amplitude (PWA) increases as we approach the atrio-caval junction (ACJ) by recording the intra- cavity and show that this method is more sensitive and specifi c compared with RX control. Reference Laleg TM, et al.: Biomed Signal Process 2007, 2:163-170. 1. Laleg TM, et al.: Biomed Signal Process 2007, 2:163-170. P123 Tackling the burden of postsurgical complications in the USA: would perioperative goal-directed therapy help? G Manecke, A Asemota, F Michard UCSD Medical Center, San Diego, CA, USA Critical Care 2014, 18(Suppl 1):P123 (doi: 10.1186/cc13313) P123 Conclusion We have shown the increase in PWA by detecting that the endo-cavity, as we proceed with the insertion of the CVC, corresponds to higher proximity to the ACJ. These results canceled the need for RX control in the future. Introduction Pay-for-performance programs and economic constraints call for solutions improving the quality of healthcare without increasing costs. Many studies have shown decreased morbidity in major surgery when perioperative goal-directed therapy (PGDT) is used. We assessed the clinical and economic burden of postsurgical complications in the University HealthSystem Consortium (UHC) in order to predict potential savings with PGDT. P125 Impact of the neutral position and rotation of the head in ultrasound-guided internal jugular vein catheterization on duration of procedure and complications M Kurt, A Ozgultekin, G Turan, F Ormancı, S Batan, O Ekinci Haydarpasa Numune Teaching and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P125 (doi: 10.1186/cc13315) y Results A total of 75,140 patients met the search criteria. In total, 8,421 patients developed one or more postsurgical complications (morbidity rate 11.2%). In-hospital mortality was 12.42% and 1.39% (P <0.001), mean hospital length of stay was 20.48 ± 20.09 days and 8.05 ± 7.11 days (P <0.001), and mean direct cost was $47,284 ± 49,170 and $17,408 ± 15,612 (P <0.001) in patients with and without complications, respectively. With PGDT, morbidity rate was projected to decrease to 6.5 to 9.0%, yielding gross costs savings of $50 million to 151 million for the study population or $670 to $1,406 per patient. Introduction In internal jugular vein (IJV) catheterization, neck rotation may enhance the visibility of anatomical landmarks; however, it may increase the risk of carotid artery (CA) puncture by replacing the position of the IJV in relation to the CA. In our study, during US- guided IJV catheterizations, we investigated the eff ects of changing the position of the IJV by the CA depending on the neutral position and 45° rotation of the head on duration of procedure and complications. Methods After obtaining hospital ethics committee approval, 100 intensive care patients aged >18 years were included in the study. Patients were randomly selected and catheterization was performed in a neutral position of the head (n = 50) and by turning the head 45° to the opposite side (n = 50). The US-guided catheterization procedure was performed in accordance with general principles. Once the needle entered the IJV and blood was aspirated, the US probe was released from the hand, and the catheter was placed and fi xed according to the Seldinger technique. The data of the intervention side for each process, the count number of successful catheter insertion, whether there is arterial access or not and the duration of procedure (from skin contact of the needle to catheter insertion) were recorded. Conclusion Postsurgical complications have a dramatic impact on mortality, hospital length of stay and costs. Potential cost savings resulting from PGDT are substantial. PGDT may be recommended to improve quality of care and decrease costs. Reference 1. Pelegatti C, et al.: Endovascular ECG to guide placement of CVC. J Vasc Acces 2011, 12:348-353. Methods Data from adults who had 10 major surgical procedures in 2011 were screened in the UHC database. Thirteen postsurgical complications were tabulated. In-hospital mortality, hospital length of stay and costs from patients with and without complications were compared. The risk ratios reported by the most recent meta-analysis were used to estimate the potential reduction in postsurgical morbidity with PGDT. Potential cost savings were calculated from the actual and anticipated morbidity rates. P124 Radiological control of central venous catheter (CVC) versus electrocardiogram-guided control inserted CVC: confi rm with transesophageal echocardiography F Righetti, G Castellano St Boniface Hospital Verona, Italy Critical Care 2014, 18(Suppl 1):P124 (doi: 10.1186/cc13314) Radiological control of central venous catheter (CVC) versus electrocardiogram-guided control inserted CVC: confi rm with transesophageal echocardiography h ll Development of a standardized method of peripherally inserted central catheter (PICC-line) bedside installation J Hobeika, K Serri, M Albert Hôpital Sacré-Coeur de Montréal, Montreal, Canada Critical Care 2014, 18(Suppl 1):P128 (doi: 10.1186/cc13318) Development of a standardized method of peripherally inserted central catheter (PICC-line) bedside installation J Hobeika, K Serri, M Albert Hôpital Sacré-Coeur de Montréal, Montreal, Canada Critical Care 2014, 18(Suppl 1):P128 (doi: 10.1186/cc13318) Introduction Bedside insertion of peripherally inserted central catheters (PICCs) results in tip malposition in up to 48% [1], with catheters frequently terminating in the internal jugular vein (IJV) or in peripheral veins [2]. In an attempt to reduce tip malposition, we developed a standardized approach to PICC installation. This study aims to validate that method. Results Sixty-fi ve CVCs were placed: 51 in the right internal jugular vein (IJV), 14 in the left IJV. The mean catheter length by intracavitary ECG was signifi cantly deeper than predicted by Lum’s formulas (18.2 ± 1.9 vs. 16.7 ± 1.7 cm, P <0.001) and not diff erent from Peres’ formulas (18.2 ± 1.9 vs. 18.2 ± 1.7 cm, P = 0.8). Methods From a 34-bed adult ICU, we retrospectively reviewed PICC insertions over a 6-month period before the intervention program (control group). We designed a prospective interventional pilot study on 40 consecutive patients (intervention group). Patients in the intervention group were positioned in a standardized fashion and the PICC length was measured from easily identifi ed anatomic landmarks. During PICC insertion, the patient’s head was either rotated ipsilaterally to the site of PICC insertion or the ipsilateral IJV was manually compressed, depending on the patient’s capacity to collaborate. Once the PICC was inserted, an ultrasound survey was conducted to identify the catheter in the subclavian vein (SC) and ensure its absence in the ipsilateral IJV. The primary endpoint was defi ned as PICC tip position, obtained from the post-procedural chest X-ray. A catheter was considered to be in an optimal position if the tip resided in the distal third of the superior vena cava (SVC), adequate if it resided between the subclavian vein (SC) and the distal third of the SVC, and aberrant if in any other location. At post-procedural CXR, 88% of the tips were in the target zone. In three cases (5%) the catheter went in the wrong direction but was immediately corrected during the procedure since the intracavitary P wave did not change its amplitude. Compared with the intracavitary ECG, the incidence of malposition would have been signifi cantly higher with Lum’s formulas (48% vs. 12%, P <0.001) and Peres’ formulas (51% vs. 12%, P <0.001). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 the localization of the IJV in relation to the CA, the anterior placement rate increasing the risk of CA puncture was signifi cantly higher in a position of rotation compared with the neutral position (P = 0.001). No signifi cant diff erence was found between procedure durations. Complications were recorded when observed. the localization of the IJV in relation to the CA, the anterior placement rate increasing the risk of CA puncture was signifi cantly higher in a position of rotation compared with the neutral position (P = 0.001). No signifi cant diff erence was found between procedure durations. Complications were recorded when observed. may not be available in emergency situations, and therefore guidelines also propose that physicians remained skilled in landmark (LM) placement. We conducted this prospective observational study to determine the learning curve of the LM technique in residents only learning the UG technique. g q Methods During the fi rst 3 months of their rotation in our ICU, residents inexperienced in CVC used only the real-time UG technique. During the following 3 months, residents were allowed to place CVC by means of the LM technique when authorized by the attending physician. Conclusion In our study, it has been shown that anterior placement of the IJV in the neutral position is less but this has no advantage in avoiding arterial puncture. The smaller area of procedure in a neutral position can cause diffi culties in practice. However, the processing times between each head position were not diff erent. Nevertheless, further studies evaluating whether there are comparable complication rates and the same duration of procedure in emergency and trauma patients in whom head rotation cannot be possible are needed. q y g p y Results A total of 172 procedures (84 UG and 88 LM) were performed by the inexperienced residents during the study. The success rate was lower (72% vs. 84%; P = 0.05) and the complication rate was higher (22% vs. 10%; P = 0.04) for LM compared with UG procedures. Comparison between the fi ve last UG procedures and the fi rst fi ve LM procedures performed demonstrated that the transition between the two techniques was associated with a marked decrease of the success rate (65% vs. 93%; P = 0.01) and an increase of the complication rate (33% vs. 8%; P = 0.01). Development of a standardized method of peripherally inserted central catheter (PICC-line) bedside installation J Hobeika, K Serri, M Albert Hôpital Sacré-Coeur de Montréal, Montreal, Canada Critical Care 2014, 18(Suppl 1):P128 (doi: 10.1186/cc13318) Conclusion The intracavitary ECG was associated with a lower incidence of tip malposition than Peres’ and Lum’s formulas. It is also the only technique allowing one to immediately correct a primary malposition during catheter insertion. g References References 1. Peres PW: Anaesth Intensive Care 1990, 18:536-539. 2. Lum P: J Vasc Access 2004, 9:80-85. 3. Pittiruti M, et al.: J Vasc Access 2011, 12:280-291. 4. Schuster M, et al.: Br J Anaesth 2000, 85:192-194. 1. Peres PW: Anaesth Intensive Care 1990, 18:536-539. Results In the retrospective control arm, 105 PICCs were reviewed for tip position. Optimal, adequate, and aberrant positions were found in 22 (21%), 49 (47%), and 34 (32%) respectively, in comparison with 17 (43%), 15 (38%), and eight (20%) in the intervention group (P <0.05 between both groups). In the control arm, 11 (10%) PICCs terminated outside the central venous system, whereas none failed to achieve central venous access in the intervention arm. 2. Lum P: J Vasc Access 2004, 9:80-85. 3. Pittiruti M, et al.: J Vasc Access 2011, 12:280-291. 4. Schuster M, et al.: Br J Anaesth 2000, 85:192-194. Minkovich et al.: Can J Anaesth 2011, 58:709-713. Trerotola et al.: J Vasc Interv Radiol 2007, 18:513e8. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 After 10 LM procedures, residents achieved a success rate and a complication rate of 81% and 6%, respectively. Radiological control of central venous catheter (CVC) versus electrocardiogram-guided control inserted CVC: confi rm with transesophageal echocardiography F Righetti, G Castellano Results The localization of the IJV in relation to the CA is 66% anterolateral, 4% anterior and 30% lateral in a neutral position, and 62% anterolateral, 28% anterior, 10% lateral position in 45° rotation. So while there is no change in a signifi cant proportion of patients in Introduction The placement of a central venous catheter (CVC) is now common practice. Control of the seat of the tip occurs through the S44 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P126 Anthropometric formulas versus intracavitary ECG for optimal tip position of central venous catheters MS Vallecoccia, M Biancone, F Cavallaro, D Settanni, C Marano, MG Annetta, M Pittiruti, M Antonelli Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P126 (doi: 10.1186/cc13316) Conclusion Residents who only learn the UG technique will not be immediately able to perform the LM technique, but require specifi c training based on at least 10 LM procedures. Whether the LM technique should still be taught when an ultrasound device is not available must therefore be addressed. Introduction Peres [1] and more recently Lum [2] developed some anthropometric formulas to correlate patient’s height (H) and ideal length of central venous catheter (CVC) in order to identify optimal tip position. The aim of this study is to compare the reliability of the anthropometric formulas with the method based on intracavitary ECG. Methods We enrolled patients admitted to our ICU candidate to elective CVC insertion, who had a detectable P wave on surface ECG. Intracavitary ECG was used to identify the optimum tip location since a maximal P wave indicates the cavo-atrial junction [3]. Post-insertion chest X-ray (CXR) was performed in all patients to verify the tip position. Assuming that the cavo-atrial junction is about 3 cm from the carina [4], the tip position was considered correct between 1 and 5 cm from the carina (between the lower 1/3 of the superior vena cava and the upper 1/3 of the right atrium). For each patient we retrospectively evaluated whether the catheter length calculated with Lum’s and Peres’ formulas on an estimated height would have been acceptable.i Residents learning ultrasound-guided catheterization are not suffi ciently skilled to use landmarks Residents learning ultrasound-guided catheterization are not suffi ciently skilled to use landmarks J Maizel, L Guyomarc’h, P Henon, S Samy Modeliar, B De Cagny, G Choukroun, M Slama CHU Amiens, France Critical Care 2014, 18(Suppl 1):P127 (doi: 10.1186/cc13317) Residents learning ultrasound-guided catheterization are not suffi ciently skilled to use landmarks J Maizel, L Guyomarc’h, P Henon, S Samy Modeliar, B De Cagny, G Choukroun, M Slama CHU Amiens, France Critical Care 2014, 18(Suppl 1):P127 (doi: 10.1186/cc13317) Conclusion Using the standardized method described above, PICC tip positioning can be greatly improved. In our results, 100% of catheters placed using the standardized method allowed for central venous access. This pilot study paves the way for a larger, multicentric evaluation of the bedside installation method of PICC. R f fi y J Maizel, L Guyomarc’h, P Henon, S Samy Modeliar, B De Cagny, G Choukroun, M Slama CHU Amiens, France Critical Care 2014, 18(Suppl 1):P127 (doi: 10.1186/cc13317) J Maizel, L Guyomarc’h, P Henon, S Samy Modeliar, B De Cagny, G Choukroun, M Slama CHU Amiens, France Critical Care 2014, 18(Suppl 1):P127 (doi: 10.1186/cc13317) evaluation o References Hendrikse KA, et al.: Low value of routine chest radiographs in a mixed medical–surgical ICU. Chest 2007, 132:823-828. 2. Oba Y, Zaza T: Abandoning daily routine chest radiography in the intensive care unit: meta-analysis. Radiology 2010, 255:386-395. 2. Oba Y, Zaza T: Abandoning daily routine chest radiography in the intensive care unit: meta-analysis. Radiology 2010, 255:386-395. 2. Oba Y, Zaza T: Abandoning daily routine chest radiography in the intensive care unit: meta-analysis. Radiology 2010, 255:386-395. p Results Eighty CVCs were placed in 78 patients, either by residents in training or by attending doctors. The vein was selected by US scan: right internal jugular (IJ) 64%, left IJ 17%, right axillary 11%, left axillary 4%, right innominate 4%. One pre-existing PNX was identifi ed by US before the procedure. Accidental arterial puncture occurred in three cases (two by residents). In fi ve cases, the wrong direction of the wire was detected by US and corrected during the procedure. The mean number of attempts was 1.65 (1.35 for attending vs. 1.95 for residents, P <0.02) and mean insertion time was 10.4 minutes (8.72 for attending vs. 12.11 for residents, P <0.005). Pleural US scan ruled out puncture-related PNX in all patients. At post-procedural CXR: no PNX was detected, 90% of tips were in the target zone, 5% were in the middle 1/3 of SVC and 5% were in the middle 1/3 of RA (all CVC with tips out of the target zone were inserted by residents).f References 1. Pittiruti M, et al.: Clin Nutr 2009, 28:365-377. 2. Schuster M, et al.: Br J Anaesth 2000, 85:192-194. Ultrasound measurement of carotid fl ow time changes with volume status Ultrasound measurement of carotid fl ow time changes with volume status DC Mackenzie1, NA Khan2, D Blehar3, CP Stowell2, VE Noble2, AS Liteplo2 1Maine Medical Center, Portland, ME, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3University of Massachusetts, Worcester, MA, USA Critical Care 2014, 18(Suppl 1):P131 (doi: 10.1186/cc13321) DC Mackenzie1, NA Khan2, D Blehar3, CP Stowell2, VE Noble2, AS Liteplo2 1Maine Medical Center, Portland, ME, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3University of Massachusetts, Worcester, MA, USA Critical Care 2014, 18(Suppl 1):P131 (doi: 10.1186/cc13321) Introduction Assessment of volume status and responsiveness guides resuscitation strategy. Non-invasive techniques are desirable. Changes in carotid fl ow time have been proposed as a marker of volume status, but few data support their use. We sought to determine whether carotid fl ow time decreased in the volume-depleted state of acute blood loss, and whether volume-depleted individuals would demonstrate an increase in carotid fl ow time after a passive leg raise (PLR) maneuver. Methods Volunteers aged 18 to 55 presenting to the hospital’s blood donor center for whole blood donation were eligible to participate. Individuals with a history of aortic or carotid artery disease, atrial fi brillation, or a contraindication to blood donation were excluded. Prior to blood donation, an investigator performed an ultrasound of the right common carotid artery with a high-frequency linear transducer, obtaining a Doppler tracing of carotid artery fl ow. Measurements of peak velocity, systole time, and carotid fl ow time were obtained. A PLR was performed for 30 seconds, followed by repeat measurements of carotid velocity and fl ow time. Whole blood was then collected according to the blood donor center’s protocol. Immediately after blood donation, repeat measurements of carotid fl ow and velocity were obtained in the supine position and after a PLR. Carotid fl ow times corrected for heart rate (FTc) were analyzed with Student’s t test. The institutional review board approved the study. Conclusion Our insertion protocol was safe and eff ective, with minor diff erences between experienced versus nonexperienced operators. Our data suggest that immediate CXR is not necessary soon after CVC insertion, since PNX can be ruled out by US and since the intracavitary ECG method allows tip location in the SVC and RA in all cases. evaluation o References Introduction An ultrasound-guided (UG) technique is the recom men- ded procedure for central venous catheterization (CVC). But ultrasound Minkovich et al.: Can J Anaesth 2011, 58:709-713. Trerotola et al.: J Vasc Interv Radiol 2007, 18:513e8. Minkovich et al.: Can J Anaesth 2011, 58:709-713. Trerotola et al.: J Vasc Interv Radiol 2007, 18:513e8. S45 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P129 and pericardial eff usion with or without cardiac tamponade. Positions of the endotracheal tube and central venous catheter were also checked. P129 Is chest X-ray necessary after central venous catheter insertion? MS Vallecoccia, F Cavallaro, M Biancone, D Settanni, C Marano, MG Annetta, M Pittiruti, M Antonelli Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P129 (doi: 10.1186/cc13319) Results Ninety-four of the 151 patients included (62%) showed abnormalities on chest X-ray (AC 9%, AIS 25%, PLAPS 42%, PE 3.3%, PTX 2%). Compared with chest X-ray, chest ultrasound had a sensitivity of 86% and a specifi city of 99% for AC, a sensitivity of 95% and a specifi city of 100% for AIS, a sensitivity of 97% and a specifi city of 98% for PLAPS, a sensitivity of 99% and a specifi city of 100% for PE, and a sensitivity and specifi city of 100% for PTX. Furthermore, chest ultrasound detected all pericardial eff usions while neither chest X-ray nor chest auscultation were able to identify them. Chest ultrasound identifi ed all cases of endotracheal tube (two patients) and central venous catheter (two patients). There was a highly signifi cant correlation between abnormalities detected by chest ultrasound and X-ray (k = 0.90), but a poor correlation between chest auscultation and X-ray abnormalities (k = 0.15). Introduction According to the European Society of Parenteral and Enteral Nutrition guidelines [1], post-insertion chest X-ray (CXR) is not necessary if the location of the tip has been verifi ed during the procedure and if pleura-pulmonary damage has been ruled out by other methods. The aim of this study is to assess feasibility and safety of an echo-ECG-guided method of central venous catheter (CVC) insertion and to evaluate whether post-insertion CXR can be avoided. Methods We enrolled only patients admitted to our ICU and candidate to elective CVC insertion, who had a detectable P wave on surface ECG. evaluation o References Our insertion protocol included: preliminary ultrasound (US) scan of central veins and pleural space; US-guided puncture and US control of the correct direction of the guidewire; intracavitary ECG method for tip location (cavo-atrial junction (CAJ) = maximal P wave); and US scan of pleural space to rule out pneumothorax (PNX). Post-insertion CXR was performed in all patients to rule out PNX and verify tip location close to the CAJ (CAJ = 3 cm below the carina [2]). Tip location between 1 and 5 cm below the carina – in the lower 1/3 of the superior vena cava (SVC) or in the higher 1/3 of the right atrium (RA) – was considered acceptable [1]. Introduction According to the European Society of Parenteral and Enteral Nutrition guidelines [1], post-insertion chest X-ray (CXR) is not necessary if the location of the tip has been verifi ed during the procedure and if pleura-pulmonary damage has been ruled out by other methods. The aim of this study is to assess feasibility and safety of an echo-ECG-guided method of central venous catheter (CVC) insertion and to evaluate whether post-insertion CXR can be avoided. Methods We enrolled only patients admitted to our ICU and candidate to elective CVC insertion, who had a detectable P wave on surface ECG. Our insertion protocol included: preliminary ultrasound (US) scan of central veins and pleural space; US-guided puncture and US control of the correct direction of the guidewire; intracavitary ECG method for tip location (cavo-atrial junction (CAJ) = maximal P wave); and US scan of pleural space to rule out pneumothorax (PNX). Post-insertion CXR was performed in all patients to rule out PNX and verify tip location close to the CAJ (CAJ = 3 cm below the carina [2]). Tip location between 1 and 5 cm below the carina – in the lower 1/3 of the superior vena cava (SVC) or in the higher 1/3 of the right atrium (RA) – was considered acceptable [1]. Conclusion Chest auscultation may help identify endotracheal misplacement and tension pneumothorax but it may miss most of major abnormalities. Chest ultrasound represents a valid alternative to chest X-ray to detect all postoperative abnormalities and misplacements. References 1. Hendrikse KA, et al.: Low value of routine chest radiographs in a mixed medical–surgical ICU. Chest 2007, 132:823-828. 1. Hendrikse KA, et al.: Low value of routine chest radiographs in a mixed medical–surgical ICU. Chest 2007, 132:823-828. 1. P130 Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation A Vezzani1, T Manca1, F Benassi1, A Gallingani1, I Spaggiari1, C Brusasco2, F Corradi3, T Gherli1 1Azienda Ospedaliero Universitaria di Parma, Italy; 2Università degli studi di Genova, Italy; 3E.O. Ospedali Galiera, Genova, Italy Critical Care 2014, 18(Suppl 1):P130 (doi: 10.1186/cc13320) Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation P132 P132 Real-time ultrasound-guided subclavian vein cannulation in cardiac surgery: comparison between short-axis and long-axis techniques F Corradi1, T Manca2, C Brusasco3, F Cocconcelli2, A Agostinelli2, F Benassi2, T Gherli2, A Vezzani2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Azienda Ospedaliero- Universitaria di Parma, Italy; 3Università degli Studi di Genova, Italy Critical Care 2014, 18(Suppl 1):P132 (doi: 10.1186/cc13322) Introduction Central venous catheters play an important role in patient care; however, their use is associated with various complications and more frequently through the subclavian vein (SCV) route. A previous study showed that ultrasound-guided cannulation of the SCV in critical care patients is superior to the landmark method and should be the method of choice in these patients [1]. The aim of this study was to compare short-axis and long-axis approaches for ultrasound-guided subclavian vein cannulation with respect to indicators of success. Methods Eighty-three patients undergoing cardiac surgery and requiring central venous cannulation were randomized to receive long- axis or short-axis ultrasound-guided cannulation of the subclavian vein by a skilled anesthesiologist. First-pass success, unsuccessful placement, number of attempts, number of needle passes, skin and vessel puncture, time to successful catheterization and complications were considered as outcomes. Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Results The subclavian vein was successfully cannulated by ultrasound- guided techniques in all patients. Central venous cannulation failed in two and 10 cases respectively with short-axis and long-axis view and the other view was used successfully. Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation y A Vezzani1, T Manca1, F Benassi1, A Gallingani1, I Spaggiari1, C Brusasco2, F Corradi3, T Gherli1 1Azienda Ospedaliero Universitaria di Parma, Italy; 2Università degli studi di Genova, Italy; 3E.O. Ospedali Galiera, Genova, Italy Critical Care 2014, 18(Suppl 1):P130 (doi: 10.1186/cc13320) Results Eighty donors were screened for participation by two investigators; 68 consented and completed donation (60.3% female, mean age 31). Donors had mean blood loss of 450 ml. The mean FTc supine after blood donation was 296 ms; this was signifi cantly diff erent from the FTc prior to donation (supine = 320 ms, PLR = 323 ms; P <0.0001). The mean FTc following blood donation and PLR was 321 ms, signifi cantly diff erent from the supine position after donation (P <0.0001), but not pre-donation measurements. Introduction Chest auscultation and chest X-ray are commonly used to detect postoperative abnormalities and complications in patients admitted to intensive care after cardiac surgery [1,2]. The aim of the study was to evaluate whether chest ultrasound represents an eff ective alternative to bedside chest X-ray to identify early postoperative abnormalities. Methods A total of 151 consecutive patients (103 male and 47 female) were studied by chest auscultation, ultrasound and X-ray upon admission to intensive care after cardiac surgery. Six pathologic entities were explored by each method: postero-lateral pleural eff usion and/ or alveolar consolidation (PLAPS), alveolar-interstitial syndrome (AIS), alveolar consolidation (AC), pneumothorax (PTX), pleural eff usion (PE), Conclusion Ultrasound measurement of carotid fl ow time was signifi cantly decreased in the setting of acute blood loss. An auto- bolus by PLR after blood loss restored FTc to pre-donation levels. Further investigation of FTc as a non-invasive predictor of volume responsiveness is warranted. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S46 i Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Transthoracic echocardiography used in conjunction with passive leg raising for assessment of fl uid responsiveness in severe sepsis or septic shock patients K Jaikriengkrai, K Chittawatanarat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2014, 18(Suppl 1):P133 (doi: 10.1186/cc13323) p p K Jaikriengkrai, K Chittawatanarat, A Limsukon g Chiang Mai University, Chiang Mai, Thailand lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93) (Figure 1). The Bland–Altman plot showed 95% limits of agreement from –8.96 to +8.83% and mean diff erence (bias) of –0.07% (Figure 2). g y g Critical Care 2014, 18(Suppl 1):P133 (doi: 10.1186/cc13323) Introduction During passive leg raising (PLR), we need a real-time device to demonstrate the hemodynamic change [1-3]. This study investigates the ability of transthoracic echocardiography (TTE) to predict fl uid responsiveness (FR) in terms of detecting change of stroke volume (ΔSV) after PLR compared with the transpulmonary thermodilution technique (TPTD) ΔSV after volume expansion (VE). f Conclusion We may use %ΔSV measured by TTE after PLR to predict FR, which is noninvasive and less time-consuming than other invasive techniques. q References 1. Monnet X, et al.: Crit Care 2013, 17:217. Methods A prospective study was carried out in a medical ICU. Eligible patients were age ≥18 years without necessarily full adaptation to the ventilator with hemodynamic instability who were considered for VE. SV assessment using the subaortic velocity–time measurement was obtained by TTE simultaneously with other hemodynamic parameters derived from TPTD at baseline, within 2 minutes of PLR and following VE (250 ml fl uid in 10 minutes). A fl uid responder was defi ned by ΔSV ≥15% after VE by TPTD. 2. Lafanechere A, et al.: Crit Care 2006, 10:R132. 3. Biais M, et al.: Crit Care 2009, 13:R195. 2. Lafanechere A, et al.: Crit Care 2006, 10:R132. P133 Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Transthoracic echocardiography used in conjunction with passive leg raising for assessment of fl uid responsiveness in severe sepsis or septic shock patients K Jaikriengkrai, K Chittawatanarat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2014, 18(Suppl 1):P133 (doi: 10.1186/cc13323) Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation The fi rst-pass success rate was signifi cantly higher in the short-axis group (73%) compared with the long-axis group (40%) (P = 0.005). The procedure time, number of attempts, needle redirection, and skin and vessel punctures were signifi cantly lower in the short-axis than long-axis group (P <0.05). The overall number of complications did not diff er signifi cantly between groups even if artery puncture and hematoma occurred more frequently in the long-axis group. Moreover, the need to change the ultrasound- guided insertion technique was more frequent in the long-axis group. Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Conclusion Ultrasound-guided subclavian vein cannulation by an experienced operator has a higher fi rst-pass success rate and lower access time using the short-axis than long-axis approach. f Reference 1. Fragou M, Gravvanis A, Dimitriou V, Papalois A, Kouraklis G, Karabinis A, Saranteas T, Poularas J, Papanikolaou J, Davlouros P, Labropoulos N, Karakitsos D: Real-time ultrasound-guided subclavian vein cannulation versus the landmark method in critical care patients: a prospective randomized study. Crit Care Med 2011, 39:1607-1612. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods Standard 12-lead and actual V3R to V5R, V7 to V9 lead ECGs at Tokyo Medical University Hospital were successfully recorded and compared with synthesized ECGs at the J point, M point that was defi ned for the point after 1/16 RR interval and T wave amplitude. ECGs of the complete right branch block, complete left branch block and pacing rhythm were excluded. (ECMO). In many circumstances, transoesophageal echocardiography (TOE) is the preferred monitoring tool. It can aid in cannula positioning, especially during double-lumen cannula placement for V-V ECMO, weaning of V-A ECMO and diagnose causes of high-access pressures and circuit fl ow problems. We use TOE as our preferred monitoring equipment before, during and after establishing ECMO. We sought to investigate how often information gained from TOE imaging had a major impact on management decisions. p g y Results A total of 1,216 ECGs were correctly recorded. The diff erences of actual and synthesized at the J point, M point and T wave amplitude were very small. Means of the diff erence ± 2SD were V3R/V4R/V5R/V7/ V8/V9: J point, 17 ± 1/14 ± 1/13 ± 1/12 ± 1/15 ± 1/18 ± 1 μV; M point, 15 ± 1/13 ± 1/12 ± 1/12 ± 1/12 ± 1/13 ± 1 μV; and T wave amplitude, 20 ± 3/32 ± 2/16 ± 2/37 ± 2/39 ± 2/43 ± 3 μV. There were positive correlations between all actual and synthesized ECGs of J point, M point and T wave amplitude (Figure 1). j p g Methods A single-centre observational study at a tertiary referral institution. All patients supported with V-A or V-V ECMO during an 18-month period were included. Routine procedures such as wire position checks during cannulation or information gained to assist weaning from V-A support were not included. g pp Results Twenty patients were supported with either V-A (all peripheral) or V-V ECMO during the observation period. In 12 patients (60%) TOE was instrumental in diagnosing potentially fatal complications or altered clinical management. In three patients on V-A support, afterload reduction and modulation of inotropic support was necessary due to extensive spontaneous echo contrast formation in the left ventricle and stagnant pulmonary blood fl ow; two out of these three patients, immediately after establishing support, required intra-aortic balloon counterpulsation to reverse clot formation around the aortic valve and root. Accuracy of synthesized right-sided/posterior chest lead electrocardiograms g Y Saitoh1, Y Goseki2, A Yamashina2 1Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium; 2Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P135 (doi: 10.1186/cc13325) Introduction Right-sided precordial leads (V3R to V5R) and posterior chest leads (V7 to V9) provide important information for the right ventricle and posterior wall. These additional lead electrocardiograms (ECGs) improve diagnostic value in acute coronary syndrome patients [1]. However, these additional electrocardiograms are not routinely recorded due to the time-consuming procedure involved. Recently these synthesized six additional lead ECGs using the standard 12-lead ECG system (Nihonkoden Co. Ltd) have been developed [2,3]. But the accuracy is not clear. The purpose of the present study was to evaluate the accuracy of synthesized ECGs at the ST part. Methods We recruited patients meeting criteria for severe sepsis and septic shock within 24 hours of admission to ICU. After haemodynamic stabilisation, PWV was recorded at inclusion and after 48 hours using dual-channel plethysmography. Severity of illness was assessed with APACHE II and serial SOFA scores, haemodynamic and infl ammatory parameters (CRP, procalcitonin and fi brinogen) recorded. A 28-day follow-up was performed to distinguish between survivors and nonsurvivors. Results Twenty consecutive general ICU patients (six with severe sepsis and 14 with septic shock) were enrolled in the study; median age 59 years (IQR 56.5 to 72), APACHE II score 17 (13 to 20.5), SOFA score 5 (IQR 4 to 9). At 28 days, six patients had died. Median initial PWV was 10.4 (IQR 6.9 to 12.1) m/second in patients with severe sepsis, and 6.8 (IQR 5.3 to 7.5) m/second in patients with septic shock (P = 0.13). After 48 hours, PWV in the severe sepsis and septic shock groups had become similar, 9.3 (IQR 7.3 to 11.1) m/second and 9.2 (IQR 7.8 to 13) m/second respectively (P = 0.96). PWV had signifi cantly increased in survivors (7.8 to 12.3 m/second) (P = 0.04) versus nonsurvivors (6 to 7.8 m/second) (P = 0.69). Higher PWV correlated with increasing systolic pressure and lower CRP levels (r = 0.73, P = 0.01).f Figure 1 (abstract P135). Correlation between actual and synthesized ECG at V7. Conclusion In early sepsis, aortic stiff ness is decreased in patients with greater disease severity, and in survivors increases to median levels within 48 hours. The main factors associated with lower pulse wave velocity are lower systolic pressure and higher CRP levels. References References 1. Zalenski RJ, et al.: Am J Cardiol 1997, 79:1579-1585. 2. Katoh T, et al.: J Nihon Med Sch 2011, 78:22-29. 3. Nakayama M, et al.: J Electrocardiol 2012, 32:221-228. 1. Zalenski RJ, et al.: Am J Cardiol 1997, 79:1579-1585. 1. Zalenski RJ, et al.: Am J Cardiol 1997, 79:1579-1585. 2. Katoh T, et al.: J Nihon Med Sch 2011, 78:22-29. 2. Katoh T, et al.: J Nihon Med Sch 2011, 78:22-29. 3. Nakayama M, et al.: J Electrocardiol 2012, 32:221-228. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Further diagnosis were Avalon cannula misplacement in the blind end of a piggyback cava following liver transplant, collapsing right atrial free wall with ‘sucking down’ into the access cannula (n = 3), pericardial clot formation and tamponade (n = 1), persistent left superior vena cava making planned cannula placement into the right atrium impossible or potentially fatal, diagnosis of patent foraminae ovale providing left atrial decompression and appropriate drainage cannula positioning during liver transplantation on ECMO. Conclusion The ST part of synthesized V3R to V5R and V7 to V9 lead ECGs appears to be highly reliable. Synthesized additional lead ECGs might be useful to diagnose ischemic heart disease. R f Aortic stiff ness in patients with early sepsis Aortic stiff ness in patients with early sepsis , , ķ , g 1Hospital of Traumatology and Orthopaedics, Riga, Latvia; 2Institute of Atomic Physics and Spectroscopy, University of Latvia, Riga, Latvia; 3Riga Stradins University, Riga, Latvia Conclusion In our practice, TOE is an indispensable tool for safe ECMO practice, both during V-A and V-V support. y g Critical Care 2014, 18(Suppl 1):P136 (doi: 10.1186/cc13326) Introduction Acute and chronic systemic infl ammatory conditions are associated with aortic stiff ening. Carotid–femoral pulse wave velocity (PWV), a marker of aortic stiff ness, increases in patients with infl ammatory diseases and independently correlates to levels of C-reactive protein (CRP). The eff ects of massive infl ammatory response in early sepsis on mechanical properties of the aorta have not been investigated. The objective of the current study was to prospectively assess aortic stiff ness in patients with early severe sepsis and septic shock and relate it to infl ammatory and haemodynamic variables and outcome. . Vlachopoulos C, et al.: Circulation 2005, 112:2193-2200. P135 P135 Accuracy of synthesized right-sided/posterior chest lead electrocardiograms Y Saitoh1, Y Goseki2, A Yamashina2 1Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium; 2Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P135 (doi: 10.1186/cc13325) Transoesophageal echocardiography and extracorporeal membrane oxygenation: fancy for enthusiasts or indispensable tool? y xygenation: fancy for enthusiasts or indispensable tool? g g p , Critical Care 2014, 18(Suppl 1):P134 (doi: 10.1186/cc13324) y Results Preliminary reports on 16 patients with satisfactory cardiac windows were analyzed. ΔSV-TTE after PLR ≥13.6% predicts FR with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted FR with sensitivity of 83.33% with Introduction Echocardiography is commonly used during both veno- arterial (V-A) and venovenous extracorporeal membrane oxygenation S47 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P136 Aortic stiff ness in patients with early sepsis S Kazune1, A Grabovskis2, E Strīķe3, I Vanags3 1Hospital of Traumatology and Orthopaedics, Riga, Latvia; 2Institute of Atomic Physics and Spectroscopy, University of Latvia, Riga, Latvia; 3Riga Stradins University, Riga, Latvia Critical Care 2014, 18(Suppl 1):P136 (doi: 10.1186/cc13326) Novel technology for non-invasive thoracic fl uid measurement: an animal model comparative study p y G Karp1, M Jonas1, T Mandelbaum2, E Kishinevsky1, N Adi1 1Kaplan Medical Center, Rehovot, Israel; 2Sheba Medical Center, Tel Aviv, Israel Critical Care 2014, 18(Suppl 1):P137 (doi: 10.1186/cc13327) p y G Karp1, M Jonas1, T Mandelbaum2, E Kishinevsky1, N Adi1 1Kaplan Medical Center, Rehovot, Israel; 2Sheba Medical Center, Tel Aviv, Israel Critical Care 2014, 18(Suppl 1):P137 (doi: 10.1186/cc13327) Introduction Approximately 15 million people worldwide suff er from congestive heart failure (CHF). The most severe symptom of CHF is pulmonary congestion – an acute increase in extravascular lung fl uid due to acute decompensation of heart failure. To date, no direct, reliable, simple and non-invasive method is available for assessment of thoracic fl uids. Continuous dependable monitoring of pulmonary edema for hospital patients can improve management and reduce duration of hospitalization. KYMA’s solution is based on a matchbox- sized monitoring device, which monitors thoracic fl uid content and trends. The μCor monitor transmits and receives electromagnetic signals that are propagated through tissue layers. Conduction is highly related to the amount of accumulated fl uids in tissues. This trial investigated the correlation between KYMA’s lung water index (LWI) and directly assessed extravascular lung water (EVLW) in the scenario of acute pulmonary edema induced in seven sheep. p p Results A total 649 hours of hemodynamic record were analyzed in 40 patients chosen at random from 121 patients monitored in the last 2 years. We found 22 drops in nCI that were noticed by the nursing staff and led to volume challenge in accordance with the protocol. One-half of these interventions was carried out outside the 2-hour interval of protocol-required monitoring and documentation entry. We identifi ed nine drops in nCI that were not reacted to. None of them happened within the fi rst 4 hours postoperatively and all of them were outside the 2-hour interval of protocol-required monitoring. In fi ve of these episodes the pulse pressure also dropped, suggesting impairment of the reading of the waveform from the arterial line. Conclusion The need for hemodynamic intervention in postoperative care was quite rare in our patients. The drop in nCI was noticed in most of the cases and the patient was treated according to the protocol. However, isolated episodes of nCI drop were missed during the night after the operation. The results were pointed out to the nursing staff and a comparative survey will be conducted. Novel technology for non-invasive thoracic fl uid measurement: an animal model comparative study As with any monitoring modality, (non)adherence to the protocol may become the limiting issue to the benefi t for the patient. Methods Acute pulmonary edema was induced by intravenous infusion of noradrenaline and dextrane, with stepwise increased dosage. KYMA measurements of LWI were compared with PiCCO extravascular lung volume as the reference gold standard. The experiment continued until maximum increase of EVLW and complete heart failure were reached. Methods Acute pulmonary edema was induced by intravenous infusion of noradrenaline and dextrane, with stepwise increased dosage. KYMA measurements of LWI were compared with PiCCO extravascular lung volume as the reference gold standard. The experiment continued until maximum increase of EVLW and complete heart failure were reached. Results All seven sheep developed pulmonary edema, which was validated by increased LVEDP and EVLW. A linear correlation was found between invasive measurements of EVLW (PiCCO) and non-invasive KLWI. Results All seven sheep developed pulmonary edema, which was validated by increased LVEDP and EVLW. A linear correlation was found between invasive measurements of EVLW (PiCCO) and non-invasive KLWI.l Conclusion KYMA’s fl uid index demonstrated excellent correlation with invasive lung water measurement (R2 = 0.96; P <0.0001) and was able to detect dynamic accumulation of EVLW in a range of 40 to 50 cm3 increments (Figure 1). Since the change in fl uid content between a normal and congested lung ranges between 250 and 500 cm3, the demonstrated sensitivity as refl ected in this study, supports its use for high-resolution and accurate thoracic fl uid monitoring. P138 P138 Adherence to the nurse-driven hemodynamic protocol during postoperative care J Klimes, J Hruda, M Lukes, P Suk, V Sramek St Anna University Hospital and International Clinical Research Center, Brno, Czech Republic Critical Care 2014, 18(Suppl 1):P138 (doi: 10.1186/cc13328) Pulse wave transit time technique for perioperative non-invasive hemodynamic monitoring J Hruda, M Chobola, M Lukes, P Suk, J Klimes, V Sramek St Anna University Hospital and International Clinical Research Center, Brno, Czech Republic Figure 1 (abstract P137). Pooled correlation between EVLW and KYMA LWI. Figure 1 (abstract P137). Pooled correlation between EVLW and KYMA LWI. p Critical Care 2014, 18(Suppl 1):P139 (doi: 10.1186/cc13329) Critical Care 2014, 18(Suppl 1):P139 (doi: 10.1186/cc13329) Introduction The aim of this work is to evaluate perioperative hemodynamic monitoring for major abdominal surgery using the estimated continuous cardiac output (esCCO) technique available with the NIHON KOHDEN Vismo monitor as compared with the LiDCOrapid hemodynamic monitoring system. y y Methods The esCCO technique is a novel noncalibrated non-invasive method of cardiac output monitoring. It is based on the estimation of pulse pressure from the speed of pulse wave propagation, which is measured as a time delay between the ECG R-wave and the corresponding peripheral pulse wave detected by pulse oximetry – the delay being called the pulse wave transit time (PWTT) [1]. LiDCOrapid estimates noncalibrated cardiac output (nCO) by pulse wave contour analysis from an arterial line and is a well-established monitoring modality in our department. The patients planned for elective major abdominal surgery with ASA score of III and expected operation duration over 90 minutes are routinely monitored by LiDCOrapid with a perioperative hemodynamic optimalization protocol. In accordance with the protocol, an arterial line was inserted and LiDCOrapid monitoring was started before the induction of anesthesia. At the same time, non-invasive esCCO monitoring was started. Accuracy of synthesized right-sided/posterior chest lead electrocardiograms The association of high serum CRP levels with low aortic stiff ness in patients with sepsis does not match data described in the literature [1]. Reference Figure 1 (abstract P135). Correlation between actual and synthesized ECG at V7. Vlachopoulos C, et al.: Circulation 2005, 112:2193-2200. S48 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P137 Methods High-risk patients planned to undergo elective major abdominal surgery were routinely monitored with LiDCOrapid; the monitoring continues overnight after surgery. The target nCI for the postoperative monitoring, as well as hemodynamic interventions in case of its drop, were prescribed by the anesthetist in the protocol upon handover of the patient to the postoperative ICU. The hemodynamic protocol in the ICU was then nurse driven with compulsory recording of patient’s hemodynamic data into the patient’s documentation every 2 hours and at the time of each intervention. Data from the memory of the LiDCOrapid monitor were analyzed using LiDCOview software and compared with patients’ documentation. P137 Novel technology for non-invasive thoracic fl uid measurement: an animal model comparative study G Karp1, M Jonas1, T Mandelbaum2, E Kishinevsky1, N Adi1 1Kaplan Medical Center, Rehovot, Israel; 2Sheba Medical Center, Tel Aviv, Israel Critical Care 2014, 18(Suppl 1):P137 (doi: 10.1186/cc13327) P140 Validation of cardiac output from Mostcare compared with a pulmonary artery catheter in septic patients S Gopal1, J Pooni1, T Do2, A Karimi1, G Martinelli1 1New Cross Hospital, Wolverhampton, UK; 2University Hopsital of North Staff ordshire NHS Trust, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P140 (doi: 10.1186/cc13330) p p Methods Five pigs weighing 25 to 29 kg were used in the study approved by the local animal use and care committee. The animals were anesthetized, medically paralyzed, intubated and mechanically ventilated. Central venous and arterial catheters were placed for injection of the indicator and connection to the PiCCO system. The PA-S was placed in contact with the skin over the saphenous artery on the animal and adjusted to receive the maximum acoustic signal. The adjustment included the depth of ultrasonic penetration and the exact location on top of the blood vessel. Each indicator dilution was accomplished using three central venous injections of 15 ml normal saline at room temperature. Variation in CO was accomplished by removing up to 50% of the blood volume after splenectomy. The PA-S results were obtained using the empirical regression method. The resulting cardiac output readings from the PA-S were compared with the PiCCO readings. Introduction The Mostcare monitor is a non-invasive cardiac output monitor. It has been well validated in cardiac surgical patients but there is limited evidence on its use in patients with severe sepsis and septic shock [1].i Methods The fi rst 22 consecutive patients with severe sepsis and septic shock in whom the fl oatation of a pulmonary artery catheter was deemed necessary to guide clinical management were included. Cardiac output measurements were simultaneously calculated and recorded from a thermodilution pulmonary artery catheter and from the Mostcare monitor respectively. The two methods of measuring cardiac output were compared by Bland–Altman statistics and linear regression analysis. A percentage error less than 30% was defi ned as acceptable for this study. Results The correlation between the PA-S and the PiCCO system was (R2 = 0.746) over a range of CO from 1 to 6 l/minute. The Bland–Altman analysis demonstrated a bias of –0.05 l/minute and precision of 0.50 l/ minute. Results Bland–Altman analysis for cardiac output showed a bias of 0.31 l/minute, precision (=SD) of 1.97 l/minute and a percentage error of 62.54%. Linear regression produced a correlation coeffi cient r2 for cardiac output of 0.403. See Figure 1. Conclusion This animal study demonstrated the feasibility of the new PA-S for determination of indicator dilution CO in a limited range. The system showed good correlation with the PiCCO system even in the case of vasoconstriction as a result of blood loss. P140 P140 Validation of cardiac output from Mostcare compared with a pulmonary artery catheter in septic patients S Gopal1, J Pooni1, T Do2, A Karimi1, G Martinelli1 1New Cross Hospital, Wolverhampton, UK; 2University Hopsital of North Staff ordshire NHS Trust, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P140 (doi: 10.1186/cc13330) P141 changes (as with any other noncalibrated cardiac output monitor), but it can still trace the trends of CO changes. Due to its non-invasiveness, esCCO might be the monitoring of choice for high-risk patients undergoing low-risk surgery. Reference Novel non-invasive technology for cardiac output determination U Borg, D Iyer, Q Huang, Y Li, C Baker Covidien, Longmont, CO, USA Critical Care 2014, 18(Suppl 1):P141 (doi: 10.1186/cc13331) 1. Sugo Y, Ukawa T, Takeda S, Ishihara H, Kazama T, Takeda J: A novel continuous cardiac output monitor based on pulse wave transit time. Conf Proc IEEE Eng Med Biol Soc 2010, 2010:2853-2856. Introduction The aim of this animal study was to determine the feasibility and accuracy of a new non-invasive system for determination of cardiac output (CO) compared with a known device (PiCCO; Pulsion Medical). In ICUs and ORs, hemodynamic management using goal- directed therapy has been shown to improve patient outcomes [1,2]. The invasiveness of current technology may prevent clinicians from obtaining hemodynamic information. We developed a non- invasive system for detection of hemodynamic variables utilizing a photo-acoustic sensor (PA-S) and indicator dilution. The PA-S utilizes a combination of a miniature ultrasound sensor, a laser diode and an optical detector to accomplish the measurement. P140 Validation of cardiac output from Mostcare compared with a pulmonary artery catheter in septic patients S Gopal1, J Pooni1, T Do2, A Karimi1, G Martinelli1 1New Cross Hospital, Wolverhampton, UK; 2University Hopsital of North Staff ordshire NHS Trust, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P140 (doi: 10.1186/cc13330) Since the PA-S utilizes transpulmonary indicator dilution, other variables such as intrathoracic blood volume, global end-diastolic volume and extravascular lung water can be calculated. Additionally, continuous CO can be obtained from the optical sensor signal utilizing arterial waveform analysis. References g Conclusion Compared with thermodilution cardiac output, cardiac output studies obtained from the Mostcare monitor have an unacceptably high error rate. The Mostcare monitor is not a reliable monitoring device to measure cardiac output in patients with severe sepsis and septic shock on an ICU. P138 Adherence to the nurse-driven hemodynamic protocol during postoperative care Adherence to the nurse-driven hemodynamic protocol during postoperative care g Results Ten patients were monitored with a total of 141 esCCO–nCO measurement pairs. The correlation coeffi cient between esCCO and nCO was 0.65 and in the Bland–Altman diff erence analysis bias was +1.2 l/minute and 95% limits of agreement were ± 2.6 l/minute. The change of cardiac output between two consecutive measurements detected by LiDCOrapid was detected by esCCO in 80% of cases. Conclusion Hemodynamic monitoring with esCCO yields cardiac output values diff erent from those measured by LiDCOrapid. The reliability of PWTT is questionable when systemic vascular resistance Results Ten patients were monitored with a total of 141 esCCO–nCO measurement pairs. The correlation coeffi cient between esCCO and nCO was 0.65 and in the Bland–Altman diff erence analysis bias was +1.2 l/minute and 95% limits of agreement were ± 2.6 l/minute. The change of cardiac output between two consecutive measurements detected by LiDCOrapid was detected by esCCO in 80% of cases. Introduction The aim of this work is to verify that low incidence of hemodynamic interventions in hemodynamically monitored patients in our postoperative ICU is not caused by insuffi cient attention being paid to the drop of noncalibrated cardiac index (nCI). In the last 2 years there was a need for hemodynamic intervention in 92% of patients with perioperative cardiac output monitoring, while only 31% of these patients needed at least one hemodynamic intervention postoperatively in the ICU. Conclusion Hemodynamic monitoring with esCCO yields cardiac output values diff erent from those measured by LiDCOrapid. The reliability of PWTT is questionable when systemic vascular resistance S49 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P143 Comparison of PiCCO and VolumeView: simultaneous measurement in sepsis pig models H Imahase1, S Inoue1, Y Sakamoto1, T Miyasho2, K Yamashita2 1Saga University Hospital, Saga City, Japan; 2Rakuno Gakuen University, Ebetsu City, Japan Critical Care 2014, 18(Suppl 1):P143 (doi: 10.1186/cc13333) Introduction The aim was to evaluate the performance of arterial pressure-based cardiac output (APCO) [1] and pulse wave transit time- based cardiac output (esCCO) [2] monitors in Thai patients undergoing coronary artery bypass graft surgery (CABG) with cardiopulmonary bypass. Introduction Transpulmonary thermodilution is useful in the critical care. Following PiCCO, VolumeView is now available. Although previous studies concluded that signifi cant correlation was found between VolumeView and PiCCO [1,2], all experiments and data analysis were done by Edwards Lifescience. The aim of the present study was to compare the new VolumeView with PiCCO to clarify their compatibility and to give a neutral answer. y Methods We studied 50 Thai surgical patients undergoing CABG with cardiopulmonary bypass and requiring pulmonary artery catheter and radial artery catheter placement as a standard of clinical care. All patients were measured for APCO using the Vigileo/FloTrac and for esCCO using the esCCO monitoring system. The data were compared with thermodilution cardiac output (TDCO) monitoring as a reference method, simultaneously at pre-induction, post-induction, and every 30 minutes thereafter until the completion of the surgery. The bias and precision were assessed using Bland–Altman analysis. g Methods Six pigs (about 10 kg) were used and we made sepsis models by LPS administration. All pigs were instrumented with a right (Pulsio- Cath) and a left (VolumeView) thermomistor-tipped femoral arterial catheter. The central venous catheter was inserted through the right jugular vein. CO, GEDV and EVLW were measured by the two systems. Results We performed measurements at 57 points. There was a good correlation between the two devices regarding CO and GEDV, but VolumeView showed higher GEDV than PiCCO. Regarding EVLW, there was no signifi cant correlation between two systems. VolumeView showed signifi cantly higher EVLW than PiCCO (Figure 1 and Table 1). Methods Six pigs (about 10 kg) were used and we made sepsis models by LPS administration. All pigs were instrumented with a right (Pulsio- Cath) and a left (VolumeView) thermomistor-tipped femoral arterial catheter. The central venous catheter was inserted through the right jugular vein. CO, GEDV and EVLW were measured by the two systems. Results In total, 310 pairs of simultaneous measurements of APCO versus TDCO and 303 pairs of esCCO versus TDCO were obtained from 50 patients. Both APCO (R = 0.53, P <0.0001) and esCCO values (R = 0.56, P <0.0001) were correlated with TDCO values. P143 P143 Comparison of PiCCO and VolumeView: simultaneous measurement in sepsis pig models H Imahase1, S Inoue1, Y Sakamoto1, T Miyasho2, K Yamashita2 1Saga University Hospital, Saga City, Japan; 2Rakuno Gakuen University, Ebetsu City, Japan Critical Care 2014, 18(Suppl 1):P143 (doi: 10.1186/cc13333) Referencesi References 1. Biancofi ore G, et al.: Br J Anaesth 2009, 102:47-54. 2. Ishihara H, et al.: J Clin Monit Comput 2004, 18:313-320. References 1. Biancofi ore G, et al.: Br J Anaesth 2009, 102:47-54. 2. Ishihara H, et al.: J Clin Monit Comput 2004, 18:313-320. Performance of pulse contour and pulse wave transit time-based continuous cardiac output analyses: clinical validation of two methods in Thai patients undergoing cardiac surgery P Wacharasint, P Kunakorn, P Pankongsap Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P142 (doi: 10.1186/cc13332) P143 p Reference 1. Franchi F, Silvestri R, Cubattoli L, et al.: Comparison between an uncalibrated pulse contour method and thermodilution technique for cardiac output estimation in septic patients. Br J Anaesth 2011, 107:202-208. 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-936. 2. Marik PE, et al.: Curr Pharm Des 2012, 18:6215-6224. 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-936. 2. Marik PE, et al.: Curr Pharm Des 2012, 18:6215-6224. Figure 1 (abstract P140). Cardiac output Bland–Altman analysis. Figure 1 (abstract P140). Cardiac output Bland–Altman analysis. S50 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P142 P144f P144 Eff ects of the restrictive fl uid strategy on postoperative pulmonary and renal function following pulmonary resection surgery M Arslantas1, H Batirel2, B Bilgili1, H Kara2, B Yıldızeli2, M Yuksel2, K Bostanci2, A Kararmaz1, I Cinel1 1Marmara University Pendik Education and Research Hospital, Istanbul, Turkey; 2Marmara University School of Medicine, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P144 (doi: 10.1186/cc13334) Introduction Goal-directed therapy used in the perioperative period of patients undergoing cardiac surgery shortens the length of ICU stay [1]. We aimed to compare the postoperative results of the liberal and restrictive fl uid strategy used in patients undergoing pulmonary resection surgery (PRC).l g y Methods We have been using the restrictive fl uid strategy since March 2013 in our institute. Patients who were on the liberal fl uid regime were analyzed retrospectively. From March 2013 until today, patients who were on restrictive fl uid strategy were analyzed prospectively. A total of 125 patients were included in the study. Age, duration of anesthesia, type of fl uids given intraoperatively, fl uid index (ml/kg/hour), fl uid intake/output balance, creatinine and lactate levels were compared with pulmonary and renal morbidity, and length of stay in ICU, using multivariate analysis.i Methods We conducted a prospective audit of FB and renal function in patients undergoing major elective surgery admitted to critical care over a 28-day period. Fluid overload was defi ned as: (positive FB) / (weight × 0.6) × 100%. Results Thirty-two patients (56% female, median age 64) were studied over a median of 3 critical care days (range 1 to 7). Total FB was +3.9 l at discharge; however, 75% of this occurred intraoperatively so that positive FB in critical care was only +390 ml/day. Patients had median 9% fl uid overload at discharge. Two patients had transient AKI stage 1. Overall SCr decreased signifi cantly from preoperatively to discharge (P = 0.003), median 73 to 55 μmol/l, with decreases occurring both postoperatively and during critical care stay (Figure 1).l Results A signifi cant correlation (P <0.05) was established between the amount of crystalloid given intraoperatively, fl uid index and fl uid balance with pulmonary morbidity (n = 52). The fl uid index and inotropes usage were correlated with the postoperative creatinine levels (P <0.05). There was no correlation between perioperative lactate levels with fl uid balance and fl uid index. Reference Reference 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-836. 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-836. References 1. Bendjelid K, et al.: Crit Care 2010, 14:R209. 2. Kiefer N, et al.: Crit Care 2012, 16:R98. P144f Intraoperative blood loss, the amount of given crystalloid, colloid, blood and FFP, fl uid balance, duration of anesthesia and postoperative blood transfusion were found to be related (P <0.05) with the length of ICU stay. Four percent of the patients required renal replacement therapy and the overall mortality was 0.8%. y y Conclusion We achieved near-neutral fl uid balances after admission, but did not resolve intraoperative fl uid accumulation. SCr fell signifi cantly, even after there was no further net fl uid accumulation. This suggests a decrease in creatinine generation after major surgery, rather than fl uid expansion, may largely account for sCr decreases in these patients. Conclusion To reduce the morbidity of patients undergoing major surgery, the protocols of using the restrictive fl uid strategy in the perioperative period and simultaneous protection of end organs, especially the kidneys, is currently the subject of this discussion. We observed that the restrictive fl uid strategy did not lead to global organ hypoperfusion, which was monitored by lactate. Even though there was a negative correlation between the fl uid index with creatinine levels and renal failure, the need for renal replacement therapy was observed only in one case. As a conclusion, the postoperative pulmonary morbidity and length of ICU stay can be reduced in patients who undergo PRC by using the restrictive fl uid strategy (4.2 ± 0.3 ml/kg/hour), without causing any vital organ dysfunction. P145 Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery R Rajendram, JR Prowle Barts Health NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P145 (doi: 10.1186/cc13335) P143 Comparison of PiCCO and VolumeView: simultaneous measurement in sepsis pig models H Imahase1, S Inoue1, Y Sakamoto1, T Miyasho2, K Yamashita2 1Saga University Hospital, Saga City, Japan; 2Rakuno Gakuen University, Ebetsu City, Japan Critical Care 2014, 18(Suppl 1):P143 (doi: 10.1186/cc13333) Either of the changes in APCO (R = 0.63, P <0.0001) or any changes in esCCO (R = 0.60, P <0.0001) were correlated with changes in TDCO. For APCO relative to TDCO, the bias, precision, and the limits of agreement were 0.70, 1.63, and –2.5 to 3.9 l/minute, while those of esCCO were 1.20, 1.59, and –1.9 to 4.3 l/ minute, respectively. Comparisons of the bias of APCO and esCCO revealed a level of signifi cance of P <0.001. j g y y Results We performed measurements at 57 points. There was a good correlation between the two devices regarding CO and GEDV, but VolumeView showed higher GEDV than PiCCO. Regarding EVLW, there was no signifi cant correlation between two systems. VolumeView showed signifi cantly higher EVLW than PiCCO (Figure 1 and Table 1). Table 1 (abstract P143). Explanation of abbreviations Table 1 (abstract P143). Explanation of abbreviations CO GEDV EVLW PiCCO PCO PGEDV PEVLW VolumeView ECO EGEDV EEVLW Table 1 (abstract P143). Explanation of abbreviations i Conclusion Despite the overestimation of cardiac output measure- ments, APCO and esCCO calibrated with patient information has shown an acceptable trend as compared with TDCO in Thai patients undergoing CABG with cardiopulmonary bypass. Compared with esCCO, APCO demonstrated no signifi cant diff erences of precision; however, a lower mean bias was exhibited. Figure 1 (abstract P143). CO, GEDV, EVLW correlation and Bland–Altman analysis. Figure 1 (abstract P143). CO, GEDV, EVLW correlation and Bland–Altman analysis. S51 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P145). Serum creatinine changes after major surgery. Conclusion Our data analysis showed that PiCCO and VolumeView were not the same. Neither the normal values of two devices nor the rates of change were same. Careful attention should be paid to interpret the data obtained from two systems. Barts Health NHS Trust, London, UK P146 Very limited usefulness of pulse pressure variation as a predictor of volume responsiveness in critically ill septic patients GG Gavriilidis, IK Kostakou, VT Tsagari, AK Kyriakoudi, NK Koulouris, AK Koutsoukou University of Athens, Sotiria Chest Hospital, Athens, Greece Critical Care 2014, 18(Suppl 1):P146 (doi: 10.1186/cc13336) Introduction The aims of the study are to assess the usefulness of pulse pressure variation (PPV) as a predictive marker of fl uid responsiveness and to estimate the value of central venous–arterial diff erence of carbon dioxide (PCO2cv-a) to predict the outcome of critically ill septic patients. The question of whether a septic patient needs fl uids or not is crucial. Although PPV is a very reliable predictor of volume responsiveness, there are many limitations for its application. Cardiac arrhythmia, spontaneous breathing and low tidal volume ventilation prevent the extended use of this index. R Rajendram, JR Prowle Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery Methods This is a post-hoc analysis of data from a prospective observational study [1], which included a population of patients with severe sepsis or septic shock as the main reason for ICU admittance. After an echo for the assessment of the cardiac systolic performance, we measured PPV, central venous oxygen saturation, blood gases from arterial and central venous lines, central venous pressure, systolic, diastolic, mean and pulse pressures, before and after volume challenge. We calculated changes in pulse pressure before and after volume challenge, and an increase of 9% was used as criterion to defi ne volume responsiveness [2]. R Rajendram, JR Prowle Barts Health NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P145 (doi: 10.1186/cc13335) Introduction The rationale for perioperative fl uid therapy has been to preserve organ perfusion. However, conservative postoperative fl uid balance (FB) is now encouraged to avoid the eff ects of iatrogenic fl uid overload. In addition, fl uid expansion has been described as a confounder of acute kidney injury (AKI) diagnosis by dilution serum creatinine (SCr). S52 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 increased the RI in the right and left kidneys, from 0.6 ± 0.04 and 0.6 ± 0.05 to 0.7 ± 0.1 and 0.68 ± 0.15 (P <0.05), respectively. Results Among 72 patients (71% men, APACHE II score 23.2) included in this study, 41 (57%) were responders. Due to spontaneous breathing and cardiac arrhythmia we were able to calculate PPV only in 18 patients (25%). Moreover, only eight (11%) calculations of PPV proved useful because of the application of low tidal ventilation (4 to 6 ml/kg ideal body weight) in the remaining 10 patients. We did not fi nd any value of the PCO2cv-a to predict the outcome of the patients (mortality 26.3%), since the diff erence between survivors and nonsurvivors was not statistically signifi cant (7.7 vs. 7.9, P >0.05). Conclusion These preliminary results showed that Doppler renal RI was aff ected equally in both kidneys during HUT, suggesting an eff ect of hemodynamic alterations during our model of central hypovolemia. References 1. Schnell D, et al.: Renal perfusion assessment by renal Doppler during fl uid challenge in sepsis. Crit Care Med 2013, 41:1214. g p 2. Lerolle N, et al.: Renal failure in septic shock: predictive value of Doppler- based renal arterial resistive index. Intensive Care Med 2006, 32:1553. Eff ects of central hypovolemia induced by tilt table on the Doppler- based renal resistive index in healthy volunteers A Sommese, A Lima, J Van Bommel, J Bakker Erasmus MC, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P147 (doi: 10.1186/cc13337) Methods We randomised 50 high-risk patients who underwent major abdominal surgery. Tissue oxygenation was monitored at the thenar eminence using near-infrared spectroscopy. All patients were treated according to a standard care algorithm. In addition, patients in the intervention group received dobutamine if necessary to keep tissue oxygenation ≥80%. Data were recorded continuously and complications were recorded during the hospital stay with a maximum of 28 days. A Sommese, A Lima, J Van Bommel, J Bakker Erasmus MC, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P147 (doi: 10.1186/cc13337) Introduction The renal resistive index (RI) determined by Doppler ultrasonography allows a semiquantitative evaluation of kidney vasculature at the bedside. Interpretation of the RI in clinical practice is diffi cult due to interaction with cardiac output, heart rate (HR) and blood pressure [1,2]. The impact of global hemodynamics on the RI remains to be evaluated. This study aims to investigate the relationship between the RI and changes in central hemodynamic during a central hypovolemia model in healthy volunteers (HV). Results The number of complications tended to be lower in the intervention group (11 vs. 20). Eleven patients in the intervention group had no complication, versus seven in the control group. There was no signifi cant diff erence between groups in length of stay in ICU or in hospital. Administration resulted in a 5% increase of tissue oxygenation. The cardiac index increased 0.3 (0.0 to 0.6) l/minute/m2 (Figure 1). The overall protocol adherence was 94%. yp y Methods Eleven healthy volunteers (27 ± 8 years; eight male) participated in this study. Two diff erent models were performed: the fi rst model was performed by applying the head-up tilt (HUT) test. The complete maneuver was done by a 10-minute step that consisted of tilting the table from a supine position (Sup) to an angle of 70° (HUT) and back to supine (Sup’). The second model was performed by applying three consecutive valsalva maneuvers. Global hemodynamics included stroke volume (SV), HR, and mean arterial pressure, which were continuously measured non-invasively with a Finometer. At least three RI readings were obtained and averaged from the right and left kidneys in all HV. g p Conclusion Intraoperative optimisation of tissue oxygenation will potentially result in better outcome after high-risk abdominal surgery. The protocol used may be considered feasible for clinical practice. Reference Tissue oxygenation as a target for goal-directed therapy in high-risk surgery 1. Gavriilidis G, et al.: Central venous oxygen saturation does not predict fl uid responsiveness in critically ill septic patients Intensive Care Med 2013, S2:0949. PA Van Beest, JJ Vos, M Poterman, AF Kalmar, TW Scheeren University Medical Center Groningen, the Netherlands PA Van Beest, JJ Vos, M Poterman, AF Kalmar, TW Scheeren University Medical Center Groningen, the Netherlands Critical Care 2014, 18(Suppl 1):P148 (doi: 10.1186/cc13338) 2. Preau S, et al.: Passive leg raising is predictive of fl uid responsiveness in spontaneously breathing patients with severe sepsis or acute pancreatitis Crit Care Med 2010, 38:819-825. 2. Preau S, et al.: Passive leg raising is predictive of fl uid responsiveness in spontaneously breathing patients with severe sepsis or acute pancreatitis Crit Care Med 2010, 38:819-825. y g Critical Care 2014, 18(Suppl 1):P148 (doi: 10.1186/cc13338) Introduction Tissue hypoxia occurs frequently during surgery and may contribute to postoperative organ dysfunction [1]. We hypothesised that intraoperative optimisation of tissue oxygenation reduces postoperative complications and evaluated the feasibility of the optimisation protocol used. Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery g 2. Lerolle N, et al.: Renal failure in septic shock: predictive value of Doppler- based renal arterial resistive index. Intensive Care Med 2006, 32:1553. i Conclusion The usefulness of PPV, the marker with the best performance in the prediction of volume responsiveness, due to its limitations, is very limited. Eff ects of central hypovolemia induced by tilt table on the Doppler- based renal resistive index in healthy volunteers Eff ects of central hypovolemia induced by tilt table on the Doppler- based renal resistive index in healthy volunteers A Sommese, A Lima, J Van Bommel, J Bakker Erasmus MC, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P147 (doi: 10.1186/cc13337) p Reference All patients were mechanically ventilated and subjected to transesophageal echocardiography in bicavale projection, calculating the DIIVC through the M-mode by measuring the change in diameter with the acts of positive pressure ventilation and using the formula: (100 × (maximum diameter – minimum diameter)) / minimum diameter. A value >18% was considered a predictor of an increase in CI >15%. The CI was measured continuously through use of the pressure recording analytical method on a Mostcare Vytech monitor. All patients were subjected to a bolus of 500 ml crystalloid through a central venous catheter only after reaching central venous pressure (CVP) ≥10 mmHg during treatment. The results were expressed as the mean with standard deviation and percentage. Results Sixteen patients (38%) had a change in DIIVC <18%, on average 11 ± 5%, and when subjected to fl uid challenge did not have an increase in CI >15%, average 8 ± 3%, not proving to be a fl uid responder. Twenty- six patients (62%) had a change in DIIVC >18%, on average 45 ± 22%, and when subjected to fl uid challenge had an increase of the CI >15%, on average 31 ± 8%, proving to be a fl uid responder. Why measurements do (not) work: the human factor A Rameau, NA Bruins, P Egbers, MA Kuiper, EC Boerma Medical Center Leeuwarden, the Netherlands Critical Care 2014, 18(Suppl 1):P149 (doi: 10.1186/cc13339) Introduction Passive leg raising (PLR) has been suggested as a simple diagnostic tool to guide fl uid administration in critically ill patients [1]. Although the basic principles of PLR have been well studied, little is known about the impact of the introduction of this technique in daily clinical practice. The aim of the study was to describe the changes in fl uid balance of ICU patients before and after the introduction of PLR, and to determine the compliance of medical personnel with a PLR- driven protocol of fl uid administration. pl Methods In this single-centre prospective study, mixed ICU patients equipped with a PiCCO system received fl uid therapy on the basis of PLR test results in the fi rst 48 hours of treatment, after careful introduction of a new PLR-driven protocol. Exclusion criteria were increased abdominal pressure, fracture of leg or pelvis, deep vein thrombosis, head trauma and pregnancy. The control group existed of patients admitted to the ICU 1 year prior to the introduction of the protocol. p Reference 1. Scheeren TW, et al.: Goal-directed intraoperative fl uid therapy guided by stroke volume and its variation in high-risk surgical patients: a prospective multicentre study. J Clin Monit Comput 2013, 27:225-233. y Results All HV had a signifi cant decrease of SV from 83 ± 17 ml to 63 ± 14 ml and an increase in HR from 67 ± 10 bpm to 88 ± 13 bpm during the HUT. Figure 1 shows the temporal changes of mean RI in both kidneys. A signifi cant decrease in the RI in both kidneys was seen during HUT. After the move back to supine, RI returned to baseline values, with a signifi cant variation of RI in the early measurements on the right kidney compared with late measurements on the left kidney (0.67 ± 0.05 vs. 0.61 ± 0.05, P <0.05). Valsalva maneuvers signifi cantly Figure 1 (abstract P148). Evolution of individual patient values (thin lines) and the average values (thick lines) of the tissue oxygenation (StO2) and the relative change of cardiac index (CI), delta CI. All graphs are synchronised at the moment of the fi rst dobutamine administration (time = 0; arrow). Values are shown from 10 minutes before dobutamine administration until 45 minutes after. Figure 1 (abstract P147). Doppler-based renal resistive index in both kidneys during HUT. Figure 1 (abstract P148). Evolution of individual patient values (thin lines) and the average values (thick lines) of the tissue oxygenation (StO2) and the relative change of cardiac index (CI), delta CI. All graphs are synchronised at the moment of the fi rst dobutamine administration (time = 0; arrow). Values are shown from 10 minutes before dobutamine administration until 45 minutes after. Figure 1 (abstract P147). Doppler-based renal resistive index in both kidneys during HUT. Figure 1 (abstract P147). Doppler-based renal resistive index in both kidneys during HUT. S53 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P149 Methods In a period of 1 year, 42 adult patients were admitted to the ICU in septic shock. All patients were treated according to the guidelines of the Surviving Sepsis Campaign International Guidelines for Management of Severe Sepsis and Septic Shock 2012 [2]. p Reference g p gl p Conclusion We have shown that the DIIVC can predict the response of patients to fl uid challenge regardless of the values of CVP and then distinguish fl uid responders from nonresponder patients. This can help us in the choice of treatment for patients in septic shock. Referencesf y Results We included 21 patients in each group. There was no signifi cant diff erences in the fl uid balance between the control and study group after 24 hours (5.0 ± 2.9 l vs. 3.6 ± 2.7 l, P = 0.11) and 48 hours (5.7 ± 3.5 l vs. 4.8 ± 3.7 l, P = 0.39). However, compliance with the protocol was poor (56%). After 2/11 positive tests, fl uid was not administered; and after 21/39 tests, fl uid was administered despite a negative test result (Figure 1). 1. Griff ee J et al.: The role of echocardiography in hemodynamic assessment of septic shock. Crit Care Clin 2010, 26:365-382. 1. Griff ee J et al.: The role of echocardiography in hemodynamic assessment of septic shock. Crit Care Clin 2010, 26:365-382. 2. Dellinger RP et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013, 41:580-637. 2. Dellinger RP et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013, 41:580-637. Figure 1 (abstract P149). SVI, stroke volume index; –, negative PLR; +, positive PLR; fl uid 250 cm3 RL solution. Reference 1. Michard F, et al.: Predicting fl uid responsiveness in ICU patients. A critical analysis of the evidence. Chest 2000, 121:2000-2008. Methods Patients admitted postoperatively and monitored with the Nexfi n monitor were enrolled in the study. Patients were included if they were spontaneously breathing and had an increase in cardiac output ≥10% after a FC. They were classifi ed according to the increase in mean arterial pressure (MAP) after FC into MAP-responders (MAP increase ≥10%) and MAP-nonresponders (MAP increase <10%). Eadyn was calculated from the PPV and SVV values obtained from the monitor. Results A total of 34 FCs from 26 patients were studied. Seventeen FCs (50%) induced a positive MAP response. Preinfusion Eadyn was signifi cantly higher in MAP-responders (1.39 ± 0.41 vs. 0.85 ± 0.23; P = 0.0001) (Figure 1). Preinfusion Eadyn predicted a positive MAP response to FC with an area under the ROC curve of 0.92 ± 0.04 of SE (95% CI: 0.78 to 0.99; P <0.0001). A preinfusion Eadyn value ≥1.06 (grey zone: 0.9 to 1.15) discriminated MAP-responders with a sensitivity and specifi city of 88.2% (95% CI: 64 to 99%).i Use of pulse pressure variation and stroke volume variation in spontaneously breathing patients to assess dynamic arterial elastance and to predict arterial pressure response to fl uid administration M Cecconi1, MI Monge García2, M Gracia Romero2, J Mellinghoff 2, F Caliandro2, RM Grounds2, A Rhodes2 1St George’s Hospital, London, UK; 2St George’s Healthcare NHS Trust and St George’s University of London, UK Critical Care 2014, 18(Suppl 1):P151 (doi: 10.1186/cc13341) Figure 1 (abstract P149). SVI, stroke volume index; –, negative PLR; +, positive PLR; fl uid 250 cm3 RL solution. Introduction Dynamic arterial elastance (Eadyn), defi ned as the pulse pressure variation (PPV) to stroke volume variation (SVV) ratio, has been suggested as a predictor of the arterial pressure response to fl uid administration. Rather than a steady-state assessment, Eadyn depicts the actual slope of the pressure–volume relationship providing a dynamic evaluation of the arterial load. So the higher the Eadyn value, the more likely arterial blood pressure is to improve after fl uid challenge (FC). The aim of this study was to assess the eff ectiveness of Eadyn, measured non-invasively in preload-dependent, spontaneously breathing patients. Conclusion The implementation of a PLR-driven protocol of fl uid administration did not change mean fl uid balances after the fi rst 48 hours. Noncompliance with the protocol was an important confounder. P151 Use of pulse pressure variation and stroke volume variation in spontaneously breathing patients to assess dynamic arterial elastance and to predict arterial pressure response to fl uid administrationf P152 Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery P153 Kinetics of volume expansion during a fl uid challenge H Aya, A Rhodes, M Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P153 (doi: 10.1186/cc13343) Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery H Merdji1, P Biston2, M Piagnerelli2 1CHU de Reims, France; 2CHU de Charleroi, Belgium Critical Care 2014, 18(Suppl 1):P152 (doi: 10.1186/cc13342) l H Merdji1, P Biston2, M Piagnerelli2 j , , g 1CHU de Reims, France; 2CHU de Charleroi, Belgium g Critical Care 2014, 18(Suppl 1):P152 (doi: 10.1186/cc13342) Introduction According to Guyton and colleagues [1], expansion of blood volume can increase cardiac output (CO) in so far as the change in volume aff ects the mean systemic fi lling pressure (Pmsf). However, rapid stress relaxation occurs after acute intravascular volume expansion so that the eff ect of volume on Pmsf and CO may disappear after a few minutes. The objective of the present study is to describe the extent of the haemodynamic changes generated by a fl uid challenge during 10 minutes on critically ill patients. Introduction Recent studies showed that the plethysmographic variability index (PVI), a dynamic index resulting from cardiopulmonary interactions, could predict fl uid responsiveness (FR) in mechanically ventilated patients during general anesthesia and in the ICU [1,2]. In this study, we aimed to compare, in patients after cardiac surgery, the clinical utility of the PVI versus traditional statics and dynamics indices to predict FR. g y Methods Patients admitted to the ICU were monitored with a calibrated LiDCOplus (LiDCO, UK) and Navigator (Applied Physiology, Australia) to estimate Pmsf analogue (Pmsa). Then 250 ml Hartmann’s solution or Volplex was infused over 5 minutes. Data were recorded automatically from baseline to 10 minutes after the end of fl uid infusion. The time to maximum response and percentage change between baseline and last value of diff erent haemodynamic parameters are also reported. Methods We prospectively enrolled 52 consecutive adult patients in sinus rhythm and mechanically ventilated (tidal volume of 8 (7.5 to 8.5) ml/kg ideal body weight) admitted to the ICU of CHU de Charleroi (Belgium) after scheduled cardiac surgery. Before and after fl uid challenge (FC), we measured: heart rate (HR), BP, PVI (Masimo Corporation, Irvine, CA, USA) and PPV. PAOP and cardiac index (CI) were estimated by Swan–Ganz catheter (Edwards Lifesciences LLC, Irvine, CA, USA). P152 Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery Patients with an increase in CI of at least 15% after FC and subsequently were considered responders (R). Continuous variables were analyzed with Mann–Whitney U tests. Categoric variables were analyzed with the Fisher’s exact test. Correlations were assessed by Spearman’s correlation. The discriminatory ability of each haemodynamic parameter to determine FR was assessed by area under the receiver operating characteristic curve (AUC), between R and nonresponders (NR). P <0.05 was considered statistically signifi cant.ii f Results Sixty fl uid challenges were studied in 34 patients, with a mean duration of 5.3 minutes (± 2.5). In 22 events (37%), CO increased more than 10% (responders). The change between baseline and last value was greater in nonresponders for heart effi ciency (Eh) (–9.2% ± 9.7 vs. –3.1% ± 13.9, P = 0.05) but not in other haemodynamic variables. Time to maximal response on CO was 2 minutes after the end of the infusion (Figure 1).fl g Conclusion Stress relaxation damps down the eff ect of a fl uid challenge after 10 minutes except in terms of heart effi ciency. The eff ect of a fl uid challenge should be assessed up to 2 minutes after the end of fl uid infusion. Failure to do so may mislead clinicians about the patient’s fl uid responsiveness. i Results Twenty-fi ve (48%) patients were classifi ed as R. Before FC, R and NR patients were comparable except for HR (91 (83 to 108) for R vs. 80 beats/minute (72 to 89) for NR; P = 0.009), PVI (18 (12 to 26) for R vs. 12% (9 to 15) for NR; P = 0.003), PAOP (8 (6 to 9) for R vs. 11 mmHg (8 to 12) for NR; P = 0.001), and PPV (13 (8 to 19) for R vs. 10% (4 to 13) for NR; P = 0.086). PVI and delta PP before FC were correlated (r = 0.53, P <0.0001). AUC showed a better prediction of FR for PVI (AUC = 0.74, P <0.001) than for PPV (AUC = 0.64, P = 0.05). Nevertheless, the sensitivity (Se) and specifi city (Sp) of both indices were low: for a PPV over 13%, Se = 48% (0.301 to 0.665),and Sp = 82% (0.627 to 0.921); for a PVI over 12%, Se = 68% (0.482 to 0.828) and Sp = 69% (0.498 to 0.835). 1. Cannesson et al.: Br J Anaesth 2008, 101:200-206. 2. Loupec et al.: Crit Care Med 2011, 39:294-299. Fluid responsiveness in septic shock F Righetti, G Castellano St Boniface Hospital Verona, Italy p , y Critical Care 2014, 18(Suppl 1):P150 (doi: 10.1186/cc13340) p y Critical Care 2014, 18(Suppl 1):P150 (doi: 10.1186/cc13340) Introduction The correct assessment of volume status and prediction of response to fi lling fl uid challenge is of crucial importance in the patient with septic shock. A patient is a fl uid responder if there is an increase in cardiac index (CI) >15% in response to the fl uid challenge. Identifying which patient will be a fl uid responder is crucial. The aim of this prospective study is to evaluate which patient is a fl uid responder in septic shock using dynamics echocardiography with the distensibility index of the inferior vena cava (DIIVC), which has been shown to be predictive of an increase in CI [1]. i Conclusion Non-invasive dynamic arterial elastance, defi ned as the PPV to SVV ratio, predicted the arterial pressure increase to fl uid administration in spontaneously, preload-dependent patients. S54 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P151). Distribution of individual arterial load parameters at preinfusion time. Figure 1 (abstract P151). Distribution of individual arterial load parameters at preinfusion time. 1. Cannesson et al.: Br J Anaesth 2008, 101:200-206. 2. Loupec et al.: Crit Care Med 2011, 39:294-299. , 2. Loupec et al.: Crit Care Med 2011, 39:294-299. P156 P156 BXL 628 ameliorates toxicity of lactated Ringer in HK-2 human renal proximal tubule cells in a hypovolemia mimicking model YT Huang, CC Cheng, TC Lin, PC Lai Buddhist Tzu Chi General Hospital, Hualien, Taiwan Critical Care 2014, 18(Suppl 1):P156 (doi: 10.1186/cc13346) p Methods Patient records (APACHE II, length of stay (LOS), CVP, DFB, vasopressor and ventilator needs) were retrospectively collaborated, and a randomly-assigned 100 (63 men and 37 women, age 64.2 ± 15.5 years) were statistically analyzed for survival function. Methods Patient records (APACHE II, length of stay (LOS), CVP, DFB, vasopressor and ventilator needs) were retrospectively collaborated, and a randomly-assigned 100 (63 men and 37 women, age 64.2 ± 15.5 years) were statistically analyzed for survival function. Results The mean APACHE II score was 23.6 ± 7.7, LOS was 9.7 ± 10.0 days, intubated period was 6.4 ± 8.6 days, vasopressor period was 4.7 ± 5.5 days, CVP was 10.5 ± 5.5 mmHg, DFB was 1,147.9 ± 1,157.6 ml, and 42 survived. Kaplan–Meier survival and COX regression analysis showed that CVP levels of 6 to 9 mmHg and DFB +800 to +900 ml, but not above, signifi cantly predicted survival, and also shorter LOS, intubated days and lower vasopressor needs with earlier discharge possibility. On the other hand, over-increased DFB and CVP levels strictly correlated with longer LOS and higher mortality rates, and the fi rst 24-hour mean fl uid balance alone was surprisingly not predictive. Conclusion Fluid resuscitation therapy is a double-edge-sword. (1) Despite lower volumes, higher volumes also increase mortality. (2) Overall, DFB seemed more important than the fi rst 24-hour DFB. References Results The mean APACHE II score was 23.6 ± 7.7, LOS was 9.7 ± 10.0 days, intubated period was 6.4 ± 8.6 days, vasopressor period was 4.7 ± 5.5 days, CVP was 10.5 ± 5.5 mmHg, DFB was 1,147.9 ± 1,157.6 ml, and 42 survived. Kaplan–Meier survival and COX regression analysis showed that CVP levels of 6 to 9 mmHg and DFB +800 to +900 ml, but not above, signifi cantly predicted survival, and also shorter LOS, intubated days and lower vasopressor needs with earlier discharge possibility. On the other hand, over-increased DFB and CVP levels strictly correlated with longer LOS and higher mortality rates, and the fi rst 24-hour mean fl uid balance alone was surprisingly not predictive. Conclusion Fluid resuscitation therapy is a double-edge-sword. P152 Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery As for PAOP, increasing values are associated with NR (AUC = 0.22, P <0.0001). Figure 1 (abstract P153). Change of CO during and after a fl uid challenge. BL, baseline; end, end of fl uid infusion. Conclusion The PVI appears to be useful to predict FR in mechanically ventilated patients after cardiac surgery. However, Se and Sp remain low in the type of patients with relative euvolemia and cardiac dysfunction. References Conclusion The PVI appears to be useful to predict FR in mechanically ventilated patients after cardiac surgery. However, Se and Sp remain low in the type of patients with relative euvolemia and cardiac dysfunction. References Figure 1 (abstract P153). Change of CO during and after a fl uid challenge. BL, baseline; end, end of fl uid infusion. S55 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P156 (1) Introduction Lactated Ringer (L/R) for resuscitation of hemorrhagic shock is suggested by the ATLS program. However, prior studies showed that resuscitation with L/R was associated with more kidney damage in rats with severe hemorrhagic shock. The direct eff ects of L/R on human renal tubule cells have not been reported. p Methods Human proximal renal tubular cell line HK-2 was used. Viability was examined by MTT assay. An additional equal volume of phosphate- buff ered saline (PBS) served as control. Addition of 200 nM dipyridyl and 1 mM H2O2 served as conditions of hypoxia and oxidative stress, respectively. Patterns of cell death were observed by fl ow cytometry. Results To imitate early resuscitation in hypovolemic shock, medium was replaced with 40% v/v of L/R with dipyridyl plus H2O2 for 4 hours, followed by replacing with complete medium for 44 hours. In such il Conclusion Fluid resuscitation therapy is a double-edge-sword. (1) Despite lower volumes, higher volumes also increase mortality. (2) Overall, DFB seemed more important than the fi rst 24-hour DFB. References 1. Dellinger RP, Levy MM, et al.: Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013, 41:580-637. Figure 1 (abstract P156). Cell death of HK-2 cells after lactated Ringer administration in a hypovolemia mimic model. 2. Cannon CM, Holthaus CV, et al.: The GENESIS Project. J Intensive Care Med 2013, 28:355-368. 3. Early Goal-Directed Therapy Collaborative Group of Zhejiang P: Chin Crit Care Med 2010, 22:331-334. 3. Early Goal-Directed Therapy Collaborative Group of Zhejiang P: Chin Crit Care Med 2010, 22:331-334. Fluid challenge with shock V Inal, O Mert itical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) Introduction The latest sepsis guideline has emphasized early resuscitative fl uid management [1]. Early goal-directed therapy (EGDT) has been shown to improve 28-day mortality in recent studies [2,3]. This strategy was based on improving tissue perfusion and oxygenation in spite of other supportive and therapeutic measures. Technically and historically, central venous pressure (CVP) measurement is one of the most dependent methods to estimate fl uid responsiveness and intravascular volume status on resuscitation. The Surviving Sepsis Campaign (SSC) guidelines recommended goal levels of CVP 8 to 12 mmHg in order to obtain appropriate tissue perfusion [1]. In this study, we objected to re-evaluate eff ectiveness of a fl uid resuscitation strategy in sepsis, comparing the eff ect of patients’ daily fl uid balances (DFB) and CVP on patients’ survival. may lead to undercorrection of fi brinogen in critical bleeding situations. The aggravated response on the FibTEM-MCF may better refl ect HES eff ects on clot structure. P154 Fluid challenge with shock V Inal, O Mert Trakya University Medical Faculty, Edirne, Turkey Critical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) P154 Fluid challenge with shock V Inal, O Mert Trakya University Medical Faculty, Edirne, Turkey Critical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) In vivo eff ect of hydroxyethyl starch solution (HES 130/0.4) on diff erent fi brinogen assays U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) In vivo eff ect of hydroxyethyl starch solution (HES 130/0.4) on diff erent fi brinogen assays U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) Reference may lead to undercorrection of fi brinogen in critical bleeding situations. The aggravated response on the FibTEM-MCF may better refl ect HES eff ects on clot structure. Figure 1 (abstract P155). Relative change of P-fi brinogen compared with preoperative values. Reference 1. Prather JW, et al.: Eff ect of blood volume, mean circulatory pressure, and stress relaxation on cardiac output. Am J Physiol 1969, 216:467-472. P154 Fluid challenge with shock V Inal, O Mert Trakya University Medical Faculty, Edirne, Turkey Critical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) P155 P155 In vivo eff ect of hydroxyethyl starch solution (HES 130/0.4) on diff erent fi brinogen assays U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) P157 P157 Hypotonic fl uids after liver transplantation may be associated with prolonged ICU stay A Nadeem, N Salahuddin, A ElHazmi, M Joseph, B Bohlega, H Sallam, Y Elsheikh, D Broering King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P157 (doi: 10.1186/cc13347) Introduction Morbidity after liver transplantation has been linked with preoperative MELD scores and transfusion volumes [1]. We hypothesized that in the immediate post-transplant period there may be modifi able risk factors associated with prolonged ICU stays. y y p y Conclusion Early application of vasopressin reduced the time of vasopressor use, progression to multiple organ dysfunction, length of stay in the ICU, and mortality rates at 14 and 28 days as compared with norepinephrine only. This observed diff erence can be attributed to early restoration of tissue perfusion in the control group making the state of septic shock shorter and reducing the potential for multiple organ dysfunction, which directly infl uenced patient survival. Reference i Methods In a retrospective, case–control study, we reviewed all liver transplant adult recipients over a 33-month period, January 2010 to September 2013. Recipients were divided into two groups based on a priori determined cutoff value of 8 days after transplantation. Signifi cant associations for prolonged ICU admission were identifi ed using chi-square analysis for categorical and ANOVA for continuous variables. P <0.05 was considered signifi cant. SPSS version 22.0 was used for all analyses. 1. Dellinger RP, Levy MM,Carlet JM, et al.: Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock. Crit Care Med 2008, 36:296-327. 1. Dellinger RP, Levy MM,Carlet JM, et al.: Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock. Crit Care Med 2008, 36:296-327. Results Total numbers of transplants performed were 162. Mean pre- transplant MELD score was 19.5 ± 7.7; viral hepatitis was the most common indication for transplant, 44 (27%). Living-related donor transplants were carried out in 87 (54%) cases. Median ICU length of stay was 4 ± 11.9 days and ICU mortality was 15 (9%). Acute kidney injury developed in 30 (19%) with 5% needing renal replacement therapy. Early complications developed in 33%. Prolonged ICU stay was related to: a higher pre-transplant INR (P = 0.000), larger volumes of RBC (2,064 ± 1,701 vs. 1,176 ± 1,454 ml, P = 0.000), platelets (453 ± 271 vs. Reference 1. Bennett-Guerrero E., et al.: Preoperative and intraoperative predictors of postoperative morbidity, poor graft function, and early rejection in 190 patients undergoing liver transplantation. Arch Surg 2001, 136:1177-1183. 1. Bennett-Guerrero E., et al.: Preoperative and intraoperative predictors of postoperative morbidity, poor graft function, and early rejection in 190 patients undergoing liver transplantation. Arch Surg 2001, 136:1177-1183. P157 365 ± 276 ml, P = 0.046) and cryoprecipitate transfusions (47 ± 98 vs. 23 ± 85 ml, P = 0.037) received in the OR and mean volumes of hypotonic crystalloids administered in the fi rst 24 hours (P = 0.002), 48 hours (P = 0.010) and 72 hours (P = 0.007) of ICU admission. Serum P159 Early Vasopressin Application in Shock study S Oliveira, F Dessa, C Rocha, F Oliveira Hospital Bangu, Rio de Janeiro, Brazil Critical Care 2014, 18(Suppl 1):P158 (doi: 10.1186/cc13348) Introduction Vasopressin is frequently used to maintain blood pressure in refractory septic shock [1]. This study is looking into the hypothesis that vasopressin compared with norepinephrine would decrease the severity of septic status, evolution to multiple organ dysfunction, length of hospitalization, and mortality among patients with septic shock. Methods In this randomized, double-blind study, we assigned patients who still needed vasopressor to restore tissue perfusion after fl uid resuscitation to receive norepinephrine (0.05 to 2.0 μg/kg/minute) or vasopressin (0.01 to 0.03 U/minute) with low doses of norepinephrine. Both groups had the vasoactive drug infusions titrated and tapered to maintain a target mean blood pressure. The analysis included the total time of use and dosage of vasopressors every 6 hours, the move to single organ dysfunction and multiple organ failure, length of ICU stay and hospitalization, and mortality 7, 14 and 28 days after the start of infusions. Figure 2 (abstract P156). Elocalcitol (BXL 628) ameliorates toxicity of lactated Ringer in HK-2 human renal tubule cells. conditions, L/R augmented cytotoxicity, and more annexin V (+) cells were observed. Co-treatment or post-treatment with BXL 628, a novel VDR agonist, reversed L/R-induced cytotoxicity. See Figures 1 and 2. Conclusion BXL 628 rescued L/R-induced apoptosis in human renal proximal tubule cells in a hypovolemia mimicking model. Results A total of 407 patients underwent randomization but 387 patients were included in this study (191 patients received vasopressin, and 196 received norepinephrine only). The total time for vasopressors was 37 hours and 68 hours in the vasopressin and norepinephrine groups with P = 0.02. Single organ dysfunction and multiple organ dysfunction using vasopressin and norepinephrine were respectively: 37.7% vs. 49.2%, P = 0.02; and 17.8% vs. 26%; P = 0.05. Length of stay in the ICU was 14 and 17 days (P = 0.29) and the time of hospitalization was 23 and 28 days (P = 0.11) respectively. There was a signifi cant diff erence between the vasopressin and norepinephrine groups in the mortality rate at 14 and 28 days (29.3% vs. 36.7%, P = 0.05; 34% and 42.3%, P = 0.03), but there were no signifi cant diff erences in the overall rates for 7-day mortality (21.2% vs. 23.9%, respectively; P = 1.1). Early Vasopressin Application in Shock study Early Vasopressin Application in Shock study S Oliveira, F Dessa, C Rocha, F Oliveira Hospital Bangu, Rio de Janeiro, Brazil Critical Care 2014, 18(Suppl 1):P158 (doi: 10.1186/cc13348) In vivo eff ect of hydroxyethyl starch solution (HES 130/0.4) on diff erent fi brinogen assays fi U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden y Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) Introduction Previous in vitro studies have shown that photometric assays may overestimate fi brinogen levels after hemodilution with HES. The in vivo eff ect of HES on fi brinogen assays was therefore studied. fi Methods Forty patients with intracranial tumor gave their consent to participate in this ethical approved study. Plasma fi brinogen levels were analyzed with ELISA, two photometric assays (Dade and Multifi bren) and one mechanical (Hook). In addition, ROTEM FibTEM- MCF was analyzed.i y Results Twenty-fi ve of the 40 patients received 1 l HES. Mean reduction of hematocrit was 17%. ELISA was lower than Hook and Multifi bren. The FibTEM relative decrease of 43% diff ered signifi cantly from the other assays. See Figure 1. Figure 1 (abstract P156). Cell death of HK-2 cells after lactated Ringer administration in a hypovolemia mimic model. y g Conclusion After in vivo hemodilution with HES, some assays overestimated fi brinogen levels. An overestimation of around 0.3 g/l Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S56 Figure 2 (abstract P156). Elocalcitol (BXL 628) ameliorates toxicity of lactated Ringer in HK-2 human renal tubule cells. sodium, chloride, lactate or creatinine levels were not signifi cantly diff erent between the two groups.li f Conclusion The administration of hypotonic fl uids in the fi rst 72 hours of liver transplantation may be a risk factor for prolonged ICU admission. This eff ect appears independent of serum electrolyte levels or renal dysfunction. 1. Dellinger RP, Levy MM,Carlet JM, et al.: Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock. Crit Care Med 2008, 36:296-327. P160 Angiotensin II may be useful for the treatment of hypotension in distributive shock, but a safe and effi cacious dose is unknown LW Busse1, E Brasha2, DL Davison2, MG Seneff 2, J Honig2, ZK Alotaibi3, LS Chawla2 1Inova Fairfax Health System, Falls Church, VA, USA; 2George Washington University, Washington, DC, USA; 3Prince Sultan Medical Military City, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P160 (doi: 10.1186/cc13350) Conclusion Vasopressin did not reduce mortality rates as compared with norepinephrine among patients with cancer and septic shock who were treated with catecholamine vasopressors. Critical Care 2014, 18(Suppl 1):P160 (doi: 10.1186/cc13350) Introduction Patients with distributive shock who require high-dose vasopressors have a high mortality. Vasopressors belong to two classes: catecholamines and vasopressin analogs. Each class has limitations. Patients receiving catecholamines often develop tachyphylaxis and metabolic complications (for example, lactic acidosis). Vasopressin analogs can cause mesenteric or myocardial ischemia and oliguria. Angiotensin II (ATII) is an endogenous peptide that increases blood pressure and aldosterone production. Preclinical data suggest a role for ATII in the treatment of sepsis-associated acute kidney injury. ATII may prove useful in patients who remain hypotensive despite catecholamine and vasopressin therapy. This is the fi rst randomized clinical trial to date which seeks to evaluate ATII for use in distributive shock. Acknowledgement Clinical Trials number: NCT 01718613. P162 Heart rate reduction with esmolol in septic shock: eff ects on myocardial performance A Morelli1, A D’Egidio1, A Orecchioni1, E Maraff a1, S Romano2 1University of Rome La Sapienza, Rome, Italy; 2University of Florence, Italy Critical Care 2014, 18(Suppl 1):P162 (doi: 10.1186/cc13352) Introduction Clinical study suggests that beta-blockers, by decreasing the heart rate (HR) together with an increase in stroke volume (SV), do not negatively aff ect cardiac output (CO), allowing an economization of cardiac work and oxygen consumption in patients with septic shock [1]. Whether this hemodynamic profi le leads to an amelioration of myocardial performance is still unclear. The objective of the present study was therefore to elucidate whether a reduction in HR with esmolol is associated with an improvement of cardiac effi ciency in patients with septic shock who remained tachycardic after hemodynamic optimization. Methods Twenty patients with distributive shock and a cardiovascular Sequential Organ Failure Assessment score ≥4 were randomized to either ATII infusion (n = 10) or placebo (n = 10) plus standard of care. Vasopressin versus norepinephrine for the management of septic shock in cancer patients Vasopressin versus norepinephrine for the management of septic shock in cancer patients C Zambolim, D Nagaoka, J Fukushima, C Park, J Carneiro, E Osawa, J Almeida, S Zeff erino, F Galas, L Hajjar Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P161 (doi: 10.1186/cc13351) Conclusion There are few case reports of terlipressin-induced hyponatraemia [1]. Hyponatraemia is considered a rare side eff ect brought about by the partial agonist eff ect of terlipressin on renal vasopressin V2 receptors. Sola and colleagues monitored serum sodium concentrations in patients with acute variceal bleeding. They found that rapid reduction in serum sodium was common (up to 36%) and one patient developed osmotic demyelination syndrome. Hyponatraemia resolved on cessation of terlipressin [2]. Close monitoring of serum sodium levels is essential in patients on terlipressin therapy so rapid drops in sodium can be identifi ed and managed to stop associated neurological complications. Introduction Patients with septic shock die mainly due to refractory shock. Vasopressin is commonly used as an adjunct to catecholamines to support blood pressure in refractory septic shock, but its eff ect on mortality is unknown. We hypothesized that vasopressin as compared with norepinephrine would decrease mortality among cancer patients with septic shock. g References References Methods In this, randomized, double-blind trial, we assigned patients who had cancer and septic shock and needed a vasopressor to receive norepinephrine or vasopressin in addition to open-label vasopressors. All vasopressor infusions were titrated and tapered according to protocols to maintain a target blood pressure. The primary endpoint was the mortality rate 28 days after the start of infusions.i 1. Hyun J, et al.: Terlipressin induced hyponatremic seizure. Scand J Gastroenterol 2010, 45:501-504. 1. Hyun J, et al.: Terlipressin induced hyponatremic seizure. Scand J Gastroenterol 2010, 45:501-504. 2. Sola E, et al.: Hyponatremia in patients treated for severe gastrointestinal bleeding due to portal hypertension. Hepatology 2010, 52:1783-1790. Results A total of 107 patients underwent randomization in this fi rst part of trial, and were infused with the study drug (53 patients received vasopressin, and 54 norepinephrine), and were included in the analysis. There was no signifi cant diff erence between the vasopressin and norepinephrine groups in the 28-day mortality rate (67.9 and 58.5%, respectively; P = 0.31). There were no signifi cant diff erences in the overall rates of serious adverse events (5.3% and 5.5%, respectively; P = 1.00). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A 28-year-old man was admitted to the ICU following intubation and sedation for seizures due to acute alcohol withdrawal. A CT scan of the brain was normal. In the ICU the patient became haemodynamically unstable and fresh blood was aspirated from the nasogastric tube. Gastroscopy showed bleeding oesphageal varicies and hypertensive portal gastropathy. The patient was treated with variceal banding, tazobactam/pipacillin (4.5 g three times daily) and terlipressin (2 g four times daily) as per protocol for variceal bleeding. He was successfully extubated 48 hours later. Results The mean age was 62.9 ± 15.8 years. Of the patients, 75% were male, 45% were Caucasian and 40% were African American. The 30-day mortality for the two groups were similar for the ATII cohort and the placebo cohort (50% vs. 60%, P = 1.00). ATII resulted in marked reduction in norepinephrine dosing in all patients. The mean norepinephrine dose for the placebo cohort was 20.1 ± 16.8 μg/ minute vs. 7.3 ± 11.9 μg/minute for the ATII cohort (P = 0.022). The most common adverse event was hypertension, which occurred in 20% of patients receiving ATII.f Conclusion ATII is an eff ective vasopressor agent in patients with distributive shock requiring multiple vasopressors. The initial dose range of ATII that appears to be appropriate for patients with distributive shock is 2 to 10 ng/kg/minute. Further studies to assess the use of ATII in patients with distributive shock are warranted. Results On admission the patient’s serum sodium was 139 mmol/l. The patient received terlipressin for 4 days in the following regimen: 2 g four times daily for 48 hours, then 1 mg four times daily for 24 hours and then 0.5 mg four times daily for a further 24 hours before stopping. On the last day of his terlipressin therapy, the patient’s GCS dropped from 15 to 11. Serum sodium had fallen acutely to 116 mmol/l. The last two doses of terlipressin were cancelled and no other treatment for hyponatraemia was administered. His serum sodium and GCS returned to normal limits within 13 hours of terlipressin cessation with no neurological consequences. P161 Terlipressin-induced hyponatraemia Introduction Terlipressin is a vasopressin (V1 receptor) agonist that causes splanchnic constriction and is used in the management of variceal bleeding. This case report demonstrates the profound hyponatraemia, suffi cient to cause a fall in conscious level, developing following terlipressin administration for variceal gastrointestinal bleeding. S57 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery g y H Araujo1, R Campbell2, D Green2, E Mills3 1King’s College London, UK; 2King’s College Hospital NHS Foundation Trust, London, UK; 3LiDCO Ltd, London, UK Critical Care 2014, 18(Suppl 1):P164 (doi: 10.1186/cc13354) g y H Araujo1, R Campbell2, D Green2, E Mills3 1King’s College London, UK; 2King’s College Hospital NHS Foundation Trust, London, UK; 3LiDCO Ltd, London, UK Critical Care 2014, 18(Suppl 1):P164 (doi: 10.1186/cc13354) g pi Results For a MAP between 65 and 75 mmHg, esmolol administration signifi cantly decreased HR (115 ± 10 to 91 ± 7 bpm), NE (0.7 ± 0.4 to 0.5 ± 0.3 μg/kg/minute), and dP/dt MAX (1.1 ± 0.3 to 0.8 ± 0.3 ms/ mmHg). Conversely, TAPSE (15 ± 3 to 20 ± 3 mm), CCE (–0.2 ± 0.4 to –0.03 ± 0.4 units) and SV (37 ± 8 to 42 ± 10 ml) signifi cantly increased at the end of the study period (all P <0.05). CO (4.1 ± 0.8 to 3.9 ± 0.8 l/ minute) and LVEF (46 ± 10 to 48 ± 10%) did not change. Introduction Fall in mean arterial blood pressure (MAP) during anaesthetic induction is common and may result in profound hypo- tension. Using the LiDCORapid (LiDCO Ltd, UK), we previously demonstrated that the fall in MAP is usually driven by a reduction in stroke volume (SV) that was presumed due to venodilation. Low- dose phenylephrine (PE), a venoconstrictor, commenced immediately prior to induction ameliorated these eff ects to a signifi cant degree [1]. However, it is important that the pre-induction administration of PE should not cause any marked changes in MAP and CO. We retrospectively studied a group of 40 high-risk patients about to undergo major peripheral vascular surgery where PE was commenced immediately pre-induction. The haemodynamic eff ects of this dose of PE on MAP, SV and CO were analysed. g Conclusion In patients with established septic shock who remained tachycardic after hemodynamic optimization, titration of esmolol to reduce the HR to a predefi ned threshold economized cardiac function, resulting in a maintained CO with a lower HR and a higher stroke volume. Such a hemodynamic profi le was characterized by an improved cardiac effi ciency, as indicated by the decrease in dP/dt MAX associated with an increase in CCE. P163 A new automatic urinometer shows lower bias, no loss of precision due to temporal deviation and higher user evaluation when compared with a manual standard urinometer in an ICU setting A Eklund, J Van der Linden Karolinska University Hospital, Stockholm, Sweden Critical Care 2014, 18(Suppl 1):P163 (doi: 10.1186/cc13353) Results Patient demographics, mean (range): age 71 (46 to 89) years, 31 male, weight 80 (55 to 115) kg, ASA 3 (2 to 4). The changes in MAP and CO were not clinically or statistically signifi cant. MAP increased by an average of only 2%, range –25 to +12; CO by 0%, range –23 to +16 (P = 0.35 and P = 0.36 respectively). Karolinska University Hospital, Stockholm, Sweden y p Critical Care 2014, 18(Suppl 1):P163 (doi: 10.1186/cc13353) y Conclusion In a previous study [1], PE administered prospectively prior to induction markedly reduced the haemodynamic changes following induction of anaesthesia in high-risk patients using remifentanil and propofol. This eff ect was achieved without signifi cant changes in MAP and CO in the 5-minute to 10-minute period between commencement of PE and induction of anaesthesia. Introduction In the intensive care setting, most physiologic parameters are monitored automatically. However, urine output (UO) is still monitored hourly by manually handled urinometers. This study evaluated an automatic urinometer (AU) and compared it with a manual urinometer (MU). Methods This was a prospective study in the ICU of a cardio-thoracic surgical clinic. In postoperative patients (n = 36) with indwelling urinary catheters and an expected stay of 24 hours or more, hourly UO samples were measured with an AU (n = 220, Sippi®; Observe Medical, Gothenburg, Sweden) or a MU (n = 188, UnoMeter™ 500; Unomedical a/s, Birkeroed, Denmark), and thereafter validated by cylinder measurements. Malposition of instrument at reading excluded measurement. Data were analyzed with the Bland–Altman method. The performance of the MU was used as minimum criteria of acceptance when the AU was evaluated. The loss of precision with the MU due to temporal deviation from fi xed hourly measurements was recorded (n = 108). A questionnaire, fi lled out by the ward staff (n = 28), evaluated the ease of use of the AU compared with the MU. . Bidd H, Tan A, Mills E, Araujo H, Green D, O’Brien T: Phenylephrine commenced prior to anaesthesia reduced the haemodynamic changes associated with induction of anaesthesia in patients undergoing high risk vascular surgery [Abstract A378]. g Reference 1. Morelli A, Ertmer C, Westphal M, et al.: Eff ect of heart rate control with esmolol on hemodynamic and clinical outcomes in patients with septic shock: a randomized clinical trial. JAMA 2013, 310:1683-1691. P163 Presented at American Society of Anesthesiologists 2012 Annual Meeting; October 13-17 2012; Washington, DC. [http://www.asaabstracts.com/strands/asaabstracts/search.htm] P164 despite adequate volume resuscitation received a continuous esmolol infusion to maintain the HR between 94 and 80 bpm. NE was titrated to achieve a MAP between 65 and 75 mmHg. To investigate myocardial performance, we simultaneously assessed LV ejection fraction (LVEF), tricuspidal annular plane solid excursion (TAPSE) by echocardiography, the dP/dt MAX and the cardiac cycle effi ciency (CCE) both estimated from the arterial pressure waveform. Data were obtained at baseline and after achieving the predefi ned HR threshold (T1). Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery H Araujo1, R Campbell2, D Green2, E Mills3 1King’s College London, UK; 2King’s College Hospital NHS Foundation Trust, London, UK; 3LiDCO Ltd, London, UK Critical Care 2014, 18(Suppl 1):P164 (doi: 10.1186/cc13354) Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P165 (doi: 10.1186/cc13355) Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery Finally, echocardiographic data suggest that reducing HR with esmolol positively aff ects right ventricular function. Reference y Methods A radial artery line was inserted prior to induction of anaesthesia, and baseline MAP, CO and SV were obtained using the LiDCORapid. The PE infusion was commenced at a rate of 1 to 2 mg/ hour. Remifentanil was then administered using a target-controlled infusion pump until a 2 ng/ml predicted eff ect site concentration (Ceff ) was reached. Propofol was then administered to induce anaesthesia. Haemodynamic changes from the pre-PE baseline to commencement of propofol were recorded at 5-second intervals. The changes in MAP, SV and CO were assessed as the percent change in values obtained immediately prior to PE and followed for at least 5 minutes until commencement of propofol. P160 ATII was started at a dose of 20 ng/kg/minute, and titrated per a protocolized schedule for a goal of maintaining a mean arterial pressure (MAP) of 65 mmHg. The infusion (either ATII or placebo) was continued for 6 hours and then titrated off . The primary endpoint was the eff ect of ATII on the standing dose of norepinephrine required to maintain a MAP of 65 mmHg. Secondary endpoints included the eff ect of ATII on urine output, serum lactate and creatinine clearance, as well as 30-day mortality. Methods After 24 to 36 hours of initial hemodynamic stabilization, 24 septic shock patients with HR >95 bpm and requiring norepinephrine (NE) to maintain mean arterial pressure (MAP) between 65 and 75 mmHg S58 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Acetaminophen-induced hypotension in the surgical ICU Z Stoecker Potential use of veno-arterial extracorporeal membrane oxygenation for cardiogenic shock refractory to mechanical assist devices: baseline physiology and mortality data C Vimalanathan, N Barrett, N Ioannou, C Langrish, C Meadows, G Salt, G Glover Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P167 (doi: 10.1186/cc13357) g Results Of the 11 patients included, six had spontaneous intracranial hemorrhage (ICH), four had traumatic ICH and one was free of neurologic injury. Following administration of 393 doses of APAP, the average change in MAP for all patients was –7.52 mmHg and SBP was –12.04 mmHg. When evaluated on an individual basis, patients with ICH had an average change in MAP of –10.64 (–5.2 to –19.08) and SBP of –17.81 (–7.54 to –35.75). The patient without neurologic injury had an average change in MAP and SBP of –3.01 and –2.42. The average change in MAP and SBP following administration of oral APAP solution (n = 357) was –7.84 and –12.39 while the change following rectal administration (n = 3) was –4.22 and –8 and tablet administration (n = 33) was –4.4 and –8.61. Introduction Mortality from cardiogenic shock remains high [1] and, despite a physiological rationale, intra-aortic balloon counterpulsation (IABP) has recently been shown to be ineff ective in reducing mortality [2,3]. Veno-arterial extracorporeal membrane oxygenation (V-A ECMO) may off er a survival advantage over IABP. The objective of this study was to describe the characteristics and outcomes of patients supported with IABP or Impella and to identify the characteristics of patients who die, despite mechanical assistance, for whom a proposed V-A ECMO programme may be benefi cial. p g yi Methods A retrospective cohort study in a 30-bed, medical–surgical ICU. All adult patients supported with IABP or Impella over 2 years to March 2013 were identifi ed and data were extracted by case-note review. Subgroup analysis was carried out for patients aged ≤65 and for those who fulfi lled the modifi ed Melbourne criteria for V-A ECMO [4]. Data collected included demographic data, physiology and organ support at baseline and at 6, 12, and 24 hours, ICU and hospital outcomes and cause of death. Comparisons between survivors and nonsurvivors were made with t test/chi-squared tests as appropriate. Results A total of 129 patients were identifi ed: 78% were male, mean age was 70 years (SD ±11.8), mean APACHE II score was 20 (±5) and ICU mortality was 44%. Comparing survivors with nonsurvivors the only statistically signifi cant diff erence was metabolic acidosis (–6.8 ± 5.3 vs. –10.9 ± 7.0 mEq/l; P <0.05). Heart rate, mean arterial pressure, lactate, central venous oxygen saturation, cardiac index, arterial blood pH and mechanical ventilation failed to show a signifi cant diff erence. Potential use of veno-arterial extracorporeal membrane oxygenation for cardiogenic shock refractory to mechanical assist devices: baseline physiology and mortality data C Vimalanathan, N Barrett, N Ioannou, C Langrish, C Meadows, G Salt, G Glover Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P167 (doi: 10.1186/cc13357) Eleven of these patients would have fulfi lled the proposed criteria for V-A ECMO, with an ICU mortality of 36%. Methods A retrospective cohort study in a 30-bed, medical–surgical ICU. All adult patients supported with IABP or Impella over 2 years to March 2013 were identifi ed and data were extracted by case-note review. Subgroup analysis was carried out for patients aged ≤65 and for those who fulfi lled the modifi ed Melbourne criteria for V-A ECMO [4]. Data collected included demographic data, physiology and organ support at baseline and at 6, 12, and 24 hours, ICU and hospital outcomes and cause of death. Comparisons between survivors and nonsurvivors were made with t test/chi-squared tests as appropriate.i Conclusion It appears from this small case series that patients with ICH may experience more isolated hypotension following administration of APAP when compared with others. These changes seemed to be greater than previously reported for intravenous administration. It may also be possible that APAP solution exudes hypotensive eff ects more commonly when compared with tablet or rectal formulations. References 1. Brown G: Acetaminophen-induced hypotension. Heart Lung 1996, 25:137-140. q pp p Results A total of 129 patients were identifi ed: 78% were male, mean age was 70 years (SD ±11.8), mean APACHE II score was 20 (±5) and ICU mortality was 44%. Comparing survivors with nonsurvivors the only statistically signifi cant diff erence was metabolic acidosis (–6.8 ± 5.3 vs. –10.9 ± 7.0 mEq/l; P <0.05). Heart rate, mean arterial pressure, lactate, central venous oxygen saturation, cardiac index, arterial blood pH and mechanical ventilation failed to show a signifi cant diff erence. Eleven of these patients would have fulfi lled the proposed criteria for V-A ECMO, with an ICU mortality of 36%. 2. Mrozek, S, et al.: [Acetaminophene-induced hypotension in intensive care unit: a prospective study]. Ann Fr Anesth Reanim 2009, 28:448-453. P167 Methods Patients admitted to the SICU over a 7-month time frame who were reported by the nursing staff to have experienced hypotension following the oral or rectal administration of APAP were included. Their electronic medical records were retrospectively reviewed to describe the change in systolic blood pressure (SBP) and mean arterial pressure (MAP) within the fi rst hour following all administrations of APAP. Additional data collected consisted of patient age, sex, admission diagnoses and formulation/route of APAP administration. Potential use of veno-arterial extracorporeal membrane oxygenation for cardiogenic shock refractory to mechanical assist devices: baseline physiology and mortality data C Vimalanathan, N Barrett, N Ioannou, C Langrish, C Meadows, G Salt, G Glover Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P167 (doi: 10.1186/cc13357) i References 1. Jeger RV, et al.: Ann Intern Med 2008, 149:618-626. 2. Thiele H, et al.: N Engl J Med 2012, 367:1287-1296. 3. Werdan K, et al.: Eur Heart J 2013. [Epub ahead of print] 4. Aubron C, et al.: Crit Care 2013, 17:R73. 1. Jeger RV, et al.: Ann Intern Med 2008, 149:618-626. 2. Thiele H, et al.: N Engl J Med 2012, 367:1287-1296. 3. Werdan K, et al.: Eur Heart J 2013. [Epub ahead of print] 4. Aubron C, et al.: Crit Care 2013, 17:R73. . e e , et a .: g J ed 0 , 36 : 8 96. 3. Werdan K, et al.: Eur Heart J 2013. [Epub ahead of print] 4. Aubron C, et al.: Crit Care 2013, 17:R73. Introduction Profound myocardial depression can occur after cardiac surgery. Use of ventricular assist support through venoarterial extracorporeal membrane oxygenation (ECMO) has been positively reported. This study will focus on the outcomes of patients who, upon suff ering hemodynamic failure after cardiac surgery, were supported by the use of ECMO during their stay in the surgical ICU of Incor FMUSP. Methods This was a retrospective, single-center and observational study. The records of 48 patients who underwent cardiac surgery and, subsequently, needed percutaneous or surgical implantation of ventricular assist devices were evaluated. The evaluation considered the following criteria: basal characteristics, indications for ventricular assistance, duration, length of ICU and hospital stay, and hospital mortality, through data collection forms. P166 y Conclusion Only metabolic acidosis was associated with mortality in patients supported with mechanical assist devices. Our data do not allow discrimination of survivors from nonsurvivors. Patients who fulfi lled the proposed criteria for V-A ECMO showed a similar mortality to a recent series treated with V-A ECMO [4]. The proposed criteria do not identify a cohort, in this population, that would expect a mortality benefi t from V-A ECMO. R f P166 Experiences of a tertiary center with use of extracorporeal membrane oxygenation support in patients with cardiogenic shock after cardiac surgery L Galas1, V Guimaraes2, M Sundin2, L Caneo2, M Jatene2, F Jatene2, L Hajjar2, J Almeida1, F Galas2 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Heart Institute, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P166 (doi: 10.1186/cc13356) P166 Experiences of a tertiary center with use of extracorporeal membrane oxygenation support in patients with cardiogenic shock after cardiac surgery L Galas1, V Guimaraes2, M Sundin2, L Caneo2, M Jatene2, F Jatene2, L Hajjar2, J Almeida1, F Galas2 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Heart Institute, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P166 (doi: 10.1186/cc13356) Acetaminophen-induced hypotension in the surgical ICU Z Stoecker Acetaminophen-induced hypotension in the surgical ICU Z Stoecker Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P165 (doi: 10.1186/cc13355) Results Analysis according to Bland–Altman showed a smaller mean bias for the AU, +1.9 ml, compared with the MU, +5.3 ml (P <0.001). There was no statistical diff erence in measurement precision between the two urinometers, defi ned by their limits of agreement (±15.2 ml vs. ±16.6 ml, P = 0.11). The mean temporal variation with the MU was ±7.4 minutes (±12.4%), limits of agreement ±23.9 minutes (±39.8%), compared with no temporal variation with the AU (P <0.001). A total 86% of the ward staff considered the AU superior to the MU (P <0.001). Conclusion The AU had a signifi cantly lower bias than the MU and the loss of precision of hourly UO due to temporal deviations using the MU was avoided with the AU. The AU was also evaluated higher by the ward staff , refl ecting perception of higher reliability, easier use, less contact with urine bags and less time usage for measurements. The features of the AU may also indicate a favorable clinical impact in the normal ward, when staffi ng does not allow hourly measurements with a MU. Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P165 (doi: 10.1186/cc13355) Introduction Acetaminophen (APAP) is commonly administered in the surgical ICU (SICU) for its analgesic and antipyretic eff ects. Case reports have described the potential for APAP to cause allergic reactions with and without hypotension. Furthermore, there have been case reports of APAP causing isolated hypotension in the absence of other allergic responses [1]. This has been well described following intravenous administration [2]. However, other routes of administration causing hypotension and associated diagnoses remain to be elucidated. The present case series describes 11 patients with APAP-induced hypotension in the SICU at a Level I trauma center. S59 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P168 Normobaric oxygen paradox and the microcirculation in the critically ill patient: a prospective observational study A Donati, E Damiani, AT Colesnicenco, E Montesi, S Ciucani, A Carsetti, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2014, 18(Suppl 1):P168 (doi: 10.1186/cc13358) Normobaric oxygen paradox and the microcirculation in the critically ill patient: a prospective observational study A Donati, E Damiani, AT Colesnicenco, E Montesi, S Ciucani, A Carsetti, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2014, 18(Suppl 1):P168 (doi: 10.1186/cc13358) Università Politecnica delle Marche, Ancona, Italy y Critical Care 2014, 18(Suppl 1):P168 (doi: 10.1186/cc13358) Introduction The normobaric oxygen paradox (NOP) is a recent concept that postulates the use of intermittent hyperoxia to stimulate erythropoietin (EPO) production [1]. Hyperoxia increases oxygen free radicals and may lead to endothelial damage and vasoconstriction [2]. We evaluated the microvascular response to transient hyperoxia and its eff ects on EPO production. y g Results Of the 48 patients included on the study, 26 (54%) were males, and 31 (64%) were younger than 18 years old. These patients developed cardiogenic shock during 72 hours after cardiac surgery. In all cases, ECMO was inserted after cardiac surgery. Of all patients, 32 (66%) were central ECMO, inserted in the operative room, and 16 were percutaneous, inserted in the ICU. The median duration of ventricular assistance was 6 days (IQR 0 to 41), the length of ICU stay was 16 days (IQR 1 to 111), and hospital stay was 29 days (IQR 1 to 198). Twenty patients survived (41%) and were discharged from our hospital. Methods Six patients with hemodynamic stability and mechanically ventilated with FiO2 <50% were included in this prospective observational study. Patients underwent a 2-hour period of hyperoxia (FiO2 100%). The sublingual microcirculation (sidestream dark-fi eld imaging (SDF)) was evaluated at baseline (t0), 2 hours after hyperoxia (t1), and 2 hours after return to basal FiO2 (t2). SDF monitoring was continuously performed also during the variation of FiO2 for 2 minutes. EPO levels were assayed at baseline and for 2 days. Conclusion The use of mechanical circulatory assists devices is an effi cient tool to manage seriously ill patients after cardiac surgery. This tool should be considered early in the diagnosis of cardiogenic shock after cardiac surgery. Predictive criteria for the development of intra-abdominal hypertension and abdominal compartment syndrome D Lyer, P Rastogi, A Aneman, S D’Amours Liverpool Hospital, Sydney, Australia Critical Care 2014, 18(Suppl 1):P169 (doi: 10.1186/cc13359) Conclusion In patients with hyperlactatemia on ICU admission, lactate-guided therapy did not reduce complications and was related to a longer ICU length of stay. This study suggests that goal-directed therapy aiming to decrease initial lactate levels does not result in clinical benefi t. Introduction This study aims to develop predictive criteria to identify which patients are at risk of developing intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) upon admission to the ICU. Early lactate-guided therapy in cardiac surgery patients: a randomized controlled trial M Sundin1, J Almeida2, E Osawa1, F Galas1, M Gaiane1, S Zeff erino1, L Camara1, LG Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P170 (doi: 10.1186/cc13360) Figure 1 (abstract P168). TVD and NOP. Results An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure 1). The TVD tended to increase without returning to baseline levels at t2. EPO increased in four patients out of 6 (P = NS). A negative correlation was found between the change in TVD after hyperoxia (t1 – t0) and the change in EPO (r = –0.88, P = 0.03). Results An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure 1). The TVD tended to increase without returning to baseline levels at t2. EPO increased in four patients out of 6 (P = NS). A negative correlation was found between the change in TVD after hyperoxia (t1 – t0) and the change in EPO (r = –0.88, P = 0.03). Introduction It is unknown whether lactate monitoring aimed to decrease levels during the fi rst hours in patients undergoing cardiac surgery improves outcome. The aim of this study was to evaluate the eff ect of lactate monitoring and resuscitation directed at decreasing lactate levels in patients admitted to the ICU in the fi rst 8 hours with lactate level ≥3.0 mEq/l. Conclusion Hyperoxia leads to vasoconstriction that seems to be reversible at hyperoxia cessation. Further data are needed to verify the effi cacy of the NOP in stimulating erythropoiesis in the critically ill. There might be a relation between hyperoxia-induced reduction in vessel density and the EPO increase. P168 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S60 Results An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure 1). The TVD tended to increase without returning to baseline levels at t2 EPO increased Figure 1 (abstract P168). TVD and NOP. Figure 1 (abstract P168). TVD and NOP. (odds ratio, 4.95; 95% CI, 1.19 to 7.24; P <0.001), hemoperitoneum/ pneumoperitoneum/intraperitoneal fl uid collection (odds ratio, 3.61; 95% CI, 1.29 to 10.12; P = 0.014), obesity (odds ratio, 3.41; 95% CI 1.97 to 5.90; P <0.001), fl uid received >2.3 l (odds ratio, 2.68; 95% CI, 1.48 to 4.84; P = 0.001), abbreviated SOFA score >4 points (odds ratio, 2.49; 95% CI, 1.49 to 4.15; P <0.001) and lactate >1.4 mmol/l (odds ratio, 2.28; 95% CI, 1.33 to 3.91; P <0.001) were identifi ed as independent predictors of IAH upon admission to ICU. The presence of three or more of these risk factors at admission predicted the onset of IAH with sensitivity of 75% and a specifi city of 76%, and the onset of grade II, III and IV IAH with a sensitivity of 91% and a specifi city of 62%. (odds ratio, 4.95; 95% CI, 1.19 to 7.24; P <0.001), hemoperitoneum/ pneumoperitoneum/intraperitoneal fl uid collection (odds ratio, 3.61; 95% CI, 1.29 to 10.12; P = 0.014), obesity (odds ratio, 3.41; 95% CI 1.97 to 5.90; P <0.001), fl uid received >2.3 l (odds ratio, 2.68; 95% CI, 1.48 to 4.84; P = 0.001), abbreviated SOFA score >4 points (odds ratio, 2.49; 95% CI, 1.49 to 4.15; P <0.001) and lactate >1.4 mmol/l (odds ratio, 2.28; 95% CI, 1.33 to 3.91; P <0.001) were identifi ed as independent predictors of IAH upon admission to ICU. The presence of three or more of these risk factors at admission predicted the onset of IAH with sensitivity of 75% and a specifi city of 76%, and the onset of grade II, III and IV IAH with a sensitivity of 91% and a specifi city of 62%. yi y Conclusion IAH is a common clinical entity in the intensive care setting. Predictive criteria, based on data readily available upon a patient’s admission to ICU, were developed and eff ectively predicted the risk of developing IAH. P170 Early lactate-guided therapy in cardiac surgery patients: a randomized controlled trial M Sundin1, J Almeida2, E Osawa1, F Galas1, M Gaiane1, S Zeff erino1, L Camara1, LG Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P170 (doi: 10.1186/cc13360) References Methods Patients were randomly allocated to two groups. In the lactate group, treatment was guided by lactate levels with the objective to decrease lactate by 20% or more per 2 hours for the initial 8 hours of ICU stay. In the control group, the treatment team had no knowledge of lactate levels (except for the admission value) during this period. The primary outcome measure was the incidence of complications in 28 days. 1. Balestra C, Germonpré P, Poortmans JR, Marroni A: J Appl Physiol 2006, 100:512-518 1. Balestra C, Germonpré P, Poortmans JR, Marroni A: J Appl Physiol 2006, 100:512-518. 2. Tsai AG, Cabrales P, Winslow RM, Intaglietta M: Am J Physiol Heart Circ Physiol 2003, 285:H1537-H1545. 2. Tsai AG, Cabrales P, Winslow RM, Intaglietta M: Am J Physiol Heart Circ Physiol 2003, 285:H1537-H1545. y Results The lactate group received more fl uids and dobutamine. However, there were no signifi cant diff erences in lactate levels between the groups. The rate of complications was similar between groups (11% vs. 7%, P = 0.087). Length of ICU stay was higher in the lactate group (3.5 vs. 2.4 days, P = 0.047) when compared with the control group. Results The lactate group received more fl uids and dobutamine. However, there were no signifi cant diff erences in lactate levels between the groups. The rate of complications was similar between groups (11% vs. 7%, P = 0.087). Length of ICU stay was higher in the lactate group (3.5 vs. 2.4 days, P = 0.047) when compared with the control group. Conclusion In patients with hyperlactatemia on ICU admission, lactate-guided therapy did not reduce complications and was related to a longer ICU length of stay. This study suggests that goal-directed therapy aiming to decrease initial lactate levels does not result in clinical benefi t. Lactate as a predictor of deterioration in emergency department patients with and without infection K Oedorf1, D Day1, Y Lior2, V Novack2, N Shapiro1, D Henning1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Soroka University Medical Center, Beersheba, Israel Critical Care 2014, 18(Suppl 1):P171 (doi: 10.1186/cc13361) y Results Thirty-nine and 2% of patients developed IAH and ACS as per consensus defi nitions. Upon ICU admission, patients that would go on to develop IAH had a signifi cantly higher APACHE III score (62 (44 to 81) vs. 50 (37 to 68); P <0.001), abbreviated SOFA score (6 (4 to 8) vs. 4 (2 to 6); P <0.001), CVP (17 (13 to 20) vs. 15 (12 to 17) mmHg; P <0.001), lactate (2.2 (1.4 to 3.8) vs. 1.5 (1.1 to 2.4) mmol/l; P <0.001), INR (1.3 (1.1 to 1.5) vs. 1.2 (1.1 to 1.3); P <0.001), bilirubin (11 (7 to 19) vs. 10 (6 to 15) μmol/l; P = 0.027), creatinine (108 (76.8 to 175) vs. 84.0 (66.0 to 118) μmol/l; P <0.001), 24-hour fl uid balance (2.47 (0.95 to 4.05) vs. 1.23 (0.29 to 2.27) l; P <0.001), vasopressor requirement (60 vs. 39%; P <0.001), mechanical ventilation requirement (71 vs. 58%; P = 0.011), PEEP (8.00 (5.00 to 10.00) vs. 5.50 (5.00 to 8.00) cmH2O; P <0.001), and peak airway pressure (24 (21 to 30) vs. 22 (19 to 24) cmH2O; P <0.001). These patients also had a signifi cantly lower abdominal perfusion pressure (61 (55 to 71) vs. 69 (60 to 77) mmHg; P <0.001), pH (7.29 (7.22 to 7.35) vs. 7.33 (7.28 to 7.37); P <0.001) and PaO2:FiO2 ratio (182 (100 to 263) vs. (264 (168 to 371); P <0.001) upon ICU admission. Abdominal distension Introduction The use of serum lactate level to risk-stratify emergency department (ED) patients with sepsis is widely used. Studies in nonsepsis populations also suggest its utility in predicting adverse outcomes. Whether lactate prognosticates equally across disease states is not clearly defi ned. This study compares the ability of lactate to identify patients at risk for deterioration (intubation, acute renal dysfunction, vasopressor use, or death) and mortality during hospitalization in infected and non-infected populations. Introduction The use of serum lactate level to risk-stratify emergency department (ED) patients with sepsis is widely used. Studies in nonsepsis populations also suggest its utility in predicting adverse outcomes. Whether lactate prognosticates equally across disease states is not clearly defi ned. P169 Predictive criteria for the development of intra-abdominal hypertension and abdominal compartment syndrome D Lyer, P Rastogi, A Aneman, S D’Amours Liverpool Hospital, Sydney, Australia Critical Care 2014, 18(Suppl 1):P169 (doi: 10.1186/cc13359) P171 Methods This is a prospective, observational study of 403 consecutively admitted ICU patients in a 3-month period, requiring the insertion of an indwelling urinary catheter. Intra-abdominal pressure was measured at least twice daily in all patients. Lactate as a predictor of deterioration in emergency department patients with and without infection K Oedorf1, D Day1, Y Lior2, V Novack2, N Shapiro1, D Henning1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Soroka University Medical Center, Beersheba, Israel Critical Care 2014, 18(Suppl 1):P171 (doi: 10.1186/cc13361) 1. Balestra C, Germonpré P, Poortmans JR, Marroni A: J Appl Physiol 2006, 100:512-518. Lactate as a predictor of deterioration in emergency department patients with and without infection This study compares the ability of lactate to identify patients at risk for deterioration (intubation, acute renal dysfunction, vasopressor use, or death) and mortality during hospitalization in infected and non-infected populations. Methods A prospective, observational cohort study of ED adult patients presenting from 11 November 2012 to 1 February 2013 who had lactate measured and abnormal vital signs (hearth rate ≥130, respiratory rate ≥24, shock index ≥1, systolic blood pressure <90 mmHg). Patients with isolated atrial tachycardia, seizure, intoxication, or psychiatric agitation S61 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 lactate (initial)) × 100%. Lactate (initial) is blood or tissue lactate within the fi rst 24 hours after ICU admission (H0). Lactate (delayed) is blood or tissue lactate at H4, H8, H12, H16, H20, H24 and H48 (H = hours). were excluded. Patients were stratifi ed into three groups by lactate <2.5 (low), 2.5 to 4 (intermediate), and >4 mmol/l (high). Chi-square test for trend was used to compare outcome rates between lactate levels for each diagnostic category. were excluded. Patients were stratifi ed into three groups by lactate <2.5 (low), 2.5 to 4 (intermediate), and >4 mmol/l (high). Chi-square test for trend was used to compare outcome rates between lactate levels for each diagnostic category.i Results A total of 112 patients having septic shock (n = 79) or severe sepsis (n = 33) were examined. Tissue lactate clearance was higher compared with blood lactate clearance at H0 to H8 (P = 0.02), H0 to H12 (P = 008), H0 to H16 (P = 0.01), H0 to H20 (P = 0.01), and H0 to H24 (P = 0.02). Tissue lactate clearance was higher in survivors compared with nonsurvivors at H0 to H12, H0 to H20 and H0 to H24 (P = 0.02, for all). Multivariate analysis showed that ARACHE II along with tissue clearances at H0 to H12, H0 to H20 and H0 to H24 <30% were independent outcome predictors. Blood lactate clearance was not related to survival. Results Of 1,152 patients identifi ed, 366 were excluded and 298 did not have lactate measurements, leaving 488 for the analysis: 289 sepsis patients and 202 nonsepsis patients. Of these, 168 (34.4%) met the deterioration outcome, and there were 61 (12.5%) deaths. Lactate as a predictor of deterioration in emergency department patients with and without infection For infected patients, 46/342 (13.5%; 95% CI 10.2 to 17.5) low, 34/100 (34.0%; 95% CI 25.4 to 43.7) intermediate, and 20/46 (43.5%; 95% CI 30.2 to 57.8) high lactate patients suff ered deterioration (P <0.01). Likewise, 6/342 (1.6%; 95% CI 0.7 to 3.9) low, 19/100 (19.0%; 95% CI 12.4 to 27.9) intermediate, and 11/46 (23.9%; 95% CI 13.8 to 38.0) high lactate patients died during hospitalization (P <0.01). For non-infected patients, 42/342 (12.3%; 95% CI 9.2 to 16.2) low, 13/100 (15.1%; 95% CI 7.6 to 21.1) intermediate, and 13/46 (28.2%; 95% CI 17.2 to 42.7) high lactate patients suff ered deterioration (P = 0.01). In the regression models, lactate was strongly associated with deterioration for both infected (odds ratio (OR) = 1.94; 95% CI 1.4 to 2.3) and non-infected (OR = 1.48; 95% CI 1.14 to 1.91) groups. In regression models for mortality, lactate had better discrimination ability in infected (OR = 1.8; 95% CI 1.4 to 2.3) patients than in the non-infected (OR = 1.11; 95% CI 0.85 to 1.47) patients. Conclusion In critically ill septic patients, after the initial resuscitation phase, adipose tissue clears lactate earlier than blood. High tissue lactate clearance, but not blood lactate clearance, is associated with a favorable clinical outcome. P173 P173 P173 Correlation between conventional and advanced hemodynamic parameters versus serum lactate in patients with severe sepsis M Kok1, A Oei2, A Karakus3, H Endeman4 1UMC Utrecht, the Netherlands; 2AMC, Amsterdam, the Netherlands; 3Diakonessenhuis, Utrecht, the Netherlands; 4OLVG, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P173 (doi: 10.1186/cc13363) Conclusion Lactate levels can be used to identify patients who are at increased risk of deterioration regardless of infection status. Lactate identifi es high-risk patients for mortality in infected patients more strongly than in non-infected patients. Critical Care 2014, 18(Suppl 1):P173 (doi: 10.1186/cc13363) P172 Introduction The aim of this study was to determine the correlation between levels of serum lactate (SL) and conventional hemodynamic parameters (HPs) (mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), urinary output (UP)) in patients with severe sepsis. In a subgroup, advanced HPs (central venous saturation (SvO2), peripheral temperature (PT), cardiac index (CI), global end-diastolic volume index (GEDI) and extravascular lung water (ELWI)) were compared with levels of SL. Adipose tissue lactate clearance but not blood lactate clearance is associated with clinical outcome in severe sepsis or septic shock during the post-resuscitation period M Theodorakopoulou, S Orfanos, N Nikitas, D Vasilliadi, S Apolonatou, A Diamantakis, G Karkouli, C Diakaki, A Armaganidis, I Dimopoulou University Hospital of Athens Greece ‘ATTIKON’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P172 (doi: 10.1186/cc13362) Delayed assessment of serum lactate in sepsis is associated with an increased mortality rate Delayed assessment of serum lactate in sepsis is associated with an increased mortality rate R Arnold1, Z Zhang1, S Patel2, S Smola1, J Isserman1, E Jackson1 1Christiana Care Health System, Newark, DE, USA; 2Cooper University Hospital, Camden, NJ, USA Critical Care 2014, 18(Suppl 1):P174 (doi: 10.1186/cc13364) Results Of the 61 patients identifi ed, fi ve were excluded as their ICU stay was <24 hours. The overall mortality in the remaining 56 patients (mean age of 76.7 ± 10.4 (SD) years) was 21.4%. In univariate analysis, mSOFA and several other variables correlated signifi cantly (P <0.05) with mortality. The area under the ROC curve for LC at 6, 12, and 24 hours was 0.601, 0.719, and 0.731, respectively. LC at 24 hours was the most accurate, and its optimum cutoff value was 37.5%. LC, lactate level, and BE at 24 hours, as well as the signifi cant factors in univariate analysis, were entered into a stepwise logistic regression model, which revealed 24-hour LC ≤37.5% (odds ratio (OR), 23.0) and mSOFA score (OR, 2.1) as independent predictive values of mortality. Introduction Lactate assessment early in the resuscitation of sepsis has been recommended as a diagnostic biomarker. An abnormal lactate, independent of blood pressure, is an indication for aggressive fl uid resuscitation and its normalization is a recommended endpoint of resuscitation. The objective of this study was to evaluate the eff ect of the timing of lactate assessment on patient outcomes in sepsis. Conclusion In patients with colorectal perforation, 24-hour LC is more accurate than LC measured at earlier time points. Patients with 24- hour LC ≤37.5% and a high mSOFA score have a high risk of in-hospital mortality. R f g p p Methods Data were compiled using the Clinical Vigilance for Sepsis electronic health record (EHR) screening tool, which identifi ed consecutive patients from two hospital systems over 12 months at a 300-bed community hospital and over 24 months from a 500-bed academic tertiary care center. CV Sepsis alert screens the EHR to identify the presence of infection based on a multifactor alert system including labs, vital signs, and treatment team documentation. A physician order for intravenous antibiotics was used as a surrogate for suspected infection. The database identifi ed 37,160 consecutive patients treated for infection from a total of 216,550. Lactate quartile concentration and prognosis in severe sepsis and septic shock M De La Torre-Prados1, A Garcia-de la Torre2, C Trujillano-Fernández1, J Perez-Vacas1, A Puerto-Morlan1, E Camara-Sola1, A Garcia-Alcantara1, A Garcia-Alcantara1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Puerto Real University Hospital, Cadiz, Spain Critical Care 2014 18(Suppl 1) P176 (doi 10 1186/cc13366) y y Results A total 5,072 of 37,160 consecutive patients (13%) had a measured lactate. Sepsis patients experienced an overall 3% (1,186/37,160) mortality rate. In total, 4,153 (82%) patients had measured lactate within 3 hours, and 919 (18%) were delayed, with a decreased morality rate (eLac 6.8 vs. dLac 24.7, P <0.0001). There was no diff erence in average lactate levels between the groups (eLac: 2.1 ± 2.6, dLac: 2.3 ± 3.0, P = NS). A larger ratio of delayed-lactate patients had a lactate ≥4 mmol/l (dLac 12.6% vs. eLac 8.7%). Introduction The Surviving Sepsis Campaign (SSC) indicates that a lactate (LT) concentration greater than 4 mmol/l indicates early resuscitation bundles. However, several recent studies have suggested that LT values lower than 4 mmol/l may be a prognostic marker of adverse outcome. The aim of this study was to identify clinical and analytical prognostic parameters in severe sepsis (SS) or septic shock (ShS) according to quartiles of blood LT concentration. Conclusion The delay in lactate assessment relative to clinical evidence of infection was associated with an increased mortality rate. The average lactate level in each group did not account for this eff ect. The timing of the assessment, not the lactate level, was prognostic of outcome. The mortality benefi t associated with lactate assessment within the 3-hour guideline suggests that an increased clinical awareness may lead to early initiation of time-sensitive interventions known to improve outcomes. Methods A cohort study was designed in a polyvalent ICU. We studied demographic, clinical and analytical parameters in 148 critically ill adults, within 24 hours from SS or ShS onset according to SSC criteria. We tested for diff erences in baseline characteristics by lactate interval using a Kruskal–Wallis test for continuous data or a chi-square test for categorical data and reported the median and interquartile ranges; SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 148 consecutive episodes of SS (16%) or ShS (84%). The median age was 64 (interquartile range, 48.7 to 71) years; male: 60%. The main sources of infection were respiratory tract 38% and intra-abdomen 45%; 70.7% had medical pathology. Mortality at 28 days was 22.7%. y Reference 1. Nates JL, et al.: Automating and simplifying the SOFA score in critically ill patients with cancer. Health Informatics J 2010, 16:35-47. P176 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 MAP 73 mmHg (13, 37 to 120), HR 101 beats/minute (22, 51 to 172), CVP 12 mmHg (5, 1 to 29), UP 55 ml/hour (62, 0 to 500), SL 3.2 mmol/l (3, 1 to 18.6), CI 4.1 ml/kg/minute (1.1, 1.6 to 6.7), GEDI 871 ml/m2 (210, 500 to 1,691), ELWI 11 ml/kg (5, 4 to 23), PT 32.1 C (2.8, 26.4 to 38), and SvO2 75% (8, 39 to 93). Relevant and signifi cant (P <0.005) PCCs between HPs and SL were respectively: MAP –0.417, HR 0.195, UP –0.237, SvO2 –0.204 and PT –0.569. Figure 1 shows the relation between two HPs with the highest correlation with SL. Methods We retrospectively analyzed the clinical data of patients who underwent emergency surgery for colorectal perforation and were admitted to the ICU of our hospital from January 2003 to August 2013. Patients with traumatic, iatrogenic, and appendicitis perforations were excluded. The primary endpoint was survival to hospital discharge. The modifi ed Sequential Organ Failure Assessment (mSOFA) score, a customized SOFA score excluding the central nervous system component [1], was used for prognostic scoring. The mSOFA score and several clinical factors were analyzed by univariate analysis as possible predictors of survival. We collected lactate levels and base excess (BE) measured during surgery and at 6, 12, and 24 hours after the fi rst measurement and calculated the respective LC values. The associations of initial blood lactate level, LC, and BE with mortality were assessed by receiver operating characteristics (ROC) curve and logistic regression analyses. g Conclusion The conventional HPs MAP, HR, UP and SvO2 are signifi cantly correlated to levels of SL, but clinical value might be limited due to the relatively low correlation coeffi cients. In a small subgroup, PT is better correlated to the level of SL. Lactate quartile concentration and prognosis in severe sepsis and septic shock Quartiles of blood LT concentration were quartile 1 (Q1): 1.87 mmol/l or less, quartile 2 (Q2): 1.88 to 2.69 mmol/l, quartile 3 (Q3): 2.7 to 4.06 mmol/l, and quartile 4 (Q4): 4.07 mmol/l or greater (Table 1). The median LT concentrations of each quartile were 1.43 (Q1), 2.2 (Q2), 3.34 (Q3), and 5.1 (Q4) mmol/l (P <0.001). The diff erences between these quartiles were that the patients in Q1 had signifi cantly lower APACHE II scores (P = 0.04), SOFA score (P = 0.024), number of organ failures (NOF) (P <0.001) and ICU mortality (P = 0.028), compared with patients in Q2, Q3 and Q4. Patients in Q1 had signifi cantly higher Delayed assessment of serum lactate in sepsis is associated with an increased mortality rate Patients with a measured lactate were divided relative to its measurement within 3 hours (eLac) or greater than 3 hours (dLac) of sepsis identifi cation as recommended by the Surviving Sepsis Campaign. The CV Sepsis alert was the reference standard for time zero. Patients were compared in each group for the occurrence of the primary outcome of in-hospital mortality. y Reference Adipose tissue lactate clearance but not blood lactate clearance is associated with clinical outcome in severe sepsis or septic shock during the post-resuscitation period g p p M Theodorakopoulou, S Orfanos, N Nikitas, D Vasilliadi, S Apolonatou, A Diamantakis, G Karkouli, C Diakaki, A Armaganidis, I Dimopoulou University Hospital of Athens Greece ‘ATTIKON’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P172 (doi: 10.1186/cc13362) p Methods An observational prospective, single-center, pilot study was performed in intensive care (IC) of a medium-sized teaching hospital. Adult patients with severe sepsis were included and received standard goal-directed therapy (Surviving Sepsis Guidelines). Every patient received an arterial line and a central venous line in the upper diaphragm position. A subgroup received pulse contour cardiac output (PiCCO)-guided resuscitation and PT measurements. Pearson correlation coeffi cients (PCCs) were calculated between HPs and SL, which were measured every 4 hours for the fi rst 48 hours after inclusion. P <0.05 was considered statistically signifi cant.i Introduction Blood lactate clearance, a surrogate of tissue hypoxia, is associated with increased mortality in septic patients. However, no study has directly measured lactate clearance at the tissue level in the post-resuscitation period of sepsis. This study aimed to examine the relative kinetics of blood and tissue lactate clearances and to investigate whether these are associated with outcome in ICU patients having severe sepsis or septic shock during the post-resuscitation phase. p Methods A microdialysis catheter was inserted in the subcutaneous adipose tissue of the upper thigh and interstitial fl uid samples were collected. Serial measurements of blood and interstitial fl uid lactate levels were performed over a 48-hour period. Lactate clearance was calculated according to the formula: (lactate (initial) – lactate (delayed) / y gi Results Twenty-fi ve patients (12 men) were included. Mean age was 68 years (30 to 93), mean APACHE II score 31 (20 to 42). The most frequent reasons for IC admission were abdominal sepsis (n = 11) and pneumosepsis (n = 7). Mean HPs (with SD and range) were respectively: Figure 1 (abstract P173). Scatterplot of (a) MAP and SL and (b) PT and SL. Figure 1 (abstract P173). Scatterplot of (a) MAP and SL and (b) PT and SL. Figure 1 (abstract P173). Scatterplot of (a) MAP and SL and (b) PT and SL. S62 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P175 It may be useful to revise the cutoff value of lactate according to the SSC (4 mmol/l). P177 Correlation between arterial lactate and venous lactate in patients with sepsis and septic shock P Theerawit, C Na Petvicharn Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P177 (doi: 10.1186/cc13367) Introduction Measurement of arterial lactate (A-LACT) levels has been used to monitor poor tissue perfusion, predicting mortality and guiding resuscitation. Peripheral venous lactate (V-LACT) has been regarded as an unreliable test, but a less invasive approach. We aimed to determine correlation between A-LACT and V-LACT and agreement of both in order to determine the usefulness of V-LACT as a biomarker for assessment in sepsis. Methods We conduct a prospective cross-sectional study during June Table 1 (abstract P176). Baseline characteristics in SS and ShS patients by quartiles of blood LT Lactate <1.87 (n = 33) Lactate 1.88 to 2.69 (n = 41) Lactate 2.7 to 4.06 (n = 34) Lactate>4.07 (n = 37) P value Age (years) 57 (45 to 71) 64 (51.5 to 74.5) 65 (48 to 69) 60 (48.5 to 71) NS APACHE II 25 (18.5 to 30) 25 (19.5 to 27) 25 (21.5 to 29.5) 27 (22 to 33) 0.04 SOFA 9 (7 to 10.5) 9 (7 to 11) 9 (8 to 11) 11(8 to 13) 0.024 NOF 3 (3 to 4) 3 (3 to 4) 4 (3 to 5) 5 (3.5 to 5) <0.001 LT (mmol/l) 1.43 (1.16 to 1.56) 2.2 (1.99 to 2.47) 3.34 (3 to 3.72) 5.1 (4.4 to 7.34) <0.001 Procalcitonin (ng/ml) 2.81 (0.76 to 20.7) 11.5 (2.88 to 37.15) 13.47 (1.91 to 42.1) 21.6 (5.2 to 5.8) 0.05 Cholesterol (mg/dl) 127 (97.5 to 165) 130 (95.5 to 152.5) 100 (72 to 128) 91 (79 to 116.7) 0.06 28-day mortality (%) 10.8 21.2 24.4 35.1 0.029 ShS (%) 83.8 85.4 81.1 87.9 NS Figure 1 (abstract P177). Conclusion The arterial lactate and venous lactate levels were strongly correlated in the condition of sepsis or septic shock. Consequently, V-LACT may be used in substitution for A-LACT particularly in lactate levels not higher than 4 mmol/l. However, trending should be generally applied instead of the absolute value. P177 l P177 Correlation between arterial lactate and venous lactate in patients with sepsis and septic shock P Theerawit, C Na Petvicharn Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P177 (doi: 10.1186/cc13367) Figure 1 (abstract P177). Figure 2 (abstract P177). Introduction Measurement of arterial lactate (A-LACT) levels has been used to monitor poor tissue perfusion, predicting mortality and guiding resuscitation. Peripheral venous lactate (V-LACT) has been regarded as an unreliable test, but a less invasive approach. We aimed to determine correlation between A-LACT and V-LACT and agreement of both in order to determine the usefulness of V-LACT as a biomarker for assessment in sepsis. p Methods We conduct a prospective, cross-sectional study during June to December 2011 at a university hospital. Septic patients in the ICU were enrolled in this research. Sepsis was defi ned according to the Surviving Sepsis Campaign: International Guidelines for Management of Severe Sepsis and Septic Shock: 2008. The exclusion criteria were: contraindication for arterial puncture; and denying inform consent. The venous lactate would be sampled at the same point in time as arterial lactate measurement. The correlation and agreement between arterial and venous lactate was the primary outcome. p y Results A total of 73 pair-samples in 45 intensive care patients were collected. Mean age was 68.33 ± 14.5 years. Fifty percent of all patients received the vasopressors to stabilize hemodynamics. The mean serum creatinine level was 2.78 mg/dl and the mean anion gap was 13.55 mmol/l. The mean arterial lactate (A-LACT) level was 3.73 ± 4.0 mmol/l, and the mean venous lactate (V-LACT) level was 4.6 ± 4.2 mmol/l. The A-LACT and V-LACT were strongly correlated as shown in Figure 1 (r = 0.927, P <0.0001, r2 = 0.859). The mean diff erence between V-LACT and A-LACT was 0.889 mmol/l. The 95% limits of the V-A diff erence in the individual patients were between –2.3 and 4.1 mmol/l. However, the agreement looks very good at lactate levels not higher than 4 mmol/l (Figure 2). The regression equation was: A-LACT = (0.877 × V-LACT) – 0.320. Figure 2 (abstract P177). Figure 2 (abstract P177). Conclusion The arterial lactate and venous lactate levels were strongly correlated in the condition of sepsis or septic shock. Consequently, V-LACT may be used in substitution for A-LACT particularly in lactate levels not higher than 4 mmol/l. However, trending should be generally applied instead of the absolute value. P175 Lactate clearance as a predictor of mortality in colonic perforation R Egashira, T Kobayashi Osaki Citizen Hospital, Miyagi, Japan Critical Care 2014, 18(Suppl 1):P175 (doi: 10.1186/cc13365) Introduction The objective of this study was to determine whether lactate clearance (LC) is a signifi cant indicator of mortality in patients with colorectal perforation. LC has been associated with mortality in heterogeneous critically ill patients, but its role as a predictor of mortality in homogeneous patients with colorectal perforation is unclear. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S63 Table 1 (abstract P176). Baseline characteristics in SS and ShS patients by quartiles of blood LT Lactate <1.87 (n = 33) Lactate 1.88 to 2.69 (n = 41) Lactate 2.7 to 4.06 (n = 34) Lactate>4.07 (n = 37) P value Age (years) 57 (45 to 71) 64 (51.5 to 74.5) 65 (48 to 69) 60 (48.5 to 71) NS APACHE II 25 (18.5 to 30) 25 (19.5 to 27) 25 (21.5 to 29.5) 27 (22 to 33) 0.04 SOFA 9 (7 to 10.5) 9 (7 to 11) 9 (8 to 11) 11(8 to 13) 0.024 NOF 3 (3 to 4) 3 (3 to 4) 4 (3 to 5) 5 (3.5 to 5) <0.001 LT (mmol/l) 1.43 (1.16 to 1.56) 2.2 (1.99 to 2.47) 3.34 (3 to 3.72) 5.1 (4.4 to 7.34) <0.001 Procalcitonin (ng/ml) 2.81 (0.76 to 20.7) 11.5 (2.88 to 37.15) 13.47 (1.91 to 42.1) 21.6 (5.2 to 5.8) 0.05 Cholesterol (mg/dl) 127 (97.5 to 165) 130 (95.5 to 152.5) 100 (72 to 128) 91 (79 to 116.7) 0.06 28-day mortality (%) 10.8 21.2 24.4 35.1 0.029 ShS (%) 83.8 85.4 81.1 87.9 NS Table 1 (abstract P176). Baseline characteristics in SS and ShS patients by quartiles of blood LT cholesterol (P = 0.06) and lower procalcitonin (P = 0.05) at enrolment. At the extremes, patients in Q1 had decreased 28-day mortality (P = 0.023) and, patients in Q4 had increased 28-day mortality, compared with the other quartiles of patients (P = 0.009). Interestingly, patients in Q2 had signifi cant increased mortality compared with patients in Q1 (P = 0.043), whereas the patients in Q2 had no signifi cant diff erence in 28-day mortality compared with patients in Q3. Conclusion Adverse outcomes and several potential risk factors, including organ failure, are signifi cantly associated with higher quartiles of LT concentrations. P175 41) Lactate 2.7 to 4.06 (n = 34) Lactate>4.07 (n = 37) P 65 (48 to 69) 60 (48.5 to 71) NS 25 (21.5 to 29.5) 27 (22 to 33) 0.04 9 (8 to 11) 11(8 to 13) 0.024 4 (3 to 5) 5 (3.5 to 5) <0.001 3.34 (3 to 3.72) 5.1 (4.4 to 7.34) <0.001 13.47 (1.91 to 42.1) 21.6 (5.2 to 5.8) 0.05 100 (72 to 128) 91 (79 to 116.7) 0.06 24.4 35.1 0.029 81.1 87.9 NS Figure 1 (abstract P177). Figure 2 (abstract P177). cholesterol (P = 0.06) and lower procalcitonin (P = 0.05) at enrolment. At the extremes, patients in Q1 had decreased 28-day mortality (P = 0.023) and, patients in Q4 had increased 28-day mortality, compared with the other quartiles of patients (P = 0.009). Interestingly, patients in Q2 had signifi cant increased mortality compared with patients in Q1 (P = 0.043), whereas the patients in Q2 had no signifi cant diff erence in 28-day mortality compared with patients in Q3. Figure 1 (abstract P177). Conclusion Adverse outcomes and several potential risk factors, including organ failure, are signifi cantly associated with higher quartiles of LT concentrations. It may be useful to revise the cutoff value of lactate according to the SSC (4 mmol/l). P179 Hyperdynamic ejection fraction in the critically ill patient JR Paonessa1, TP Brennan2, LA Celi1 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Massachusetts Institute of Technology, Cambridge, MA, USA Critical Care 2014, 18(Suppl 1):P179 (doi: 10.1186/cc13369) Introduction Pulmonary regurgitation (PR) that develops after right ventricular (RV) outfl ow reconstruction including the Rastelli and Norwood procedure may often result in serious cardiac events early after surgery. We hypothesized that PR may be associated with pulmonary vascular resistance (PVR) and RV contraction. Accordingly, we assessed the impact of PVR on PR and RV function using a swine model. 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Massachusetts Institute of Technology, Cambridge, MA, USA Critical Care 2014, 18(Suppl 1):P179 (doi: 10.1186/cc13369) Introduction The hyperdynamic left ventricular ejection fraction (HDLVEF) in the ICU is a common fi nding thought to be associated with critical illness and possibly sepsis. The exact etiology of hyperdynamic ejection fraction has yet to be determined, and the prognosis of these patients has not been well defi ned. Methods Eight pigs (14 ± 2 kg) underwent total resection of the pulmonary valve cusps under cardiopulmonary bypass (PR group). This was compared with a control group (n = 6) that underwent only bypass. In both groups, the pulmonary regurgitant fraction (PRF) and cardiac output were measured by a pulsed Doppler fl ow meter, and the percent segmental shortening of RV (%RVSS) and RV end-diastolic dimension (RVDd) were measured by sonomicrometry. We also performed dobutamine stress evaluation as well as changing the PVR by carbon dioxide (PaCO2) and inhaled nitric oxide (NO). pi Methods The cohort consisted of 2,632 adults admitted to the ICU with echocardiogram reports using the MIMIC-II database, and was divided into those with HDLVEF and those with normal left ventricular ejection fraction (NLVEF). Those with impaired ejection fraction were excluded from the analysis. Baseline comparisons were performed using chi- squared tests for equal proportion with results reported as numbers, percentages, and 95% CIs. Continuous variables were compared using t tests and reported as means with 95% CIs, while non-normally distributed data were compared using Wilcoxon rank-sum tests and reported as medians. 2 Results All bypass time was 18 ± 3 minutes. In the PR group, the PRF was 40 ± 4% and the RVDd was 53 ± 9 mm (vs. control 34 ± 6 mm). P <0.05. P177 l Conclusion The arterial lactate and venous lactate levels were strongly correlated in the condition of sepsis or septic shock. Consequently, V-LACT may be used in substitution for A-LACT particularly in lactate levels not higher than 4 mmol/l. However, trending should be generally applied instead of the absolute value. S64 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P179) P178 ab e (abst act 9) Normal Hyperdynamic (n = 2,373, 90%) (n = 259, 10%) P value Male 1,159 (49) 98 (38) <0.001 Service type MICU 1,234 (52) 138 (53) 0.695 CCU 364 (15) 28 (11) 0.052 SICU 572 (24) 69 (27) 0.367 CSRU 188 (8) 23 (9) 0.590 Primary outcome Twenty-eight-day mortality 458 (19) 79 (31) <0.001 One-year mortality 892 (38) 129 (50) <0.001 ICU mortality 296 (12) 60 (23) <0.001 Hospital mortality 433 (18) 80 (31) <0.001 Treatment RRT 363 (15) 55 (21) 0.013 Vasopressor 1,063 (45) 139 (54) 0.006 Ventilated 1,453 (61) 186 (72) <0.001 Comparison of the eff ects of histidine–triptophan–ketoglutarate solution and crystalloid cardioplegia on myocardial protection during pediatric cardiac surgery S Kuslu, P Zeyneloglu, A Pirat, A Camkiran, M Ozkan, G Arslan Baskent University Faculty of Medicine, Ankara, Turkey Critical Care 2014, 18(Suppl 1):P178 (doi: 10.1186/cc13368) Introduction The major components of myocardial protection during cardiac surgery are the combination of cardioplegia solutions with hypothermia. The primary endpoint of this study is to compare the eff ects of histidine–triptophan–ketoglutarate (HTK) solution and crystalloid cardioplegia on release of cardiac troponin-I (cTn-I) and creatine kinase-myocardial band (CK-MB), which are perioperative determinants of myocardial protection; the secondary endpoint is to evaluate the intraoperative and postoperative hemodynamic variables and clinical outcome parameters. p Methods A total of 66 children aged 1 month to 6 years undergoing elective congenital heart surgery were randomly allocated to HTK solution (Group H, n = 32) or crystalloid cardioplegia (Group C, n = 34) after aortic cross-clamping. Blood samples for cTn-I and CK-MB levels were measured before the surgical incision, at the end of surgery and at 4, 16, 24 and 48 hours postoperatively. Results Demographic features were similar in both groups. Duration of surgery, aortic clamp and cardiopulmonary bypass times, amounts of intraoperative fl uids used and urine outputs were similar between the groups. P177 l The groups were not signifi cantly diff erent in terms of cTn-I and CK-MB levels at the intraoperative and postoperative period (P >0.05 for all). The dose of positive inotropic drug at the end of surgery was signifi cantly high in Group H (P = 0.01). The requirements for defi brillation were similar in both groups. There were no signifi cant diff erences between the groups regarding postoperative hemodynamic parameters, positive inotropic requirements, amounts of fl uids and blood given and pacemaker requirements (P >0.05 for all). Duration of mechanical ventilation, lengths of ICU and hospital stay were similar in both groups. Conclusion Patients with hyperdynamic LVEF in the ICU clearly have increased mortality. Hyperdynamic LVEF may be a result of increased catecholamines during cytokine storm. It is unclear whether hyperdynamic LVEF itself worsens outcomes. Further investigation is needed. g p Conclusion The present study demonstrated that there is no superiority of HTK solution and crystalloid cardioplegia to each other for myocardial protection during pediatric cardiac surgery. P180 Impact of nitric oxide on pulmonary regurgitation and cardiac function in the acute stage after right ventricular outfl ow surgery Y Ko, K Morita, R Nagahori, T Abe, K Hashimoto Jikei University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P180 (doi: 10.1186/cc13370) , , g , , Jikei University School of Medicine, Tokyo, Japan y , y , p Critical Care 2014, 18(Suppl 1):P180 (doi: 10.1186/cc13370) P182 P182 Intraoperative dexamethasone on left atrial function and postoperative atrial fi brillation in cardiac surgical patients K Jacob, S Dieleman, H Nathoe, D Van Osch, E De Waal, M Cramer, J Kluin, D Van Dijk Utrecht University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P182 (doi: 10.1186/cc13372) Figure 1 (abstract P180). same as catecholamine during the acute stage after RV outfl ow surgery with PR. Introduction Postoperative new-onset atrial fi brillation (PNAF) is very common after cardiac surgery. The infl ammatory response due to surgery and cardiopulmonary bypass (CPB) may contribute to PNAF by inducing atrial dysfunction [1]. Corticosteroids reduce the infl ammatory response and may thus reduce atrial dysfunction and PNAF [2]. The aim of this study was to determine whether dexamethasone protects from left atrial dysfunction and PNAF in cardiac surgical patients. same as catecholamine during the acute stage after RV outfl ow surgery with PR. P181 Cardiogenic oscillation in pediatric patients after cardiac surgery H Imanaka, N Okuda, T Itagaki, M Onodera, M Nishimura Tokushima University Hospital, Tokushima, Japan Critical Care 2014, 18(Suppl 1):P181 (doi: 10.1186/cc13371) Methods Patients undergoing cardiac surgery were randomized to a single dose of dexamethasone (1 mg/kg) or placebo after inducing anesthesia. Transesophageal echocardiography was performed in patients after CPB. The primary outcome was left atrial total ejection fraction (LA-TEF) after sternal closure; secondary outcomes included left atrial diameter and PNAF, detected by Holter monitoring. Introduction Cardiogenic oscillation is the fl uctuation in fl ow tracing in mechanically ventilated patients. Large cardiogenic oscillation may cause autotriggering in adult patients after cardiac surgery [1] and inaccurate volume monitoring [2]. However, it is unknown how cardiogenic oscillation is problematic in pediatric patients. Therefore, we prospectively surveyed cardiogenic oscillation in pediatric patients after cardiac surgery. y g Results Sixty-two patients were included. Baseline characteristics were well balanced. Postoperative LA-TEF was 36.4% in the dexamethasone group and 40.2% in the placebo group (P = 0.15) (Figure 1). Secondary echocardiographic outcomes were also insignifi cant (Table 1). The incidence of PNAF was 30% in the dexamethasone group and 39% in the placebo group (P = 0.47). g y Methods We enrolled 17 pediatric patients who underwent cardiac surgery using cardiopulmonary bypass. They were mechanically ventilated with pressure-controlled ventilation. We measured the amplitude in cardiogenic oscillation and compared them between their admission to the ICU and before extubation. P179 A signifi cant reduction in the %RVSS (18 ± 1% vs. control 22 ± 1%) and the cardiac output (2.1 ± 0.2 l/minute* vs. control 2.5 ± 0.3 l/minute) were observed. The PRFs were 60 ± 5% (PaCO2 >80 mmHg), 37 ± 2% (PaCO2 <20 mmHg), 24 ± 2% (NO 20 ppm; PaCO2 40 mmHg), and were positively correlated with the PVR (Figure 1A). During the dobutamine stress, the %RVSS was increased (baseline 18 ± 1%, 5γ 21 ± 2%, 10γ 26 ± 3%), and was negatively correlated with the PRFs (Figure 1B). Results Patients with HDLVEF had increased mortality in hospital, at 28 days and at 1 year when compared with patients with NLVEF. HDLVEF patients more frequently required renal replacement therapy (RRT), vasopressors and mechanical ventilation. Of the 2,632 patients, 1,220 were septic. There was an increased proportion of HDLVEF in the septic compared with the nonseptic groups (11.2% vs. 8.6%, P = 0.026). Interestingly, other statistically signifi cant associated comorbidities were cancer, CHF, arrhythmias, and hypertension, which were more commonly seen in the HDLVEF group. See Table 1. Conclusion These results indicated that massive PR resulted in marked deterioration of RV performance; however, low PVR and high RV contractility may contribute to reduce the severity of PR and improve cardiac function. Nitric oxide may be a useful treatment modality the Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S65 same as catecholamine during the acute stage after RV outflow surgery Figure 1 (abstract P180). of inotropes and intravascular volume might have contributed to the decrease in cardiogenic oscillation. of inotropes and intravascular volume might have contributed to the decrease in cardiogenic oscillation. Figure 1 (abstract P180). Conclusion In pediatric patients after cardiac surgery, cardiogenic oscillation was initially large but was decreasing at the extubation. References 1. Crit Care Med 2000, 28:402. 2. Crit Care Med 2004, 32:1546. P183 White blood cell count and new-onset atrial fi brillation after cardiac surgery S Dieleman, K Jacob, H Nathoe, M Ten Berg, D Van Osch, J Frencken, D Van Dijk Utrecht University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P183 (doi: 10.1186/cc13373) Introduction Ranolazine, a piperazine derivative, is used as an anti- anginal drug to treat patients with chronic angina in clinical practice [1] and may improve coronary blood fl ow by reducing compression eff ects of ischemic contracture, and by improving endothelial function [2,3]. In the present study we investigate the vascular eff ects of ranolazine on the endothelium, adrenergic system and Ca2+ in isolated rat aorta. Introduction Postoperative new-onset atrial fi brillation (PNAF) is the most common complication after cardiac surgery. Infl ammation as an underlying mechanism has been studied by various infl ammatory markers, and white blood cell count (WBC) is the only present consequent infl ammatory marker predicting PNAF [1]. This study aimed to determine the association between perioperative WBC and PNAF. Methods Rat aortic segments (3 mm long) with and without endothelium were mounted for isometric tension recording in organ baths containing Krebs–Henseleit solution. Electrical fi eld stimulation (2, 4 and 8 Hz, 20 V, 0.25 ms duration for 30 seconds) was provided by a Grass S88 stimulator via two platinum electrodes positioned on each side and parallel to the axis of the aortic segment. Concentration– response curves of ranolazine (10–7 to 10–4 M) were obtained in a cumulative manner using endothelin-1, noradrenaline, thromboxane A2 and KCl as constrictor agents. Methods Patients >18 years undergoing elective cardiac surgery with a sinus rhythm preoperatively were recruited from the Dexamethasone for Cardiac Surgery-PNAF trial for this post-hoc cohort study. The WBC was prospectively measured preoperatively and once during each of the fi rst four postoperative days. Development of PNAF was evaluated with continuous 12-lead ECG monitoring the fi rst 5 days postoperatively. Results The contractile responses to electrical fi eld stimulation were abolished by tetrodotoxin, guanethidine and prazosin, indicating that the contractile eff ect is due to the action of noradrenaline on alpha adrenoreceptors. Ranolazine diminished (P <0.05) neurogenic adrenergic contractions induced by electrical fi eld stimulation in aortic rings with and without endothelium. Ranolazine produced concentration-dependent relaxation in rings precontracted with noradrenaline (Emax 86 ± 6%, n = 10; P <0.05) but not in rings precontracted with endothelin-1, thromboxane A2 and KCl. Neither L-NAME (10–4 M), an inhibitor of nitric oxide synthase, nor indomethacin (10–5 M), an inhibitor of cyclooxygenase, modifi ed the relaxation induced by ranolazine. The calcium antagonist nifedipine (10–6 M) reduced the relaxation induced by ranolazine. P182 We performed statistical analysis with the t test and considered P <0.05 signifi cant. Table 1 (abstract P182). Secondary postoperative echocardiographic parameters in both groups Table 1 (abstract P182). Secondary postoperative echocardiographic parameters in both groups Parameter Dexamethasone Placebo P value LA-TEF 36.4 40.2 0.15 LA diameter 4.6 4.3 0.19 LA area 16.0 16.4 0.81 Conclusion Intraoperative high-dose dexamethasone did not have any protective eff ect on postoperative LA-TEF or dimension and did not reduce the risk of PNAF in cardiac surgical patients. Parameter Dexamethasone Placebo P value LA-TEF 36.4 40.2 0.15 LA diameter 4.6 4.3 0.19 LA area 16.0 16.4 0.81 y gi Results Cardiogenic oscillation was 2.1 ± 0.6 l/minute just after the surgery (Figure 1). Autotriggering occurred in seven of 17 patients when triggering sensitivity was set at 1 l/minute. Before the extubation, cardiogenic oscillation signifi cantly decreased to 1.4 ± 0.4 l/minute when autotriggering disappeared. Intensive care including adjustment Conclusion Intraoperative high-dose dexamethasone did not have any protective eff ect on postoperative LA-TEF or dimension and did not reduce the risk of PNAF in cardiac surgical patients. Figure 1 (abstract P181). Cardiogenic oscillation. Figure 1 (abstract P182). Primary outcome preoperatively and postoperatively in dexamethasone and placebo groups. Figure 1 (abstract P182). Primary outcome preoperatively and postoperatively in dexamethasone and placebo groups. Figure 1 (abstract P181). Cardiogenic oscillation. S66 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References 1. Haff ajee et al.: JACC Cardiovasc Imaging 2011, 4:833-840. 2. Chaney et al.: Chest 2002, 121:921-923. P183 White blood cell count and new-onset atrial fi brillation after cardiac surgery S Dieleman, K Jacob, H Nathoe, M Ten Berg, D Van Osch, J Frencken, D Van Dijk Utrecht University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P183 (doi: 10.1186/cc13373) Results A total of 657 patients were included in this trial, 277 developed PNAF. The WBC was signifi cantly higher in the PNAF group on day 2 and day 4 (Figure 1). However, multivariate analysis showed that preoperative and postoperative WBC, days 1 to 3, were not associated with PNAF (Table 1). Older age (OR: 1.05; CI: 1.03 to 1.07; P <0.001), CABG plus valve surgery (OR: 2.95; CI: 1.78 to 4.88), single valve surgery (OR: 3.09; CI: 2.03 to 4.69; P <0.001) and other surgery (OR: 2.21; CI: 1.23 to 3.97; P <0.001) were correlated with the occurrence of PNAF. Table 1 (abstract P183). Multiple regression analysis of association between high WBC and developing PNAF Time point OR 95% CI Baseline 1.04 0.96 to 1.13 Day 1 1.03 0.98 to 1.08 Day 2 1.03 0.99 to 1.08 Day 3 1.03 0.96 to 1.11 Day 4 1.09 1.01 to 1.16 Table 1 (abstract P183). Multiple regression analysis of association between high WBC and developing PNAF Conclusion These results indicate that ranolazine diminished the contractile response induced by adrenergic stimulation, suggesting an eff ect as an adrenergic blocker. The relaxant eff ects of ranolazine on rat aortic vessels is not dependent on the endothelium-derived factors (nitric oxide or dilator prostanoids) but involves an interference with the entry of calcium through dihydropyridine calcium channels. References 1. Chaitman BR.: Circulation 2006, 113:2462-2472. 2. Stone PH, et al.: J Am Coll Cardiol 2010, 56:934-942. 3. Deshmukh SH, et al.: Coron Artery Dis 2009, 20:343-347. 1. Chaitman BR.: Circulation 2006, 113:2462-2472. 2. Stone PH, et al.: J Am Coll Cardiol 2010, 56:934-942. 3. Deshmukh SH, et al.: Coron Artery Dis 2009, 20:343-347. 1. Chaitman BR.: Circulation 2006, 113:2462-2472. Conclusion Preoperative and postoperative WBC were not associated with development of PNAF. Reference P184 References 1. Haff ajee et al.: JACC Cardiovasc Imaging 2011, 4:833-840. 2. Chaney et al.: Chest 2002, 121:921-923. Anti-adrenergic eff ects of ranolazine in isolated rat aorta P Marchio, MD Mauricio, FB El Amrani, S Guerra, D Aguirre-Rueda, SL Vallés, JM Vila, M Aldasoro University of Valencia, Spain Critical Care 2014, 18(Suppl 1):P184 (doi: 10.1186/cc13374) Eff ects of perfusion pressure on the splanchnic circulation after cardiopulmonary bypass: a randomized double cross-over study Conclusion Isofl urane prevents experimental TCM and preserves LV function, an eff ect not mediated via opening of K-ATP channels. The eff ect cannot be explained entirely by attenuation of myocardial stress. Isofl urane sedation in the ICU might be an interesting approach for patients suff ering from hyperadrenergic conditions at risk of developing TCM. Introduction No randomized trial has assessed the eff ects of diff erent mean arterial pressure (MAP) targets in postcardiac surgery intensive care. We investigated the short-term eff ects of MAP of 65 or 85 mmHg on splanchnic oxygen fl ux, metabolic function, mucosal perfusion and cytokine regulation. P185 Patient demographics aff ecting the apex causing apical ballooning. TCM is frequent in patients with adrenergic overstimulation and is probably common in ICU patients [1]. In a TCM rat model we evaluated whether diff erent anesthetic agents could attenuate LV akinesia in TCM. g Methods Isoprenaline was intraperitoneal (i.p.) injected, which induces LV akinesia and apical ballooning within 90 minutes [2]. We performed the study in two diff erent settings. In the fi rst setting, spontaneously breathing rats (n = 12 in each group) were sedated with either pentobarbital, ketamine, isofl urane or no anesthetic before i.p. isoprenaline. One additional group received the K-ATP blocker glyburide before sedation with isofl urane. In the second setting, rats were anaesthetized with ketamine + midazolam, mechanically ventilated and the carotid artery was cannulated. Before i.p. isoprenaline, animals were randomized to either no isofl urane (0 MAC), isofl urane 0.5 MAC or isofl urane 1.0 MAC (n = 12 in each group). Arterial blood gas was obtained before isoprenaline and 60 minutes after isoprenaline. The heart rate (HR), systolic blood pressure (SBP) and body temperature (BT) were recorded continuously. After 90 minutes, echocardiography was performed. Extent of akinesia was expressed as the percentage of total LV endocardial length. End-diastolic and end-systolic LV volumes were measured, and stroke volume (SV) and cardiac output (CO) were calculated. Conclusion Administering morphine prior to extubation causes signifi cant delays in weaning from mechanical ventilation. We plan to introduce intraoperative and postoperative protocols to facilitate rapid weaning from mechanical ventilation for elective cardiac surgical patients. Results In spontaneously breathing rats, the degree of akinesia was signifi cantly lower with pentobarbital and isofl urane (± glyburide) but not with ketamine compared with controls. The degree of akinesia was lowest with isofl urane. In ventilated rats, the degree of apical akinesia (%) was signifi cantly lower at 0.5 MAC (8.7 ± 7.3) and 1 MAC (5.7 ± 7.4) versus 0 MAC (17.7 ± 8.0). This was accompanied by a higher CO and SV. HR was lower at 1 MAC (6%) and SBP was lower at 0.5 MAC (106 ± 7) and 1 MAC (98 ± 7) versus 0 MAC (126 ± 8). BT and pH was lower in both isofl urane groups. In a multivariate model, isofl urane was the only variable that was independently associated with the degree of LV akinesia. P188 Preoperative therapy with angiotensin-converting enzyme inhibitors in cardiac surgery patients: is there any impact on postoperative renal function? P185 Reference 1. Abdelahdi et al.: Am J Cardiol 2004, 93:1176-1178. Reference 1. Abdelahdi et al.: Am J Cardiol 2004, 93:1176-1178. , , , , g Glenfi eld Hospital, University Hospitals of Leicester, UK Glenfi eld Hospital, University Hospitals of Leicester, UK Critical Care 2014, 18(Suppl 1):P185 (doi: 10.1186/cc13375) i p , y p , Critical Care 2014, 18(Suppl 1):P185 (doi: 10.1186/cc13375) Figure 1 (abstract P183). WBC at baseline (t = 0) and in the four postoperative days (t = 1 to 4), PNAF versus no PNAF. Introduction Early extubation post coronary artery bypass grafting does not increase perioperative morbidity and reduces the length of stay (LOS) in the ICU and in hospital [1]. Use of low-dose opioid- based general anaesthesia and time-directed protocols for fast- track interventions does not increase mortality or postoperative complications in low–moderate-risk patients and has been found to have a reduced time to extubation and shortened ICU stay [2]. Our mean time to extubation is 6 hours, although patients are assessed to be safe to be weaned from mechanical ventilation at 2 hours following arrival in the ICU. This study aims to identify factors that delay extubation in patients undergoing routine cardiac surgery at our institution. g g g y Methods A prospective analysis was performed on all patients post adult cardiac surgery from 14 May 2013 to 10 July 2013. Emergency surgical patients and those with intraoperative complications were excluded. Results A two-sample t test was used to analyse the data. Patient demographics are presented in Table 1. There were signifi cant delays in time of extubation in those who received morphine prior to extubation compared with those that did not (P = 0.0184) (Table 2). There were no Figure 1 (abstract P183). WBC at baseline (t = 0) and in the four postoperative days (t = 1 to 4), PNAF versus no PNAF. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S67 signifi cant diff erences in LOS in ICU or hospital. Factors such as age, EUROSCORE and type of operation did not have an infl uence on time Table 1 (abstract P185). Patient demographics Age (years) 66 (10.1) EUROSCORE (%) 2.90 (1.84) Table 2 (abstract P185). Morphine versus no morphine Morphine No morphine Patient number 51 20 Time to extubation (hours) 7:48 (4:42) 4:48 (2:19) LOS ICU (hours) 51:21 (55:16) 40:06 (28:47) LOS hospital (days) 9.83 (5.01) 10.8 (13.5) Table 1 (abstract P185). References References 1. Park JH, et al.: Chest 2005, 128:296-302. 2. ShaoY, et al.: Int J Cardiol 2013, 168:1943-1950. Methods A single-center, randomized controlled, double cross-over trial was performed. Patients were randomized to: HLH (high–low– high) where MAP targets were 85–65–85 mmHg in sequence, with each lasting 2 hours, or LHL (low–high–low) where MAP targets were 65– 85–65 mmHg. Blood pressure was adjusted with noradrenalin infusion. Results Six + six patients were included in the study. MAP targets were achieved in all patients at all time points (64 ± 3, 84 ± 4; 65 ± 5 mmHg in the LHL group and 84 ± 3; 66 ± 2; 85 ± 5 mmHg in the HLH group at the fi rst, second and third time points), with corresponding changes in fi lling pressures. Cardiac output did not change over time. Hepatic venous saturation was 41 ± 15; 58 ± 24; 56 ± 21% in the LHL group and 50 ± 19; 43 ± 20; 41 ± 18% in the HLH group at the fi rst, second and third time points, with a signifi cant time group interaction (P <0.05). No changes were observed in global or trans-splanchnic lactate levels and cytokine levels or in gastric tonometry CO2.f Preoperative therapy with angiotensin-converting enzyme inhibitors in cardiac surgery patients: is there any impact on postoperative renal function? Preoperative therapy with angiotensin-converting enzyme inhibitors in cardiac surgery patients: is there any impact on postoperative renal function? p p F Ampatzidou, M Sileli, K Diplaris, C Koutsogiannidis, T Karaiskos, G Drossos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P188 (doi: 10.1186/cc13378) Introduction Preoperative therapy with angiotensin-converting enzyme inhibitors (ACEI) is common in patients undergoing cardiac surgery. The aim of this study was to evaluate the still-debated impact of preoperative use of ACEI on postoperative renal function in cardiac surgery patients [1,2]. y g y 2 Conclusion Increasing MAP with norepinephrine has some eff ects splanchnic oxygenation, but has no impact on metabolic or biochemical function and key cytokine removal or release. MAP targets of 60 to 65 mmHg or 80 to 85 mmHg appear physiologically equivalent for the splanchnic circulation. y Methods A total of 624 consecutive patients, who underwent cardiac surgery from July 2012 to October 2013, were evaluated. Data were prospectively collected in our clinic’s electronic database and were retrospectively analyzed as to preoperative ACEI therapy. The chi- square test was used for correlations. Endpoints of the study were the development of postoperative acute kidney injury (AKI) and the diff erence between hospital admission and discharge glomerular fi ltration rate (GFR). The AKI defi nition was based on modifi ed RIFLE classifi cation. GFR values were estimated by the MDRD formula. p References 1. Cheng DC, et al.: J Thorac Cardiovasc Surg 1996, 112:755-764. 2. Zhu F, et al.: Cochrane Database 2012, 10:CD003587. 1. Cheng DC, et al.: J Thorac Cardiovasc Surg 1996, 112:755-764. 2 Zhu F et al : Cochrane Database 2012 10:CD003587 Tissue-aggressive infl ammatory response defi nes the tissue aggressiveness of the post-infarction milieu y Methods To conduct the study we analyzed the database of a CICU of a medium-sized hospital in the city of Presidente Prudente, Brazil. Admissions that occurred during the period 1 September 2010 to 31 August 2013 were analyzed. The information was collected from the EPIMED MONITOR system and statistically analyzed using EPI INFO, version 3.5.2 software. P <0.05 two-tailed was considered signifi cant, and confi dence intervals at 95% (95% CI) were used for the logistic regression multivariate estimated in the sample. Introduction A potential measure of post-ischemic milieu in myocardium subject to acute ischemic injury is to assess the so- called myocardial salvage index (SI) by relating the fi nal infarct size to the initial myocardium at risk (MaR). Cardiac magnetic resonance (CMR) has previously been shown to enable determination of both infarct size using late gadolinium enhancement and MaR using T2- weighted imaging. This technique could thus potentially be used to identify infl ammatory responses that could be targeted to accomplish cardioprotection. The aim of this study was to relate SI, as determined by CMR, to the infl ammatory response in patients with acute myocardial infarction.i g Results A total of 2,098 admissions were recorded, of which 42.1% were female and 57.9% male. The average age was 66.99 ± 13.30 years. The prognosis for SAPS 3 admission score averaged 40.8 ± 15.48 points and the mean unit length of stay was 3.5 ± 5.01 days. The main admission diagnoses were unstable angina (13.06%), non-ST-segment elevation myocardial infarction (8.29%), ST-segment elevation myocardial infarction (8.10%) and supraventricular cardiac tachyarrhythmia (6.91%). We observed a higher risk of death among patients who had, at admission, congestive heart failure NYHA 2, 3 or 4 (odds ratio (OR) = 2.81, 95% CI: 2.07 to 3.83, P = 0.001), chronic renal failure (OR = 2.48, 95% CI: 1.68 to 3.67, P = 0.001), peripheral artery disease (OR = 1.93, 95% CI: 1.04 to 3.58, P = 0.033), severe chronic obstructive pulmonary disease (OR = 3.17, 95% CI: 1.82 to 5.51, P = 0.002), and infection at admission (OR = 7.88, 95% CI: 5.27 to 11.78, P = 0.001). Methods Fifteen patients with fi rst-time ST-elevation myocardial infarction were included in the study. All patients underwent primary PCI due to an acute occlusion in one branch of the left coronary artery. Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) p J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) p J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) p J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) i Results A total of 354 patients (56.7%) were treated with ACEI preoperatively. Overall, 95 patients (15.3%) developed postoperative AKI. Preoperative use of ACEI was not associated with the development Introduction Tako-tsubo cardiomyopathy (TCM) is an acute cardiac syndrome with regional hypokinesia in the left ventricle (LV), often Introduction Tako-tsubo cardiomyopathy (TCM) is an acute cardiac syndrome with regional hypokinesia in the left ventricle (LV), often Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S68 of postoperative AKI (P = 0.981). Mean GFR values on admission day were 65.23 ± 16.89 for ACEI users, and 65.06 ± 19.62 for the rest of the cohort. Mean GFR values on discharge day were 66.77 ± 21.25 and 67.35 ± 25.64 respectively. The diff erence in GFR at the time of hospital admission and on discharge day had no statistical diff erence, P = 0.511 (Table 1). Overall, 25 patients (4%) needed dialysis. Conclusion The impact of preoperative ACEI treatment on postoperative renal function after cardiac surgery is still debated. We found no association, neither protective nor harmful, between preoperative ACEI therapy and renal function impairment. References 1. Arora P, et al.: Clin J Am Soc Nephrol 2008, 3:1266-1273. 2. Ouzounian M, et al.: Ann Thorac Surg 2012, 93:559-564. Table 1 (abstract P188). 2. Polanczyk CA, et al.: Fatores de Risco Cardiovascular no Brasil: os próximos 50 Anos. Arq Bras Cardiol 2005, 84:199-201. 1. Roger VL: Heart disease and stroke statistics – 2013 update. Circulation 2013, 127:e6-e245. Hospital visit pattern and its eff ect on reperfusion time and clinical outcomes in ST-segment elevation acute myocardial infarction Introduction The reperfusion time is critical in ST-segment elevation myocardial infarction (STEMI), and it makes a diff erence to clinical outcomes. This study was designed to investigate the hospital visit pattern and its eff ect on reperfusion time and clinical outcomes of STEMI patients. Methods A total of 199 STEMI patients were registered in this study from three university hospitals in Kyungsang-do area, Korea, and were divided into two groups; group 1 (n = 69) who directly visited the hospitals capable of percutaneous coronary intervention (PCI), and group 2 (n = 130) who fi rst visited local hospitals and then transferred to PCI-capable hospitals. We analyzed the estimated distance and time to the hospitals using a driving navigation system, elapsed time from chest pain to primary PCI hospitals, chest pain to reperfusion time, and in-hospital outcomes. 1. Arora P, et al.: Clin J Am Soc Nephrol 2008, 3:1266-1273. 2. Ouzounian M, et al.: Ann Thorac Surg 2012, 93:559-564. Results There was no diff erence in fi rst medical contact time between groups 1 and 2. But the time from chest pain to PCI hospital was shorter in group 1 (206.2 ± 268.5 vs. 370.3 ± 415.2 minutes, P = 0.001). Sixty patients in group 1 and 108 patients in group 2 underwent reperfusion therapy (P = 0.473). The chest pain to reperfusion time was shorter in group 1 (294.6 ± 255 vs. 397.2 ± 341.9 minutes, P = 0.045). The diff erence in estimated time by navigator and actual hospital visit time was also shorter in group 1 (194.2 ± 269.9 vs. 321.6 ± 411.0 minutes, P = 0.009). In-hospital mortality was higher in group 2 (0 vs. 4.6%, P = 0.094). Characterization of the profi le and clinical variables associated with mortality in a Brazilian coronary ICU Characterization of the profi le and clinical variables associated with mortality in a Brazilian coronary ICU CE Bosso1, VF Paula1, OA Souza2, GE Valerio2, SV Ferreira2 1Instituto do Coração de Presidente Prudente, Brazil; 2UNOESTE – Universidade do Oeste Paulista, Presidente Prudente, Brazil Critical Care 2014, 18(Suppl 1):P189 (doi: 10.1186/cc13379) y Conclusion A primary visit to a local hospital was associated with longer reperfusion time and was associated with higher mortality. Therefore, the reperfusion time could be reduced by patient education and management of the community healthcare system. Introduction Cardiovascular disease is the leading cause of death worldwide and the outlook for 2020 data is even more alarming [1,2]. In this context, the coronary ICUs (CICUs) have become increasingly numerous. However, data concerning this are still very limited in the literature. This study aims to characterize the profi le of CICU admissions in Brazil and the main clinical variables associated with increased mortality. P191 P191 Tissue-aggressive infl ammatory response defi nes the tissue aggressiveness of the post-infarction milieu E Grins, L Algotsson, H Engblom, M Carlsson, H Arheden, S Jovinge Scania University Hospital, Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P191 (doi: 10.1186/cc13381) Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model AKI and GFR diff erence ACEI users (n = 354) ACEI nonusers (n = 270) P value AKI 54 (15.3%) 41 (15.2%) 0.981 GFR diff erence (mean) +1.43 +2.3 0.511 Table 1 (abstract P188). AKI and GFR diff erence ACEI users (n = 354) ACEI nonusers (n = 270) P value AKI 54 (15.3%) 41 (15.2%) 0.981 GFR diff erence (mean) +1.43 +2.3 0.511 Table 1 (abstract P188). AKI and GFR diff erence ACEI users (n = 354) ACEI nonusers (n = 270) P value AKI 54 (15.3%) 41 (15.2%) 0.981 GFR diff erence (mean) +1.43 +2.3 0.511 Table 1 (abstract P188). AKI and GFR diff erence ACEI users (n = 354) ACEI nonusers (n = 270) P value Rhabdomyolysis following cardiac surgery: from prevalence to prevention y g y Methods TTE was performed before anesthesia induction and upon ICU arrival in 15 patients with preoperative diagnosis of LV dysfunction defi ned as an EF <35%. In-ICU measurements of Ees, Ea and VAc were taken at diff erent steps of PEEP application lasting 3 minutes as follows: 0 cmH2O PEEP (ZEEP), 5 cmH2O PEEP, 10 cmH2O PEEP and 15 cmH2O PEEP. TTE and hemodynamic parameters were recorded and analyzed. Results All patients were uncoupled preoperatively (VAc >1.31, average VAc (AVAc) = 1.56) before anesthesia induction and showed worsened uncoupling postoperatively at ZEEP. PEEP application altered VAc by modifying both Ees and Ea at all steps and all patients showed further uncoupling at any level of PEEP application (AVAc at ZEEP = 1.97, at 5 cmH2O PEEP = 1.61, at 10 cmH2O PEEP = 1.87, at 15 cmH2O PEEP = 2.23) A PEEP of 5 cmH2O provided the more favorable VAc. p AS Omar1,2, HA Ewila1,3, SR Aboulnaga1,4, AK Tuli1 1Hamad Medical Corporation, Doha, Qatar; 2Beni Suef University, Egypt; 3Suez Canal University, Egypt; 4Ain Shams University, Egypt Critical Care 2014, 18(Suppl 1):P194 (doi: 10.1186/cc13384) p AS Omar1,2, HA Ewila1,3, SR Aboulnaga1,4, AK Tuli1 AS Omar1,2, HA Ewila1,3, SR Aboulnaga1,4, AK Tuli1 1Hamad Medical Corporation, Doha, Qatar; 2Beni Suef University, Egypt; 3Suez Canal University, Egypt; 4Ain Shams University, Egypt Critical Care 2014, 18(Suppl 1):P194 (doi: 10.1186/cc13384) Introduction In the view of robust consequences following cardiac surgery, acute kidney injury (AKI) remains a major concern. Rhabdomyolysis (RML) following cardiac surgery and its relation to AKI need to be investigated. We aim to study the prevalence of RML development following cardiac surgery and the perioperative risk factors that may expedite the occurrence of RML. 2 p Conclusion Sb evaluation of Ea/Ees shows that following CABG the patients with depressed LV remain uncoupled, and that in such patients the application of PEEP leads to further decoupling. In this preliminary experience, 5 cmH2O PEEP seems to be the most appropriate in preventing further worsening of the VAc. A large RCT is needed to draw conclusions on the PEEP eff ect on VAc following CABG in a depressed heart. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P = 0.5742) response. However, the proportion of TH1 (CD4+ IFNγ+) cells was higher in the group with the lowest SI (57.47 ± 4.805 vs. 38.10 ± 6.514, P = 0.0349). P = 0.5742) response. However, the proportion of TH1 (CD4+ IFNγ+) cells was higher in the group with the lowest SI (57.47 ± 4.805 vs. 38.10 ± 6.514, P = 0.0349). predictive of mortality. The recently introduced high-sensitivity cardiac troponin T (HS cTnT) assay has resulted in an increased detection of elevated cTnT in ICU patients [1]. The aim of this study was to determine the prevalence of elevated cTnT using the HS assay and its relationship with mortality. Conclusion This is the fi rst study that identifi es infl ammatory cell patterns which infl uence the hostility of the infarction milieu in vivo. The study indicates that SI can be used as an evaluator of post- ischemic milieu and that TH1 response is associated with unfavorable post-infarction injury and could therefore be a possible target for future studies to limit infarction size. The current study is, by its size and observational character, important; an argument for rather than a substitute for future interventional studies in this area. y Methods A retrospective observational study was performed on all ICU admissions over a 12-month period. Data were obtained from the clinical information system (ICIP; Philips) and the ICU audit databases (AcuBase). Data collected included patient demographics, peak cTnT value, APACHE II score, requirement for organ support and mortality. The primary outcome measure was hospital mortality. Data were analysed using SPSS v.17.0. cTnT levels were divided into categories for analysis: normal (<14 ng/l) and elevated. The elevated category was further subdivided into quartiles. Univariate analysis was performed between potential risk factors and mortality followed by multivariate regression analysis to ascertain independent predictors of mortality. Results There were 417 admissions to the ICU during the study period, 89 of whom were excluded because of an absent cTnT value, leaving 328 patients included in the analysis. cTnT was elevated in 85% of patients. ICU mortality was 19% and hospital mortality was 28%. Hospital mortality (%) per cTnT category was: <14 ng/l = 2%; 14 to 38 ng/l = 19%; 39 to 90 ng/l = 26%; 91 to 252 ng/l = 39%; >252 ng/l = 43%. On univariate analysis, cTnT levels, age, ventilation and APACHE II score were signifi cantly associated with mortality. 1. Hajjar L, Grande S, Galas F, Roquim A, Sampaio L, Auler J: Risk factors and outcome of rhabdomyolysis after cardiac surgery. Crit Care 2008, 12(Suppl 2):P470. Rhabdomyolysis following cardiac surgery: from prevalence to prevention Methods All patients undergoing cardiac surgeries in our hospital were enrolled in the study during the period of 1 year in a prospective descriptive study measuring the occurrence of RML and its association with AKI, where all patients in the study underwent serial assessment of serum creatinine kinase (CK) and serum myoglobin. Serial renal function, prior statin treatment, cardiac injury, lengths of ventilation, and lengths of stay in the ICU and hospital were monitored. References Results We recruited 202 patients in our study, 185 males and 17 females with mean age 52 ± 12.4 years. According to the existence of RML (CK 2,500 U/ml or more) [1], patients were divided into group 1 where RML was identifi ed in 17 patients (8.4%), which was associated with AKI in seven patients (41%), and group 2 without RML (185 patients), where AKI occurred in 34 patients (18.4%) (P = 0.025). We observed a signifi cantly longer duration of ventilation and lengths of stay in the ICU and in hospital in the RML group (P <0.01 for all observations). 1. Guarracino F, et al.: Crit Care 2013, 17:213. 2. Chen CH, et al.: JACC 2001, 38:2028-2034. 3. Franchi F, et al.: Biomed Res Int 2013, 2013:918548. P193 Prevalence of elevated cardiac troponin T in ICU patients using the high-sensitivity assay and the relationship with mortality S O’Sullivan1, M Sutton2, G Fitzpatrick1 1Tallaght Hospital & Trinity College Dublin, Ireland; 2Health Research Board, Dublin, Ireland Critical Care 2014, 18(Suppl 1):P193 (doi: 10.1186/cc13383) Prevalence of elevated cardiac troponin T in ICU patients using the high-sensitivity assay and the relationship with mortality p g p Conclusion Early increase in the serum CK and myoglobin in postoperative high-risk cardiac surgeries may predict the concomitance of early AKI, where proper intervention may prevent the sequelae of logistic organ dysfunction. 1Tallaght Hospital & Trinity College Dublin, Ireland; 2Health Research Board, Dublin, Ireland Critical Care 2014, 18(Suppl 1):P193 (doi: 10.1186/cc13383) Tissue-aggressive infl ammatory response defi nes the tissue aggressiveness of the post-infarction milieu Final infarct size and MaR was determined by CMR performed 1 week after the acute event. The ischemic time was defi ned as the time from pain onset to opening of the occluded vessel. Blood samples were taken for assessment of infl ammatory response. Infl ammatory cells were analyzed by fl ow cytometry in a BD FACS Aria. Parameters were gated against control antibody and fl uorescence minus one strategy. Cytokine patterns were analyzed by BioRad BioPlex multiplex protein analysis technology. Conclusion The results reported may direct healthcare professionals to profi le the patient hospitalized in a CICU, paying attention to the most disturbing and lethal comorbidities present on admission. References Conclusion The results reported may direct healthcare professionals to profi le the patient hospitalized in a CICU, paying attention to the most disturbing and lethal comorbidities present on admission. References Results The SI did not correlate with MaR (P = 0.2720, R2 = 0.09191). The population was divided into the lowest half of the SI (representing the most hostile milieu; SI: 23 to 57%) and the upper half of the SI (representing the friendliest post-infarction milieu; SI: 71 to 95%). The patients’ profi le of adaptive infl ammatory response was characterized by fl ow cytometry. The two groups did not diff er with regard to their T-regulatory response (CD25+FoxP3+, P = 0.7203) or NK-cell (CD3–CD56+, 1. Roger VL: Heart disease and stroke statistics – 2013 update. Circulation 2013, 127:e6-e245. 2. Polanczyk CA, et al.: Fatores de Risco Cardiovascular no Brasil: os próximos 50 Anos. Arq Bras Cardiol 2005, 84:199-201. S69 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 cTnT levels were signifi cant in multivariate regression independent of age and ventilation but did not reach signifi cance (P = 0.06) in a multivariate analysis that included the APACHE II score. y Methods A retrospective observational study was performed on all ICU admissions over a 12-month period. Data were obtained from the clinical information system (ICIP; Philips) and the ICU audit databases (AcuBase). Data collected included patient demographics, peak cTnT value, APACHE II score, requirement for organ support and mortality. The primary outcome measure was hospital mortality. Data were analysed using SPSS v.17.0. cTnT levels were divided into categories for analysis: normal (<14 ng/l) and elevated. The elevated category was further subdivided into quartiles. Univariate analysis was performed between potential risk factors and mortality followed by multivariate regression analysis to ascertain independent predictors of mortality. Impact of positive end-expiratory pressure application on ventriculo-arterial coupling in decompensated left ventricles after cardiac surgery: a non-invasive echocardiographic study P Bertini, V Simone, R Baldassarri, L Doroni, F Guarracino University Hospital, Pisa, Italy Critical Care 2014, 18(Suppl 1):P192 (doi: 10.1186/cc13382) Impact of positive end-expiratory pressure application on ventriculo-arterial coupling in decompensated left ventricles after cardiac surgery: a non-invasive echocardiographic study P Bertini, V Simone, R Baldassarri, L Doroni, F Guarracino University Hospital, Pisa, Italy Critical Care 2014, 18(Suppl 1):P192 (doi: 10.1186/cc13382) g y y Results There were 417 admissions to the ICU during the study period, 89 of whom were excluded because of an absent cTnT value, leaving 328 patients included in the analysis. cTnT was elevated in 85% of patients. ICU mortality was 19% and hospital mortality was 28%. Hospital mortality (%) per cTnT category was: <14 ng/l = 2%; 14 to 38 ng/l = 19%; 39 to 90 ng/l = 26%; 91 to 252 ng/l = 39%; >252 ng/l = 43%. On univariate analysis, cTnT levels, age, ventilation and APACHE II score were signifi cantly associated with mortality. cTnT levels were signifi cant in multivariate regression independent of age and ventilation but did not reach signifi cance (P = 0.06) in a multivariate analysis that included the APACHE II score. Introduction The management of ICU patients following heart surgery can be hustling when coping with severe left ventricular (LV) dysfunction. Single beat (Sb) measurements of ventriculo- arterial coupling (VAc) can be used by the intensivist when dealing with altered hemodynamic states [1]. LV elastance (Ees) and arterial elastance (Ea) can be measured by trasthoracic echocardiography (TTE) in a Sb fashion [2], so allowing quick assessment of VAc. PEEP application is common practice in the ICU but can have hemodynamic consequences and lead to instability. Speckle tracking analysis by TTE has been recently reported to help titrating PEEP in critically ill patients [3]. However, this can be demanding and require specifi c ultrasound tools. In this study we aimed to assess whether standard TTE can be useful in evaluating the eff ect of respiratory treatment with PEEP on cardiovascular effi ciency by measuring VAc after coronary artery bypass surgery (CABG). Conclusion In 85% of general ICU patients, troponin measured by HS cTnT assay was elevated. cTnT levels were signifi cantly associated with mortality and are predictive of mortality independent of age and mechanical ventilation, but not independently of APACHE II score. There was a high correlation between troponin levels and APACHE II scores. Reference 1. Abubaker et al.: Intensive Care Med 2013, 39(Suppl 2):0531. P194 Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review Methods We searched the ICNARC database from 2006 to 2013 for patients aged 80 years or over, admitted from theatre or after surgery. Data were referenced against the electronic theatre management system, and patients not undergoing abdominal cavity surgery were excluded. The data were analysed using an Excel spreadsheet (Microsoft) and Medcalc software. Results Eighty-fi ve patients were included, with ages ranging from 80 to 99 years. Fifty-one (60%) patients were male and 79 (93%) patients were categorized as having an emergency operation. ICU mortality was 34/85 (40%) and hospital mortality was 48/85 (56%). Variables assessed for association with hospital mortality can be seen in Table 1. Only invasive ventilation and time (days) from hospital admission to ICU admission were signifi cant predictors of hospital mortality. Table 1 (abstract P195). Survivors versus nonsurvivors Survivors Nonsurvivors P value Table 1 (abstract P195). Survivors versus nonsurvivors Table 1 (abstract P195). Survivors versus nonsurvivors Survivors Nonsurvivors P value Age 83 83 0.82 Time 1, 1 to 8 4.5, 1 to 8 0.004 APACHE II 17, 14 to 20 17, 14 to 21 0.96 Sex (%) M57, F43 M63, F37 0.65 Ventilation (n, %) 31, 83 48, 100 0.005 RRT (n, %) 6, 16 11, 23 0.58 Data presented as median, IQR or n, %. Table 1 (abstract P195). Survivors versus nonsurvivors Table 1 (abstract P195). Survivors versus nonsurvivors Conclusion The development of ARI in SAP was associated with hemodynamic instability, whereas excessive volume expansion does not prevent ARI. Severe acute pancreatitis in ICU: a 5-year audit y R Durrani, O Murphy, A Kibeida, G Fitzpatrick Introduction Severe acute pancreatitis (SAP) is associated with signifi cant mortality and morbidity. The objective of this study is to examine the profi le, outcome and resource utilization for patients with SAP admitted to the ICU in a university teaching hospital over a 5-year period. Conclusion ICU and hospital mortality rates were high at 40% and 56% respectively. Increasing age did not correlate with mortality. However, invasive ventilation and time between hospital and ICU admission were associated with a higher mortality. This may be due to a delay in diagnosis or surgical intervention. We suggest that one considers early intervention in patients aged over 80. A functional outcome measure at discharge may be a more clinically relevant endpoint. Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review p Results Of 145 patients, 24 patients who developed ARI at any time during hospitalization (ARI group) were contrasted with 24 patients without ARI (control group). The patients were older in the ARI group: age 57 ± 13 versus 49 ± 16, P = 0.046. Although none of the patients in the ARI group had creatinine values ≥1.5-fold from the estimated baseline, the creatinine values on admission were higher in this group: 100 ± 38 versus 68 ± 16, P = 0.001. The severity of pancreatitis was similar. On admission, the APACHE II and SOFA scores were higher in the ARI group (12.7 ± 3.7 vs. 8.6 ± 3.4, P = 0.001 and 5.6 ± 3.4 vs. 2.8 ± 1.9, P = 0.002, respectively). The patients in the ARI group had higher intra- abdominal pressure and SOFA respiratory score on admission and after 72 hours (P <0.01). Although on admission the cardiovascular SOFA score was similar in both groups, it increased signifi cantly after 72 hours in the ARI group from 1.1 ± 1.7 to 2.1 ± 1.8, P = 0.012 and became higher compared with the control group: 2.1 ± 1.8 versus 0.6 ± 1.2, P = 0.001. Lactate was higher in the ARI group on admission and after 72 hours (P <0.05). The percentage of patients requiring vasopressors over 72 hours was greater in the ARI group: 66.7% versus 29.2%, P = 0.01. Positive fl uid balance after 72 hours in ICU was higher in the ARI group: 8,594 ± 7,044 ml versus 4,192 ± 4,467 ml, P = 0.004; however, the infused volume of crystalloids did not diff er between groups. Central venous pressure was higher on admission (P = 0.036) and after 72 hours (P = 0.001) in the ARI group. Multivariable logistic regression analysis revealed that development of ARI was independently associated with cardiovascular SOFA score after 72 hours: odds ratio = 1.475, 95% CI 1.049 to 2.073, P = 0.025. Royal Liverpool University Hospital, Liverpool, UK Critical Care 2014, 18(Suppl 1):P195 (doi: 10.1186/cc13385) Introduction This review was undertaken to establish the ICU and hospital mortality rates in patients aged 80 and over admitted to our ICU following abdominal cavity surgery. Intensive care mortality increases progressively with age [1] and as the population changes we are likely to see increasing numbers of patients over the age of 80 admitted to critical care units. Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review p Methods A retrospective observational study was carried out of all patients admitted to the ICU from 1 January 2008 to 31 December 2012 with SAP. Data were collected from the ICU database (AcuBase), the medical records and the ICU clinical information system. Data collected included patient demographics, etiology of SAP, data for APACHE II, Imrie, Ranson and Acute Kidney Injury Network (AKIN) scores, and requirement for organ support. Outcomes recorded were length of stay, ICU mortality and hospital mortality. Cost of ICU care was calculated based on previously reported methodology.i 1. Paul E, et al.: Management of the critically ill geriatric patient. Crit Care Med 2006, 34:S176-S182. p y p gy Results Thirty-eight eligible patients were identifi ed. Mean age was 51.4 years (range 24 to 86), 68% were male. The commonest etiologies were alcohol (53%) and gallstone pancreatitis (24%). The mean APACHE II score was 18.5 (IQR 14 to 23). Twenty-eight patients (74%) required mechanical ventilation, three of whom required high-frequency oscillation (all three survived). Twenty-two patients (58%) had evidence of an acute kidney injury on admission (AKIN criteria). Eighteen (47%) required renal replacement therapy and 60% required inotropes. The ICU mortality and the hospital mortality were 26%. There was no signifi cant diff erence in age, APACHE II, Imrie, or Ranson scores between survivors and nonsurvivors. The median length of stay in the ICU was 11 days (IQR 5.25 to 28.5) and the median hospital stay was 45.4 days (IQR 22.25 to 104.5). Nine patients (24%) required multiple ICU admissions and the mortality was signifi cantly higher in this group (P <0.05, chi-square test). In total, 834 ICU bed-days were taken up by 38 patients. Based on a median cost for an ICU bed-day of €2,205 [1], the total cost of ICU care for these patients is estimated at €1,838,970 or almost €50,000 per patient. g g Reference 1. Hajjar L, Grande S, Galas F, Roquim A, Sampaio L, Auler J: Risk factors and outcome of rhabdomyolysis after cardiac surgery. Crit Care 2008, 12(Suppl 2):P470. Introduction Elevated cardiac troponin levels are common in ICU patients even in the absence of acute coronary syndromes and may be Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S70 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P195 was composed of randomly selected patients with SAP who did not develop ARI. 95 Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review A Roberts, P Hampshire Royal Liverpool University Hospital, Liverpool, UK Critical Care 2014, 18(Suppl 1):P195 (doi: 10.1186/cc13385) Bacterial infection in severe acute pancreatitis patients admitted to the ICU Results Of the 803 APAP-ALF patients, the median age was 37 (29 to 47) years and 76% were female. A total of 238 (30%) patients were listed for and 87 (11%) received LT. A total of 531 (66%) patients recovered without LT at 21 days. Using standard logistic regression methods for all patients with complete data (n = 679), KCC (INR, creatinine, coma grade 3/4, pH) yielded an AC of 69% (Sn 90%, Sp 27%) at admission. For late-stage data (n = 341), KCC provided similar AC (70%) and Sn (97%), but suff ered poor Sp (15%). CART analysis using the KCC variables on admission off ered predictive AC (66%) and Sn (65%), with increased Sp (67%). Using day 3 to 7 data, the KCC-CART model had increased AC (82%) and Sn (86%), with improved Sp (46%) compared with logistic KCC. New CART models were developed with 18 variables based on previous literature. A new CART model on admission (MELD, lactate and MV: test AC 72%, Sn 71%, Sp 77%) performed better than KCC-CART. For days 3 to 7, a CART model (MELD, lactate, coma grade) off ered superior prediction (test AC 86%, Sn 91%, Sp 46%) compared with days 3 to 7 KCC-CART. Introduction Controversy surrounds the empirical use of antibiotics in severe acute pancreatitis (SAP). There are concerns that the widespread use of antibiotic therapy in the absence of documented infection may lead to selection of drug-resistant organisms [1]. The aim of this study was to review the profi le of pancreatic fl uid isolates in patients with SAP admitted to the ICU. Methods Data were reviewed for 38 patients admitted to the ICU over a 5-year period. We evaluated organisms cultured from pancreatic specimens, as well as the prevalence of drug-resistant organisms in this group of patients. Results Aspirate of pancreatic material for culture was obtained in 55% of patients (n = 21). The mean time to acquisition of samples for culture from admission to ICU was 15.5 days. Fluid was sterile in 67% (n = 14) of initial samples. Gram-positive organisms were cultured from 43% (n = 9) of samples, Gram-negative organisms from 5% (n = 1) and yeasts from 5% (n = 1). Antibiotic therapy was administered in 95% of patients prior to samples being obtained for culture. P200 P200 A multicenter retrospective cohort analysis of therapeutic hypothermia in acute liver failure C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA Critical Care 2014, 18(Suppl 1):P200 (doi: 10.1186/cc13390) A multicenter retrospective cohort analysis of therapeutic hypothermia in acute liver failure yp C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA y Conclusion In the majority of patients, initial aspirates of pancreatic material were sterile. This may be a result of prior antibiotic usage. Where organisms were cultured from initial aspirates, Gram-positive organisms predominated, possibly as a result of prior anti-Gram- negative antibiotic use. Therefore, in patients with ongoing sepsis who are receiving broad-spectrum antibiotic therapy, consideration needs to be given to the empiric treatment of Gram-positive infection, and in particular drug-resistant organisms such as VRE. Local epidemiology should be taken into account. Rationale use of antibiotics, in accordance with best-practice guidelines, may limit development of drug resistance; however, other risk factors for resistance may exist in this group and this would need to be further evaluated. Reference y Critical Care 2014, 18(Suppl 1):P200 (doi: 10.1186/cc13390) Introduction Cerebral edema is a severe and life-threatening complication in acute liver failure (ALF). Concerns exist that therapeutic hypothermia (TH) may increase the risk of infection, worsen coagulopathy and inhibit hepatic regeneration. We therefore reviewed the experience in use of TH in participating US Acute Liver Failure Study Group (ALFSG) centers. The aims were to determine utilization of TH in ALF patients at high risk for cerebral edema (grade III or IV hepatic encephalopathy (HE)) and to determine its eff ect on survival and complication rates. Introduction Cerebral edema is a severe and life-threatening complication in acute liver failure (ALF). Concerns exist that therapeutic hypothermia (TH) may increase the risk of infection, worsen coagulopathy and inhibit hepatic regeneration. We therefore reviewed the experience in use of TH in participating US Acute Liver Failure Study Group (ALFSG) centers. A multicenter retrospective cohort analysis of therapeutic hypothermia in acute liver failure The aims were to determine utilization of TH in ALF patients at high risk for cerebral edema (grade III or IV hepatic encephalopathy (HE)) and to determine its eff ect on survival and complication rates. 1. Tenner S, Baillie J, DeWitt J, Vege SS; American College of Gastroenterology: Am J Gastroenterol 2013, 108:1400-1415. 1. Tenner S, Baillie J, DeWitt J, Vege SS; American College of Gastroenterology: Am J Gastroenterol 2013, 108:1400-1415. Methods A retrospective cohort study of all ALF patients enrolled in the US ALFSG registry between January 1998 and September 2013 with grade III or IV HE. TH using an external cooling device was used in 97 (8%) patients while in 1,135 (92%) patients it was not (controls). Results TH ALF patients were younger (36 vs. 40, P = 0.03) and had acetaminophen etiology (63 vs. 47%, P = 0.04). Admission MELD (32 vs. 34) and lactate (5.4 vs. 5.0 mmol/l, P >0.2 for all) were similar. More TH ALF patients received renal replacement therapy (63 vs. 40%), vasopressors (61 vs. 43%) and ICP monitoring (40 vs. 22%, P <0.0002 for all). Overall (38% vs. 40%, P = 0.7) and 21-day transplant survival (45 vs. 49%, P = 0.5) were similar. There were no diff erences in bleeding (12% vs. 12%) or bloodstream infections (17 vs. 18%, P >0.7 for both). More TH ALF patients had arrhythmias (38 vs. 27%, P = 0.03). There were no diff erences in listing (43 vs. 40%, P = 0.5) or receipt of transplant (18 vs. 25%, P = 0.1). After controlling for MELD, requirement for organ support on multivariable analysis, hypothermia was not independently Methods A retrospective cohort study of all ALF patients enrolled in the US ALFSG registry between January 1998 and September 2013 with grade III or IV HE. TH using an external cooling device was used in 97 (8%) patients while in 1,135 (92%) patients it was not (controls). i References 1. Harrison et al.: Crit Care 2007, 11(Suppl 1):S1. 1. Harrison et al.: Crit Care 2007, 11(Suppl 1):S1. 2. Pavlidis et al.: Crit Care Res Pract 2013, 2013:897107. 2. Pavlidis et al.: Crit Care Res Pract 2013, 2013:897107. 96 Risk factors for acute renal impairment in patients with severe acute pancreatitis M Serpytis, N Scupakova, J Sabliauskas, A Sileikis, J Sipylaite, K Strupas Vilnius University, Vilnius, Lithuania Critical Care 2014, 18(Suppl 1):P196 (doi: 10.1186/cc13386) Introduction The aim of this study was to clarify the risk factors for acute renal impairment (ARI) in patients with severe acute pancreatitis (SAP). Methods We conducted a retrospective observational case–control study. The data of all patients admitted to the ICU at a university hospital with the diagnosis of SAP from January 2008 to December 2012 were abstracted from the hospital database. ARI was defi ned according to RIFLE criteria. Patients with any signs of renal impairment (serum creatinine value ≥1.5-fold from the estimated baseline) on admission or history of kidney disease were excluded. A control group S71 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion A hospital mortality rate of 26% is similar to that reported recently from a specialist unit in the UK [2] but less than the 42% reported in the UK in 2007 [1], suggesting some improvement in recent years. SAP is associated with prolonged ICU and hospital stay and signifi cant resource utilization. admission but not in later phases of illness. The aim was to improve determinations of prognosis on and after admission in APAP- ALF patients using the classifi cation and regression tree (CART) methodology to construct optimal binary splits on independent variables to predict outcome. Methods CART models were applied to US ALFSG registry data for prediction of 21-day spontaneous survival on admission and late stage (days 3 to 7). Analyses were carried out using R software (package rpart) for all (n = 803) APAP-ALF patients enrolled between January 1998 and September 2013 with complete outcome data. Training data were used to build CART trees and test data were used to evaluate prediction accuracy (AC), sensitivity (Sn) and specifi city (Sp). Bacterial infection in severe acute pancreatitis patients admitted to the ICU On review of all samples received from patients (including nonpancreatic specimens), vancomycin-resistant enterococci (VRE) were isolated in 13 patients. Linezolid-resistant enterococci (LRE) were isolated in six patients, fi ve of whom had VRE isolated prior to the culture of LRE. Extended- spectrum beta-lactamase organisms were isolated in two patients, and carbapenem nonsusceptible Gram-negative organisms in three patients. The mean APACHE II score was 18.5 and overall hospital mortality was 26%. Conclusion CART analysis increased predictive performance compared with traditional KCC. KCC-CART trees have higher Sp and similar predictive AC compared with traditional KCC, with newer CART trees providing marginal improvement over KCC-CART models. Acknowledgment Supported in part by a U-01 58369-014 from NIDDK to the US Acute Liver Failure Study Group. P200 P198 98 Bacterial infection in severe acute pancreatitis patients admitted to the ICU AR Prior, S Egan, R Durani, PG Murphy, J Febbell, G Fitzpatrick Tallaght Hospital, Dublin, Ireland Critical Care 2014, 18(Suppl 1):P198 (doi: 10.1186/cc13388) References 1. Choi NK, et al.: Hepatogastroenterology 2012, 59:1189-1193. 2. Monsel A, et al.: Crit Care 2011, 15:R234. 1. Choi NK, et al.: Hepatogastroenterology 2012, 59:1189-1193. 2. Monsel A, et al.: Crit Care 2011, 15:R234. pp p Critical Care 2014, 18(Suppl 1):P202 (doi: 10.1186/cc13392) g References 1. Shawcross DL, Austin MJ, Abeles RD, et al.: The impact of organ dysfunction in cirrhosis: survival at a cost? J Hepatol 2012, 56:1054-1062. 1. Shawcross DL, Austin MJ, Abeles RD, et al.: The impact of organ dysfunction in cirrhosis: survival at a cost? J Hepatol 2012, 56:1054-1062. 2. Alexopoulos S, Matsuoka L, Cho Y, et al.: Outcomes after liver transplantation in patients achieving a model for end-stage liver disease score of 40 or higher. Transplantation 2013, 95:507-512. 3. Oberkofl er CE, Dutkowski P, Stocker R, et al.: Model of end stage liver disease (MELD) score greater than 23 predicts length of stay in the ICU but not mortality in liver transplant recipients. Crit Care 2010, 14:R117. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 associated with 21-day spontaneous survival (P = 0.93) while MELD (P <0.0001) and vasopressors (P <0.02) were. Results Twenty patients (27.1%) developed 33 episodes of infection. Among them 35% suff ered from liver dysfunction, 40% took large doses of immunosuppression and 50% were re-operated. Five out of them showed signs of infection promptly after transplantation. Medium duration of mechanical ventilation was 16.5 ± 12.75 days (vs. 3.24 ± 5.5 of those without an infection) and medium length of stay was 22.14 ± 18.35 days (vs. 6.79 ± 8.86). The medium APACHE II score was 17.8 ± 5.5 (vs. 16.4 ± 4.37, P = 0.256) and medium SOFA was 11.32 ± 2.62 (vs. 9.9 ± 2.62, P = 0.054). The majority of patients was transfused with many blood units, FFP and cryoprecipitates and was hemodynamically unstable (56.3%). MELD score and MELD-Na were higher (22.57 and 27.5 relatively vs. 19.02 and 22.5). Eight cases had VAP from Gram- negative bacteria, 15 had bacteraemia (mainly Gram-negative, but also fungaemia), 10 had intraabdominal infection (most of them with two or three re-operations) and urine infection. The majority of pathogens were multidrug resistant. Regarding Klebsiella pneumoniae Carbapenemase, 60% was sensitive to colimycine, 20% to tobramycin, 70% to gentamycin and 40% to tygecycline. In four cases the donors had an infection that was transmitted to the recipient as much as 75%. Fourteen patients died (10 in ICU and four in the transplantation clinic) soon after with multiorgan failure. associated with 21-day spontaneous survival (P = 0.93) while MELD (P <0.0001) and vasopressors (P <0.02) were. p Conclusion TH was not associated with increased bleeding/infection rates nor diff erences in survival in ALF patients with high-grade HE. ICP monitoring was not universally used in TH ALF patients (~40%). There is a need for a prospective trial to clarify the use of TH in patients with ALF. Acknowledgement Supported in part by a U-01 58369-014 from NIDDK to the US ALFSG. Extracorporeal membrane oxygenation before and after adult liver transplantation: worth the eff ort? Results On the day of LT, 5% (28/519) of patients had a MELD score ≥40. These patients had longer fi rst ICU stay after LT (14 vs. 2 days; P <0.001). MELD score ≥40 at transplant was independently associated with fi rst ICU stay after transplant ≥10 days (OR, 3.21). These patients had longer fi rst hospital stay after LT (45 vs. 18 days; P <0.001); however, there was no signifi cant diff erence in the rate of ICU readmission (18% vs. 22%; P = 0.58) or re-transplant rate (4% vs. 4%; P = 1.00). Cumulative survival at 1 month, 3 months, 1 year, 3 years, and 5 years was 98%, 96%, 90%, 79%, and 72%, respectively. There was no signifi cant diff erence in cumulative survival stratifi ed by MELD score ≥40 versus <40 at transplant (P = 0.59). Introduction Extracorporeal membrane oxygenation (ECMO) is increasingly used for the treatment of refractory but potentially reversible respiratory and/or cardiac failure. Data on perioperative support with veno-arterial (V-A) and veno-venous (V-V) ECMO for adult liver transplant recipients are scarce [1,2]. We report our experience of ECMO support in patients with acute liver failure (ALF) as a bridge to transplant and postoperative ECMO use following complications after surgery. Methods A retrospective study in a specialist tertiary referral ICU. Patients supported with V-V or V-A ECMO before, during, or after orthotopic liver transplant (OLT) were identifi ed. Conclusion Cirrhotic patients with MELD score ≥40 at transplant utilize greater postoperative health resources; however, they derive similar long-term survival benefi t with LT. Results In total, four patients were supported during a 12-month period. Two patients required V-V and two patients V-A support. Two patients with ALF were bridged to OLT, one patient V-V ECMO for refractory respiratory failure and the other patient required emergency V-A support for treatment of intraoperative arrest. Both patients were successfully transplanted but died subsequently on ECMO: disseminated aspergillosis and haemophagocytic syndrome, and anoxic brain injury respectively. Two patients received postoperative ECMO support. The fi rst was treated with V-V ECMO for refractory persistent hypoxaemia following OLT for hepato-pulmonary syndrome, the second received emergency V-A support (eCPR) following cardiac tamponade and arrest on postoperative day 2. Both patients made a full recovery. gi References Determining late predictors of outcome for acetaminophen- induced acute liver failure using classifi cation and regression tree modeling analysis C Karvellas1, J Speiser2, W Lee3 1University of Alberta, Edmonton, Canada; 2Medical University of South Carolina, Charlston, SC, USA; 3University of Texas Southwestern, Dallas, TX, USA C Karvellas1, J Speiser2, W Lee3 1University of Alberta, Edmonton, Canada; 2Medical University of South Carolina, Charlston, SC, USA; 3University of Texas Southwestern, Dallas, TX, USA Critical Care 2014, 18(Suppl 1):P199 (doi: 10.1186/cc13389) Introduction Liver transplantation (LT) in acetaminophen-induced acute liver failure (APAP-ALF) patients often presents signifi cant challenges. The King’s College Criteria (KCC) have been validated on S72 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P204 Is cirrhotic cardiomyopathy a risk factor for post-reperfusion syndrome during liver transplantation? E Scarlatescu, G Droc, D Tomescu Fundeni Clinical Institute, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P204 (doi: 10.1186/cc13394) Is cirrhotic cardiomyopathy a risk factor for post-reperfusion syndrome during liver transplantation? E Scarlatescu, G Droc, D Tomescu Fundeni Clinical Institute, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P204 (doi: 10.1186/cc13394) P204 Introduction Patients with liver transplantation suff er a major risk of infections immediately after the surgery due to heavy immunosuppression, severe stress and underlying pathology. The aim of this study is to fi nd their causes and consequences in our ICU. Introduction Patients with liver transplantation suff er a major risk of infections immediately after the surgery due to heavy immunosuppression, severe stress and underlying pathology. The aim of this study is to fi nd their causes and consequences in our ICU. Causes and consequences of infections in patients after liver transplantation: 2-year study in the only ICU that hospitalizes these cases in Greece Conclusion Emergency ECMO support before and after liver transplant is feasible. Despite the poor outcome in patients with ALF, we consider ECMO a valuable option to bridge selected patients to transplant. References A Karapanagiotou, C Kydona, S Papadopoulos, T Theodoridou, A Karapanagiotou, C Kydona, S Papadopoulos, T Theodoridou, E Mouloudi, C Dimitriadis, G Imbrios, N Gritsi-Gerogianni Hippokrateion Hospital of Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P202 (doi: 10.1186/cc13392) Postoperative resource utilization and survival among liver transplant recipients with Model for End-stage Liver Disease score ≥40: a retrospective cohort study Introduction Cirrhotic patients with Model for End-stage Liver Disease (MELD) score ≥40 have high risk of death without liver transplant (LT) [1]. This study aimed to evaluate these patients’ outcomes after transplant. Conclusion Patients after liver transplantation with organ dysfunction and multiple transfusions, due to surgical complications, have a major risk of developing an infection inside the ICU that aff ects their survival. Methods The retrospective cohort included 519 adult cirrhotic patients who underwent LT at one Canadian center between 2002 and 2012. Primary exposure was severity of end-stage liver disease measured by MELD score at transplant (≥40 vs. <40) [2]. The primary outcome was duration of fi rst ICU stay after LT [3]. Secondary outcomes were duration of fi rst hospital stay after LT, rate of ICU readmission, re-transplant rate, and survival rates. P203 p References 1. Cannon RM, et al.: Transpl Int 2012, 25:1223-1228. 2. Daly RC, et al.: Transplantation 2013, 95:e2-e4. Results In our study the criteria used to defi ne post-reperfusion syndrome relied on the hemodynamic changes that occurred at reperfusion. Preoperative echocardiography showed normal systolic and diastolic function at rest in all of the patients. For the identifi cation of patients at risk for CCM we used two of the supportive criteria from the recent defi nition of CCM: prolonged QTc interval and increased BNP levels. The study group included 28 men (53.8%) and 24 women. Mean (± SD) age was 50.5 (± 11.4). Mean MELD and MELD Na scores were respectively 15.51 (± 5.43) and 18.9(± 6.22). The value of BNP correlated well with the length of the QTc interval (P = 0.005), and with MELD and MELD Na scores (P = 0.025 and P = 0.001). In our study, post-reperfusion syndrome occurred in 63.4% of the patients. We could not fi nd a correlation between post-reperfusion syndrome and the BNP levels (P = 0.85) or the prolonged QTc interval (P = 0.38). The post-reperfusion syndrome did not correlate with the severity of the liver disease as assessed by MELD and MELD Na scores. The severity of post-reperfusion syndrome did not correlate with QTc prolongation or BNP levels. 1. Cannon RM, et al.: Transpl Int 2012, 25:1223-1228. 2. Daly RC, et al.: Transplantation 2013, 95:e2-e4. Reference 1. Paugham-Burtz C, et al.: Liver Transpl 2009, 15:522-529. 1. Paugham-Burtz C, et al.: Liver Transpl 2009, 15:522-529. y Results We enrolled 101 patients; 87 patients survived, and 14 died. Mortality was signifi cantly higher in the high TAT/PIC group (0%, 19% and 24% for low, middle and high TAT/PIC groups, respectively; P = 0.011). In addition, SOFA and APACHE II scores were signifi cantly higher in the low FDP/D-dimer group (APACHE II = 22.3, 18.9 and 15.3; P <0.01, SOFA = 8.6, 6.5 and 5.1; P <0.01, for low, middle and high FDP/ D-dimer groups, respectively). See Figure 1. Impaired balance between coagulation and fi brinolysis plays a prominent role in patients with sepsis , , p Critical Care 2014, 18(Suppl 1):P206 (doi: 10.1186/cc13396 Introduction The balance between coagulation and fi brinolysis was a prominent factor in the pathophysiology of sepsis, but this mechanism has been poorly understood. We aimed to determine whether collapsing this balance during the fi rst day of sepsis correlates with progression of organ dysfunction and subsequent death. Methods This study included all patients with sepsis admitted to a tertiary referral hospital in Japan. Global coagulation tests and hemostatic molecular markers such as fi brin/fi brinogen degradation products (FDP), D-dimer, thrombin–antithrombin complex (TAT) and plasmin-alpha 2 plasmin inhibitor complex (PIC) were measured within 12 hours after admission, and then SOFA and APACHE II scores and in-hospital mortality were evaluated. Patients were divided into three groups based on the levels of TAT/PIC and FDP/D-dimer and diff erences of clinical outcome between groups were assessed by chi-square analysis and ANOVA. Conclusion Reperfusion is a critical time during liver transplantation. The clinical predictors of post-reperfusion syndrome are still under debate [1]. Our study showed that the post-reperfusion syndrome is not correlated with the severity of the liver disease or with the presence of risk factors indicating CCM. P206 P206 Impaired balance between coagulation and fi brinolysis plays a prominent role in patients with sepsis Y Umemura1, K Yamakawa2, T Kiguchi1, H Ogura2, T Shimazu2, S Fujimi1 1Osaka General Medical Center, Osaka, Japan; 2 Osaka University Graduate School of Medicine, Osaka, Japan Critical Care 2014, 18(Suppl 1):P206 (doi: 10.1186/cc13396) Is cirrhotic cardiomyopathy a risk factor for post-reperfusion syndrome during liver transplantation? y E Scarlatescu, G Droc, D Tomescu Fundeni Clinical Institute, Bucharest, Romania Methods We studied 72 cirrhotic patients who were transplanted in our hospital during the years 2011 and 2012. The cases that developed intrahospital infection, its source and microbiology, the surgical complications, the function of the transplant, the duration of mechanical ventilation and their outcome were fully examined. Fundeni Clinical Institute, Bucharest, Romania Critical Care 2014 18(Suppl 1):P204 (doi: 10 1 , , Critical Care 2014, 18(Suppl 1):P204 (doi: 10.1186/cc13394) Introduction A period of hemodynamic instability following revascularization of the liver graft during liver transplantation is S73 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion CHLT is a life-saving operation that is performed with relatively low mortality and can be successfully performed in select patients with congenital heart disease. Patients undergoing CHLT at our institution had relatively preserved hepatic function but limited cardiac function often requiring inotropic support. Cardiac transplantation typically precedes liver transplantation during CHLT given the decreased ischemic tolerance of the cardiac graft [2]. However, liver transplantation prior to cardiac transplantation may serve to mitigate high-titer donor-specifi c antibodies. Various aforementioned operative approaches may be successfully utilized for the liver transplantation portion of these procedures. We attribute the favorable outcomes and perioperative courses to the multidisciplinary approach to care that CHLT patients receive at our institution. f frequently observed and is termed post-reperfusion syndrome. Recent studies showed the existence of a specifi c heart disease associated with cirrhosis termed cirrhotic cardiomyopathy (CCM). The aim of this study was to investigate whether the CCM has an infl uence on the development or the severity of post-reperfusion syndrome. Methods Fifty-two consecutive liver transplant patients were included in a retrospective observational study. The variables recorded were: age, etiology of the liver disease, MELD and MELD Na scores, the associated pathologies, the length of the QT interval, and plasma levels of brain natriuretic peptide (BNP). The patients with known renal or heart disease and the recipients of organs from extended criteria donors were excluded from the study. The QT interval was corrected for the heart rate using Bazett’s formula (QTc). Statistical analysis was performed using SPSS Statistics v.19.1.i P207 Clinical usefulness of measurement of plasma soluble fi brin levels in critically ill patients Introduction Hemodynamic disorders in critically ill patients are often connected with bacterial load. Bacterial load is usually associated with bacteremia, LPS, high level of IL-6, PCT and also with aromatic microbial metabolites [1-3], and so forth. In our opinion, microbial metabolites can participate in hemodynamic disorders in critically ill patients, particularly due to their infl uence on NO production [4] and intestinal permeability. Methods In a prospective study we observed critically ill patients on the day of admission to a polyvalent ICU, severe cardiac pathology was excluded. The level of phenylpropionic, phenyllactic, p-OH- phenyllactic, p-OH-phenylacetic acids and total phenylcarboxylic acids (PhCAs) were measured in blood serum using gas chromatography (GC-FID). The level of PCT and NT-proBNP were measured using Elecsys 2010. Comparison between patients with hypotension (on vasopressor support) (group A) and without (group B) was performed.f Results The SF values in the DIC and the Subclinical DIC groups were signifi cantly higher than in the No DIC group. We created the receiver operating characteristic curve of SF value for DIC onset (JAAM-DIC score ≥4) and the SF value of 35 μg/ml was set as the cutoff SF value. The high Methods In a prospective study we observed critically ill patients on the day of admission to a polyvalent ICU, severe cardiac pathology was excluded. The level of phenylpropionic, phenyllactic, p-OH- phenyllactic, p-OH-phenylacetic acids and total phenylcarboxylic acids (PhCAs) were measured in blood serum using gas chromatography (GC-FID). The level of PCT and NT-proBNP were measured using Elecsys 2010. Comparison between patients with hypotension (on vasopressor support) (group A) and without (group B) was performed.f Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Figure 2 (abstract P207). ROC curve of the SF value for DIC. Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. pp g p g p p Results We studied 50 ICU patients with diff erent diseases: pneumonia (n = 15), severe kidney failure (n = 13), abdominal surgical pathology (n = 10), alcoholic cirrhosis (n = 5), soft-tissue infection (n = 7). In group A (24/50) the median of PhCAs was 17.8 (IR 11.4 to 30.0) μmol/l, and in group B (26/50) it was 7.2 (IR 3.7 to 13.2) μmol/l, P = 0.003 (t test). Perioperative management of patients undergoing combined heart–liver transplantation p DW Barbara, KH Rehfeldt, JK Heimbach, CB Rosen, RC Daly, JY Findlay Mayo Clinic, Rochester, MN, USA DW Barbara, KH Rehfeldt, JK Heimbach, CB Rosen, RC Daly, JY Findlay Mayo Clinic, Rochester, MN, USA Conclusion We demonstrated that the balance between coagulation and fi brinolysis, assessed with FDP/D-dimer and TAT/PIC ratios, was correlated with disease severity and clinical outcomes in sepsis, suggesting that impaired balance of the hemostatic system might play a pivotal role in progression of sepsis pathophysiology. Introduction Combined heart–liver transplantation (CHLT) is an uncommonly performed procedure for patients with coexisting cardiac and liver disease [1]. The purpose of this study was to examine and describe the perioperative management of patients undergoing CHLT. Methods A retrospective review was performed of patients undergoing CHLT at our institution from 1999 to 2013. Figure 1 (abstract P206). Comparison of the characteristics between the three groups. Results Twenty-seven CHLTs were performed, with 4/27 including simultaneous kidney transplantation. Familial amyloidosis was the indication for 21 CHLTs (78%), and 12 of these explanted livers were used for domino transplantations. Nineteen patients (70%) were receiving inotropic infusions at the time of organ availability. The median preoperative MELD score was 12, and elevations in preoperative international normalized ratio were due to warfarin in all but one patient. Liver transplantation immediately preceded cardiac transplantation in 2/27 cases to reduce high-titer donor-specifi c antibodies. Venovenous bypass was utilized in 14 operations (52%) performed with the caval interposition liver transplantation approach, cardiopulmonary bypass during liver transplantation in two cases (7%), and no bypass in 11 operations (41%) performed with a caval sparing (piggyback) surgical technique. Postoperatively, the median duration of mechanical ventilation, ICU stay, and hospital stay until discharge were 1 day, 5.5 days, and 15 days, respectively. Transfusions in the fi rst 48 hours following CHLT were not substantial in the majority of patients. One patient died within 30 days of CHLT. Figure 1 (abstract P206). Comparison of the characteristics between the three groups. S74 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P207 SF group (SF ≥35 μg/ml) had signifi cantly higher JAAM-DIC score, SOFA score and APACHE II score than the low SF group (SF <35 μg/ml). Mainly in the high SF group except DIC patients on admission, we found that SF increased before the JAAM-DIC score changed. P207 Clinical usefulness of measurement of plasma soluble fi brin levels in critically ill patients In group A, all patients (with or without documented infections) had symptoms of infection manifestation [5], 20/24 (83.3%) of them died. In group B, the symptoms of infection manifestation were revealed in 12/26 (46%) cases, and the mortality was signifi cantly lower, 3/26 (11.5%) (P <0.05). General mortality was 23/50 (46%). The profi le of PhCAs diff ered in groups A versus B. f g p Conclusion The total level of PhCAs in critically ill patients with hypotension was considerably higher than in hemodynamically stable patients. The participation of microbial factor in pathogenesis of hemodynamic disorders in the presence of systemic infl ammation may be validated with the load of microbial metabolites (PhCAs). Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Acknowledgement This work was supported by the Russian Foundation for Basic Research (project number 13-04-01758/13). References 1. Beloborodova N, et al.: Crit Care 2009, 13:41. Figure 2 (abstract P207). ROC curve of the SF value for DIC. Figure 2 (abstract P207). ROC curve of the SF value for DIC. 2. Sarshor Yu, et al.: Shock 2013, 40(Suppl 1):31. 3. Beloborodova N, et al.: Biochemistry 2009, 74:1350-1355. 4. Ogiwara T, et al.: Anticancer Res 2003, 23:1317-1323. 5. Dellinger RP, et al.: Crit Care Med 2013, 41:580-637. P207 Clinical usefulness of measurement of plasma soluble fi brin levels in critically ill patients P207 Clinical usefulness of measurement of plasma soluble fi brin levels in critically ill patients T Masuda, T Kobayashi, H Takahashi, K Yoshikawa, M Takahashi, E Isotani Tokyo Women’s Medical University Medical Center East, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P207 (doi: 10.1186/cc13397) y T Masuda, T Kobayashi, H Takahashi, K Yoshikawa, M Takahashi, E Isotani Tokyo Women’s Medical University Medical Center East, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P207 (doi: 10.1186/cc13397) y T Masuda, T Kobayashi, H Takahashi, K Yoshikawa, M Takahashi, E Isotani Tokyo Women’s Medical University Medical Center East, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P207 (doi: 10.1186/cc13397) Introduction The soluble fi brin monomer fi brinogen complex (SF) is a complex coupling fi brin monomer and fi brinogen molecules. As the level of SF refl ects the thrombin generation activity in plasma, we may estimate the early-activated state of blood coagulation by the measurement of SF. The aim of this study is to evaluate the clinical usefulness of SF for the hypercoagulated state. P208 P208 Value of microbial metabolites in blood serum as criteria for bacterial load in the pathogenesis of hemodynamic disorders in critically ill patients Y Sarshor, N Beloborodova, V Moroz, A Osipov, A Bedova, E Chernevskaya, M Getsina Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2014, 18(Suppl 1):P208 (doi: 10.1186/cc13398) y g Methods We measured the plasma level of SF in 63 patients within 48 hours after admission and on the 1st, 3rd, 5th and 7th days after admission. Underlying disease mainly includes sepsis, shock, and so on. According to the disseminated intravascular coagulation diagnostic criteria established by the Japanese Association of Acute Medicine, we defi ned the DIC group as JAAM-DIC score more than 3 within 48 hours after admission, the Subclinical DIC group as score more than 3 within 7 days beyond 48 hours after admission, and the No DIC group as score less than 4 during the entire study period. The SF value of each group was compared with the Mann–Whitney U test. Introduction Hemodynamic disorders in critically ill patients are often connected with bacterial load. Bacterial load is usually associated with bacteremia, LPS, high level of IL-6, PCT and also with aromatic microbial metabolites [1-3], and so forth. In our opinion, microbial metabolites can participate in hemodynamic disorders in critically ill patients, particularly due to their infl uence on NO production [4] and intestinal permeability. Perioperative management of patients undergoing combined heart–liver transplantation See Figures 1 and 2. Conclusion We think measurement of the plasma SF level may be clinically useful in evaluating the severity of critically ill patients such as those with sepsis. Plasma platelet-derived microparticles to platelet count ratio as a marker of mortality in critically ill patients l Results Upon ICU admission, levels of sRAGE, HMGB1, S100A12 and CRP were higher as compared with healthy levels. HMGB1, S100A12 and CRP remained elevated throughout the ICU stay but sRAGE decreased to levels lower than in healthy volunteers by day 7. sRAGE and CRP showed distinct time profi les during the ICU stay in patients undergoing cardiac versus other surgery and in patients with versus without sepsis upon admission. Elevated sRAGE upon admission, unlike CRP, was associated with need for renal replacement therapy, liver dysfunction, circulatory failure and mortality. Except for mortality, these associations remained in multivariate logistic regression analysis correcting for baseline risk factors. Introduction While the crosstalk between coagulopathy and infl ammation plays a key role in the development of multiple organ dysfunction in critically ill patients, the mechanisms governing this crosstalk have yet to be established. Microparticles (MPs) are submicron vesicles shed from a variety of cells that are considered to have proinfl ammatory and prothrombotic properties. Although platelet- derived MPs (PDMPs) are the main form of MPs, the role of PDMPs in critically ill patients remains unclear [1]. The aims of this study were to investigate serum PDMP levels in critically ill patients and to assess their prognostic value. Conclusion Critical illness alters several components of the RAGE axis. Elevated sRAGE levels upon admission to the ICU were associated with adverse outcome, independent of baseline pathology. Conclusion Critical illness alters several components of the RAGE axis. Elevated sRAGE levels upon admission to the ICU were associated with adverse outcome, independent of baseline pathology. Reference p g Methods This study comprised 119 critically ill patients who were admitted to the ICU. PDMPs were measured by ELISA three times a week, and 372 samples were obtained. We calculated both the mean PDMP value and the mean PDMP/platelet (PDMP/PLT) ratio (converted to plasma PDMP levels per 104 platelets) during the course of the ICU stay. Baseline patient data, including APACHE II score, SOFA score, Japanese Association for Acute Medicine DIC score, International Society for Thrombosis and Haemostasis overt DIC score, blood coagulation parameters, C-reactive protein and lactate, were collected at the time of admission to the ICU. The primary outcome was in- hospital mortality. Potential predictors were analyzed for possible association with outcomes.if 1. Creagh-Brown BC, et al.: The RAGE axis in systemic infl ammation, acute lung injury and myocardial dysfunction: an important target? P209 P209 Fibrinogen at admission is an independent predictor of mortality in severe sepsis and septic shock M Azfar, M Khan, M Khurshid King Saud University, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P209 (doi: 10.1186/cc13399) M Azfar, M Khan, M Khurshid g y y Critical Care 2014, 18(Suppl 1):P209 (doi: 10.1186/cc13399) Introduction Coagulation abnormalities are common in severe sepsis or septic shock [1]. Introduction Coagulation abnormalities are common in severe sepsis or septic shock [1]. Methods A prospective observational cohort study of 100 patients above 18 years of age diagnosed with severe sepsis or septic shock on admission. The fi rst blood sample collected on admission was analyzed. Data were collected through a predesigned pro forma. Those with previous history of any coagulation disorders were excluded. Figure 2 (abstract P207). ROC curve of the SF value for DIC. S75 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Univariate analysis showed signifi cant correlation of APACHE II, platelet, PT, aPTT, fi brinogen and D-dimer with mortality in patients with severe sepsis or septic shock. Multivariate analysis showed APACHE II >20 (P = 0.001), fi brinogen <2 (P = 0.019) and D-dimer >1(P = 0.06) are independent predictors of mortality in severe sepsis or septic shock. See Table 1. in-hospital mortality. The AUCs were calculated as 0.768 ± 0.073 for the mean PDMP/PLT ratio (P = 0.00) and 0.811 ± 0.048 for the APACHE II score (P = 0.00). in-hospital mortality. The AUCs were calculated as 0.768 ± 0.073 for the mean PDMP/PLT ratio (P = 0.00) and 0.811 ± 0.048 for the APACHE II score (P = 0.00). Conclusion The PDMP/PLT ratio is a good predictor of in-hospital mortality in critically ill patients, especially in patients with DIC. Reference 1. Delabranche X, et al.: Intensive Care Med 2013, 39:1695-1703 Table 1 (abstract P209). Receptor for advanced glycation end products axis in critically ill patients Introduction Systemic infl ammation caused by infection or trauma often leads to adverse outcome in critically ill patients. Binding of ligands to the receptor for advanced glycation end products (RAGE) activates several pathways, including the nuclear factor-kappa B pathway, which generates infl ammatory cytokines, proteases and oxidative stress. RAGE activation has been suggested to link amplifi cation and perpetuation of infl ammation to subsequent organ damage and adverse outcome in sepsis, acute lung injury and myocardial dysfunction [1]. The soluble receptor, sRAGE, is thought to act as a decoy, thus protecting against further RAGE activation. High mobility group box 1 (HMGB1) is a nuclear protein that is released during cellular stress and damage. S100A12 is a neutrophil-derived protein that acts as a proinfl ammatory danger signal. Both are ligands for RAGE. We hypothesized that excessive RAGE activation is linked to adverse outcome in critically ill patients and that a diff erent pattern of RAGE activation and infl ammation may be present in patients according to underlying pathology. Conclusion The fi brinogen level at admission is an independent predictor of mortality in patients with sepsis or septic shock. It also confi rms correlation of other coagulation abnormalities with the outcome. Reference 1. Levi M, et al.: Coagulation abnormalities in critically ill patients. Crit Care 2006, 10:222. P212 Usefulness of the endotoxin activity assay as a biomarker to assess severity in ICU patients P210 y y Methods We measured sRAGE, HMGB1 and S100A12 serum levels upon admission, day 7 and the last day in the ICU in 405 critically ill surgical patients who needed intensive care for at least 7 days and in 69 matched healthy controls. We assessed the relation of these levels with clinical complications and outcome, in comparison with C-reactive protein (CRP) as a routinely measured clinical parameter of infl ammation. Plasma platelet-derived microparticles to platelet count ratio as a marker of mortality in critically ill patients M Ohuchi, K Hashimoto, A Ushiba, T Kishimoto, T Yamane, T Hamamoto, T Tabata, Y Tsujita, M Matsushiga, K Takahashi, K Matsumura, K Fujino, Y Eguchi Shiga University of Medical Science, Otsu-shi, Japan Critical Care 2014, 18(Suppl 1):P210 (doi: 10.1186/cc13400) P209 Died Survived 95% CI Variable (n = 65) (n = 35) ARR of ARR P value Platelet <150 33 (50.8) 10 (28.6) 1.37 0.03, 1.84 0.03 PT >16 41 (63.1) 14 (40) 1.40 1.02, 1.91 0.027 aPTT >40 41 (63.1) 14 (40) 1.40 1.02, 1.91 0.027 Fibrinogen <2 40 (61.5) 15 (42.9) 1.31 0.96, 1.70 0.07 D-dimer >1 64 (98.5) 31 (88.6) 3.37 0.48, 19.5 0.03 APACHE II >20 53 (81.5) 18 (51.4) 1.80 1.15, 2.84 0.002 Age >65 27 (41.5) 8 (22.9) 1.60 1.04, 2.46 0.02 Conclusion The fi brinogen level at admission is an independent predictor of mortality in patients with sepsis or septic shock. It also confi rms correlation of other coagulation abnormalities with the outcome. Reference 1. Levi M, et al.: Coagulation abnormalities in critically ill patients. Crit Care 2006, 10:222. Plasma platelet-derived microparticles to platelet count ratio as a marker of mortality in critically ill patients Intensive Care Med 2010, 36:1644-1656. Usefulness of presepsin and procalcitonin levels in the diagnosis of sepsis in patients with acute kidney injury Y Nakamura, H Ishikura, R Ichiki, K Hoshino, M Mizunuma, J Tanaka, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P213 (doi: 10.1186/cc13403) Introduction The presepsin (PSEP) and procalcitonin (PCT) levels are useful biomarkers for diff erentiating between sepsis and non- infectious systemic infl ammatory response syndrome (SIRS). The PSEP and PCT levels have been reported to be abnormally high in patients with chronic renal failure. However, there have been no signifi cant investigations regarding the relationships between these biomarkers and the presence of acute kidney injury (AKI) in the diagnosis of sepsis. The purpose of this study was to clarify the diagnostic accuracy of PSEP and PCT levels in patients with AKI. Conclusion We concluded that although serum leptin may not be benefi cial in early diff erentiation between sepsis and non-infectious SIRS on admission, it may be highly specifi c on the second day. p Reference Reference 1. Ulevitch RJ, Tobias PS: Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol 1999, 11:19-22. y y Results Our patients had mean age of 52.3 ± 18.6 years, 10 males (33.3%). There were no signifi cant diff erences regarding baseline demographic and clinical data apart from blood pressures, which were lower in sepsis group. Serum leptin on day 2 only was higher in the sepsis group (44.2 ± 17.7 μg/l vs. 31.1 ± 2.1 μg/l, P = 0.008) with no diff erence on days 0 and 4 of admission. We detected a serum leptin level of 38.05 μg/l on day 2 to be 93% sensitive and 100% specifi c to diagnose sepsis. The three serum CRP levels were higher in the sepsis group compared with the SIRS group (61.2 ± 9 mg/l vs. 48.9 ± 7.1 mg/l, P <0.001 on day 0, 71.5 ± 9.6 mg/l and 196.8 ± 39.8 mg/l in the sepsis group vs. 56.9 ± 8 mg/l and 73.7 ± 32.5 mg/l in the SIRS group for days 2 and 4 respectively, P <0.001 for both). We found a CRP of 67.5 mg/l on day 2 having 87% sensitivity and 93% specifi city for the diagnosis of sepsis. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 analysis; the area under the curve (AUC) for the PSEP and PCT levels was 0.883 and 0.870, respectively, in the non-AKI group. In addition, the AUC values for the PSEP and PCT levels in the Risk, Injury and Failure groups were 0.843 and 0.843, 0.818 and 0.922 and 0.669 and 0.804, respectively. In the Failure group, the AUC for the PSEP and PCT levels was 0.828 and 0.852, respectively, after dividing the PSEP and PCT levels by the creatinine (Cr) level. The optimal cutoff values for the PSEP and PCT levels for diagnosing sepsis were 409 pg/ml/Cr (sensitivity: 66.0%, specifi city: 91.7%) and 1.5 ng/ml/Cr (sensitivity: 63.6%, specifi city: 95.8%), respectively. the endotoxin activity assay (EAA), a newly developed rapid assay of endotoxin. Blood endotoxin levels (EA levels) of 314 patients admitted to our university hospital ICU were measured within 24 hours from admission, and their correlation with disease severity and outcome was examined. Methods The study is a single-center retrospective analysis of critically ill patients admitted to our university hospital ICU from November 2006 to March 2012. All patients whose EA and procalcitonin levels were measured and severity criteria of disease recorded were enrolled. A total of 314 patients were analyzed. i y y Conclusion The diagnostic accuracy of PSEP and PCT levels for detecting sepsis decreased in the Failure group; however, the value of these parameters for diagnosing sepsis in patients meeting the Failure criteria increased after dividing them by the Cr level. Results The mean ± SD of all ICU-admitted patients (n = 314) was 0.39 ± 0.25, and that of healthy volunteers (n = 61) was 0.10 ± 0.09. The mean APACHE II score at admission increased in parallel with increased EA levels. The mean (± SD) APACHE II score in the very low group of patients (EA <0.2) was 17.3 ± 8.9, while that in the low group (0.2 ≤EA <0.4) was 20.6 ± 9.2; it was 22.6 ± 8.1 in the intermediate group (0.4 ≤EA <0.6) and 25.3 ± 8.5 in the high group (0.6 ≤EA). The diff erence between the groups was statistically signifi cant. The diff erence between the groups was statistically signifi cant. Usefulness of the endotoxin activity assay as a biomarker to assess severity in ICU patients Usefulness of the endotoxin activity assay as a biomarker to asses severity in ICU patients M Noguchi, T Ikeda, K Ikeda, S Suda, A Kodaira, T Ueno, A Ohmi Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P212 (doi: 10.1186/cc13402) y p M Noguchi, T Ikeda, K Ikeda, S Suda, A Kodaira, T Ueno, A Ohmi okyo Medical University, Tokyo, Japan M Noguchi, T Ikeda, K Ikeda, S Suda, A Kodaira, T Ueno, A Ohmi Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P212 (doi: 10.1186/cc13402) Results The mean PDMP/PLT ratio was signifi cantly diff erent when comparing hospital survivors (n = 98; median, 1.95) and nonsurvivors (n = 21; median 8.40; P = 0.00). The mean PDMP/PLT ratio was signifi cantly higher in patients with (median, 4.28) than in those without DIC (median, 1.57; P = 0.00). In DIC patients, the mean PDMP/ PLT ratio was signifi cantly higher in nonsurvivors (median, 9.39) than in survivors (median, 3.65; P = 0.02). Multivariate logistic regression analysis revealed that both the mean PDMP/PLT ratio (P = 0.04) and APACHE II score (P = 0.00) were independently associated with Introduction Endotoxin is a component of the membrane of Gram- negative bacteria and plays a key role in the pathogenesis of sepsis [1]. Measurement of endotoxin levels in patient blood is important for the early diagnosis of and appropriate determination of the treatment strategy for sepsis. The aim of this study was to investigate the prevalence of endotoxemia in Japanese critically ill patients using Introduction Endotoxin is a component of the membrane of Gram- negative bacteria and plays a key role in the pathogenesis of sepsis [1]. Measurement of endotoxin levels in patient blood is important for the early diagnosis of and appropriate determination of the treatment strategy for sepsis. The aim of this study was to investigate the prevalence of endotoxemia in Japanese critically ill patients using S76 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Diff erentiating sepsis from non-infective systemic infl ammatory response syndrome: comparison between C-reactive protein and leptin N Farag1, K Taema2, E Abdel-Latif2, G Hamed2 Conclusion Our patients’ EA levels were signifi cantly correlated with disease severity criteria and 28-day mortality of the patients. When the EA level and procalcitonin level were used concomitantly, disease severity could be assessed more precisely than when either marker was used alone. These results suggest that the EA level is a useful marker for disease severity assessment and outcome prediction in critically ill patients. Introduction Diff erentiation of sepsis from non-infectious SIRS is important in improving sepsis outcome. We intended in this study to evaluate the role of serum leptin and to compare it with CRP in diff erentiating sepsis from non-infectious SIRS. Introduction Diff erentiation of sepsis from non-infectious SIRS is important in improving sepsis outcome. We intended in this study to evaluate the role of serum leptin and to compare it with CRP in diff erentiating sepsis from non-infectious SIRS. f g Methods We included 30 patients with SIRS. According to the presence or absence of infection, our patients were classifi ed into the SIRS group and the sepsis group. Leptin and CRP were evaluated in all patients on admission, day 2 and day 4. P213 Usefulness of presepsin and procalcitonin levels in the diagnosis of sepsis in patients with acute kidney injury Y Nakamura, H Ishikura, R Ichiki, K Hoshino, M Mizunuma, J Tanaka, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P213 (doi: 10.1186/cc13403) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 The percentages of patients diagnosed with severe sepsis or septic shock were 19.3%, 34.5%, 50.0% and 81.3% in the very low, low, intermediate and high groups, respectively.i P214 Diff erentiating sepsis from non-infective systemic infl ammatory response syndrome: comparison between C-reactive protein and leptin N Farag1, K Taema2, E Abdel-Latif2, G Hamed2 1Alsahel Teaching Hospital, Cairo, Egypt; 2Cairo University, Cairo, Egypt Critical Care 2014, 18(Suppl 1):P214 (doi: 10.1186/cc13404) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods The observational study in ICU ventilated septic patients with peritonitis (65%), pancreonecrosis (20%) and mediastinitis (15%) was performed in 2010 and 2013. ARDS was diagnosed and staged according to the V.A. Negovsky Research Institute criteria [1,2] and the Berlin defi nition. Plasma SPA was measured on ARDS diagnosis (day 0) and days 3 and 5 by the immunoenzyme essay (BioVendor, USA). Patients were treated according to the international guidelines. Data were statistically analyzed by STATISTICA 7.0, ANOVA method, and presented as median and 25th to 75th percentiles, ng/ml; P <0.05 was considered statistically signifi cant. Areas under the receiver operating curves were calculated. Results The WBC and PCT median values were signifi cantly (P <0.05) higher in infected than control patients, during 10 postoperative days and on the third and fourth postoperative day, respectively, both variables showing a peak at 3 days in infected patients. The number of times that the WBC count surpassed its second postoperative median value (13,000 cells/mm3) during the fi rst 3 postoperative days plus the number of times that PCT surpassed its own (1.7 ng/ ml) was a parameter (ranging from 1 to 6) with three categories (R1= surpassed zero to one time; R2 = two to three times; R3 = four to six times) signifi cantly associated with risk of infection, which increased with increasing number of times (R2: OR = 2.48 (1.32 to 4.65) and R3: OR = 7.06 (3.17 to 15.71)).i Results Eighty-fi ve patients (out of 300 screened) were enrolled in the study according to the inclusion/exclusion criteria. Patients were assigned into groups: ARDS (n = 50, 48 ± 5.7 years old, M/F 35/15, mortality 26%) and noARDS (n = 35, 46 ± 8.7 years old, M/F 30/5, mortality 23%). Groups were comparable in APACHE II scores. In the ARDS group SPA was higher at all points than in the noARDS group (day 0: 32.6, 25 to 75 IQR 18.2 to 60.7 vs. 23.4, 25 to 75 IQR 17.8 to 28.6; day 3: 31.5, 25 to 75 IQR 20.9 to 31.5 vs. 24.6, 25 to 75 IQR 19.7 to 24.6; day 5: 32.5, 25 to 75 IQR 17.3 to 66.4 vs. 22.5, 25 to 75 IQR 13.4 to 29.5, P <0.05). The plasma SPA was signifi cantly lower in surviving versus dead patients with ARDS (day 0: 20.9, 25 to 75 IQR 13.0 to 35.7 vs. Reference 1. Sablotzki A, Friedrich I, Muhling J, Dehne MG, Spillner J, Silber RE, et al.: The systemic infl ammatory response syndrome following cardiac surgery: diff erent expression of proinfl ammatory cytokines and procalcitonin in patients with and without multiorgan dysfunctions. Perfusion 2002, 17:103-109. 1. Sablotzki A, Friedrich I, Muhling J, Dehne MG, Spillner J, Silber RE, et al.: The systemic infl ammatory response syndrome following cardiac surgery: diff erent expression of proinfl ammatory cytokines and procalcitonin in patients with and without multiorgan dysfunctions. Perfusion 2002, 17:103-109. Single pro-adrenomedullin determination in septic shock and 28-day mortality 28 day mortality A Garcia-de la Torre1, M De La Torre-Prados2, J Parez-Vacas2, C Trujillano-Fernandez2, A Puerto-Morlan2, E Camara Sola2, A Garcia-Alcantara2 1University Hospital Puerto Real, Cadiz, Spain; 2Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2014, 18(Suppl 1):P216 (doi: 10.1186/cc13406) Conclusion Plasma SPA level ≥38.8 ng/ml on the day of ARDS diagnosis is a sensitive and specifi c candidate biomarker of mortality prediction in ARDS septic patients. P218 Club Cell protein: a candidate diagnostic biomarker of Pseudomonas aeruginosa nosocomial pneumonia V Moroz1, A Kuzovlev1, S Polovnikov2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology, RAMS, Moscow, Russia; 2N.N. Burdenko Main Military Hospital, Moscow, Russia Critical Care 2014, 18(Suppl 1):P218 (doi: 10.1186/cc13408) Club Cell protein: a candidate diagnostic biomarker of Pseudomonas aeruginosa nosocomial pneumonia V Moroz1, A Kuzovlev1, S Polovnikov2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology, RAMS, Moscow, Russia; 2N.N. Burdenko Main Military Hospital, Moscow, Russia Critical Care 2014, 18(Suppl 1):P218 (doi: 10.1186/cc13408) Introduction Early etiological diagnosis of nosocomial pneumonia (NP) determines prompt targeted treatment. The aim of this study was to investigate the role of Club Cell protein (CCP) as a candidate diagnostic biomarker of Pseudomonas aeruginosa (PA) NP. g Methods The observational study in ICU ventilated septic patients with peritonitis (65%), pancreonecrosis (20%) and mediastinitis (15%) was performed in 2010 and 2013. Diagnosis of NP was made according to the standard clinical criteria. Associations of multiresistant Gram- negative bacteria were detected in sputum of all patients. PA was detected in 75% of patients. Plasma CCP was measured on the day of NP diagnosis (day 0) and days 3, 5 and 7 by the immunoenzyme essay (BioVendor, USA). Data were statistically analyzed by STATISTICA 7.0, ANOVA method, and presented as Me and 25 to 75 percentiles, ng/ml; P <0.05 was considered signifi cant. Areas under the receiver operating curves (ROC) were calculated. Conclusion Pro-adrenomedullin is an important prognostic biomarker of survival when measured on admission of septic shock patients to the ICU. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 45.7, 25 to 75 IQR 23.5 to 67.9; day 3: 25.5, 25 to 75 IQR 11.8 to 35.5 vs. 45.0, 25 to 75 IQR 29.6 to 68.4; day 5: 24.5, 25 to 75 IQR 11.4 to 33.6 vs. 49.6, 25 to 75 IQR 31.3 to 79.0, P <0.05). Plasma SPA on day 0 had a good capacity for prediction of mortality in ARDS patients: SPA on day 0 ≥38.8 ng/ml yielded a sensitivity of 65% and specifi city of 80% (AUC 0.74; 95% CI 0.577 to 0.866; P = 0.0026). Conclusion Daily assessment of WBC and PCT during the fi rst 3 postoperative days may be of value to diagnose and predict infection in cardiac surgery patients. f References Critical Care 2014, 18(Suppl 1):P216 (doi: 10.1186/cc13406) 1. Kuzovlev A, et al.: Semin Cardiothorac Vasc Anesth 2010, 14:231-241. 2. Kuzovlev A, et al.: Crit Care 2013, 17(Suppl 2):P21800. Introduction The early phase of septic shock is dominated by severe alterations of the cardiovascular system. The prognostic value of pro- adrenomedullin (pADM), a vasoactive pro-hormone, measured within 24 hours from septic shock onset was assessed. p Methods A prospective, observational study in 100 patients >18 years with septic shock in a polyvalent ICU of a university hospital. Demographic, clinical parameters and pADM, C-reactive protein (CRP) and procalcitonin (PCT) were studied during the fi rst 24 hours after admission in 2011. Descriptive and comparative statistical analysis was performed using the statistical software packages StatSoft STATISTICA 7.1 and MedCalc 9.2.1.0. Results We analyzed 100 consecutive episodes of septic shock in the ICU. The median age of the study sample was 64 years, interquartile range 16.8 years, 59% were men; the main sources of infection were respiratory tract (48%) and intra-abdominal (24%). The 28-day mortality was 36%. The profi le of dead patients showed signifi cantly higher clinical severity scores, APACHE II (27 vs. 25; P <0.001) and SOFA (12 vs. 10; P <0.001). The area under the curve was 0.72 for pADM, signifi cantly higher than those for CRP (0.62) and PCT (0.65), but similar to those for APACHE II score (0.69) and SOFA score (0.78). Kaplan–Meier survival analysis was signifi cant (P = 0.0012) for patients with pADM <1.2 nmol/l. Cox regression analysis also showed statistical signifi cance (P = 0.0004) and a likelihood ratio =1.26 per each 1 nmol/l increase in pADM. Use of procalcitonin and white blood cells as combined predictors of infection in cardiac surgery patients p Methods This study was conducted as a single-center retrospective study. Blood samples were collected from patients immediately after admission to the Department of Emergency and Critical Care Medicine, Fukuoka University Hospital between June 2010 and August 2013. We enrolled 629 patients in whom both the PSEP and PCT levels were measured on admission. We classifi ed the patients into two groups according to the RIFLE criteria (Risk, Injury, Failure, Loss of kidney function and End-stage kidney disease: Loss and ESKD): the AKI group and the non-AKI group. The patients in the AKI group were further classifi ed into the sepsis group and the nonsepsis group according to each stage of AKI. We subsequently investigated the diagnostic accuracy of the PSEP and PCT levels for detecting sepsis in these groups. Results We evaluated 254 patients with AKI and 375 patients without AKI. The AKI group included 103 patients who met the Risk criteria, 65 who met the Injury criteria, 66 who met the Failure criteria and 18 who met the Loss and ESKD criteria. The mean PSEP and PCT levels were signifi cantly higher in the sepsis group than in the nonsepsis group among the non-AKI patients and those meeting the Risk, Injury and Failure criteria (P <0.01). The diagnostic accuracy of the PSEP and PCT levels for detecting sepsis was determined according to a ROC Critical Care 2014, 18(Suppl 1):P215 (doi: 10.1186/cc13405 Introduction The aim of this study is to identify a marker able to predict sepsis in cardiac surgery patients and to diff erentiate SIRS from infectious and non-infectious origin. The occurrence of sepsis after cardiac surgery increases the mortality risk. Sepsis may be mistaken for the cardiac surgery-associated systemic infl ammatory response syndrome (SIRS) [1]. Methods A prospective, observational study was carried out to compare procalcitonin (PCT), C-reactive protein (CRP) and white blood cells (WBCs) during the fi rst 10 postoperative days after cardiac surgery with cardiopulmonary bypass (CPB) between 122 patients with infection (Infection group) and 301 without (Control group) to identify predictors of infection. S77 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P217 f Results Ninety patients (out of 350 screened) were enrolled in the study according to the inclusion/exclusion criteria. Patients were assigned to groups: NP (n = 50, 45 ± 4.3 years old, M/F 36/14) and noNP (n = 40, 48 ± 7.2 years old, M/F 30/10). Groups were comparable in APACHE II and CPIS scores. In patients with PA NP (n = 30), plasma CCP was signifi cantly lower at all points than in the patients with no PA detected (n = 20; Figure 1). Plasma CCP on day 0 had a good capacity for the diagnosis of PA NP: CCP on day 0 ≤17.5 ng/ml yielded a sensitivity of 86.5% and specifi city of 66.7% (AUC 0.74; 95% CI 0.630 to 0.829; P = 0.0001; Figure 2).i P217 Surfactant protein A: a candidate predictive biomarker of mortality in acute respiratory distress syndrome in sepsis V Moroz, A Kuzovlev, A Goloubev V.A. Negovsky Scientifi c Research Insitute of General Reanimatology, RAMS, Moscow, Russia Critical Care 2014, 18(Suppl 1):P217 (doi: 10.1186/cc13407) Introduction Prediction of mortality in septic acute respiratory distress syndrome (ARDS) is a problem of great signifi cance. The aim of this study was to investigate the role of surfactant protein A (SPA) as a candidate predictive biomarker of mortality in ARDS. Conclusion Plasma CCP level ≤17.5 ng/ml is a sensitive and specifi c candidate diagnostic biomarker of PA NP. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S78 P219 Plasma cholinesterase activity as diagnostic marker for systemic infl ammation AR Zivkovic, K Schmidt, T Brenner, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2014, 18(Suppl 1):P219 (doi: 10.1186/cc13409) Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure Therefore early diagnosis of systemic inflammation Figure 1 (abstract P218). Club Cell protein concentration in patients with Pseudomonas aeruginosa NP. Figure 2 (abstract P218). ROC curve for Club Cell protein (day 0) for the diagnosis of Pseudomonas aeruginosa NP. Figure 1 (abstract P218). Club Cell protein concentration in patients with Pseudomonas aeruginosa NP. practice, but a defi nitive diagnostic tool for an early detection of systemic infl ammation remains to be identifi ed. The neurotransmitter acetylcholine (Ach) has been shown to play an important role in the infl ammatory response. Serum cholinesterase (butyrylcholinesterase (BChE)) is synthesized in the liver and acts as the major Ach hydrolyzing enzyme in plasma. P217 f Hence, BChE activity has been widely used as a biomarker for liver function. However, the role of this enzyme in the infl ammatory pathomechanism has not yet been fully understood. Here, we describe a strong correlation between the BChE activity and the systemic infl ammatory response. yl y p Methods In this study we measured BChE activity in healthy subjects and in critically ill patients, clinically diagnosed with systemic infl ammation or sepsis. Furthermore, we measured the levels of routine infl ammation biomarkers and liver function parameters in blood of critically ill patients. Data were statistically analyzed using unpaired Student t test, Pearson correlation or ANOVA followed by Dunnett’s post hoc analysis. P <0.05 was considered statistically signifi cant. y yi Results We found that the activity of BChE in patients with systemic infl ammation was dramatically reduced in comparison with healthy subjects and negatively correlated with the serum levels of conventional infl ammatory biomarkers. BChE synthesis depends on hepatic function. Surprisingly, the observed reduction in the BChE activity during systemic infl ammation does not correlate with liver failure. Conclusion Our results suggest that BchE activity might play an important role in the diagnosis of systemic infl ammation independent of overall hepatic function. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods In 183 patients, in a prospective observational study, serum CRP and PCT values were collected every day starting on postoperative day 1 through day 5. The defi nition of SIRS includes two or more of the following: temperature >38 or <36°C; heart rate >90 beats/minute; respiratory rate >20/minute; arterial carbon dioxide pressure <32 mmHg; white blood cell count >12,000/mm3 and <4.00/mm3. The ability of PCT to predict sepsis and other postoperative complications were determined by performing receiver operative characteristic curve analysis. Methods In 183 patients, in a prospective observational study, serum CRP and PCT values were collected every day starting on postoperative day 1 through day 5. The defi nition of SIRS includes two or more of the following: temperature >38 or <36°C; heart rate >90 beats/minute; respiratory rate >20/minute; arterial carbon dioxide pressure <32 mmHg; white blood cell count >12,000/mm3 and <4.00/mm3. The ability of PCT to predict sepsis and other postoperative complications were determined by performing receiver operative characteristic curve analysis. count with conventional markers in patients presenting to the ED of a UK hospital with suspected sepsis. count with conventional markers in patients presenting to the ED of a UK hospital with suspected sepsis. Methods A retrospective review was undertaken of adult patients presenting to the ED (annual census 90,000) with pyrexial illness over a 12-month period, 2011 to 2012. Data included white cell count (WCC), neutrophil count (NC), lymphocyte count (LC) and C-reactive protein (CRP). The results were compared. Sensitivity and specifi city were calculated for each parameter and receiver operating characteristic (ROC) curves were constructed. y y Results All patients were divided post hoc into patients with SIRS (Group 1, n = 83) and patients without SIRS (Group 2, n = 100). A PCT threshold value of 2.79 ng/ml on postoperative day 1 was able to discriminate postoperative complications in patients with or without SIRS with a sensitivity of 82.5% and a specifi city of 70% (area under curve: 0.76, P <0.01).i Results All patients were divided post hoc into patients with SIRS (Group 1, n = 83) and patients without SIRS (Group 2, n = 100). A PCT threshold value of 2.79 ng/ml on postoperative day 1 was able to discriminate postoperative complications in patients with or without SIRS with a sensitivity of 82.5% and a specifi city of 70% (area under curve: 0.76, P <0.01). P222 1. Venet F, Filipe-Santos O, et al.: Decreased T-cell repertoire diversity in sepsis: a preliminary study. Crit Care Med 2013; 41:111-119. P222 Use of procalcitonin for identifi cation of postoperative complications after coronary artery bypass surgery with cardiopulmonary bypass A Baysal1, M Doğukan2, H Toman3 1Kartal Kosuyolu High Speciality Research and Training Hospital, Istanbul, Turkey; 2Adıyaman University Training and Research Hospital, Adıyaman, Turkey; 3Çanakkale 18 Mart Research Hospital, Çanakkale, Turkey Critical Care 2014, 18(Suppl 1):P222 (doi: 10.1186/cc13412) P221 P223 Altered T-cell repertoire diversity in septic shock patients 2 2 2 2 Altered T-cell repertoire diversity in septic shock patients A Kutz, E Grolimund, B Mueller, P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P221 (doi: 10.1186/cc13411) N Takeyama1, A Tomino2, M Hashiba2, A Hirakawa2, T Hattori2, H Miyabe2 1Fujita Health University, Aichi, Japan; 2Fuita Health University, Aichi, Japan Critical Care 2014, 18(Suppl 1):P223 (doi: 10.1186/cc13413) N Takeyama1, A Tomino2, M Hashiba2, A Hirakawa2, T Hattori2, H Miyabe2 1Fujita Health University, Aichi, Japan; 2Fuita Health University, Aichi, Japan Critical Care 2014, 18(Suppl 1):P223 (doi: 10.1186/cc13413) Introduction The occurrence of sepsis-induced immune suppression is associated with multiple organ dysfunctions, although the exact role of T-cell malfunction is obscure. We investigated the impact of sepsis on the adaptive immune system and to monitor T-cell receptor (TCR) diversity. Methods TCR diversity was analyzed in peripheral blood mononuclear cells (PBMCs) isolated from septic shock patients at three time points (days 1, 3 and 7 after diagnosis of septic shock). TCR diversity was measured in genomic DNA isolated from PBMCs using the Human Immun TraCkeRb test (ImmunID Technologies, Grenoble, France) [1]. Multi-N-plex PCR was performed using a primer specifi c to a V gene family and several primers specifi c to J segments. The signal is measured as a function of the fl uorescence intensity of the reference marker. Rearrangement validation and map generation were detected and analyzed using the Constel’ID software (ImmunID Technologies). HLA-DR expression on CD14 cells was measured by fl ow cytometry. Results TCR diversity was markedly decreased in septic patients at day 1 compared with healthy volunteers. A recovery of TCR diversity was observed at days 3 and 7 except for dead patients. HLA-DR expression was signifi cantly decreased in septic patients at day 1. The total lymphocyte count reduced in septic patients at day 1, but the lymphocyte count recovered at days 3 and 7 except for dead patients. Conclusion We observed a lower TCR diversity and lymphopenia in the early stages of septic shock. A quick recovery of TCR diversity and lymphocyte count was observed in live patients. On the other hand, dead patients stayed on the lower level over the course of the study. These results suggest that altered TCR diversity and lymphopenia might participate in increased mortality and susceptibility to secondary nosocomial infections. Introduction The occurrence of sepsis-induced immune suppression is associated with multiple organ dysfunctions, although the exact role of T-cell malfunction is obscure. Altered T-cell repertoire diversity in septic shock patients 2 2 2 2 The total lymphocyte count reduced in septic patients at day 1, but the lymphocyte count recovered at days 3 and 7 except for dead patients. Results TCR diversity was markedly decreased in septic patients at day 1 compared with healthy volunteers. A recovery of TCR diversity was observed at days 3 and 7 except for dead patients. HLA-DR expression was signifi cantly decreased in septic patients at day 1. The total lymphocyte count reduced in septic patients at day 1, but the lymphocyte count recovered at days 3 and 7 except for dead patients. Conclusion We observed a lower TCR diversity and lymphopenia in the early stages of septic shock. A quick recovery of TCR diversity and lymphocyte count was observed in live patients. On the other hand, dead patients stayed on the lower level over the course of the study. These results suggest that altered TCR diversity and lymphopenia might participate in increased mortality and susceptibility to secondary nosocomial infections. Conclusion The infl uence of pre-analytic factors on the examined biomarkers is marginal and not clinically relevant. Our observations reinforce the concept of using biomarkers in algorithms with widely- separated cutoff values and overruling criteria considering the entire clinical picture, without adjustment for pre-analytic factors. Reference 1. Schuetz et al.: JAMA 2009, 302:1059-1066. Altered T-cell repertoire diversity in septic shock patients 2 2 2 2 We investigated the impact of sepsis on the adaptive immune system and to monitor T-cell receptor (TCR) diversity. Methods TCR diversity was analyzed in peripheral blood mononuclear cells (PBMCs) isolated from septic shock patients at three time points (days 1, 3 and 7 after diagnosis of septic shock). TCR diversity was measured in genomic DNA isolated from PBMCs using the Human Immun TraCkeRb test (ImmunID Technologies, Grenoble, France) [1]. Multi-N-plex PCR was performed using a primer specifi c to a V gene family and several primers specifi c to J segments. The signal is measured as a function of the fl uorescence intensity of the reference marker. Rearrangement validation and map generation were detected and analyzed using the Constel’ID software (ImmunID Technologies). HLA-DR expression on CD14 cells was measured by fl ow cytometry. Introduction Blood biomarkers are increasingly used to diagnose, guide therapy in, and risk-stratify community-acquired pneumonia (CAP) patients in emergency departments (EDs). How pre-analytic factors aff ect these markers’ initial levels in this population is unknown. Methods In this secondary analysis of consecutive ED patients with CAP from a large multicentre antibiotic stewardship trial [1], we used adjusted multivariate regression models to determine the magnitude and statistical signifi cance of diff erences in mean baseline concentrations of fi ve biomarkers (procalcitonin (PCT), C-reactive protein (CRP), white blood cell count, proadrenomedullin (ProADM), copeptin) associated with six pre-analytic factors (antibiotic or corticosteroid pretreatment, age, gender, chronic renal failure or liver insuffi ciency). fi y Results Of 925 CAP patients (median age 73 years, 58.8% male), 25.5% had antibiotic pretreatment, 2.4%, corticosteroid pretreatment, 22.3% chronic renal failure, and 2.4% chronic liver insuffi ciency. Diff erences associated with pre-analytic factors averaged 6.1 ± 4.6%; the three largest statistically signifi cant changes (95% CI) were: PCT, +14.2% (+2.1 to +26.4%, P = 0.02) in patients with liver insuffi ciency; ProADM, +13.2% (+10.2 to +16.1%, P <0.01) in patients with age above median; and CRP, –12.8% (–25.4 to –0.2%, P = 0.05) with steroid pretreatment.l l Results TCR diversity was markedly decreased in septic patients at day 1 compared with healthy volunteers. A recovery of TCR diversity was observed at days 3 and 7 except for dead patients. HLA-DR expression was signifi cantly decreased in septic patients at day 1. Reference Reference 1. deJager et al.: Crit Care 2010, 14:R192. Pre-analytic factors and initial biomarker levels in community- acquired pneumonia patients Altered T-cell repertoire diversity in septic shock patients N Takeyama1, A Tomino2, M Hashiba2, A Hirakawa2, T Hattori2, H Miyabe2 1Fujita Health University, Aichi, Japan; 2Fuita Health University, Aichi, Japan Critical Care 2014, 18(Suppl 1):P223 (doi: 10.1186/cc13413) Lymphopenia as a predictor of bacteremia in the emergency department p R Lowsby1, C Gomes1, I Jarman2, P Nee1 1St Helens and Knowsley Teaching Hospital NHS Trust, Liverpool, UK; 2Liverpool John Moores University, Liverpool, UK Critical Care 2014, 18(Suppl 1):P220 (doi: 10.1186/cc13410) Figure 2 (abstract P218). ROC curve for Club Cell protein (day 0) for the diagnosis of Pseudomonas aeruginosa NP Introduction Bloodstream infection (BSI) is associated with a reduction in circulating lymphocytes. Lymphopenia has been proposed as an early marker of BSI in pyrexial adults in the emergency department (ED) setting [1]. The aim of this study was to compare lymphocyte Figure 1 (abstract P220). ROC curve comparing blood test parameters in predicting bacteremia. Figure 2 (abstract P218). ROC curve for Club Cell protein (day 0) for the diagnosis of Pseudomonas aeruginosa NP. Figure 2 (abstract P218). ROC curve for Club Cell protein (day 0) for the diagnosis of Pseudomonas aeruginosa NP. P219 Plasma cholinesterase activity as diagnostic marker for systemic infl ammation AR Zivkovic, K Schmidt, T Brenner, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2014, 18(Suppl 1):P219 (doi: 10.1186/cc13409) Plasma cholinesterase activity as diagnostic marker for systemic infl ammation l AR Zivkovic, K Schmidt, T Brenner, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2014, 18(Suppl 1):P219 (doi: 10.1186/cc13409) Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure. Therefore, early diagnosis of systemic infl ammation would be of great therapeutic and prognostic importance. Various infl ammation biomarkers have been used in clinical and experimental Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure. Therefore, early diagnosis of systemic infl ammation would be of great therapeutic and prognostic importance. Various infl ammation biomarkers have been used in clinical and experimental Figure 1 (abstract P220). ROC curve comparing blood test parameters in predicting bacteremia. S79 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results A total of 2,515 patient records were screened. Patients on chemotherapy were excluded, as were those under 18 years old. In total, 1,954 patients (53% female, median age 66 years) were included in the analysis. Blood cultures were positive in 13.7% of cases. There were signifi cant diff erences between all variables measured, with the exception of WCC, between bacteremic and nonbacteremic patients. The area under the curve of 70.8 was best for lymphocyte count (Figure 1). Conclusion (1) Serum PCT values increased signifi cantly after cardiopulmonary bypass in the SIRS group in comparison with patients without SIRS on postoperative day 1 and remain elevated until postoperative day 5. (2) Serum CRP values also follow a similar pattern, but a CRP threshold value was not obtained to diff erentiate between postoperative complications in patients with or without SIRS. A PCT threshold value of 2.79 ng/ml on postoperative day 1 is a valuable marker to discriminate between patients with or without SIRS. Conclusion In adult patients presenting to the ED with pyrexial illness, lymphopenia predicts bacteremia better than the usual markers of infection. P224 P224 Association between DNA haplogroups and severe sepsis in patients who underwent major surgery E Gomez1, M Heredia1, S Resino2, M Jimenez-Sousa2, J Gomez-Herreras1, E Tamayo1, P Jorge1, M Lorenzo3, P Ruiz1 1Hospital Clínico Universitario de Valladolid, Spain; 2Instituto de Salud Carlos III, Madrid, Spain; 3Hospital Río Hortega, Valladolid, Spain Critical Care 2014, 18(Suppl 1):P224 (doi: 10.1186/cc13414) P224 Association between DNA haplogroups and severe sepsis in patients who underwent major surgery E Gomez1, M Heredia1, S Resino2, M Jimenez-Sousa2, J Gomez-Herreras1, E Tamayo1, P Jorge1, M Lorenzo3, P Ruiz1 1Hospital Clínico Universitario de Valladolid, Spain; 2Instituto de Salud Carlos III, Madrid, Spain; 3Hospital Río Hortega, Valladolid, Spain Critical Care 2014, 18(Suppl 1):P224 (doi: 10.1186/cc13414) p y yp A Baysal1, M Doğukan2, H Toman3 Association between DNA haplogroups and severe sepsis in patients who underwent major surgery E Gomez1, M Heredia1, S Resino2, M Jimenez-Sousa2, J Gomez-Herreras1, E Tamayo1, P Jorge1, M Lorenzo3, P Ruiz1 1Hospital Clínico Universitario de Valladolid, Spain; 2Instituto de Salud Carlos III, Madrid, Spain; 3Hospital Río Hortega, Valladolid, Spain Critical Care 2014, 18(Suppl 1):P224 (doi: 10.1186/cc13414) A Baysal1, M Doğukan2, H Toman3 1Kartal Kosuyolu High Speciality Research and Training Hospital, Istanbul, Turkey; 2Adıyaman University Training and Research Hospital, Adıyaman, Turkey; 3Çanakkale 18 Mart Research Hospital, Çanakkale, Turkey Critical Care 2014, 18(Suppl 1):P222 (doi: 10.1186/cc13412) A Baysal , M Doğukan , H Toman 1Kartal Kosuyolu High Speciality Research and Training Hospital, Istanbul, Turkey; 2Adıyaman University Training and Research Hospital, Adıyaman, Turkey; 3Çanakkale 18 Mart Research Hospital, Çanakkale, Turkey Critical Care 2014, 18(Suppl 1):P222 (doi: 10.1186/cc13412) Introduction The values of C-reactive protein (CRP) and procalcitonin (PCT) were investigated to determine their eff ects on postoperative complications in patients with or without systemic infl ammatory response syndrome (SIRS). Introduction The aim of this study was to analyze whether mitochondrial DNA haplogroups are associated with severe sepsis and S80 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 were measured in supernatant from peripheral blood mononuclear cells stimulated with anti-CD3 and anti-CD3+anti-CD28, incubated for 4 days. Cytokine concentrations of IL-1β, IL-5, IL-6, IL-8, IL-10, IL-13, IL-17A, IFNγ and TNFα were determined by multiplex cytometric bead array. mortality after major surgery in European populations. Mitochondrial DNA (mtDNA) variants may play an important role for predicting clinical outcome in sepsis [1]. P224 Methods We carried out a case–control study on 240 septic patients (severe sepsis or septic shock; Case group) and 267 patients with systemic infl ammatory response syndrome (SIRS; Control group). Furthermore, a longitudinal substudy for analyzing survival was performed in septic patients. mtDNA genotyping was performed by Sequenom’s MassARRAY platform. y Results Plasma levels of IFNγ and IL-13 were lower in septic patients compared with healthy participants. In contrast, plasma levels of IL-6 (see Figure 1) and IL-8 were increased in septic patients compared with both surgical patients and healthy participants. Plasma levels of IL-10 were signifi cantly higher only in comparison with surgical patients. Following incubation with anti-CD3 and anti-CD3+anti-CD28, concentrations of IL-1β, IL-5, IL-6 (see Figure 1), IL-13, IL-17A, IFNγ and TNFα were markedly decreased in samples from septic patients. In addition, stimulation with anti-CD3+anti-CD28 resulted in lower production of IL-10 in septic patients. Lower concentrations of IL-8 were detected in septic patient samples stimulated with only anti-CD3. We found cytokine levels of IL-12p70 remained unaff ected across all groups and stimuli. Results Regarding cardiac surgery, patients with clusters JT and haplogroup J had higher likelihoods of sepsis than patients with clusters HV (OR = 2.76 (95% CI = 1.27; 6.02); P = 0.010) and haplogroup H (OR = 3.68 (95% CI = 1.17; 11.54); P = 0.026), respectively. No signifi cant association was found for abdominal surgery. When analyzing survival, 45.4% patients died with a survival median of 39 (95% CI = 31.4; 46.62) days. When the clusters were analyzed for all patients, 41% (55/134) of patients within cluster HV died versus 71.4% (10/14) patients within cluster IWX (P = 0.018). The adjusted Cox regression showed that patients within cluster IWX had a higher risk of dying than patients within cluster HV (hazard ratio (HR) = 2.24 (95% CI = 1.10; 4.56); P = 0.027). No signifi cant association between haplogroups and mortality was found when patients were stratifi ed by the type of surgery. Conclusion We demonstrated a proinfl ammatory cytokine profi le in blood from septic patients, preceding a pan downregulation of all assessed cytokines following in vitro T-cell stimulation. To our knowledge, this study is the fi rst to perform an immune functional assay across these three groups. Conclusion European mitochondrial haplogroups are associated with sepsis development in patients who underwent major cardiac surgery, but not in patients who underwent major abdominal surgery. P225 T-cell receptor activation-associated cytokine release is impaired in septic patients with faecal peritonitis DG Gore1, L Preciado-Llanes2, GH Mills1, AW Heath2, RC Read3 1Sheffi eld Teaching Hospitals NHS Foundation Trust, Sheffi eld, UK; 2The University of Sheffi eld, UK; 3University of Southampton, UK Critical Care 2014, 18(Suppl 1):P225 (doi: 10.1186/cc13415) Activated protein C consumption and coagulation parameters in severe sepsis and septic shock Activated protein C consumption and coagulation parameters in severe sepsis and septic shock M De La Torre-Prados1, A Garcia-de la Torre2, C Trujillano-Fernandez1, J Perez-Vacas1, A Puerto-Morlan1, E Camara-Sola1 1Hospital Virgen de la Victoria, Málaga, Spain; 2University Hospital Puerto Real, Cadiz, Spain Critical Care 2014, 18(Suppl 1):P226 (doi: 10.1186/cc13416) 1. Yang Y, Shou Z, Zhang P, He Q, Xiao H, Xu Y, Li C, Chen J: Mitochondrial DNA haplogroup R predicts survival advantage in severe sepsis in the Han population. Genet Med 2008, 10:187-192. Introduction Activated protein C (APC) defi ciency is prevalent in severe sepsis and septic shock patients. The aim of the study was to relate the anticoagulation activity evaluated by APC with other coagulation parameters adjusted to 28-day mortality. P224 Besides, mtDNA haplogroups also infl uence mortality. Haplogroups HV and H were related to low risk of sepsis and death, while JT and J were related to high risk of sepsis, and IWX was associated with death. f P226 T-cell receptor activation-associated cytokine release is impaired in septic patients with faecal peritonitis p p p DG Gore1, L Preciado-Llanes2, GH Mills1, AW Heath2, RC Read3 1Sheffi eld Teaching Hospitals NHS Foundation Trust, Sheffi eld, UK; 2The University of Sheffi eld, UK; 3University of Southampton, UK Critical Care 2014, 18(Suppl 1):P225 (doi: 10.1186/cc13415) Methods A cohort study of 150 critically ill adults. Age, sex, sources of infection and coagulation markers within 24 hours from severe sepsis or septic shock onset, defi ned according to Surviving Sepsis Campaign (SSC) criteria, were studied. We analyzed APC activity using a hemostasis laboratory analyzer (BCS® XP; Siemens). A descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Introduction Sepsis is associated with immune hyporesponsiveness but the immunological processes behind this are ill defi ned.if Introduction Sepsis is associated with immune hyporesponsiveness but the immunological processes behind this are ill defi ned.if Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) admitted to the UCI. The median age of the study sample was 64 (interquartile range (IQR): 22.3 years; male: 60%). The main sources of infection were: respiratory tract 38%, intra-abdomen i Methods This study quantifi ed diff erences in plasma concentrations of cytokines between septic patients with faecal peritonitis, age and gender-matched surgical patients (without sepsis) and age and gender-matched healthy participants. In addition, cytokine levels Figure 1 (abstract P225). Concentration of IL-6 in plasma on day 0 (a) and supernatant on day 4 (b). igure 1 (abstract P225). Concentration of IL-6 in plasma on day 0 (a) and supernatant on day 4 (b). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S81 Figure 1 (abstract P226). Relationship between activated protein C and antithrombin III and INR. Figure 1 (abstract P226). Relationship between activated protein C and antithrombin III and INR. Table 2 (abstract P227). Flow-cytometric data Normal value WBC (×109/l) 10.67 ± 4.82 4 to 10 Total lymphocytes (×106/l) 1,123.15 ± 547.02 1,200 to 3,000 CD3+ lymphocytes (×106/l) 67.11 ± 11.86 1,100 to 1,700 CD3+CD4+ lymphocytes (×106/l) 493.07 ± 313.92 600 to 1,400 CD3+CD8+ lymphocytes (×106/l) 250.65 ± 171.94 300 to 900 CD4+DR+ lymphocytes (×106/l) 23.19 ± 15.09 CD8+DR+ lymphocytes (×106/l) 16.26 ± 20.83 Table 2 (abstract P227). Flow-cytometric data Table 2 (abstract P227). Flow-cytometric data 45%, and 70.7% had medical pathology. The 28-day mortality was 22.7%. P227 Methods We retrospectively analyzed data from 54 patients, including 10 patients with isolated brain injury and 44 patients with brain and extracranial injuries. The fl ow-cytometric analysis was performed within 48 hours of trauma. Collected data are shown in Table 1. P228 Polymorphonuclear cell surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, Z Gueldue, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany Critical Care 2014, 18(Suppl 1):P228 (doi: 10.1186/cc13418) Table 1 (abstract P227). Collected data Age (years), mean ± SD 55.43 ± 23.12 Sex (M/F) 44/10 Body mass index, mean ± SD 25.51 ± 2.69 ISS, mean ± SD 29.04 ± 26.57 RTS, mean ± SD 5.76 ± 1.53 TRISS, mean ± SD 68.73 ± 28.77 Admission SAPS II, mean ± SD 44.96 ± 14.87 Admission GCS, mean ± SD 7.63 ± 3.88 Flow-cytometric analysis day (days), mean ± SD 1.69 ± 3.34 Discharge GCS, mean ± SD 10.48 ± 3.19 ICU LOS, mean ± SD 10.67 ± 8.08 Table 1 (abstract P227). Collected data Flow-cytometric analysis in traumatic brain injury to evaluate immunosuppression pp S Di Valvasone, L Perretta, M Bonizzoli, F Liotta, F Annunziato, F Socci, P Ruggiano, A Peris Careggi Hospital, Firenze, Italy Critical Care 2014, 18(Suppl 1):P227 (doi: 10.1186/cc13417) Results Preliminary analysis is limited to descriptive statistics that show an immediate immunosuppression condition after TBI, as established by reduction of CD4+ T lymphocytes and CD8+ T lymphocytes. Signifi cant data are collected in Table 2. Introduction Flow-cytometric analysis is still restricted to cancer and immunocompromised patients. There are no clinical studies that evaluate the immunological changes in traumatic brain injury (TBI) patients. The objective of this study is to determine whether patients with severe TBI (GCS <9) manifest early (<48 hours after injury) signs of immunosuppression and whether this condition increases the incidence of infection during the ICU stay. gi Conclusion In severe TBI patients, an immunosuppression state is early developed. It is relevant to establish whether this condition could aff ect the course and prognosis of ICU patients. P228 T-cell receptor activation-associated cytokine release is impaired in septic patients with faecal peritonitis Nonsurvivors had a signifi cantly higher consumption of APC than survivors, 56% (IQR: 38.5) versus 68.6 (IQR: 41.4); P = 0.023. The profi le of lower levels of APC was a surgery patient with septic shock, neurological focus or catheter-related infection and Gram-negative pathogens from blood cultures. Spearman’s showed relationship with antithrombin III, r = 0.674 (P <0.001) and International Normalized Ratio (INR), r = –0.611 (P >0.001). See Figure 1. Conclusion Low levels of PC are associated with poor outcome and severity in severe sepsis, and it is well correlated with antithrombin III and INR. Lymphocyte surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, L Brach, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany C i i l C 2014 18(S l 1) P229 (d i 10 1186/ 13419) y p y Critical Care 2014, 18(Suppl 1):P229 (doi: 10.1186/cc13419) Methods A total of 434 sepsis and severe sepsis patients hospitalized in the ICU of two centers between 2006 and 2012 were included in the study. Three groups were formed as patients having low (<0.75), normal (>0.75 to <0.80) and high (>0.80) Cl:Na ratio within the fi rst 24 hours in the ICU. Patients’ age, gender, APACHE II score, SOFA score, pH, PaCO2, HCO3, base excess, Na, K, Cl, Ca, lactate, strong ion diff erence, anion gap, length of ICU stay and mortality were recorded. Logistic regression analysis was used to calculate odd ratios and 95% CIs for the association of Cl:Na with mortality. In the fully adjusted model, pH, BE, AG, lactate and Cl:Na ratio were entered into the model. P <0.05 was considered statistically signifi cant. Introduction Lymphocytes show diff erent cell surface molecules depending on the degree of their activation or suppression. We used this surface expression to look at infl ammatory or infectious states in patients with polytrauma and patients with sepsis. The present study was performed to clarify: what is the expression profi le of lymphocyte surface markers in polytraumatized patients over time; and are there diff erences in the expression patterns of polytraumatized patients compared with healthy controls and with patients with severe sepsis and septic shock? Methods In a prospective observational study in a surgical ICU, surface expression of CD3, CD4, CD8, IL7R, CD4/25, CD8/25, CD2/86, CD28, CD3/56, CD2, CD39, CD244, CD11b, and CD56 on lymphocytes was measured by fl ow cytometry. We collected data from six healthy controls, from eight patients with severe sepsis or septic shock, and, longitudinally from eight polytraumatized patients on admission (0 hours), and at 4, 12, 24, 48, 120, and 240 hours. ISS, SAPS 3 and SOFA score refl ect severity of injury, of disease and of organ dysfunctions of the polytraumatized patients. yi Results The distribution of the patients was as follows: low Cl:Na (75, 17%), normal Cl:Na (243, 56%), high Cl:Na (116, 27%). Univariate analysis revealed that in low and high Cl:Na ratio patients, mortality was higher by 1.56-fold (0.87 to 2.81) and 2.22-fold (1.36 to 3.61) (P = 0.135 and P <0.01). In multivariate analysis, increased mortality by 1.95-fold (0.92 to 4.12) and 2.02-fold (1.01 to 4.03) was found in low and high Cl:Na ratios (P = 0.081 and P = 0.046) (Figure 1). References 1. Stewart PA: Can J Physiol Pharmacol 1983, 61:1441-1461. 2. Durward A, et al.: Intensive Care Med.2001, 5:828-835. Dysfunction of peroxisomes as one of the possible causes of multiple organ dysfunction syndrome development AN Osipenko1, AV Marochkov2 p g Critical Care 2014, 18(Suppl 1):P231 (doi: 10.1186/cc13421) P229 Lymphocyte surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, L Brach, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany Critical Care 2014, 18(Suppl 1):P229 (doi: 10.1186/cc13419) Stewart’s strong ion theory can be used in the interpretations of acid– base abnormalities [1]. The Cl:Na ratio is a simple alternative test that obviates the need to solve the complex strong ion gap (SIG) equations [2]. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 septic shock, and longitudinally, on admission (0 hours), and 4, 12, 24, 48, 120 and 240 hours thereafter, eight polytraumatized patients were monitored. ISS, SAPS3 and SOFA score refl ect severity of injury, of disease and of organ dysfunctions. Figure 1 (abstract P230). In the multivariate model, eff ect of Cl:Na ratio group was adjusted for pH, lactate, anion gap and base excess. septic shock, and longitudinally, on admission (0 hours), and 4, 12, 24, 48, 120 and 240 hours thereafter, eight polytraumatized patients were monitored. ISS, SAPS3 and SOFA score refl ect severity of injury, of disease and of organ dysfunctions. g y Results In polytraumatized patients (24 hours measurement), but also in septic patients, CD88+ expression and CD33+/CD66b– expression were signifi cantly lower and CD33–/CD66b+ higher than in healthy controls. Results In polytraumatized patients (24 hours measurement), but also in septic patients, CD88+ expression and CD33+/CD66b– expression were signifi cantly lower and CD33–/CD66b+ higher than in healthy controls. Especially, the lowest values of CD88+ occurred in patients with polytrauma developing septic complications. signifi cantly lower and CD33 /CD66b higher than in healthy controls. Especially, the lowest values of CD88+ occurred in patients with polytrauma developing septic complications. Furthermore, the polytraumatized patients revealed higher levels of CD66b, CD11b and CD16, whereas CD16+ was lower. Considering the post-traumatic course, polytraumatized patients with better prognostic evaluation in SOFA and SAPS3 initially expressed higher levels of TLR-2. Polytrauma patients could be divided into two subgroups, one with and the other without septic complications. The septic subgroup presented predominantly higher values in SOFA, SAPS3 and ISS scoring. Conclusion Cell surface molecules on PMN of polytraumatized patients diff er from those of healthy volunteers. Besides, distinct expression patterns resemble those of patients with sepsis. Patents with higher ISS values go along with higher scores in SOFA and SAPS3, and are at risk to develop septic complications. Table 1 (abstract P227). Collected data Introduction Each cell of the immune system shows a characteristic expression of cell surface molecules depending on the grade of activation or suppression. We examined the cell surface profi le on polymorphonuclear cells (PMN) of polytraumatized patients and compared them with those of healthy controls and furthermore with patients with severe sepsis or septic shock. Moreover, we wanted to fi nd out in the polytraumatized patients whether there are associations of PMN surface marker patterns with severity of injury, of disease and of organ dysfunctions. Methods In a prospective observational study, cell surface expression of CD16, CD88, CD64, CD66b, CD11b, CD95, CD33, CD39, IL-17RA, TLR- 2, TLR-4 and HLA-DR on PMN was determined using fl ow cytometry. Thirteen healthy volunteers, eight patients with severe sepsis and S82 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Lymphocyte surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, L Brach, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany C i i l C 2014 18(S l 1) P229 (d i 10 1186/ 13419) Conclusion Stewart’s strong ion theory provides assessment for the etiology of acid–base disturbances. This evaluation can be performed easier and faster with the Cl:Na ratio. This study demonstrates that the disturbed Cl:Na ratio is associated with increased mortality in sepsis and severe sepsis patient groups. Results In septic and polytraumatized patients, the expression of CD3, CD28 and CD2 was signifi cantly lower than in controls. In polytraumatized patients, surface expression of CD244, CD39 and CD8/25 was signifi cantly higher than in controls. Within the polytraumatized patients, we found two diff erent subgroups, group A with septic progression and Group B without septic progression of disease. Especially, the septic polytraumatized patients revealed very low levels of CD3, CD2 and CD28. Moreover, their levels of CD39 and CD8/25 were less increased and their ISS higher than those of the nonseptic subgroup. Dysfunction of peroxisomes as one of the possible causes of multiple organ dysfunction syndrome development AN Osipenko1, AV Marochkov2 1A. Kuleshov Mogilev State University, Mogilev, Belarus; 2Mogilev Regional Hospital, Mogilev, Belarus Critical Care 2014, 18(Suppl 1):P231 (doi: 10.1186/cc13421) g Conclusion Surface expression molecules of polytraumatized patients diff er from those of the healthy controls and within the group. The more heavily injured polytraumatized patients are more likely to develop severe sepsis and septic shock with surface expression profi les on lymphocytes comparable with those of septic patients. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods Whole blood was taken from healthy volunteers and was placed in tubes containing EDTA and immediately transferred to the laboratory. Heparinized blood samples were diluted 1:10 in RPMI 1640 culture medium (100 μl whole blood added in 900 μl RPMI 1640). Samples were preincubated with 0‚ 5‚ 12.5‚ 25‚ 50‚ 100 and 200 mM alcohol (EtOH) for 10 minutes at room temperature. After incubation, 500 pg LPS was added to each sample for 4 hours at 37°C. At the end of the process, samples were centrifuged (1,800 rpm, 5 minutes, r.t.). Culture supernatants were collected and stored at –70°C until measurements. TNFα and TNFR levels were determined in culture supernatant using the ELISA method [2]. volunteers aged 37.7 ± 3.2 years served as control. The preanalytical phase was to prepare a solution of ethyl esters of fatty acids (FAc) and diethylacetals of fatty aldehydes (FAl) of blood plasma samples by acidic ethanolysis. Analysis of FAl and FAc in plasma was carried out using capillary gas–liquid chromatography. Quantitative evaluation of the analytes was performed as a mass percentage of the FA and FAl amount. Statistical analysis was performed using the Mann–Whitney U test (P <0.05). Results In contradistinction to triglycerides, diacylphospholipids and Pls participate in exchange of polyunsaturated fatty acids (PUFA) with a large number of double bonds, such as primarily arachidonic and docosahexaenoic PUFA, acting as an intermediate station, through which these fatty acids are transported to cell membranes [1]. Thus, the ratio of level of FAl to the level of aforementioned blood plasma PUFA may refl ect a change in the share of Pls relative to diacylphospholipids. According to our data, at MODS the ratio of fatty aldehydes to amount of arachidonic and docosahexaenoic PUFA is 0.16, while in the control group the fi gure is 0.27 (P <0.001). Thus, we can conclude that the level of Pls towards diacylphospholipid plasma levels of the experimental group decreased by approximately 40%. Currently, there is evidence that there is a reduction of cholesterol levels in blood plasma of patients with the diseases associated with peroxisome biogenesis disorders [2]. In our study, patients had low levels of plasma total cholesterol (124.6 ± 28.7 mg/dl), which may also indicate peroxisome dysfunction in MODS patients. p g [ ] Results We studied 24 healthy males volunteers aged 36.5 ± 1.4 (X ± SEM). P232f P232 Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo A Gavala1, K Venetsanou1, P Myrianthefs1, E Manolis2, C Kittas3, G Baltopoulos1 1ICU ‘Agioi Anargyroi’ General Hosrital, School of Nursing, Athens University, Athens, Greece; 2Dentistry School, Athens University, Athens, Greece; 3Medical School, Athens University, Athens, Greece Critical Care 2014, 18(Suppl 1):P232 (doi: 10.1186/cc13422) Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo P233 Neutrophil phenotype model for extracorporeal treatment of sepsis A Malkin1, R Sheehan1, S Mathew1, H Redl2, G Clermont1 1University of Pittsburgh, PA, USA; 2Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria Critical Care 2014, 18(Suppl 1):P233 (doi: 10.1186/cc13423) y References References 1. Braverman N, Moser A: Bioch Biophys Acta 2012, 1822:1442-1452. 2. Kovacs W, et al.: J Mol Cell Biol 2004, 24:1-13. References 1. Cook RT‚ et al.: Alcohol Clin Exp Res 1998, 22:1927. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. 1. Cook RT‚ et al.: Alcohol Clin Exp Res 1998, 22:1927. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 TNFα was not detected in samples treated without alcohol in the absence of LPS stimulation (control) or in the presence of alcohol alone (data not shown). TNFα production was signifi cantly decreased at a dose of 25 mM alcohol after LPS stimulation (P <0.0001) compared with LPS-challenged samples (Figure 1A). Alcohol had no eff ect on TNFR I production when incubated with or without LPS (data not shown). Alcohol at lower doses (<50 mM) seemed to decrease TNFR II levels, but an increase in TNFR II levels was observed at higher doses (>50 mM) of alcohol, which was statistically signifi cant at doses of 100 and 200 mM alcohol after LPS stimulation ex vivo (P <0.001) (Figure 1B). Conclusion Our observations indicate a suppression of pro infl am- matory response, but also a diff erential eff ect of alcohol on TNFR II production of whole blood in the presence of LPS challenge depending on the degree of alcohol intoxication. Conclusion On the basis of determined defi ciency of analyzed compound levels, we concluded the possibility of peroxysomal dysfunction in MODS. Cl:Na ratio on ICU admission as a prognostic indicator of mortality in sepsis patients Cl:Na ratio on ICU admission as a prognostic indicator of mortality in sepsis patients HK Atalan1, B Gucyetmez2, N Cakar3 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Istanbul Medical Faculty, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P230 (doi: 10.1186/cc13420) Introduction This study demonstrates that low level of plasmalogens (Pls) is an important marker of peroxisomal dysfunction. The primary- OH group in glycerol Pls was not substituted by the acyl group (fatty acid) as in diacylphospholipids but by the aldehydogenic alkenyl group (fatty aldehyde) found in the form of vinyl ether [1]. It is known that there is disruption of several organ systems in diseases connected with peroxisomal dysfunction, as in the case of multiple organ failure. Cl:Na ratio on ICU admission as a prognostic indicator of mortality in sepsis patients HK Atalan1, B Gucyetmez2, N Cakar3 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Istanbul Medical Faculty, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P230 (doi: 10.1186/cc13420) HK Atalan1, B Gucyetmez2, N Cakar3 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Istanbul Medical Faculty, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P230 (doi: 10.1186/cc13420) Introduction The aim of the study is to investigate the relationship between Cl:Na ratio disturbances and mortality in sepsis patients. Introduction The aim of the study is to investigate the relationship between Cl:Na ratio disturbances and mortality in sepsis patients. p y p g Methods The objects of study were 18 people with multiple organ failure (35.6 ± 8.7 years) of various etiologies. The blood of 16 healthy S83 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data K Vardas1, K Apostolou1, K Psarra2, E Botoula2, S Tsagarakis2, E Magira1, C Routsi1, E Briassouli1, D Goukos1, S Nanas1, G Briassoulis3 1Medical School, NKUA, Athens, Greece; 2Evangelismos Hospital, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2014, 18(Suppl 1):P234 (doi: 10.1186/cc13424) Results AI prevented Cr, BUN and CysC from increasing after CLP (0.43 ± 0.18, 0.2 ± 0.02, P = 0.0003; 62.2 ± 10.8, 43.7 ± 17.7, P = 0.01; 103.9 ± 54.8, 49.3 ± 30.8, P = 0.01, respectively). AICAR decreased cytokine release after CLP (IL-6: 1,818 ± 344 vs. 1,374 ± 268, P = 0.04; IL- 10: 7,592 ± 5,038 vs. 2,245 ± 2,668, P = 0.05; TNFα: 213 ± 172 vs. 39 ± 16, P = 0.03) and LPS in macrophage culture (IL-6: 241.7 ± 1.5 vs. 7.2 ± 1.5, P = 0.055; TNF: 1,882 ± 583 vs. 82 ± 28, P = 0.04). However, AI lost its protective signal when administered 4 hours pre or 2 hours after CLP (Cr CLP vs. CLP+AI: 0.2 vs. 0.2, P = 0.1 and 0.45 ± 0.3 vs. 0.56 ± 0.3, P = 0.5, respectively). AI also decreased ICAM-1 expression in vivo and in vitro, vascular leak (0.09 ± 0.01 vs. 0.06 ± 0.008, P = 0.003) and PMN adhesion (1,055 ± 179 vs. 751 ± 242, P = 0.04). Introduction Besides cortisol, prolactin might also be important in maintaining normal immune response in stress states. Its role in the hypothalamus–pituitary–adrenal axis hormonal and immune stress response in sepsis has hardly been investigated in a critically ill setting. The aims of the study were to evaluate the early (fi rst 48 hours) ACTH, cortisol and prolactin plasma levels in ICU patients with severe sepsis/ septic shock (SS) or systemic infl ammatory response syndrome (SIRS) compared with healthy control subjects (C) and to correlate their expression with heat shock proteins (HSPs), interleukins (ILs) and outcome. Conclusion AMPK activation by AICAR prevented sepsis-induced AKI. AICAR decreased cytokine release, endothelial activation and vascular leak, suggesting that organ protection may be mediated by modulation of the infl ammatory response and protection of the microvasculature. However, AICAR’s protective eff ect may be conditional to timing of administration. Methods Thirty consecutively admitted patients with SS, 22 with SIRS, and 15 C were enrolled in the study. P235 AMP-protein kinase may protect against sepsis-induced acute kidney injury through modulation of immune response and endothelial activation AMP-protein kinase may protect against sepsis-induced acute kidney injury through modulation of immune response and endothelial activation H Gomez, D Escobar, A Botero, BS Zuckerbraun University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P235 (doi: 10.1186/cc13425) p p y Results Of 16 baboons, 11 (69%) died, six (38%) within 1 day of bacterial infusion. The model was well calibrated to the data from survivors and nonsurvivors. Sensitivity analysis identifi ed six model parameters, out of 21, as key determinants of mortality. Predictions indicate best eff ect with introduction of the proposed extracorporeal intervention within 1 hour of infection for a 72-hour duration, results in the survivor population increasing from 31% to 61%. The treatment can result in harm if initiated <10 hours from infection and continued <24 hours. A delay of 10 hours increases survival by 7% on average. Treatment effi cacy quickly diminishes if not introduced within 15 hours of infection. H Gomez, D Escobar, A Botero, BS Zuckerbraun University of Pittsburgh, PA, USA Introduction Organ injury is a hallmark of sepsis, in particular acute kidney injury (AKI). Yet the mechanisms involved in sepsis-induced AKI are not well understood. Energy prioritization is an important cell defense mechanism, and thus we hypothesized that exogenous activation of AMP-protein kinase (AMPK), a master regulator of cellular energy metabolism, protects against sepsis-induced AKI. Conclusion These fi ndings support the continued development of an extracorporeal treatment that modulates CXCR-1/2 levels. Further development of the mathematical model will help guide device development and determine which patient populations should be targeted by treatment. gy p g p Methods Sixty C57BL/6 male mice, 6 to 8 weeks of age, weighing 20 to 25 g were divided into six groups: 1, cecal ligation and puncture (CLP); 2, CLP+AICAR (AI, AMPK activator, 100 mg/kg 24 hours before CLP); 3, CLP+compound C (AMPK inhibitor, 30 mg/kg; CoC); 4, sham; 5, sham+AI; 6, sham+CoC. Blood/tissue samples were collected 8 hours after CLP. Renal function (creatinine (Cr, mg/dl), BUN (mg/dl) and cystatin C (CysC, ng/ml)), cytokine expression (ELISA), endothelial activation (ICAM- 1 expression), neutrophil adhesion (PMN, fl uorescence, anti-CD11b mAb-tagged PMNs) and vascular leak (Evan’s blue) were assessed. The eff ect of AI given 4 hours (n = 12) before and 2 hours after (n = 11) CLP was also evaluated. Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo A Gavala1, K Venetsanou1, P Myrianthefs1, E Manolis2, C Kittas3, G Baltopoulos1 p 1ICU ‘Agioi Anargyroi’ General Hosrital, School of Nursing, Athens University, Athens, Greece; 2Dentistry School, Athens University, Athens, Greece; 3Medical School, Athens University, Athens, Greece Critical Care 2014, 18(Suppl 1):P232 (doi: 10.1186/cc13422) p 1ICU ‘Agioi Anargyroi’ General Hosrital, School of Nursing, Athens University, Athens, Greece; 2Dentistry School, Athens University, Athens, Greece; 3Medical School, Athens University, Athens, Greece Critical Care 2014, 18(Suppl 1):P232 (doi: 10.1186/cc13422) Introduction Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage. Interleukin-8 (IL-8), a key modulator of neutrophil function, signals through neutrophil specifi c surface receptors CXCR-1 and CXCR-2. Expression of these surface receptors can be altered by perfusion through an extracorporeal device. Extracorporeal methods of immune modulation have shown promise for treatment of sepsis; however, achieving an appropriate response is a major challenge [1]. In this study a mechanistic mathematical model was used to evaluate and deploy an extracorporeal sepsis treatment that modulates CXCR-1/2 levels. Introduction Acute alcohol exposure suppresses proinfl ammatory response, which may be related to increased susceptibility to infections [1].The purpose of the study was to investigate the eff ect of acute exposure to alcohol on TNFα production capacity and TNFα receptors (TNFRs) in an ex vivo model of whole-blood stimulation with lipopolysaccharide (LPS). Figure 1 (abstract P232). Figure 1 (abstract P232). S84 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A simplifi ed mechanistic mathematical model of IL-8- mediated activation of CXCR-1/2 receptors was developed. Receptor-level dynamics and systemic parameters are coupled with multiple neutrophil phenotypes to generate dynamic populations of activated neutrophils that reduce pathogen load, and/or primed neutrophils that cause adverse tissue damage when misdirected. The mathematical model was calibrated using experimental data from baboons administered a 2-hour infusion of E. coli and followed for a maximum of 28 days. An extracorporeal intervention was implemented by introducing a trapped receptor state that limits IL-8 signaling through CXCR-1/2. Time of onset, duration, and capture effi cacy of the extracorporeal device were explored to provide probabilistic predictions of the impact on mortality. Conclusion Prolactin seems to be a stress hormone, probably related to the severity of illness, increased along with IL-6 and eHSP90 levels in SS. P234 Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data K Vardas1, K Apostolou1, K Psarra2, E Botoula2, S Tsagarakis2, E Magira1, C Routsi1, E Briassouli1, D Goukos1, S Nanas1, G Briassoulis3 1Medical School, NKUA, Athens, Greece; 2Evangelismos Hospital, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2014, 18(Suppl 1):P234 (doi: 10.1186/cc13424) Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data Patients’ demographics, laboratory examinations, and APACHE II, SOFA and SAPS III scores were recorded on ICU admission. Blood sampling was performed between 9:00 and 10:00 a.m. Prolactin and cortisol were measured using the ADVIA centaur system, ACTH using the Immulite 2000. Intracellular monocyte HSP (iHSP) was determined after staining with surface antigens CD33– PE/Cy5 and CD45 PE/Cy7 followed by either HSP72-FITC or HSP90a-PE intracellular staining (four-color fl ow cytometry). Mean fl uorescence intensity values for each HSP were noted and analyzed. ELISA was used to determine extracellular (e) HSP, IL-6 and IL-10 levels. Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo Also in these patients, cortisol correlates to lactate and eHSP90, but in contrast to SIRS the iHSP72 and iHSP90 immune response is depressed. Acknowledgements This research has been co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. Reference Reference Reference 1. Peng Z-Y, et al.: Kidney Int 2012, 81:363-369. P235 AMP-protein kinase may protect against sepsis-induced acute kidney injury through modulation of immune response and endothelial activation The experiment was reproduced in vitro using cell culture (renal epithelial, endothelial cells and macrophages), with similar groups: 1, control; 2, control+AI (1 mM/1 hour pre LPS); 3, LPS (100 ng/ml for 4 hours); 4, LPS+AI; 5, control+CoC (10 μM/1 hour pre LPS); 6, LPS+CoC. Cytokines were measured with ELISA. Data are presented as mean ± SD and as LPS/CLP and LPS/CLP+AI. P236 The same procedures were done for sham-operated mice and for mice subject only to femur crush and to pneumothorax. Figure 2 (abstract P236). Ex vivo proinfl ammatory cytokine release after whole-blood LPS stimulation in older patients. with a group with arterial disease, expressed by proinfl ammatory cytokine release after ex vivo whole-blood LPS stimulation [2]. with a group with arterial disease, expressed by proinfl ammatory cytokine release after ex vivo whole-blood LPS stimulation [2]. Methods The study comprised 16 polytrauma patients admitted to the ICU, aged 78 ± 8 (Group I) and 16 with arterial disease, aged 74 ± 5 (Group II). Ten milliliters of peripheral blood were collected from each patient, divided into two tubes with/without anticoagulant. Diluted 1:10 whole-blood samples were stimulated with 500 pg/ml LPS, at 37°C, for 4 hours. Serum and cell culture supernatants (CCSP) were removed and stored at –70°C. TNFα and IL-6 were measured in serum and CCSP by ELISA. j y p Results Initial experiments with 21 mice showed that the overall death rate for this model of multitrauma was 66.7% with most deaths occurring in the fi rst 48 hours. In the second set of experiments, 12 mice remaining alive 72 hours post injury were challenged with P. aeruginosa; mortality was 37.5% compared with 75% of 12 non- injured and infected mice (log-rank: 5.77, P = 0.016). Respective mean production of TNFα from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 651 and 523 pg/ml (P = NS); post femur crush 217 and 916 pg/ml (P = 0.010); post pneumothorax 286 and 1,056 pg/ml (P = 0.018); and post both femur crush and pneumothorax 207 and 1,011 pg/ml (P = 0.019). Respective mean production of IFNγ from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 324 and 230 pg/ml (P = NS); post femur crush 278 and 840 pg/ml (P = 0.021); post pneumothorax 377 and 2,356 pg/ml (P = 0.025); and post both femur crush and pneumothorax 252 and 908 pg/ml (P = 0.019). Mean production of TNFα from splenocytes of injured mice sacrifi ced 24 hours after bacterial challenge was 1,184 pg/ ml compared with 484 pg/ml non-injured and infected mice (P = 0.024). Similar diff erences were not found for IFNγ or for quantitative tissue cultures. See Figure 1. p References 1. Bruno L: Proc Nutr Soc 1999, 58:85-98. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. 1. Bruno L: Proc Nutr Soc 1999, 58:85-98. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. P236 y Methods The study comprised 16 polytrauma patients admitted to the ICU, aged 78 ± 8 (Group I) and 16 with arterial disease, aged 74 ± 5 (Group II). Ten milliliters of peripheral blood were collected from each patient, divided into two tubes with/without anticoagulant. Diluted 1:10 whole-blood samples were stimulated with 500 pg/ml LPS, at 37°C, for 4 hours. Serum and cell culture supernatants (CCSP) were removed and stored at –70°C. TNFα and IL-6 were measured in serum and CCSP by ELISA. y Results Serum proinfl ammatory cytokines were signifi cantly elevated after severe trauma against control group (TNFα, P <0.001 and IL-6, P <0.001). Ex vivo cytokine release showed the opposite direction. There was a signifi cantly lower TNFα and IL-6 release for Group I (TNFα, P <0.05 and IL-6, P <0.01) compared with Group II. TNFα ex vivo release from the samples of Group II was >300 pg/ml. See Figures 1 and 2. Conclusion Older patients showed adequate immunological response, considering the limit of 300 pg/ml. The incidence of severe trauma was involved in the downregulation of immune activity and should be considered. Group I patients do not have the opportunity to precondition their immune status. Group II patients can better compensate operative therapies. Conclusion Mice survivors from multiple trauma become resistant to subsequent infection. This is probably linked with the induction of tolerance of the innate immunity to substances released after sterile tissue injury. P236 Study of the ex vivo immune response of polytrauma older patients in the ICU on admission: preliminary results L Filippou, K Venetsanou, G Voulalas, D Markopoulou, D Chroni, C Maltezos, I Alamanos KAT Hospital Athens, Kifi sia, Athens, Greece Critical Care 2014, 18(Suppl 1):P236 (doi: 10.1186/cc13426) Results Prolactin (P <0.034), cortisol (P <0.0001), and ACTH (P <0.02) levels diff ered among groups. Prolactin, cortisol, IL-6, and eHSP90 were higher in SS (P <0.03) and SIRS (P <0.04) compared with C. Cortisol and eHSP90 were higher in SS compared with SIRS (P <0.005; P <0.03); iHSP72 and iHSP90 were higher in SIRS compared with SS (P <0.05; P <0.05) or C (iHSP72, P <0.02). Cortisol related with eHSP90 (r = 0.45, P <0.02), lactate (r = 0.33, P <0.02) and HCO3 (r = –0.45), and prolactin with SAPS III (r = 0.34, P <0.02), but not with mortality or LOS. Introduction Immunological status is diff erentiated with age, infl uencing treatment and outcome [1]. The aim is to determine the immune response of severely traumatized older patients compared Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S85 Figure 1 (abstract P236). Serum proinfl ammatory cytokines in older patients. Figure 2 (abstract P236). Ex vivo proinfl ammatory cytokine release after whole-blood LPS stimulation in older patients. Figure 1 (abstract P236). Serum proinfl ammatory cytokines in older patients. Figure 1 (abstract P237). Cytokine stimulations after sterile injury. Figure 1 (abstract P236). Serum proinfl ammatory cytokines in older patients. Figure 2 (abstract P236). Ex vivo proinfl ammatory cytokine release after whole-blood LPS stimulation in older patients. Methods Ninety-seven C56Bl6 male mice were subject to multitrauma after crush of the femur and chemical pneumothorax by turpentine. Mice surviving 72 hours after the injuries were challenged intravenously with one 7log10 log-phase inoculum of P. aeruginosa and survival was recorded. In separate experiments, mice were sacrifi ced post injury; splenocytes were isolated and stimulated with 10 ng/ ml LPS and cytokines were measured in supernatants by an enzyme immunoassay. Quantitative cultures of the right lung, kidney and liver were performed. The same procedures were done for sham-operated mice and for mice subject only to femur crush and to pneumothorax. Results Initial experiments with 21 mice showed that the overall death rate for this model of multitrauma was 66.7% with most deaths occurring in the fi rst 48 hours. P236 In the second set of experiments, 12 mice remaining alive 72 hours post injury were challenged with P. aeruginosa; mortality was 37.5% compared with 75% of 12 non- injured and infected mice (log-rank: 5.77, P = 0.016). Respective mean production of TNFα from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 651 and 523 pg/ml (P = NS); post femur crush 217 and 916 pg/ml (P = 0.010); post pneumothorax 286 and 1,056 pg/ml (P = 0.018); and post both femur crush and pneumothorax 207 and 1,011 pg/ml (P = 0.019). Respective mean production of IFNγ from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 324 and 230 pg/ml (P = NS); post femur crush 278 and 840 pg/ml (P = 0.021); post pneumothorax 377 and 2,356 pg/ml (P = 0.025); and post both femur crush and pneumothorax 252 and 908 pg/ml (P = 0.019). Mean production of TNFα from splenocytes of injured mice sacrifi ced 24 hours after bacterial challenge was 1,184 pg/ ml compared with 484 pg/ml non-injured and infected mice (P = 0.024). Similar diff erences were not found for IFNγ or for quantitative tissue cultures. See Figure 1. Methods Ninety-seven C56Bl6 male mice were subject to multitrauma after crush of the femur and chemical pneumothorax by turpentine. Mice surviving 72 hours after the injuries were challenged intravenously with one 7log10 log-phase inoculum of P. aeruginosa and survival was recorded. In separate experiments, mice were sacrifi ced post injury; splenocytes were isolated and stimulated with 10 ng/ ml LPS and cytokines were measured in supernatants by an enzyme immunoassay. Quantitative cultures of the right lung, kidney and liver were performed. The same procedures were done for sham-operated mice and for mice subject only to femur crush and to pneumothorax. Methods Ninety-seven C56Bl6 male mice were subject to multitrauma after crush of the femur and chemical pneumothorax by turpentine. Mice surviving 72 hours after the injuries were challenged intravenously with one 7log10 log-phase inoculum of P. aeruginosa and survival was recorded. In separate experiments, mice were sacrifi ced post injury; splenocytes were isolated and stimulated with 10 ng/ ml LPS and cytokines were measured in supernatants by an enzyme immunoassay. Quantitative cultures of the right lung, kidney and liver were performed. Tissue oxygenation in patients with severe sepsis A Oei1, M Kok2, A Karakus3, H Endeman4 Tissue oxygenation in patients with severe sepsis A Oei1, M Kok2, A Karakus3, H Endeman4 1Academic Medical Center, Amsterdam, the Netherlands; 2University Medical Center, Utrecht, the Netherlands 3Diakonessenhuis, Utrecht, the Netherlands; 4Onze Lieve Vrouwen Gasthuis, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P240 (doi: 10.1186/cc13430) Introduction The aim of this study was to determine the correlation between regional tissue oxygenation (SrO2) measured by near-infrared spectroscopy (NIRS), central venous oxygen saturation (SvO2) and levels of serum lactate (SL) in patients with severe sepsis. Methods An observational pilot study was performed in an ICU of a medium-sized teaching hospital. Adult patients admitted with severe sepsis were included and three NIRS electrodes were attached: on the left side of the forehead (LF), the right side of the forehead (RF) and the right forearm (RA). SL and SvO2 measured in a jugular or subclavian central venous line sample were determined every 4 hours. Descriptive statistics were calculated. Pearson correlation coeffi cients (PCC) were calculated between SrO2, SvO2 and SL. Normal values for SrO2 were defi ned as the median ± 1 interquartile range in patients with SL <1.7 mmol/l. Diff erences between groups were calculated by Pearson chi-square tests. P <0.05 was considered statistically signifi cant.i f Conclusion While clopidogrel may not increase the risk of bleeding in these patients, these data suggest that clopidogrel does not reduce adverse outcomes in patients with sepsis. A potential benefi t in patients with APACHE II scores ≥25 may need further study. Future research into the mechanism of ticagrelor’s benefi t reported in the PLATO trial may be directed at the non-P2Y12 receptor mechanism. q y gi Results Twenty-fi ve patients (12 men) were included. Mean age was 68 years, mean APACHE score 31. The most frequent reasons for ICU admission were abdominal sepsis (n = 11) and pneumosepsis (n = 7). Statistically signifi cant correlations were found between SvO2 and SrO2: PCC SrO2 LF, 0.46; PCC SrO2 RF, 0.50; PCC SrO2 RA, 0.21. Low, although statistically signifi cant, correlations were found between SrO2 and SL: PCC SrO2 LF, –0.16; PCC SrO2 RF, –0.15; and PCC SrO2 RA, –0.20. Calculated normal values for SrO2 LF were 60 to 80%, for SrO2 RF were 60 to 76% and for SrO2 RA were 64 to 84%. An out-of-range SrO2 had a high positive predictive value (PPV) for an increased SL. P239 P239 Continuous administration of corticosteroids in septic shock can reduce risk of hypernatremia L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, I Grintescu1, D Mirea3 1Clinical Emergency Hospital of Bucharest, Romania; 2CI Parhon National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P239 (doi: 10.1186/cc13429) Multiple trauma is linked with reversal of immunoparalysis and provides survival benefi t from Pseudomonas aeruginosa E Mandragos1 A Pistiki2 DI Droggiti2 M Georgitsi2 Does clopidogrel change morbidity and mortality in ICU sepsis patients? DG Klepser, PP Dobesh, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13428) Multiple trauma is linked with reversal of immunoparalysis and provides survival benefi t from Pseudomonas aeruginosa E Mandragos1, A Pistiki2, DI Droggiti2, M Georgitsi2, E Giamarellos-Bourboulis2 1Attikon Hospital, Chaidari, Attiki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2014, 18(Suppl 1):P237 (doi: 10.1186/cc13427) E Mandragos , A Pistiki , DI Droggiti , M Georgitsi , E Giamarellos-Bourboulis2 1Attikon Hospital, Chaidari, Attiki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2014, 18(Suppl 1):P237 (doi: 10.1186/cc13427) Introduction The purpose of this study was to evaluate whether patients with sepsis exposed to clopidogrel have lower mortality and fewer days on mechanical ventilation compared with patients not exposed to clopidogrel. A recent post-hoc analysis of the PLATO trial suggests that the antiplatelet agent ticagrelor provided a signifi cant Introduction Patients with multiple injuries are prone to hospital- acquired infections. We hypothesized that exposure to multiple injuries may modulate the innate immune response and the subsequent outcome of infections. S86 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P = 0.0041) and slightly higher when HHS was delivered as a bolus (RR 3.08, 1.32 <OR <7.25, P = 0.0071). P = 0.0041) and slightly higher when HHS was delivered as a bolus (RR 3.08, 1.32 <OR <7.25, P = 0.0071). reduction in sepsis-related mortality and pulmonary adverse events compared with clopidogrel. It is unknown whether clopidogrel also provides benefi t in patients with sepsis compared with no therapy. Knowing this information may help focus future research on determining the mechanism of ticagrelor’s benefi t. reduction in sepsis-related mortality and pulmonary adverse events compared with clopidogrel. It is unknown whether clopidogrel also provides benefi t in patients with sepsis compared with no therapy. Knowing this information may help focus future research on determining the mechanism of ticagrelor’s benefi t. Conclusion Continuous administration of hydrocortisone hemi- succinate in septic shock is associated with a lower risk of hypernatremia than bolus administration. Continuous administration of corticosteroids in septic shock can reduce risk of hypernatremia L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, I Grintescu1, D Mirea3 L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, I Grintescu1, D Mirea3 1Clinical Emergency Hospital of Bucharest, Romania; 2CI Parhon National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania 1Clinical Emergency Hospital of Bucharest, Romania; 2CI Parhon National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P239 (doi: 10.1186/cc13429) Introduction Although the administration of hydrocortisone in septic shock generates adverse eff ects, the risk of corticosteroid-induced hypernatremia may be reduced by continuous administration of the drug [1,2]. g Methods A total of 171 patients with septic shock were randomized into three study groups: group A (n = 58), 200 mg/day hydrocortisone hemisuccinate in four doses; group B (n = 59), same dose of hydrocortisone hemisuccinate in continuous administration; group C (n = 54), no hydrocortisone hemisuccinate. Mean serum sodium values, the number of hypernatremia episodes and variations in serum sodium (Na var) were investigated for 7 days. The local ethics committee approved the study. Conclusion This pilot study shows a statistically signifi cant correlation between SvO2 and SrO2 in patients with severe sepsis. An out-of-range SrO2 LF or RF had a high predictive value for increased serum lactate. We therefore conclude that NIRS could have a role in goal-directed therapy of patients with severe sepsis. References Methods This retrospective cohort study included all patients over the age of 40 with a confi rmed diagnosis of severe sepsis and an ICU stay at our academic medical center from 1 January 2005 to 31 March 2011. Clopidogrel use, patient demographics, and APACHE II score at the time of sepsis were collected from patient charts. Clinical outcomes included in hospital mortality, development of acute respiratory distress syndrome (ARDS), days on mechanical ventilation, in-hospital cardiac events, hospital cost, and length of stay.i 1. Sprung CL, Annane D, Keh D, et al.: Hydrocortisone therapy fo ith ti h k N E l J M d 2008 358 111 124 1. Sprung CL, Annane D, Keh D, et al.: Hydrocortisone therapy for patients with septic shock. N Engl J Med 2008, 358:111-124. 1. Sprung CL, Annane D, Keh D, et al.: Hydrocortisone therapy for patients with septic shock. N Engl J Med 2008, 358:111-124. 2. Annane D, Bellissant E, Bollaert PE, et al.: Corticosteroids in the treatment of severe sepsis and septic shock in adults: a systematic review. JAMA 2009, 301:2362. 2. Annane D, Bellissant E, Bollaert PE, et al.: Corticosteroids in the treatment of severe sepsis and septic shock in adults: a systematic review. JAMA 2009, 301:2362. Tissue oxygenation in patients with severe sepsis A Oei1, M Kok2, A Karakus3, H Endeman4 The PPV for out-of-range SrO2 LF was 85% versus 58% for normal SrO2 LF (OR 4.27; 95% CI 2.09 to 8.72), the PPV for out-of-range SrO2 RF was 77% versus 60% for normal SrO2 RF (OR 2.32; 95% CI 1.25 to 4.31) and the PPV for out-of-range SrO2 RA 75% versus 60% for normal SrO2 RA (OR 1.94; 95% CI 0.95 to 4.00).i P240 Results We identifi ed 824 patients who met the inclusion criteria for this study. Of these patients, 76 (9.2%) had been exposed to clopidogrel. The mean APACHE II score was similar for patients receiving clopidogrel and those who did not (23.4 vs. 22.6; P = 0.426). Patients exposed to clopidogrel had a similar rate of in-hospital mortality (26.3% vs. 33.2%; P = 0.223) and ARDS (43.4% vs. 38.6%; P = 0.415). While mortality was also similar between the groups for patients with a low APACHE II score (<25), mortality was lower in clopidogrel-exposed patients with an APACHE II score ≥25 (27.3% vs. 45.6%; P = 0.045). Patients exposed to clopidogrel did not have a higher use of blood products (65.8% vs. 65.9%; P = 0.983). Patients exposed to clopidogrel did not have signifi cantly more days on mechanical ventilation, or ventilation-free days. Cardiac events during the hospital stay were not lower in patients on clopidogrel (4.0 vs. 2.4; P = 0.432). Hospital costs and length of stay did not diff er between the groups. P241 y Results There were no diff erences between the three groups at the beginning of the study regarding demographic data and the clinical characteristics. Mean values of natremia were normal in group C (140.35 ± 7.390 mEq/l to 144.79 ± 8.338 mEq/l) during the study period. High mean values appeared on day 4 in group A (147.21 ± 8.470 mEq/l to 149.37 ± 8.973 mEq/l on day 7) and on day 5 in group B (146.36 ± 8.272 mEq/l to 147.70 ± 8.865 mEq/l). Na var was 8.59 ± 5.960 mEq/l (–8 and 21 mEq/l) in group A, 6.63 ± 7.609 mEq/l (–17 and 23 mEq/l) in group B and 4.54 ± 7.455 mEq/l (–12 and 22 mEq/l) in group C. This variation is statistically signifi cant when groups A and B are compared with group C (P = 0.012) and when only group A is compared with group C (P = 0.0019). The risk of hypernatremia after hydrocortisone hemisuccinate was almost three times higher than that of patients who did not receive this drug (RR 2.82, 1.35 <OR <5.90, Delayed admission to the ICU is associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock A Schnegelsberg, J Mackenhauer, M Pedersen, H Nibro, H Kirkegaard Aarhus University Hospital, Aarhus C, Denmark Critical Care 2014, 18(Suppl 1):P241 (doi: 10.1186/cc13431) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 24 hours has been found to be signifi cantly higher compared with patients admitted directly to the ICU from the ED [2]. 24 hours has been found to be signifi cantly higher compared with patients admitted directly to the ICU from the ED [2]. pyelonephritis or intraabdominal infection were blindly assigned to placebo or clarithromycin for four consecutive days as adjunctive treatment to standard of care. Clarithromycin was administered at a dose of 1 g once daily in 1 hour of continuous infusion. Organ failures before allocation to blind treatment were defi ned according to the Surviving Sepsis Campaign 2003 defi nitions. Cox regression analysis was done to verify the eff ect of clarithromycin as a moderator. Hazard ratios (HRs) and 95% confi dence intervals (CIs) were calculated. pyelonephritis or intraabdominal infection were blindly assigned to placebo or clarithromycin for four consecutive days as adjunctive treatment to standard of care. Clarithromycin was administered at a dose of 1 g once daily in 1 hour of continuous infusion. Organ failures before allocation to blind treatment were defi ned according to the Surviving Sepsis Campaign 2003 defi nitions. Cox regression analysis was done to verify the eff ect of clarithromycin as a moderator. Hazard ratios (HRs) and 95% confi dence intervals (CIs) were calculated. Methods A retrospective cohort study of patients admitted with community-acquired sepsis to a 12-bed, tertiary ICU at a university- affi liated teaching hospital, November 2008 to October 2010. Patients were divided into two groups based on their ICU admission pattern: direct transfer from the ED (direct group); and one or more ward transfers between the ED and ICU within 48 hours (delayed group). Our primary outcome measure was mortality.i i Results Forty-nine patients of the placebo arm and 55 patients of the clarithromycin arm had acute lung injury (ALI); mortality was 51.0% and 30.9% respectively (P = 0.046). Forty-seven patients of the placebo arm and 54 patients of the clarithromycin arm had acute coagulopathy; mortality was 44.7% and 44.4% respectively (P = 1.000). Twenty patients of the placebo arm and 19 patients of the clarithromycin arm had metabolic acidosis; mortality was 55.0% and 52.6% respectively (P = 1.000). Twenty-nine patients of the placebo arm and 39 patients of the clarithromycin arm had acute oliguria; mortality was 55.2% and 48.7% respectively (P = 0.631). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ALI (HR = 2.42; 95% CI = 1.45 to 4.03, P = 0.001), acute coagulopathy (HR = 2.65; CI = 1.69 to 4.16) and cardiovascular failure (HR = 3.36, CI = 2.09 to 5.39) were independently associated with unfavorable outcome. Adding treatment with clarithromycin in the equation reduced the risk for death by ALI by 1.86-fold (HR = 0.54; CI = 0.29 to 0.99, P = 0.049). Results We identifi ed 277 patients admitted to the ICU within 48 hours from arrival in the ED. Of these, 186 were admitted directly from the ED, and 91 patients had more than one ward transfer between the ED and the ICU. In-hospital mortality in the delayed group was 32% compared with 21% in the direct group (P = 0.0477). Patients with delayed admission had signifi cantly lower APACHE II scores: 21 (16; 26) and 24 (18.75; 31) respectively (P = 0.0016). The in-hospital LOS was similar in the two groups. For patients in the delayed group, we found a trend toward increased 30-day mortality (P = 0.0723), 90-day mortality (P = 0.0838) and higher Charlson Comorbidity Index (P = 0.0609). g y Conclusion We found that patients admitted with community- acquired severe sepsis or shock are more likely to die in-hospital if they experience redundant ward transfers within 48 hours of admission. However, the delayed group was signifi cantly lower risk stratifi ed in the ICU based on their APACHE II score. Identifi cation of severe sepsis and risk assessment in the ED is crucial for patient outcome. References Conclusion Clarithromycin is a major moderator of the physical course of Gram-negative sepsis complicated with ALI. Reference References 1. Vallés J, et al.: Chest 2003, 123:1615-1624. 2. Parkhe M, et al.: Emergency Med 2002, 14:50-57 1. Vallés J, et al.: Chest 2003, 123:1615-1624. 2. Parkhe M, et al.: Emergency Med 2002, 14:50-57. 1. Giamarellos-Bourboulis EJ, et al.: J Antimicrob Chemother 2013. [Epub ahead of print] , , 2. Parkhe M, et al.: Emergency Med 2002, 14:50-57. print] P242f Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial E Giamarellos-Bourboulis1, K Lymberopoulou2, I Tsangaris3, A Antonopoulou3, A Marioli2, L Leonidou4, E Douzinas3, I Koutelidakis5, A Armaganidis3 1University of Athens, Medical School, Athens, Greece; 2Sismanogleion General Hospital, Athens, Greece; 3University of Athens, Athens, Greece; 4University of Patras, Rion, Greece; 5Aristotle University, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P242 (doi: 10.1186/cc13432) Benefi t profi le of recombinant human soluble thrombomodulin in sepsis-induced DIC K Yamakawa1, H Ogura1, S Fujimi2, M Morikawa3, Y Ogawa1, Y Umemura2, Y Inoue3, H Tanaka3, T Hamasaki1, T Shimazu1 1Osaka University Graduate School of Medicine, Osaka, Japan; 2Osaka General Medical Center, Osaka, Japan; 3Juntendo University Urayasu Hospital, Chiba, Japan Critical Care 2014, 18(Suppl 1):P243 (doi: 10.1186/cc13433) Delayed admission to the ICU is associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock Introduction The aim of this study is to determine whether ward transfers causing delayed ICU admission are associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock. Community-acquired infections are among the leading causes of ICU admission [1]. The 30-day mortality for adult emergency department (ED) patients with a delay in ICU admission of up to Introduction The aim of this study is to determine whether ward transfers causing delayed ICU admission are associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock. Community-acquired infections are among the leading causes of ICU admission [1]. The 30-day mortality for adult emergency department (ED) patients with a delay in ICU admission of up to S87 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P246 Results The EHR screen identifi ed 216,550 patients over a total of 36 months from the two hospitals. A total of 37,160 (17%) patients were treated with i.v. antibiotics. Sepsis patients experienced a 3% (1,186/37,160) mortality rate relative to 0.5% (448/216,550) in patients without infection at any time. Sepsis at any time represented 73% (1,186/1,634) of all in-hospital deaths. ICU transfer occurred in 18% (6,865/37,160) of patients, with septic shock (vasopressor requirement) occurring in 10% (3,837), and 13% (5,072) requiring mechanical ventilation. Outcomes of neutropenic patients with severe sepsis on a specialist cancer ICU Introduction The hospital survival rate for patients with septic shock remains at approximately 50% [1]. Critically ill cancer patients have often been considered poor candidates for ICU management due to the perception of poor outcomes for this group, in particular in the presence of neutropenia. There is a paucity of data supporting this. The objective of this study was to describe clinical outcomes for a group of septic neutropenic patients admitted to a cancer ICU. y Results Eligible were 162 patients with sepsis-induced DIC; 68 patients received rhTM and 94 did not. After adjusting for imbalances, rhTM administration was signifi cantly associated with reduced mortality only in patents in the stratum II group (APACHE II, 22 to 27) (adjusted hazard ratio, 0.20; 95% confi dence interval, 0.05 to 0.74; P = 0.016), while not signifi cant in stratum I and stratum III (Figure 1). A similar tendency was observed in analysis for SOFA score (stratum I (SOFA, –10), P = 0.368; stratum II (SOFA, 11 to 12), P = 0.012; stratum III (SOFA, 13–), P = 0.673). Conclusion A survival benefi t with rhTM treatment was observed in sepsis-induced DIC and a high risk of death according to baseline APACHE II and SOFA scores. Methods All neutropenic patients (neutrophils <1,000/mm3) admitted to the ICU at the Royal Marsden hospital (London, UK) between October 2010 and December 2012 with a diagnosis of severe sepsis/septic shock were included retrospectively. Data were collated from patients’ electronic records after approval by the hospital audit committee. Data are presented as the absolute value (%) or median (IQR). Fisher’s exact test or the Mann–Whitney U test was used as appropriate. Conclusion A survival benefi t with rhTM treatment was observed in sepsis-induced DIC and a high risk of death according to baseline APACHE II and SOFA scores. Comprehensive assessment of the true sepsis burden using electronic health record screening augmented by natural language processing R Arnold, J Isserman, S Smola, E Jackson Christiana Care Health System, Newark, DE, USA Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) R Arnold, J Isserman, S Smola, E Jackson Christiana Care Health System, Newark, DE, USA Table 1 (abstract P245). Patient characteristics ICU survivors ICU nonsurvivors n 47 (73%) 17 (27%) Age (years) 53 (38 to 65) 49 (33 to 62) BM transplant 24 (51%) 7 (41%) Table 2 (abstract P245). Characteristics on admission to the ICU ICU survivors ICU nonsurvivors Neutrophil count 0.5 (0.3) 0.2 (0.3)* Days of neutropenia 3 (0 to 8) 6 (0 to 19) P <0.05. Table 1 (abstract P245). Patient characteristics ICU i ICU i Table 1 (abstract P245). Patient characteristics Table 1 (abstract P245). Patient characteristics y , , , Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) y Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) Introduction Interventional trials for sepsis have not shown an improvement in patient outcomes, often due to the lack of a diagnostic gold standard resulting in large heterogeneity of the patients enrolled. Electronic health record (EHR) screening tools have been applied to the sepsis population but limited to vital sign and laboratory data to identify target patients. Our objective was to describe an investigational database created through the application of a new EHR screening tool that applies natural language processing (NLP) analysis to clinical documentation to augment the identifi cation of infection. i Methods We acquired data from the Clinical Vigilance for Sepsis EHR screen on consecutive patients from two hospital systems over 12 months at a 300-bed community hospital and 24 months from a 500- bed academic tertiary care center. A physician order for intravenous antibiotics was used as a surrogate for suspected infection, removing patients receiving a single dose of antibiotics without subsequent administration. Each patient’s in-hospital course was tracked from arrival to fi nal disposition, identifying vital signs, laboratory values, radiological results, and interventions as they occurred. Patients were followed for the primary outcome of mortality, with secondary outcomes of transfer to the ICU, vasopressor initiation and mechanical ventilation. Conclusion In a group of oncology patients admitted to the ICU with neutropenia and severe sepsis/septic shock, we found an in-hospital mortality of less than 50%. This is similar to the general population. Reference 1. Quenot et al.: Crit Care 2013, 17:R65. References y Results Sixty-four neutropenic patients were admitted to the ICU during this period. Forty-seven (73%) patients survived to ICU discharge and 34 (53%) patients to hospital discharge. Twenty-two (34%) patients were alive at 6 months and 18 (28%) patients were alive at 12 months. Seventy-seven percent of patients had microbiologically documented infections. There was no signifi cant diff erence between ICU survivors and nonsurvivors in duration of neutropenia, the presence of, or removal of, indwelling catheters, or the source of sepsis. Mechanical ventilation and need for vasopressors were associated with worse outcomes. Patient characteristics are presented in Tables 1 and 2. 1. Vincent JL, et al.: Crit Care Med 2013, 41:2069-2079. 1. Vincent JL, et al.: Crit Care Med 2013, 41:2069-2079. 2. Yamakawa K, et al.: Intensive Care Med 2013, 39:644-652. 2. Yamakawa K, et al.: Intensive Care Med 2013, 39:644-652. Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial E Giamarellos-Bourboulis1, K Lymberopoulou2, I Tsangaris3, A Antonopoulou3, A Marioli2, L Leonidou4, E Douzinas3, I Koutelidakis5, A Armaganidis3 1University of Athens, Medical School, Athens, Greece; 2Sismanogleion General Hospital, Athens, Greece; 3University of Athens, Athens, Greece; 4University of Patras, Rion, Greece; 5Aristotle University, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P242 (doi: 10.1186/cc13432) E Giamarellos-Bourboulis1, K Lymberopoulou2, I Tsangaris3, g 1University of Athens, Medical School, Athens, Greece; 2Sismanogleion General Hospital, Athens, Greece; 3University of Athens, Athens, Greece; 4University of Patras, Rion, Greece; 5Aristotle University, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P242 (doi: 10.1186/cc13432) Introduction Recombinant human soluble thrombomodulin (rhTM) demonstrated promising evidence suggestive of effi cacy in a phase IIb, randomized, controlled trial [1] and is currently under evaluation in a phase III trial. However, the benefi t profi les of rhTM have not been elucidated. The purpose of this study was to explore whether patients with a high disease severity (according to Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores) might have a treatment benefi t from rhTM administration. Introduction A recent randomized trial of our group showed that blind treatment with clarithromycin decreased mortality from septic shock and multiple organ dysfunction, shortened time until resolution of infection in patients with severe sepsis/shock and decreased hospitalization costs [1]. The effi cacy of clarithromycin in relation with the type of failing organs is analyzed.l Methods Six hundred patients with systemic infl ammatory response syndrome due to primary Gram-negative bacteremia or acute Figure 1 (abstract P243). Estimated survival curves for each of three disease severities. Figure 1 (abstract P243). Estimated survival curves for each of three disease severities. S88 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods This was a post-hoc, subgroup analysis of a multicenter retrospective cohort study [2] conducted in three tertiary referral hospitals in Japan. All patients with sepsis-induced DIC who required ventilator management were included. We stratifi ed all patients with diff erent disease severity, as defi ned by APACHE II and SOFA scores to three strata. Intervention eff ects estimated as hazard ratios were analyzed by Cox regression analysis adjusted for propensity model to detect subgroup heterogeneity of the eff ects of rhTM on in-hospital mortality. Comprehensive assessment of the true sepsis burden using electronic health record screening augmented by natural language processing Comprehensive assessment of the true sepsis burden using electronic health record screening augmented by natural language processing R Arnold, J Isserman, S Smola, E Jackson Christiana Care Health System, Newark, DE, USA Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) Dynamic myocardial depression in septic cardiomyopathy A Darawshe, D Levingston, Y Haviv The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) Introduction Benefi cial eff ects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on vascular diseases have been demonstrated in several clinical trials. Recently discovered anti-infl ammatory eff ects of statins seem to have an important role in counteracting the harmful eff ects of sepsis on the coagulation system. Many epidemiologic studies evidence that statin treatment may be associated with a better prognosis in severe bacterial infections, and a recent randomized trial found a reduced mortality in the statin group. We decided to perform a meta-analysis of all randomized controlled trials ever performed on statin therapy in septic patients to evaluate their eff ect on survival. g The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) Introduction Depression of left ventricular contractility appears in many diseases resulting from various etiology factors. One of the most interesting features of septic cardiomyopathy is the signifi cant dynamic myocardial depression that is commonly observed. In this context, the objective of the present work is to characterize clinically, by laboratory, by echocardiography, and by invasive measures the patients with septic cardiomyopathy. f Methods Four trained investigators searched and assessed pertinent studies in BioMed Central, PubMed, Embase and the Cochrane Central Register (divergences resolved by consensus). Inclusion criteria were: random allocation to treatment; comparison of statins versus any comparator in septic patients. Exclusion criteria were: duplicate publications; nonadult studies; no data on main outcomes. Results Data from 650 patients in fi ve randomized controlled studies were analyzed. Overall analysis showed no diff erence in mortality between patients receiving statins (44/322 (14%)) versus control (50/328 (15%)), RR = 0.90 (95% CI 0.65 to 1.26), P = 0.6. Conclusion Scientific evidence for the role of statins in septic patients f Methods Four trained investigators searched and assessed pertinent studies in BioMed Central, PubMed, Embase and the Cochrane Central Register (divergences resolved by consensus). Inclusion criteria were: random allocation to treatment; comparison of statins versus any comparator in septic patients. Effi cacy of early administration of thrombomodulin alfa in patients with sepsis-induced disseminated intravascular coagulation: subanalysis from post-marketing surveillance data Y Eguchi1, S Gando2, H Ishikura3, D Saitoh4, J Mimuro5, H Takahashi6, I Kitajima7, H Tsuji8, T Matsushita9, Y Sakata5 1Shiga University of Medical Science, Shiga, Japan; 2Hokkaido University Graduate School of Medicine, Hokkaido, Japan; 3Fukuoka University, Fukuoka, Japan; 4National Defense Medical College, Saitama, Japan; 5Jichi Medical University, School of Medicine, Tochigi, Japan; 6Niigata Prefectural Kamo Hospital, Niigata, Japan; 7Graduate School of Medical and Pharmaceutical Science, University of Toyama, Japan; 8Kyoto Prefectural University of Medicine, Kyoto, Japan; 9Nagoya University Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P248 (doi: 10.1186/cc13438) Results Most patients showed vitamin D levels below 20 ng/ml, and we have not demonstrated a statistical signifi cance correlation in the univariate regression between vitamin D level and both SAPS (P = 0.300) or length of stay in hospital (P = 0.154). Also our data do not demonstrate a statistical signifi cance diff erence at t test between the value of vitamin D in the dead group and the alive group. Introduction We hypothesize that the early administration of thrombo- modulin alfa (TM-alfa) (Recomodulin® injection; Asahi Kasei Pharma Corporation, Tokyo, Japan) could improve mortality in patients with sepsis-induced disseminated intravascular coagulation (DIC). TM-alfa has been approved for use as a curative medicine for DIC in Japan from 2008 that was examined in a multicenter, randomized, clinical trial [1]. Introduction We hypothesize that the early administration of thrombo- modulin alfa (TM-alfa) (Recomodulin® injection; Asahi Kasei Pharma Corporation, Tokyo, Japan) could improve mortality in patients with sepsis-induced disseminated intravascular coagulation (DIC). TM-alfa has been approved for use as a curative medicine for DIC in Japan from 2008 that was examined in a multicenter, randomized, clinical trial [1]. Methods DIC was diagnosed based on the Japanese Association for Acute Medicine (JAAM) criteria. From May 2008 to April 2010, a total of 1,787 patients with sepsis-induced DIC from post-marketing surveillance data [2] were analyzed. The survival rates on day 28 after the last TM-alfa administration were evaluated. Methods DIC was diagnosed based on the Japanese Association for Acute Medicine (JAAM) criteria. From May 2008 to April 2010, a total of 1,787 patients with sepsis-induced DIC from post-marketing surveillance data [2] were analyzed. The survival rates on day 28 after the last TM-alfa administration were evaluated. Conclusion Several groups have reported an inverse association between vitamin D levels in critically ill patients, severity of disease, outcome length of ICU stay and mortality [3,4]. Effi cacy of early administration of thrombomodulin alfa in patients with sepsis-induced disseminated intravascular coagulation: subanalysis from post-marketing surveillance data In our experience we have not found an evident correlation between low vitamin levels and ICU outcomes. However, considering the prevalence of low vitamin D levels among the medical patients admitted to the ICU, further studies should be performed. Results The 28-day survival rate was 64.5%. Use of other anticoagulants before and after TM-alfa administration did not aff ect the survival rate (present vs. absent: 63.8% vs. 65.2% (P = 0.782) and 62.5% vs. 65.3% (P = 0.606) respectively). The survival rate decreased signifi cantly in proportion to the duration of DIC before TM-alfa administration (P <0.001). More precisely, the 28-day survival rate was 66.4% when TM- alfa was injected on the same day that DIC was diagnosed, whereas it was 48.5% and 31.1% when TM-alfa was started 4 days later and 7 days or more after DIC was diagnosed, respectively. These diff erences were signifi cant (P = 0.033 and P <0.001, respectively).f p References 1. Holick MF: Vitamin D defi ciency. N Engl J Med 2007, 357:266-281.i 2. Lucidarme O, et al.: Incidence and risk factors of vitamin D defi ciency in critically ill patients: results from a prospective observational study. Intensive Care Med 2010, 36:1609-1611. 3. McKinney JD, et al.: Relationship between vitamin D status and ICU outcomes in veterans. J Am Med Dir Assoc 2011, 12:208-211.i 3. McKinney JD, et al.: Relationship between vitamin D status and ICU outcomes in veterans. J Am Med Dir Assoc 2011, 12:208-211.i 4. Higgins DM, et al.: Relationship of vitamin D defi ciency to clinical outcomes in critically ill patients. JPEN J Parenter Enteral Nutr 2012, 36:713–720. 4. Higgins DM, et al.: Relationship of vitamin D defi ciency to clinical outcomes in critically ill patients. JPEN J Parenter Enteral Nutr 2012, 36:713–720. i Conclusion The early administration of TM-alfa may be eff ective for patients with sepsis-induced DIC diagnosed based on the JAAM criteria. References Vitamin D and ICU outcome in septic patients: a diffi cult connection? Introduction Vitamin D, a secosteroid hormone, has roles in the optimal functioning of many organ systems and illnesses [1]. Recent reports show that most patients admitted to the ICU are vitamin D insuffi cient [2]. Introduction Vitamin D, a secosteroid hormone, has roles in the optimal functioning of many organ systems and illnesses [1]. Recent reports show that most patients admitted to the ICU are vitamin D insuffi cient [2]. Conclusion Application of this novel EHR screening tool to identify sepsis patients utilizes NLP applied to clinical documentation, providing greater clinical context than laboratory and vital sign screening alone. This database represents the entire sepsis acuity spectrum, allowing for a more granular description of the infectious process as well as subgroups with adequate sample size. The dataset collected as a patient-centered time series will enable future studies to focus on the trajectory of clinical deterioration and shock before its occurrence. Methods In a 10-bed general ICU at the emergency department of a tertiary teaching hospital in Florence (Italy), 71 medical patients with severe sepsis or septic shock (51% men, 40% women) admitted to the ICU between January 2013 and September 2013 were studied. Vitamin D levels were measured by radioimmunoassay at admission, as well Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S89 Table 1 (abstract P246). topic. Published data, summarized by this meta-analysis of randomized trials, show that statin therapy has no detrimental eff ect on survival in the overall population of adult septic patients. P248fi Dynamic myocardial depression in septic cardiomyopathy f y y Critical Care 2014, 18(Suppl 1):P247 (doi: 10.1186/cc13437) f y y Critical Care 2014, 18(Suppl 1):P247 (doi: 10.1186/cc13437) A meta-analysis of randomized controlled trials on the use of statins in septic patients 1. Saito H, et al.: J Thromb Haemost 2007, 5:31-41. 1. Saito H, et al.: J Thromb Haemost 2007, 5:31-41. 2. Mimuro J, et al.: Thromb Res 2013, 131:436-443. 2. Mimuro J, et al.: Thromb Res 2013, 131:436-443. G Landoni, L Nobile, D Febres, E Frati, N Villari, AL Di Prima, R Dossi, L Pasin Vita-Salute San Raff aele University, Milan, Italy G Landoni, L Nobile, D Febres, E Frati, N Villari, AL Di Prima, R Dossi, L Pasin Vita-Salute San Raff aele University, Milan, Italy G Landoni, L Nobile, D Febres, E Frati, N Villari, AL Di Prima, R Dossi, L Pasin Vita-Salute San Raff aele University, Milan, Italy Dynamic myocardial depression in septic cardiomyopathy A Darawshe, D Levingston, Y Haviv The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) Exclusion criteria were: duplicate publications; nonadult studies; no data on main outcomes.i Methods A single-center database investigates all patients who were admitted and treated for severe sepsis or septic shock in the ICU over a period of 2 years (November 2011 to October 2013), and who were discharged with a diagnosis of septic cardiomyopathy or new left ventricular dysfunction. The clinical, laboratory, echocardiography, and invasive measures, and clinical outcome were recorded. Results From the 105 patients that were investigated, 15 patients were found with septic cardiomyopathy. Septic cardiomyopathy is more prevalent among men (60%). Patients with septic cardiomyopathy have an increased prevalence of immune compromised disease (46%), and hypertension (40%). There was a need for mechanical respiratory support for 86% of patients. The improvement in cardiac function Results Data from 650 patients in fi ve randomized controlled studies were analyzed. Overall analysis showed no diff erence in mortality between patients receiving statins (44/322 (14%)) versus control (50/328 (15%)), RR = 0.90 (95% CI 0.65 to 1.26), P = 0.6.i Conclusion Scientifi c evidence for the role of statins in septic patients is still limited and larger randomized trials should be performed on this S90 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 diagnostic and therapeutic effi cacy of these CXRs is now known to be low [1]. Routine CXRs may only be benefi cial for certain indications and the discussion regarding these indications and the safety of abandoning routine CXRs is still continuing [2]. occurred at an average of 6.9 days. E. coli is the commonest bacterial pathogen (33%). Laboratory fi ndings show elevated liver enzyme and kidney function impairment in all patients. Thirty-three percent of patients were treated with N-acetyl cysteine, and 46% were treated with renal replacement therapy. High CRP was observed in all patients. Paroxysmal atrial fi brillation was diagnosed in 46%. Invasive measures in 50% of the patients have demonstrated high cardiac index (CI) and low systemic vascular resistant index (SVRI) on their admission, and 93% demonstrate low CI and high SVRI a few hours later. Hospitalization stay was between 3 and 42 days with an average of 14.6 days. Methods We prospectively included all patients who underwent major cardiac surgery in the year 2012. A direct postoperative CXR was performed only for certain specifi ed indications. Evaluation of early graft function in a case series of lung-transplanted patients J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, L Rosso, D Chiumello, F Valenza, L Gattinoni Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, L Rosso, D Chiumello, F Valenza, L Gattinoni Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, p L Rosso, D Chiumello, F Valenza, L Gattino Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) Introduction The aim of the study was to investigate early graft function in terms of biological and radiological variables in a case series of lung transplant (Ltx). Conclusion The strategy of daily routine CXRs for critically ill patients has developed from a common practice (63%) in 2006 [2] to a rare practice (7%) nowadays. Intensivists still assume the value of routine CXRs to be higher than the effi cacy that is reported in the literature. This might be due to the clinical value of negative fi ndings, which has not been studied before. Methods We performed a prospective analysis of patients that underwent Ltx at Fondazione IRCCS Cà Granda Policlinico of Milan from 1 December 2012 to 10 December 2013. Donors’ lung parameters were recorded. Recipients’ clinical data were collected daily and lung computed tomography (CT) at end expiration was performed within 7 days after Ltx. On the same day, bronchoalveolar lavage was collected [1]. Quantitative CT analysis was run on separate lungs for each patient. Results Out of 25 LTx, 12 paired data for donors and recipients were analyzed. P252 Results Of the 83 ICUs that were contacted, 69 (83%) responded to the survey. Only 7% of ICUs still perform daily routine CXRs for all patients while 65% of ICUs say never to perform CXRs on a routine basis. A daily meeting with a radiologist is established in the majority of ICUs and is judged to be important or even essential. The therapeutic effi cacy of routine CXRs was assumed by intensivists to be lower than 10% or to be between 10 and 20%. The effi cacy of on-demand CXRs was assumed to be between 10 and 60%. There is consensus between intensivists to perform a routine CXR after endotrachial intubation, chest tube placement or central venous catheterization. P251 Table 1 (abstract P252). Quantitative CT scan analysis on transplanted lungs Table 1 (abstract P252). Quantitative CT scan analysis on transplanted lungs Lung CT Mean ± SD (n = 16) Volume (ml) 1,639 ± 437 Weight (g) 761 ± 175 Hyper infl ated (%) 0.0 ± 0.0 Normal (%) 38.9 ± 17.1 Poorly (%) 29.4 ± 8.4 Not infl ated (%) 31.7 ± 14.9 P250i Signifi cant change in the practice of chest radiography in Dutch ICUs M Tolsma1, TA Rijpstra2, MJ Schultz3, NJ Van der Meer2 1University Medical Center Utrecht, the Netherlands; 2Amphia Hospital, Breda, the Netherlands; 3Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P250 (doi: 10.1186/cc13440) Conclusion Stating clear indications for CXRs following cardiac surgery increases the effi cacy of these CXRs and safely reduces the total number of CXRs signifi cantly. Introduction We performed a survey under Dutch intensivists on the current practice of chest radiography in their departments. Chest radiographs (CXRs) are obtained frequently in ICU patients, despite the diagnostic and therapeutic effi cacy of routine CXRs being known to be low. The discussion regarding specifi c indications for CXRs in critically ill patients and the safety of abandoning routine CXRs is still continuing [1]. Methods A web-based questionnaire was sent to the medical director of all adult ICUs in the Netherlands. This survey contained questions regarding ICU characteristics, ICU patients, daily CXR strategies, indications for routine CXRs and the practice of radiologic evaluation. Introduction We performed a survey under Dutch intensivists on the current practice of chest radiography in their departments. Chest radiographs (CXRs) are obtained frequently in ICU patients, despite the diagnostic and therapeutic effi cacy of routine CXRs being known to be low. The discussion regarding specifi c indications for CXRs in critically ill patients and the safety of abandoning routine CXRs is still continuing [1]. Methods A web-based questionnaire was sent to the medical director of all adult ICUs in the Netherlands. This survey contained questions regarding ICU characteristics, ICU patients, daily CXR strategies, indications for routine CXRs and the practice of radiologic evaluation. Results Of the 83 ICUs that were contacted, 69 (83%) responded to the survey. Only 7% of ICUs still perform daily routine CXRs for all patients while 65% of ICUs say never to perform CXRs on a routine basis. A daily meeting with a radiologist is established in the majority of ICUs and is judged to be important or even essential. The therapeutic effi cacy of routine CXRs was assumed by intensivists to be lower than 10% or to be between 10 and 20%. The effi cacy of on-demand CXRs was assumed to be between 10 and 60%. There is consensus between intensivists to perform a routine CXR after endotrachial intubation, chest tube placement or central venous catheterization. References 1. Tolsma M, Kröner A, van den Hombergh CL, et al.: The clinical value of routine chest radiographs in the fi rst 24 hours after cardiac surgery. Anesth Analg 2011, 112:139-142. 1. Tolsma M, Kröner A, van den Hombergh CL, et al.: The clinical value of routine chest radiographs in the fi rst 24 hours after cardiac surgery. Anesth Analg 2011, 112:139-142. g 2. Ganapathy A, Adhikari NK, Spiegelman J, Scales DC: Routine chest X-rays in intensive care units: a systematic review and meta-analysis. Crit Care 2012, 16:R68. References 1. Ganapathy A, Adhikari NK, Spiegelman J, Scales DC: Routine chest X-rays in intensive care units: a systematic review and meta-analysis. Crit Care 2012, 16:R68. doi: 10.1186/cc11321. 2. Graat ME, Hendrikse KA, Spronk PE, Korevaar JC, Stoker J, Schultz MJ: Chest radiography practice in critically ill patients: a postal survey in the Netherlands. BMC Med Imaging 2006, 18:6:8. Evaluation of early graft function in a case series of lung-transplanted patients Lung donors’ PaO2/FiO2 was 392 ± 118, Oto score was 5 ± 3, and ICU length of stay was 2 ± 1 days. Recipients’ age was 56 ± 11 years, and body mass index was 24 ± 4 kg/m2. Four patients received double Ltx, and the warm ischemia time was 85 ± 16 and 94 ± 14 respectively for right lung and left lung. When the CT scan was performed (on Dynamic myocardial depression in septic cardiomyopathy A Darawshe, D Levingston, Y Haviv The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) An on-demand CXR could be obtained during the postoperative period according to other specifi ed indications. For all patients who did not have a CXR taken before the morning of the fi rst postoperative day, a control CXR was then performed. All CXR fi ndings were noted and classifi ed, including whether or not they led to an intervention. Diagnostic and therapeutic effi cacy values were calculated. Conclusion Septic cardiomyopathy is more common among immune compromised patients. It is characterized by dynamic changes in the cardiac function based on echocardiography and invasive measures. A persistent hyperkinetic state was associated with high mortality rate. In addition to echocardiography follow-up, invasive monitoring even in their admission is of great importance for more eff ective and adequate treatment. fi Results A total of 1,351 patients were included who mainly underwent coronary artery bypass grafting, valve surgery or a combination of both. Eighteen percent of patients underwent a minimally invasive cardiac surgery. The diagnostic effi cacy for major abnormalities was clearly higher for the postoperative and on-demand CXRs, performed on indication, when compared with the next-morning routine CXR (6.7% and 6.9% vs. 2.9%) (P = 0.002). The therapeutic effi cacy was also clearly higher for the postoperative and on-demand CXRs (2.9% and 4.1%), while the need for intervention after the morning control CXR was now reduced to be minimal (0.6%) (P = 0.002). Stating clear indications for chest radiographs after cardiac surgery increases their effi cacy and safely reduces costs y p Lung CT Mean ± SD (n = 16) fi M Tolsma1, TA Rijpstra2, PM Rosseel2, M Bentala2, HA Dijkstra2, NJ Van der Meer2 1University Medical Center Utrecht, the Netherlands; 2Amphia Hospital, Breda, the Netherlands 1University Medical Center Utrecht, the Netherlands; 2Amphia Hospital, Breda, the Netherlands Critical Care 2014, 18(Suppl 1):P251 (doi: 10.1186/cc13441) Critical Care 2014, 18(Suppl 1):P251 (doi: 10.1186/cc13441) Introduction We investigated the effi cacy and safety of chest radiographs (CXRs) performed on specifi ed indications only, directly after cardiac surgery. CXRs in the ICU are frequently obtained routinely for postoperative cardiosurgical patients, despite the fact that the S91 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 day 4 ± 1), four patients were mechanically ventilated, PaO2/FiO2 was 270 ± 93 and PaCO2 was 44.6 ± 10.0 mmHg. Primary graft dysfunction grade 2 or 3 at 72 hours post Ltx was diagnosed in six patients. All patients were discharged from ICU. Table 1 presents quantitative analysis of transplanted lungs. The alveolar protein concentration and not aerated tissue weight are signifi cantly related (R2 =0.69; P = 0.001). Conclusion Alveolar protein concentration is related to morphological features of lungs early after Ltx. However, the early postoperative period when the rate of complications is highest has been scarcely examined. Our study aimed to compare the residual pulmonary function of pulmonary lobectomy patients after VATS and the standard thoracotomy approach in the immediate postoperative period. day 4 ± 1), four patients were mechanically ventilated, PaO2/FiO2 was 270 ± 93 and PaCO2 was 44.6 ± 10.0 mmHg. Primary graft dysfunction grade 2 or 3 at 72 hours post Ltx was diagnosed in six patients. All patients were discharged from ICU. Table 1 presents quantitative analysis of transplanted lungs. The alveolar protein concentration and not aerated tissue weight are signifi cantly related (R2 =0.69; P = 0.001). Conclusion Alveolar protein concentration is related to morphological features of lungs early after Ltx. R f p p p Methods This prospective study included 14 VATS and 13 thoracotomy lobectomy (THOR) patients (age 58.8 ± 10.9 vs. 59 ± 8.9 years, P = 0.96; preoperative FVC 3.4 ± 1 vs. 3.4 ± 0.95 l, P = 0.98 and preoperative FEV1 2.7 ± 0.96 vs. 2.7 ± 0.6 l, P = 0.9, respectively). All patients received standard surgical and postoperative care with standardized pain management including i.v. References 1. Fang WF, et al.: Application and comparison of scoring indices to predict outcomes in patients with healthcare-associated pneumonia. Crit Care 2011, 15:R32. 1. Fang WF, et al.: Application and comparison of scoring indices to predict outcomes in patients with healthcare-associated pneumonia. Crit Care 2011, 15:R32. fi y Methods We present preliminary data of a prospective observational ongoing study. Sixteen patients undergoing NIV treatment outside the ICU for ARF of any origin were evaluated with LUS at three times: before NIV application (T0), and at 5 minutes (T5) and 60 minutes (T60) of NIV treatment. US scan was performed in six regions for each emithorax. LUS patterns were defi ned as: consolidation (C); multiple coalescent B-lines (B+); multiple irregularly spaced B-lines (B) and normal aeration (A). A LUS-ReS [2] was calculated detecting changes in the US pattern when comparing T0 to T5 and T0 to T60 assessments. Outcome was defi ned as NIV failure in the case of tracheal intubation or death within 1 week from ARF outset, otherwise NIV success.i 2. Jarvis S, et al.: Combining the National Early Warning Score with an early warning score based on common laboratory test results better discriminates patients at risk of hospital mortality. In Rapid Response Systems and Medical Emergency Teams; May 13–14 2013; London. P253 Can laboratory blood tests be used to risk stratify patients admitted with pneumonia? L Apps, S Hutchinson, T Leary, L Perry Norfolk & Norwich University Hospitals NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P253 (doi: 10.1186/cc13443) P253 Can laboratory blood tests be used to risk stratify patients admitted with pneumonia? L Apps, S Hutchinson, T Leary, L Perry Norfolk & Norwich University Hospitals NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P253 (doi: 10.1186/cc13443) Lung ultrasound reaeration score: a useful tool to predict non-invasive ventilation eff ectiveness L Nobile, P Beccaria, M Zambon, L Cabrini, G Landoni, A Zangrillo Ospedale San Raff aele, Milan, Italy Critical Care 2014, 18(Suppl 1):P255 (doi: 10.1186/cc13445) Conclusion Observational results suggest an EWS based on laboratory tests can be used to risk stratify patients with pneumonia and could be used to treat those with higher scores more aggressively earlier in their illness. Further analysis is required to determine whether age is a contributing factor and how this may modify the EWS. We need to determine whether laboratory-based EWS risk stratifi cation can be used in isolation, or whether it contributes suffi ciently to an existing physiological EWS that a combined system would improve outcome in our patients. Introduction The aim of our study is to evaluate the lung ultrasound (LUS) reaeration score (ReS) as a predictive tool for non-invasive ventilation (NIV) effi cacy in general wards for acute respiratory failure (ARF) treatment. Even if ICUs are considered the safest place to perform NIV, a shortage of intensive beds has lead to NIV application outside the ICU. With appropriate patient selection, treatment-timing choice, adequate monitoring and staff training, NIV application in general wards can allow one to safely treat patients at an early stage with better cost-eff ectiveness [1]. Few data assessing the right tool to evaluate NIV treatment effi cacy are available. References 1. Cajipe MD, et al.: Am J Surg 2012, 204:607-612. 1. Cajipe MD, et al.: Am J Surg 2012, 204:607-612. 2 Ceppa DP et al : Ann Surg 2012 256:487-493 1. Cajipe MD, et al.: Am J Surg 2012, 204:607-612. 2. Ceppa DP, et al.: Ann Surg 2012, 256:487-493. 2. Ceppa DP, et al.: Ann Surg 2012, 256:487-493. pp , g , 3. Varela G, et al.: Eur J Cardiothor Surg 2006, 30:644-648. 3. Varela G, et al.: Eur J Cardiothor Surg 2006, 30:644-648. g Results Of 1,598 patients, 538 died during this admission. It is uncertain whether death was due to pneumonia as only admission diagnosis is recorded but overall mortality was 35%. Analysis of data showed a strongly positive relationship between increasing EWS and increased risk of mortality with a correlation coeffi cient of 0.97. Can laboratory blood tests be used to risk stratify patients admitted with pneumonia? L Apps, S Hutchinson, T Leary, L Perry L Apps, S Hutchinson, T Leary, L Perry Norfolk & Norwich University Hospitals NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P253 (doi: 10.1186/cc13443) Results The nFVC and nFEV1 values were signifi cantly higher in the VATS group in the fourth and eighth postoperative hours compared with the THOR group (84.3 ± 14.4 vs. 64.3 ± 23.4, P = 0.013 and 84 ± 18.8 vs. 64 ± 22, P = 0.017, respectively). There was no statistically signifi cant diff erence between the groups in the 24th, 48th and 72nd hours, although VATS patients showed higher values at each time points. The length of ICU stay was similarly 1 day, but the length of hospital stay was signifi cantly longer in the THOR group (5 (3 to 6) vs. 7 (3 to 42) days (median and range) (P = 0.011)). Introduction Our objective was to determine whether an Early Warning Score (EWS) based on laboratory tests could risk stratify patients admitted to the Norfolk & Norwich University Hospitals NHS Foundation Trust with pneumonia. Physiological EWS systems producing an escalated response to an increasing score are used eff ectively within the Trust, yet mortality is high in patients admitted with a diagnosis of pneumonia. The CURB-65 score may not be an eff ective risk stratifi cation tool for predicting mortality in the older patient nor to guide intensive care admission [1]. Jarvis and colleagues developed an EWS based on laboratory blood tests and used it in conjunction with physiological EWS to eff ectively risk stratify all medical patients [2]. g Conclusion Preoperative lung function prediction severely over- estimates the real lung capacity of lobectomy patients in the immediate postoperative period. VATS lobectomy seems more benefi cial from the point of early postoperative lung function. VATS lobectomy should be considered in patients with poor preoperative spirometry results to ensure better postoperative outcome. Methods Of 2,158 patients admitted with pneumonia during 12 months from August 2012, data were collected for 1,598 who had received the required blood tests. Data included dates of admission, discharge and death if appropriate, gender, haemoglobin (g/dl), white cell count (109/l), sodium (mmol/l), potassium (mmol/l), urea (mmol/l), creatinine (mmol/l) and albumin (g/l) on admission. A composite EWS was calculated and measured against outcome. Reference 1. Matute-Bello G, Liles WC, Radella F, Steinberg KP, Ruzinski JT, Jonas M, Chi EY, Hudson LD, Martin TR: Neutrophil apoptosis in the acute respiratory distress syndrome. Am J Respir Crit Care Med 1997, 156:1969-1977. P255 Lung ultrasound reaeration score: a useful tool to predict non-invasive ventilation eff ectiveness L Nobile, P Beccaria, M Zambon, L Cabrini, G Landoni, A Zangrillo Ospedale San Raff aele, Milan, Italy Critical Care 2014, 18(Suppl 1):P255 (doi: 10.1186/cc13445) Stating clear indications for chest radiographs after cardiac surgery increases their effi cacy and safely reduces costs diclofenac combined with epidural administration of bupivacaine and fentanyl. Spirometry was performed with a bedside MIR Spirolab II spirometer (Rome, Italy) preoperatively and 4, 8, 24, 48 and 72 hours after the surgery. FVC, FEV1, PaO2, PaCO2, complication rate, and length of ICU and hospital stay were recorded. Postoperatively measured FVC and FEV1 values were divided by preoperatively predicted values and multiplied by 100 to give the normalized FVC (nFVC) and FEV1 (nFEV1) [3].i Lung function in the immediate postoperative period after video- assisted thoracoscopic and thoracotomy pulmonary resection T Végh, R Nemes, B Fülesdi p T Végh, R Nemes, B Fülesdi University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Critical Care 2014, 18(Suppl 1):P254 (doi: 10.1186/cc13444) University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Critical Care 2014, 18(Suppl 1):P254 (doi: 10.1186/cc13444) Results NIV treatment failed in fi ve patients. Eleven patients have been successfully treated with NIV. Mean LUS-ReS (SD) at T0 to T5 was 0 (± 3.1) in group 0 and 2.5 (± 2.5) in group 1 (P = 0.15). Mean LUS-ReS (SD) at T0 to T60 was –1.2 (± 3.9) in group 0 and 4.2 (± 3.4) in group 1 (P = 0.03). ROC curves were obtained for the two LUS-ReS at T0 to Introduction Previous studies reported that video-assisted thoraco- scopic surgery (VATS) is more benefi cial for pulmonary lobectomy concerning the late postoperative period than open thoracotomy [1,2]. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S92 Figure 1 (abstract P255). Methods Physician sonographers accredited by the German Medical Ultrasound Society (DEGUM) for ultrasonography in surgery, anaesthesia or medicine were invited to participate in an online survey. Frequency of exposure to patients with suspected pneumothorax, frequency of LUS use, assessment of diagnostic accuracy of LUS for ruling-out or ruling-in pneumothorax and preferences regarding technical aspects were enquired about. Results Eighty-nine physicians responded. Average exposure to pneumothorax cases was 1/week. Fifty-fi ve per cent of respondents used LUS ‘always’ or ‘frequently’. Thirty-four per cent of physicians rated LUS as ‘always accurate’, and a further 54% as ‘accurate in a majority of cases’ in ruling out pneumothorax. Twenty-one per cent rated LUS as ‘always accurate’ and 69% as ‘accurate in a majority of cases’ in ruling in pneumothorax. Physicians reporting frequent exposure to pneumothorax patients used LUS in a higher proportion of cases (‘high caseload sonographers’) and were more confi dent to rule out pneumothorax (Figure 1). In total, 16 diff erent combinations of transducers, probe orientations and scanning modes were reported. Linear transducers, sagittal probe-orientation, and B-mode and M-mode scanning were most often selected (38%). g Conclusion Physicians’ use of LUS in the diagnosis of pneumothorax was modest. Assessment of diagnostic accuracy gave markedly lower scores than reported in clinical trials [1]. Correlation between frequency of exposure, likelihood of LUS usage and confi dence in diagnostic accuracy warrants further research into the nature of the learning curve. Lung function in the immediate postoperative period after video- assisted thoracoscopic and thoracotomy pulmonary resection T Végh, R Nemes, B Fülesdi Considerable variations regarding technical aspects of LUS refl ect the ambiguity of current recommendations [2]. More research is required to establish the most effi cient way of performing LUS for suspected pneumothorax and eff orts need to be made to promote its use in these scenarios. Figure 1 (abstract P255). Figure 1 (abstract P255). T5 (AUC 0.72) and T0 to T60 (AUC 0.83) (Figure 1). A LUS-ReS cutoff value of 0 can predict NIV eff ectiveness, with a sensibility of 91% and a specifi city of 80%.i pi y Conclusion If confi rmed, our preliminary results suggest that LUS-ReS could be a useful tool in predicting NIV eff ectiveness for ARF treatment. Caution has to be applied when interpreting our results considered the small amount of patients enrolled. References References 1. Alrajhi K, et al.: Test characteristics of ultrasonography for the detection of pneumothorax. A systematic review and meta-analysis. Chest 2012, 141:703-708. 1. Alrajhi K, et al.: Test characteristics of ultrasonography for the detection of pneumothorax. A systematic review and meta-analysis. Chest 2012, 141:703-708. 2. Volpicelli G, et al.: International Liaison Committee on Lung Ultrasound. International evidence-based recommendations for point-of-care lung ultrasound. Intensive Care Med 2012, 38:577-591. 2. Volpicelli G, et al.: International Liaison Committee on Lung Ultrasound. International evidence-based recommendations for point-of-care lung ultrasound. Intensive Care Med 2012, 38:577-591. 1. Cabrini L, et al.: Medical emergency team and non-invasive ventilation d f f l d 1. Cabrini L, et al.: Medical emergency team and non-invasive ventilation outside ICU for acute respiratory failure. Intensive Care Med 2009, 35:339-343. 2. Bouhemad B, et al.: Bedside ultrasound assessment of positive end- expiratory pressure-induced lung recruitment. Am J Respir Crit Care Med 2011, 183:341-347. Eff ects of sitting on the respiratory pattern, mechanics and work of breathing in mechanically ventilated patients F Ruiz-Ferron1, J Serrano-Simon2 1Complejo Hospitalario de Jaen, Spain; 2Hospital Reina Sofi a, Cordoba, Spain Critical Care 2014, 18(Suppl 1):P259 (doi: 10.1186/cc13449) g Results The mean age of the patients was 59 years, 32 of the patients were men. The mean (SD) diameter of the bronchi in SII patients measured was 3.9 (1.5) mm (range 0.9 to 9.0 mm). There were statistically signifi cant positive associations between wall thickening (expressed as T/D ratio) and luminal narrow (expressed as LA%) and the developed pneumonia (T/D ratio: R2 = 0.56, P <0.01 and LA%: R2 = 0.19, P = 0.005) and mechanical ventilation days (T/D ratio: R2 = 0.37, P <0.0001 and LA%: R2 = 0.32, P <0.001, respectively). No statistically signifi cant associations were identifi ed between T/D ratio or LA% and initial P/F ratio, infusion volume initial 24 hours, ICU stay days, and outcome. The mean T/D ratio and LA% were 0.25 (0.04) and 25.9% (7.6) for patients with SII and 0.35 (0.04) and 44.7% (5.6) for controls. g y F Ruiz-Ferron1, J Serrano-Simon2 1Complejo Hospitalario de Jaen, Spain; 2Hospital Reina Sofi a, Cordoba, Spain Critical Care 2014, 18(Suppl 1):P259 (doi: 10.1186/cc13449) Introduction The eff ect of sitting in an armchair on mechanically ventilated patients has not been studied enough. We study a group of patients ready for weaning for the respiratory pattern, mechanics and work of breathing during reclining in bed and after sitting in an armchair. Methods Thirteen patients who needed mechanical ventilation after 18 days (1 to 60) were studied during volume assist-control mechanical ventilation and spontaneous breathing (O2T, CPAP or PSV) in both positions. Airways, esophageal pressures and fl ow were registered for posterior analysis. Passive respiratory mechanics were measured by multiple linear regression methods, respiratory drive in esophageal pressure (P01) and respiratory eff ort using the pressure time product (PTP). Conclusion We have shown with the use of HRCT scanning on admission that patients with SII have airway wall thickening compared with normal controls. Furthermore, airfl ow obstruction due to bronchial wall edema related with developed pneumonia and mechanical ventilation days in SII patients. Results On controlled mechanical ventilation the respiratory system and chest wall elastance were signifi cantly higher in sitting compared with reclining positions (Ers 39 ± 24 vs. 33 ± 25 cmH2O/l and 10 ± 3 vs. 7 ± 3 cmH2O/l), respiratory resistances were similar (15 ± 3 vs. 14 ± 5 cmH2O/l/second) in both positions. Breathing pattern did not change signifi cantly: tidal volume (0.406 ± 0.108 vs. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods We prospectively studied 42 patients with a diagnosis of SII, according to visualized bronchoscopic fi ndings at admission, and 15 control subjects. The thoracic high-resolution computed tomography (HRCT) scan was obtained within a few hours of admission to our hospital. Airway wall dimensions were calculated using a validated method. The images were viewed on a workstation using a magnifi cation of ×5, and measurements of overall (D) and internal (L) diameter of the bronchi were made using electronic calipers, with wall thickness (T) being derived from these measurements (T = (D – L) / 2). Luminal area (Ai, mm2) and total airway wall area (Ao) were calculated from L and D, respectively, using the formula: A = r2. We used both the ratio of airway wall thickness to total diameter (T/D ratio) and the percentage luminal area (LA% = (Ai / Ao + Ai) × 100). Eff ects of sitting on the respiratory pattern, mechanics and work of breathing in mechanically ventilated patients F Ruiz-Ferron1, J Serrano-Simon2 1Complejo Hospitalario de Jaen, Spain; 2Hospital Reina Sofi a, Cordoba, Spain Critical Care 2014, 18(Suppl 1):P259 (doi: 10.1186/cc13449) 0.394 ± 0.118 l), inspiratory fl ow (0.74 ± 0.27 vs. 0.69 ± 0.23 l/second), inspiratory (0.97 ± 0.23 vs. 0.97 ± 0.28 seconds) and expiratory (1.43 ± 0.55 vs. 1.39 ± 0.44 seconds) times and respiratory frequency (27 ± 7 vs. 26 ± 7 bpm). Respiratory drive and eff ort tend to be higher in the sitting position, but not signifi cantly. P01: 3.7 ± 1.9 versus 3.0 ± 1.4 cmH2O, Δpes: 19 ± 10 versus 15 ± 8 cmH2O, PTPmin: 373 ± 192 versus 284 ± 162 cmH2O/secondminute. References Speelberg B, van Beers F: Artifi cial ventilation in the semi-recumbent position improves oxygenation and gas exchange [abstract]. Chest 2003, 124:203S. 2. van Beers F, Speelberg B: Eff ect of body positioning in ventilated obese patients [abstract]. In 18th Annual Congress of the European Society of Intensive Care Medicine; Amsterdam; September 25-28 2005. 3. van Beers F, Speelberg B: The eff ect of semi-recumbency in ventilated morbid obese patients [abstract]. In 19th Annual Congress of the European Society of Intensive Care Medicine, Barcelona; September 24-27 2006. P256 j y H Yamamura, T Yamamoto, S Kaga, K Kaneda, Y Mizobata H Yamamura, T Yamamoto, S Kaga, K Kaneda, Y Mizobata Osaka City University, Osaka, Japan Critical Care 2014, 18(Suppl 1):P257 (doi: 10.1186/cc13447) P256 Ultrasound in the diagnosis of pneumothorax: a survey of current practice T Berlet, T Fehr, T Merz Inselspital/University Hospital Bern, Switzerland Critical Care 2014, 18(Suppl 1):P256 (doi: 10.1186/cc13446) y y, , p Critical Care 2014, 18(Suppl 1):P257 (doi: 10.1186/cc13447 Introduction Smoke inhalation injury (SII) is progress to pulmonary edema, pneumonia, and acute respiratory distress syndrome. SII may cause bronchial mucosal edema; we hypothesized that narrowing of luminal air bronchus due to bronchial wall edema correlated with respiratory deterioration of SII patients. Introduction The purpose of this study was to survey current practice for the use of lung ultrasound (LUS) in the diagnosis of pneumothorax. Figure 1 (abstract P256). Accuracy of LUS in pneumothorax. S93 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Semi-upright position improves ventilation and oxygenation in mechanically ventilated intensive care patients F Van Beers, P Vos St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P258 (doi: 10.1186/cc13448) Semi-upright position improves ventilation and oxygenation in mechanically ventilated intensive care patients F Van Beers, P Vos St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P258 (doi: 10.1186/cc13448) Introduction A semi-upright position (45° position) in ventilated patients is recommended to prevent ventilator-associated pneumonia (VAP) and is one of the fi rst steps in progressive early mobility. We studied ventilated intensive care patients in a semi-upright position compared with a supine position to explore whether there was an improvement of oxygenation and ventilation. 2 Conclusion A sitting position for mechanically ventilated patients increased the rigidity of chest wall and the respiratory system. The eff ects of this mobilization must be evaluated because some patients show higher respiratory drive and eff ort in this position. yg Methods We retrospectively studied 60 patients in a mixed medical, surgical, neurological ICU during 2003 and 2007 [1-3]. In this study the eff ects of 45° position on the peripheral oxygen saturation (SpO2) and the end-tidal carbon dioxide (ETCO2) were measured. Body position was changed with a Total Care® bed (Hill-Rom) after results for the supine position (10°) were obtained. Half an hour after the body position was changed, measurements were taken, which included SpO2 and ETCO2. The results of body position change in the individual patients were analysed with paired-samples t test. A signifi cance level <0.05 was considered signifi cant. P260 The win ratio method: a novel hierarchical endpoint for pneumonia trials in patients on mechanical ventilation A Montgomery1, T Abuan1, M Kollef2 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) The win ratio method: a novel hierarchical endpoint for pneumonia trials in patients on mechanical ventilation A Montgomery1, T Abuan1, M Kollef2 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) Physiologic comparison between NAVA, PAV+ and PSV in critically ill patients E Ak i ki G P i i ki E K dili D G l E Akoumianaki, G Prinianakis, E Kondili, D Georgopoulos University Hospital of Heraklion, Greece Critical Care 2014, 18(Suppl 1):P264 (doi: 10.1186/cc13454) y Methods We retrospectively reviewed charts of all patients admitted through the ED with a diagnosis of pneumonia requiring intubation in the fi rst 24 hours between January 2011 and November 2012. Patients were classifi ed as SC collected or not collected. We recorded demographic data, SC results, antibiotic choice and de-escalation, ventilator-free days (up to day 14), and mortality in ICU and hospital. Inferential statistics were performed using SPSS version 20.0, with P <0.05 considered signifi cant. Introduction The aim of the present study was to compare, in a group of diffi cult to wean critically ill patients, the short-term eff ects of PSV, PAV+ and NAVA on breathing pattern, patient eff ort and patient– ventilator interaction. Introduction The aim of the present study was to compare, in a group of diffi cult to wean critically ill patients, the short-term eff ects of PSV, PAV+ and NAVA on breathing pattern, patient eff ort and patient– ventilator interaction. Methods Seventeen patients were studied during NAVA, PAV+ and PSV with and without artifi cial increase in ventilator demands (challenge) using either dead space (DS, n = 10) or chest elastic load (CL, n = 7) application. Airway and transdiaphragmatic (Pdi) pressures, electrical activity of the diaphragm (EAdi), volume and fl ow were measured breath by breath, while inspiratory integral of Pdi (PTPPdi) and EAdi (EAdi) were calculated. i Results Of 50 patients we reviewed, 43 (86%) were intubated in the ED, 45 had SC ordered (only eight (18%) by ED physicians), and 37 (74%) had SC collected. There was no diff erence in age, gender or severity of illness as measured by APACHE score between the two groups. ICU mortality was lower in the SC collected group (24% vs. 69%, P = 0.007), as was hospital mortality (30% vs. 77%, P = 0.007) and antibiotics were de-escalated more often (89% vs. 8%, P <0.001). Patient with SC collected showed a trend toward signifi cantly more ventilator-free days (6.5 vs. 0, P = 0.053). Results At resting conditions all modes provided equal support as indicated by a similar PTPPdi per breath, per minute and per liter of ventilation. Physiologic comparison between NAVA, PAV+ and PSV in critically ill patients E Ak i ki G P i i ki E K dili D G l Apart from triggering delay, which with and without the challenge was signifi cantly higher with PAV+ than that with NAVA and PSV, patient–ventilatory synchrony did not diff er among modes. Independent of challenging conditions, inspiratory eff ort to trigger the ventilator was signifi cantly higher with PAV+ than with NAVA and PSV. Compared with PSV, PAV+ and NAVA favored a more variable breathing pattern as indicated by the signifi cantly higher coeffi cient of variation of tidal volume (VT). CL increased PTPPdi signifi cantly less with PAV+ than with PSV and NAVA, while the increase of PTPPdi after DS did not diff er among modes. The relationship between VT and PTPPdi was weaker with NAVA (median (IQR) r2 = 25.6% (2.7 to 58.1%)) than with PAV+ (55.6% (34.4 to 61.6%)) and PSV (53.9% (23.2 to 77.4%)) on account of a poor EAdi–PTPPdi relationship (r2 = 16.2% (1.4 to 30.9%)) during NAVA. Conclusion Sputum cultures were rarely ordered by ED physicians, and when not obtained in intubated patients with pneumonia, ICU and hospital mortality was higher, there was less antibiotic de-escalation, and a trend toward fewer ventilator-free days. Eff orts to improve collection of sputum cultures in these patients are warranted. Failure to obtain admission sputum culture is associated with higher mortality and fewer ventilator-free days for intubated pneumonia patients: a quality improvement project f Conclusion The results of this study revealed that among the NIMV nonresponsive hypercapnic patients, nonventilatory factors such as thyroid dysfunction and increased dead space ventilation should be considered. Y Mahal, K Smith, R Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P261 (doi: 10.1186/cc13451) Introduction The primary objective was to assess the impact of failure to obtain sputum culture (SC) among patients requiring intubation for pneumonia. For patients admitted to an ICU with severe pneumonia, guidelines recommend obtaining a lower respiratory tract sample for culture. Our experience suggested this is rarely ordered from the emergency department (ED). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 These simulation results assumed results within pairs were uncorrelated. If a positive correlation for each endpoint within pairs is assumed, power for the win ratio endpoint increases. g y Results Among the 41 patients, 28 (68%) were classifi ed as Group 1 and 13 (32%) as Group 2. No diff erences were identifi ed among the ventilatory parameters of the two groups (P >0.05). Patients in Group 1 were younger, had higher admission PaCO2 levels, higher free T3 levels and ejection fraction in echocardiography (P <0.05). VD/VT values of Group 1 measured at admission and on the second and third days of NIMV were lower than Group 2 (P <0.05). Similarly, they had lower FEV1 percent predicted values in the stable period and on the fi rst and second days of NIMV (P <0.05). FEV1/FVC was lower in Group 1 when measured in the stable period and on the third and fourth days of NIMV. In Group 2, CRP values measured during the fi rst day (P = 0.014) and third day (P = 0.031) of NIMV were identifi ed as higher. In multivariate regression analysis, admission PaCO2 (OR: 1.59, 95% CI: 1.1 to 2.3, P = 0.014), free T3 (OR: 12, 95% CI: 1.51 to 101, P = 0.019), and VD/VT (OR: 1.23, 95% CI: 1.01 to 1.52, P = 0.048) were identifi ed as independent risk factors aff ecting NIMV success. Conclusion The win ratio method is both clinically meaningful and straightforward to explain. This method could provide a new approach to powering both superiority and non-inferiority trials of novel antibiotics. In particular, this method allows for well-powered phase 2 trials, and potentially decreases the required size of phase 3 trials. Reference 1. Pocock et al.: Eur Heart J 2012, 22:176-182. P261 P264 Physiologic comparison between NAVA, PAV+ and PSV in critically ill patients Physiologic comparison between NAVA, PAV+ and PSV in criticall ill patients E Akoumianaki, G Prinianakis, E Kondili, D Georgopoulos University Hospital of Heraklion, Greece Critical Care 2014, 18(Suppl 1):P264 (doi: 10.1186/cc13454) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A total of 41 patients were included in this prospective cohort study, who were followed for at least 96 hours in the ICU between January 2010 and November 2012 with the diagnosis of hypercapnic respiratory failure. Patients with ≥10 mmHg decrease in PaCO2 in the fi rst 72 hours were accepted as successful (Group 1) and those without this decrease were accepted as unsuccessful (Group 2). Among the patients with similar NIMV application characteristics, the eff ect of age, APACHE II score, infection, bronchospasm (daily respiratory function tests were performed with portable spirometry), heart failure, thyroid dysfunction and physiologic dead space ventilation (VD/VT) on success were evaluated. In statistical analysis, t test, Mann–Whitney U test and regression analysis were used. loser, or is a draw. The pair is fi rst compared on mortality; if no diff erence, then ventilator-free days (VFD) are compared. If the outcomes are the same for both endpoints, a draw results. Active versus placebo groups will then be compared using the win ratio, defi ned as the number of pairs in which the active group was the winner divided by the number of pairs that did not result in a draw. We examined sample size and power characteristics of the win ratio endpoint using trial simulations. Results Assuming a 15% 28-day mortality rate in the active arm and 20% in the control arm, to have 80% power with a two-tailed 0.05-level test for mortality would require 906 subjects per arm. Under the same assumptions with a diff erence in mean VFDs of 3 favoring the active arm (with common SD of 6), 130 subjects per arm provides 80% power. In approximately 32% of simulations, the win ratio result for each pair was determined by mortality. These simulation results assumed results within pairs were uncorrelated. If a positive correlation for each endpoint within pairs is assumed, power for the win ratio endpoint increases. p p g Results Assuming a 15% 28-day mortality rate in the active arm and 20% in the control arm, to have 80% power with a two-tailed 0.05-level test for mortality would require 906 subjects per arm. Under the same assumptions with a diff erence in mean VFDs of 3 favoring the active arm (with common SD of 6), 130 subjects per arm provides 80% power. In approximately 32% of simulations, the win ratio result for each pair was determined by mortality. The win ratio method: a novel hierarchical endpoint for pneumonia trials in patients on mechanical ventilation g y 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) i Results Mean SpO2 supine was 96.55% ± SD 2.404 versus mean SpO2 semi-upright 97.4% ± SD 2.423, and mean ET-CO2 supine was 4.62% ± SD 1.988 versus mean ET-CO2 semi-upright 4.31% ± SD 1.060. The SpO2 (P <0.0001) and the ETCO2 (P <0.0001) improved signifi cantly for the 45° position compared with <10° position.i Introduction Development of novel antibiotics for VAP is hampered by the need for large phase 3 trials with mortality endpoints. With all- cause mortality rates decreasing, the large sample size for these trials, in particular for non-inferiority trials, has become a barrier to rapid drug development. Recently, a hierarchical approach to defi ning a composite endpoint for CV trials (win ratio method) has been described [1]. Conclusion We demonstrated a signifi cant increase in oxygen saturation and a signifi cant decrease in end-tidal CO2 when the head of the bed was elevated during mechanical ventilation. We believe positional therapy in intensive care patients is very important. The semi-upright position is an easy, eff ective and safe treatment in ICU patients. This position is eff ective in the bundle prevention of VAP, is the fi rst step in progressive early mobility and is also eff ective in oxygenation and ventilation in mechanically ventilated patients. This clinical benefi t of head-of-bed elevation >30° must lead to a standard of care in mechanically ventilated patients. Since 2009 the semi-upright position is a standard of care in our hospital. Methods We have adapted this approach in an ongoing superiority trial (Clinicaltrials.gov NCT01969799) comparing adjuvant use of a combination of fosfomycin and amikacin aerosol delivered by the PARI eFlow® Inline nebulizer in patients with Gram-negative pneumonia who are on mechanical ventilation. Both groups are receiving standard- of-care i.v. antibiotics. Patients from the active and placebo groups are matched in pairs based on presence of MDR Gram-negative bacteria, and disease severity by APACHE II score. Each pair has a winner and a S94 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Determining the mechanical ventilation mode and pressure support combination that is best compatible with the rapid shallow breathing index calculated in spontaneous ventilation Determining the mechanical ventilation mode and pressure support combination that is best compatible with the rapid shallow breathing index calculated in spontaneous ventilation S Demirtas Yilmaz, G Gürsel, M Aydogdu , , y g Gazi University Medical Faculty, Ankara, Turkey y y y Critical Care 2014, 18(Suppl 1):P267 (doi: 10.1186/cc13457) Introduction Weaning from the mechanical ventilation (MV) composes about 40 to 50% of the total length of MV. Besides, there is no single reliable parameter indicating that patient will tolerate extubation safely. The rapid shallow breathing index (RSBI) is relatively the best predictive parameter for the initial assessment of readiness for discontinuation of MV support. But evaluation of the RSBI is valuable during T-tube ventilation; and in clinical practice it is not always possible to perform this assessment. In this study, we aimed to determine the best MV mode and pressure combinations that can predict successful RSBI closest to values calculated in spontaneous ventilation and estimate the patients’ readiness for weaning. Results When we analyzed patients according to their body mass indexes (BMI), pH and pCO2 values of the patients with BMI ≤30 showed a greater improvement at all three measurements in the AVAPS compared with BIPAP (P <0.001). When patient compliance was examined, the number of patients regarded as comfortable in the BIPAP period was 20 (66.7%), but this fi gure was 25 (83.3%) for the AVAPS. Conclusion Patient comfort was higher and need for sedation was lower in AVAPS. According to the results obtained from this study, the AVAPS had positive eff ects on pH, gas variation and patient comfort; therefore, it can be confi dently used in clinical practice. Results When we analyzed patients according to their body mass indexes (BMI), pH and pCO2 values of the patients with BMI ≤30 showed a greater improvement at all three measurements in the AVAPS compared with BIPAP (P <0.001). When patient compliance was examined, the number of patients regarded as comfortable in the BIPAP period was 20 (66.7%), but this fi gure was 25 (83.3%) for the AVAPS. Methods In this prospective cohort study, 25 mechanically ventilated patients were included. After 24 hours of MV, if the patients can successfully pass the daily screening test a spontaneous breathing trial (SBT) was initiated. RSBI and other weaning parameters were calculated in diff erent combinations (PS:5 PEEP:5, PS:0 PEEP:5, PS:5 PEEP:0, PS:0 PEEP:0) before T-tube trial in all patients. Oxygenation index outperforms the P/F ratio for mortality prediction Oxygenation index outperforms the P/F ratio for mortality prediction K Davies1, C Bourdeaux1, T Peiris2, T Gould1 1Bristol Royal Infi rmary, Bristol, UK; 2University of Southampton, UK Critical Care 2014, 18(Suppl 1):P266 (doi: 10.1186/cc13456) p K Davies1, C Bourdeaux1, T Peiris2, T Gould1 1Bristol Royal Infi rmary, Bristol, UK; 2University of Southampton, UK Critical Care 2014, 18(Suppl 1):P266 (doi: 10.1186/cc13456) p K Davies1, C Bourdeaux1, T Peiris2, T Gould1 1Bristol Royal Infi rmary, Bristol, UK; 2University of Southampton, UK Critical Care 2014, 18(Suppl 1):P266 (doi: 10.1186/cc13456) Introduction The P/F ratio is widely used clinically and as part of research to categorise severity of respiratory failure [1]. However, no account is made of an important determinant of oxygenation; mean airway pressure (MAP). The oxygenation index (OI) incorporates the MAP and has been suggested as a more accurate means of determining severity of respiratory failure [2]. In addition, the optimal time for this assessment is unclear. We sought to answer these questions by analysing a large database of patient and ventilator data.i Conclusion Although there was a correlation between RSBI measured in the T-tube and RSBI measured in diff erent mode and pressure combinations, especially with the combination of PS:5 PEEP:0, a threshold value for RSBI cannot be detected during MV to predict SBT success. Does average volume-assured pressure support make any diff erence compared with BIPAP? Does average volume-assured pressure support make any diff erence compared with BIPAP? f p G Canpolat, A Ozgultekin, G Turan, A Iskender, E Adıyeke, O Ekinci Haydarpasa Numune Teaching and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P265 (doi: 10.1186/cc13455) p g Conclusion Our analysis suggests that the OI is a more sensitive descriptor of the severity of respiratory failure than the P/F ratio and that this calculation should be performed using data from the fi rst 12 hours of ventilation. Introduction Average volume-assured pressure support (AVAPS) has been developed to ensure a more fi xed tidal volume along with the convenience and advantages of pressure support ventilation. In this study we compared the AVAPS with the BIPAP. References y p Methods Approval was obtained from the hospital’s ethics committee for the study. Thirty-three patients over 18 years of age with acute respiratory failure as a result of either internal or surgical reasons were included in the study. This study was conducted with the Philips V 60 ventilator, which includes both BIPAP and AVAPS mode. The implementation protocol for non-invasive ventilation (NIV) included, fi rstly, a 2-hour BIPAP application (Period Bi) and then, without inter- ruption, a 2-hour AVAPS application (Period AV). After measuring the basal blood gas analysis, patients were informed of how NIV would be practiced and what function it would have. In BIPAP mode, the ventilator parameters were adjusted as follows; EPAP: 5 to 7 cmH2O, IPAP: 15 to 20 cmH2O. Patient comfort was analyzed with a scale ranging from 0 to 2 (0: compatible, 1: medium-compatible, 2: noncompatible). During BIPAP ventilation, levels of arterial blood gases (pH, pO2, pCO2 and SPO2), comfort scale and hemodynamic data were recorded at the 30th minute, fi rst hour and second hour. After the patient was monitored for 2 hours in BIPAP mode, the mode was changed to the AVAPS by setting the required rates without removing the mask. EPAP settings were adjusted as follows for AVAPS: 5 to 7 cmH2O, Pmin to max: 10 to 25 cmH2O, tidal volume: 6 to 8 ml/kg. As in the BIPAP mode, we analyzed and recorded the rates of arterial blood gases, comfort scale and hemodynamic parameters at the 30th minute, fi rst hour and second hour. In case of agitation that prevents NIV, patients were sedated by dexmedetomidine. 1. Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, et al.: Acute respiratory distress syndrome: the Berlin Defi nition. JAMA 2012, 307:2526-2533. 1. Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, et al.: Acute respiratory distress syndrome: the Berlin Defi nition. JAMA 2012, 307:2526-2533. 2. Seeley E, McAuley DF, Eisner M, Miletin M, Matthay MA, Kallet RH: Predictors of mortality in acute lung injury during the era of lung protective ventilation. Thorax 2008, 63:994-998. 2. Seeley E, McAuley DF, Eisner M, Miletin M, Matthay MA, Kallet RH: Predictors of mortality in acute lung injury during the era of lung protective ventilation. Thorax 2008, 63:994-998. Nonventilatory factors aff ecting noninvasive mechanical ventilation success in hypercapnic critical care patients yp p p S Demir, Y Aldağ, M Aydogdu, G Gürsel yp p p S Demir, Y Aldağ, M Aydogdu, G Gürsel ğ y g Gazi University Medical Faculty, Ankara, Turkey Gazi University Medical Faculty, Ankara, Turkey y y y Critical Care 2014, 18(Suppl 1):P263 (doi: 10.1186/cc13453) Conclusion Compared with PSV proportional modes favored breathing variability, while in the face of changing respiratory system mechanics PAV+ might be superior. However, signifi cant drawbacks of both NAVA and PAV+ limit the eff ectiveness of these modes to proportionally assist the inspiratory eff ort. Critical Care 2014, 18(Suppl 1):P263 (doi: 10.1186/cc13453) Introduction The aim was to determine the nonventilatory factors that aff ect the noninvasive mechanical ventilation (NIMV) success in patients with hypercapnic respiratory failure. S95 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P265 In comparison with survivors, nonsurvivors were older, with higher OI and 24-hour SOFA scores and lower P/F ratios. The optimal time for calculation of both OI and P/F ratio for mortality prediction is the fi rst 12 hours of ventilation. The models using worst OI are better predictors of ICU and hospital mortality than those using worst P/F ratio (area under receiver operating curve 0.840 vs. 0.822). In comparison with survivors, nonsurvivors were older, with higher OI and 24-hour SOFA scores and lower P/F ratios. The optimal time for calculation of both OI and P/F ratio for mortality prediction is the fi rst 12 hours of ventilation. The models using worst OI are better predictors of ICU and hospital mortality than those using worst P/F ratio (area under receiver operating curve 0.840 vs. 0.822). P265 Does average volume-assured pressure support make any diff erence compared with BIPAP? G Canpolat, A Ozgultekin, G Turan, A Iskender, E Adıyeke, O Ekinci Haydarpasa Numune Teaching and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P265 (doi: 10.1186/cc13455) Determining the mechanical ventilation mode and pressure support combination that is best compatible with the rapid shallow breathing index calculated in spontaneous ventilation Measurements in the spontaneous ventilation was performed with the COSMOPLUS Novometrix device that has both capnography and respiratory monitorisation function; other measurements were performed with ventilators. i Conclusion Patient comfort was higher and need for sedation was lower in AVAPS. According to the results obtained from this study, the AVAPS had positive eff ects on pH, gas variation and patient comfort; therefore, it can be confi dently used in clinical practice. Results The mean age of the study group was 73 ± 10 years; 11 of them were female and mean APACHE II score was 19 ± 6. RSBI did not diff er signifi cantly between spontaneous mode and other combinations, but the best correlation with spontaneous mode was found with PS:5 PEEP:0 (P = 0.0001, r = 0.719), and the worst with PS:0 PEEP:5 combination. RSBI calculated in each combination showed no predictive value for weaning success. Respiration rate (f) was higher in the SBT failure group than the SBT success group. When measured at PS:0 PEEP:5 and PS:5 PEEP:0 combinations, the threshold value of f was found to be 27/minute (P = 0.03). P268 P268 New setting of neurally adjusted ventilatory assist during mask noninvasive ventilation F Longhini1, C Pan2, G Cammarota1, R Vaschetto1, J Xie2, L Liu2, Y Yian2, F Della Corte1, P Navalesi1, H Qiu2 1Università del Piemonte Orientale, Novara, Italy; 2Southeast University, Nanjing, China Critical Care 2014, 18(Suppl 1):P268 (doi: 10.1186/cc13458) P268 New setting of neurally adjusted ventilatory assist during mask noninvasive ventilation F Longhini1, C Pan2, G Cammarota1, R Vaschetto1, J Xie2, L Liu2, Y Yian2, F Della Corte1, P Navalesi1, H Qiu2 1Università del Piemonte Orientale, Novara, Italy; 2Southeast University, Nanjing, China Critical Care 2014, 18(Suppl 1):P268 (doi: 10.1186/cc13458) y g g Methods The ICU of the Bristol Royal Infi rmary has used an electronic clinical information system (CIS) since 2008, with every hour of care available for analysis as a result. Ventilated patients were identifi ed, the P/F ratio and OI were calculated and the worst values for these determined for four time periods (fi rst 12, 24, 36 and 48 hours of ventilation). Logistic regression analysis was used to create models to predict unit and hospital mortality. y Results Data for over 150,000 hours of care in 4,886 patients was available for analysis. Excluding nonventilated patients and those transferred ventilated from another ICU, 2,156 patients provided data. Introduction Noninvasive ventilation through a mask is commonly applied in pressure support ventilation (nPSV). Recent studies S96 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 showed that noninvasive neurally adjusted ventilatory assist (nNAVA) improves patient–ventilator interaction and synchrony. More recently we described a new setting for nNAVA (nNAVA15) able to reduce the peak of electrical activity of the diaphragm (EAdipeak) and dyspnea (assessed by a visual analogue scale, VASd), compared with both nPSV and nNAVA, in patients undergoing NIV through a helmet, by improving the rate pressurization. We therefore designed a physiological study to evaluate and compare the eff ects of nNAVA15 with nPSV and nNAVA on VASd, EAdipeak, pressurization rate and arterial blood gases (ABGs). P268 Methods Fourteen patients undergoing noninvasive ventilation because of acute respiratory failure underwent three randomized 30-minute trials: nPSV (inspiratory support above positive end- expiratory pressure (PEEP) ≥8 cmH2O, fastest rate of pressurization); nNAVA (NAVA level to achieve a comparable EAdipeak as during nPSV); nNAVA15 (NAVA level at 15 cmH2O/μV and the maximum inspiratory airway pressure (Paw) set at the value corresponding to PEEP + inspiratory support during nPSV). Oxygen inspiratory fraction and PEEP remained unmodifi ed throughout the study period. The last minute of each trial was analyzed. Paw-time products of the initial 200 ms from the onset of ventilator pressurization (PTP200), of the initial 300 and 500 ms from the onset of the EAdi swing indexed to the ideal PTP (PTP300i and PTP500i, respectively), and of the triggering area (PTPt) were computed. ABGs and VASd were assessed at the end of each trial. Results nNAVA15 reduced VASd (3.0 (2.0; 3.0)), compared with both nPSV (5.0 (4.0; 5.2)) and nNAVA (4.0 (3.0; 5.0)) (P <0.001), without aff ecting ABGs and EAdipeak. nNAVA15 improved PTP300i and PTP500i (42% (32.5; 46.5) and 63% (54; 68)%, respectively) compared with nPSV (25 (4; 33)% and 44 (23; 52)%, P <0.001) and nNAVA (25 (20; 34)% and 46 (33; 57)%, P <0.001). PTP200 was lower in nNAVA (62 (46; 82) cmH2Osecond) with respect to both nPSV (87 (77; 112) cmH2Osecond) and nNAVA15 (85 (70; 127) cmH2Osecond) (P = 0.001). PTPt was signifi cantly improved by both nNAVA (–00.9 (–3.2; –0.2) cmH2Osecond) and nNAVA15 (–0.6 (–2.3; –0.2) cmH2Osecond) as opposed to nPSV (–9.4 (–12.3; –5.9) cmH2Osecond, P <0.001). Results hNAVA15 reduced the EAdipeak (10.2 (7.1; 16.2) μV) with respect to both hPSV (16.9 (12.7; 19.8) μV, P <0.001) and hNAVA (15.3 (10.7; 18.8) μV, P <0.001), while decreasing VASd (3.0 (3.0; 4.0) in hPSV, 3.0 (2.0; 4.0) in hNAVA and 1.0 (1.0; 2.0) in hNAVA15; P <0.01). PTP200 and PTP500i were improved by hNAVA15 (36 (28; 57) cmH2Osecond and 40 (30; 47)%, respectively) compared with hPSV (31 (24; 45) cmH2Osecond and 17 (9; 26)%, respectively) and hNAVA (23 (16; 30) cmH2Osecond and 23 (17; 37)%, respectively) (P <0.01). PTPt was lower in hNAVA15 (2.9 (1.6; 4.4) cmH2Osecond, P <0.01) compared with both hPSV and hNAVA, and lower in hNAVA (6.0 (2.7; 11.6) cmH2Osecond), compared with hPSV (18.5 (11.2; 22.5) cmH2Osecond, P <0.01). ABGs were no diff erent between trials. P268 f Conclusion In comparison with hPSV and hNAVA, hNAVA15 signifi cantly reduced EAdipeak and VASd, improving the pressurization and triggering performance, without aff ecting ABGs. Reference 1. Cammarota G, et al.: Intensive Care Med 2011, 37:1943-1950. 1. Cammarota G, et al.: Intensive Care Med 2011, 37:1943-1950. P270 Is neurally adjusted ventilatory assist feasible during anesthesia? F Campoccia Jalde, P Sackey, P Radell, M Wallin Karolinska University Hospital Solna, Sweden Critical Care 2014, 18(Suppl 1):P270 (doi: 10.1186/cc13460) Is neurally adjusted ventilatory assist feasible during anesthesia? F Campoccia Jalde, P Sackey, P Radell, M Wallin Karolinska University Hospital Solna, Sweden Critical Care 2014, 18(Suppl 1):P270 (doi: 10.1186/cc13460) Introduction Neurally adjusted ventilatory assist (NAVA) has so far been used in minimally sedated intensive care patients. NAVA has not been applied in patients in the operation room. The eff ect of diff erent sedatives/anesthetics on the electrical activity of the diaphragm (Edi) has not so far been studied. The aim of our study was to compare the eff ect of sevofl urane and propofol on the Edi signal and breathing pattern during sedation and anesthesia and also in combination with remifentanil. Methods A randomized cross-over study comparing sevofl urane and propofol sedation and anesthesia in 10 juvenile pigs. Remifentanil 0.1 μg/kg/minute was added after a period of anesthetic agent administration. The animals were ventilated with NAVA with fi xed level throughout the study. Respiratory variables were measured for the last 5 minutes of each 15-minute exposure. 2 Conclusion Compared with nPSV and nNAVA, nNAVA15 through a mask reduces VASd, assuring an optimal pressurization rate and triggering performance, without aff ecting the breathing pattern, neural drive and ABGs. Results The Edi signal and spontaneous breathing were preserved with both anesthetics. The breathing variability, expressed as the coeffi cient of variation (SD/mean) of the tidal volume (CVvt), was high with both drugs. CVvt was greater during with propofol than with sevofl urane (CVvt 32 vs. 18% during sedation and CVvt 23 vs. 14% during anesthesia). The frequency of sighs was higher with propofol both during sedation (29 vs. 12 sighs/hour) and anesthesia (21 vs. 1 sighs/hour) than with sevofl urane. A new setting to improve noninvasive neurally adjusted ventilatory assist by helmet A new setting to improve noninvasive neurally adjusted ventilatory assist by helmet y F Longhini, G Cammarota, C Olivieri, R Perucca, R Vaschetto, D Colombo, A Messina, F Della Corte, P Navalesi Università del Piemonte Orientale, Novara, Italy Critical Care 2014, 18(Suppl 1):P269 (doi: 10.1186/cc13459) l Conclusion NAVA can be applied during propofol and sevofl urane anesthesia in pigs, with well-preserved Edi and spontaneous breathing. The natural variability is maintained with NAVA even during anesthesia. In contrast to sevofl urane, propofol sedation and anesthesia is associated with a high frequency of sighs and post-sigh apneas, probably due to a centrally induced mechanism. Our data warrant studies of NAVA in humans undergoing anesthesia and surgery when neuromuscular blockade is not required. Introduction Noninvasive neurally adjusted ventilatory assist by helmet (hNAVA) was shown to improve, compared with pressure support ventilation by helmet (hPSV), patient–ventilator interaction and synchrony in patients with acute respiratory failure without aff ecting peak electrical activity of the diaphragm (EAdipeak) [1]. Recently, a new helmet is available, which improves pressurization during hPSV. We propose a new setting of hNAVA (hNAVA15) to achieve further improvement. We compare hPSV, hNAVA and hNAVA15, all delivered using the new helmet, with respect to patient’s dyspnea, assessed by a visual analogue scale (VASd), arterial blood gases (ABGs), EAdipeak, rate of ventilator pressurization and triggering performance. P271 P271 Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial A Leme1, L Hajjar1, M Amato1, J Fukushima1, C Hashizume1, E Nozawa1, E Osawa1, R Nakamura1, J Almeida2, R Ianotti1, J Auler Jr1, F Galas1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P271 (doi: 10.1186/cc13461) Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial Using respiratory data from an animal study we suggest a classifi cation into resistance shape categories based on the slope of the R(V) profi les. g Results A total of 320 patients were enrolled in the study, 163 patients in the standard protocol group and 157 in the intensive alveolar recruitment group. Patients of the interventional group presented a higher incidence of pneumonia than patients for the control group (5 (3.3%) vs. 19 (22%), P = 0.004). The length of the hospital stay was shorter among patients receiving intensive alveolar recruitment than among those receiving standard care (10.9 (9.9 to 11.9) vs. 12.4 days (11.3 to 13.6); P = 0.045). There was no diff erence between groups according to extrapulmonary complications and mortality. i Methods Fifteen pigs with lavage-induced lung damage were ventilated at two PEEP levels (0 and 12 mbar) and three tidal volumes (8, 12 and 16 ml/kg bodyweight). Compliance (C(V)) and resistance (R(V)) profi les for each individual animal and ventilation setting were calculated from respiratory data using the gliding-SLICE method [3]. C(V) profi les were associated with one of the six suggested shape categories. The dependency of the mean R(V) slope of all animals on the ventilation setting was used as a basis for classifi cation into resistance shape categories. Resistance shape categories were compared with compliance shape categories for each individual animal to test whether similar PEEP suggestions result from both methods. y y Conclusion In this trial, an intensive alveolar recruitment protocol associated with a protective mechanical ventilation strategy reduced pulmonary complication and length of hospital stay in patients undergoing cardiac surgery (NCT01502332). 1. Futier E, Constantin JM, Paugam-Burtz C, et al.: A trial of intraoperative low- tidal-volume ventilation in abdominal surgery. N Engl J Med 2013, 369:428-437. 1. Futier E, Constantin JM, Paugam-Burtz C, et al.: A trial of intraoperative low- tidal-volume ventilation in abdominal surgery. N Engl J Med 2013, 369:428-437. Results Small PEEP and VT were typically associated with increasing C(V), and decreasing C(V) corresponds to large PEEP and VT. A classifi cation of each C(V) profi le into one of six compliance shape categories was possible. The shapes of the R(V) profi les of individual animals were remarkably similar. The R(V) slope was typically largest for a PEEP and VT setting at which derecruitment was likely and smallest where overdistension was likely. Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial Methods Fifteen patients underwent three randomized 30-minute trials: hPSV, set with an inspiratory support above positive end- expiratory pressure (PEEP) ≥10 cmH2O and the fastest rate of pressurization; hNAVA, setting the NAVA level to achieve the same EAdipeak as during hPSV; hNAVA15 setting the NAVA level at 15 cmH2O/ μV and the maximum inspiratory airway pressure (Paw) at the value corresponding to PEEP + inspiratory support during nPSV. Oxygen inspiratory fraction and PEEP remained unmodifi ed throughout the study period. Paw-time products of the initial 200 ms from the onset of ventilator pressurization (PTP200), of the initial 500 ms from the onset of the EAdi swing indexed to the ideal PTPaw (PTP500i), and of the triggering area (PTPt) were computed. ABGs and VASd were assessed at the end of each trial. Introduction Protective mechanical ventilation has been associated with lower incidence of pulmonary and extrapulmonary complications in major surgery. The aim of the present study is evaluate whether adding an intensive alveolar recruitment protocol improves clinical outcomes and reduces healthcare utilization in patients undergoing cardiac surgery. S97 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods In this single-center, parallel-group trial, we randomly assigned adult patients presenting signals of defi cient gas exchange (PaO2/FIO2 <250 at a PEEP of 5 cmH2O) in the immediate postoperative period to either intensive alveolar recruitment or a standard protocol, both using low-tidal volume ventilation (6 ml/kg/ibw). Our hypothesis was that an aggressive alveolar recruitment protocol will be translated to better lung compliance, better gas exchange, fewer pulmonary complications and reduced length of hospital stay when compared with the control group. PEEP titration on the basis of intratidal resistance–volume profi les S Buehler1, S Schumann1, M Lichtwarck-Aschoff 2, J Guttmann1 1University Medical Center Freiburg, Germany; 2Uppsala University, Uppsala, Sweden Critical Care 2014, 18(Suppl 1):P273 (doi: 10.1186/cc13463) Critical Care 2014, 18(Suppl 1):P273 (doi: 10.1186/cc13463) Introduction Lung-protective mechanical ventilation requires positive end-expiratory pressure (PEEP) and tidal volume (VT) to be chosen with regard to the individual state of the lung. The shape of the intratidal compliance–volume profi les might refl ect the state of the lung (atelectatic, open, overdistended) and could therefore be classifi ed into shape categories that translate into PEEP suggestions [1]. Intratidal resistance–volume profi les might refl ect intratidal opening and collapse of the lung [2]. References 1. Mols et al.: Intensive Care Med 1999, 25:1084-1091. 2. Mols et al.: Br J Anaesth 2001, 86:176-182. 3. Schumann et al.: Physiol Meas 2009, 30:13415. 1. Mols et al.: Intensive Care Med 1999, 25:1084-1091. 2. Mols et al.: Br J Anaesth 2001, 86:176-182. 3. Schumann et al.: Physiol Meas 2009, 30:13415. f Methods We investigated two groups of 13 rats each. Animals were ventilated with ZEEP or PEEP of 5 mbar, FiO2 of 1.0 and tidal volume of 10 ml/kg. A double low-fl ow manoeuvre was designed, consisting of two consecutive low-fl ow manoeuvres with a peak pressure of 30 mbar, interrupted by a 5-second plateau phase at diff erent pressures (2, 4, 8 and 12 mbar). Alveolar size at the peak pressures and during the plateau levels was analyzed from the recorded videos frame by frame. Therefore the alveolar outline of 10 alveoli was marked manually and the outlined area was calculated [2]. Compliance of tidal breaths before and after the manoeuvres was calculated using two-point compliance. Results In both groups, analysis of the alveolar area revealed that alveolar size at the second peak of the manoeuvre did not diff er signifi cantly compared with the fi rst peak (100.97% in ZEEP group, 102.37% in the PEEP group). During the plateau phases there was a slight increase in alveolar size at higher plateau pressures (slope of linear regression at plateau 4 mbar: 0.1 %/500 ms for ZEEP group, 0.18%/500 ms for PEEP group; at plateau 8 mbar: 0.42%/500 ms for ZEEP group, 0.565%/500 ms for PEEP group). After the manoeuvres, compliance increased to 137.73% in the ZEEP group and 119.91% in the PEEP group. 1. Mols et al.: Intensive Care Med 1999, 25:1084-1091 2. Mols et al.: Br J Anaesth 2001, 86:176-182. 2. Schwenninger D, et al.: IEEE Trans Biomed Eng 2010, 57:415-421. 1. Schwenninger D, et al.: J Biomed Opt 2011, 16:046002. 1. Futier E, Constantin JM, Paugam-Burtz C, et al.: A trial of intraoperative low- tidal-volume ventilation in abdominal surgery. N Engl J Med 2013, 369:428-437. P274 P274 US study of gliding in nondependent lung regions: the dark side of the moon E De Blasio1, M Venditto1, A Federico1, G Azan1, C Pellegrini1, C Di Maria1, P De Luca1, G Rossi2 1Hospital G. Rummo, Benevento, Italy; 2Hospital ‘San Giovanni Battista’, Turin, Italy Critical Care 2014, 18(Suppl 1):P274 (doi: 10.1186/cc13464) Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial Based on this a classifi cation scheme was defi ned: 10 <slope <21 mbar s/L2 (category 1, ‘increase PEEP’), slope ≥21 mbar s/L2 (category 2, ‘keep PEEP’) and slope ≤10 mbar s/L2 (category 3, ‘decrease PEEP’). Comparison of resistance and compliance shape categories for single animals shows a good correlation. US study of gliding in nondependent lung regions: the dark side of the moon Introduction A protective ventilatory strategy should prevent VILI, but in patients with larger nonaerated areas hyperinfl ation may occur during tidal ventilation even during a protective ventilatory strategy [1]. The gliding sign is used as a marker of pneumothorax and, in a study [2], to quantify preoperatively the degree of pleural adhesion in thoracic surgery patients. In our study we measured the variations of gliding (G) and static compliance (Cstat) according to incremental/ decremental variations of PEEP in patients with hypoxic respiratory failure. Conclusion In the healthy lung, once recruited, alveoli stay stable in size over wide pressure ranges. Further recruitment manoeuvres do not lead to further increase of alveolar size, but increase of compliance. During plateau phases, alveolar size increases dependent on pressure. This leads to the assumption that recruitment is not only pressure dependent but also time dependent. Methods Ten patients with hypoxic respiratory failure (P/F <300) were ventilated in VCV (Vt of 7 ml/kg, FiO2 100%, RR 10/minute); keeping Vt constant, PEEP was gradually increased from ZEEP to 22 cmH2O, unless there was occurrence of hypotension or SpO2 <90% or Pplat >45 cmH2O or G no more visible, and then similarly reduced from 22 cmH2O to ZEEP. The gliding was assessed at six points of intercostal P272 Endomicroscopic analysis of time-dependent and pressure- dependent recruitment of subpleural alveoli H Runck, D Schwenninger, S Schumann, J Haberstroh, J Guttmann University Medical Center, Freiburg, Germany Critical Care 2014, 18(Suppl 1):P272 (doi: 10.1186/cc13462) Conclusion Resistance shape categories might provide additional guidance for PEEP setting. Combining compliance and resistance shape categories could improve lung-protective ventilation. References Introduction We used transthoracic endoscopy [1] to continuously record images of subpleural alveoli during recruitment manoeuvres and diff erent steady plateau pressures between the manoeuvres. References 1. Chiumello D, et al.: Crit Care Med 2013. [Epub ahead of print] 2. Karbing DS, et al.: Med Eng Phys 2011, 33:240-248. 1. Chiumello D, et al.: Crit Care Med 2013. [Epub ahead of print] 2. Karbing DS, et al.: Med Eng Phys 2011, 33:240-248. 1. Chiumello D, et al.: Crit Care Med 2013. [Epub ahead of print] 2. Karbing DS, et al.: Med Eng Phys 2011, 33:240-248. References 1. Terragni PP, et al.: Am J Respir Crit Care Med 2007, 175:160-166. 2. Masato S, et al.: Ann Thorac Surg 2005, 80:439-442. 1. Terragni PP, et al.: Am J Respir Crit Care Med 2007, 175:160-166. 2. Masato S, et al.: Ann Thorac Surg 2005, 80:439-442. p ρ Results PEEP improved V/Q in four patients, shunt reducing 7 to 42% with no/small increase in ΔAcPCO2. Two deteriorated, with large ΔAcPCO2 or shunt increase. No systematic changes in ΔAcPO2 were seen. Figure 1 shows response to PEEP in two patients. Changes in nonaerated regions and shunt were correlated (ρ = 0.94, P = 0.002). No correlations were found between poorly aerated regions and ΔAcPO2 (ρ = –0.09, P = 0.84) or hyperinfl ated regions and ΔAcPCO2 (ρ = 0.07, P = 0.88). p References Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S98 Figure 1 (abstract P274). PCC between mean G (cm) and mean Cstat at diff erent PEEP, in axis Pplat. Table 1 (abstract P275). P. aeruginosa log mean, wet to dry ratio, and PaO2/ FiO2 at 5 hours Figure 1 (abstract P274). PCC between mean G (cm) and mean Cstat at diff erent PEEP, in axis Pplat. Reference 1. N Engl J Med 2000, 342:1301-1308. Table 1 (abstract P275). P. aeruginosa log mean, wet to dry ratio, and PaO2/ FiO2 at 5 hours P. aeruginosa (cfu/g) W/D PaO2/FiO2 at 5 hours (mmHg) Prot-V 3.5 ± 0.7 1.7 ± 0.2 434 ± 62 Control 4.2 ± 0.7 2.7 ± 1.3 343 ± 61 Data presented as mean ± SD. P. aeruginosa (cfu/g) W/D PaO2/FiO2 at 5 hours (mmHg) Prot-V 3.5 ± 0.7 1.7 ± 0.2 434 ± 62 Control 4.2 ± 0.7 2.7 ± 1.3 343 ± 61 Data presented as mean ± SD. P276 P276 Changes in computed tomography and ventilation/perfusion mismatch with positive end-expiratory pressure DS Karbing1, M Panigada2, N Bottino2, E Spinelli2, A Protti2, SE Rees1, L Gattinoni2 1Aalborg University, Aalborg, Denmark; 2Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P276 (doi: 10.1186/cc13466) spaces bilaterally and the movement of a hyperechoic point of pleura or a b-line was observed during tidal ventilation. For each step, the excursion of G during the inspiratory phase was measured and compared with the Cstat values. Statistical analysis was performed with the Pearson correlation coeffi cient (PCC). Introduction The purpose was to compare eff ects of PEEP on computed tomography (CT) and estimated ventilation/perfusion (V/Q) mismatch. Previously, an oxygenation-based method was shown more related to the CT-measured eff ect of PEEP than lung mechanics [1], indicating lung aeration is better quantifi ed using V/Q mismatch. Pulmonary shunt and low and high V/Q mismatch can be estimated from varying FIO2 and measuring ventilation and blood gas contents [2]. Results All patients completed the study without adverse events. In all patients we observed a reduction of G and Cstat at the increase of PEEP and specularly an increase of G and Cstat during the reduction of PEEP (Figure 1). In fi ve patients at the lower levels of PEEP (from 0 to 10) an increase of Cstat and G was observed. For all patients the PCC of Cstat and G and was >0.5 (P <0.03), ranging from 0.537 (P = 0.017) to 0.964 (P <0.0001). 2 Methods Preliminary results in six ARDS patients. CT scans were taken in static conditions at PEEP 5, 45 and 15 to 20 cmH2O. V/Q was estimated at 5 and 15 to 20 cmH2O as: shunt, low V/Q as alveolar to lung capillary PO2 diff erence (ΔAcPO2), high V/Q as alveolar to lung capillary PCO2 diff erence (ΔAcPCO2) [2]. Nonaeration, poor aeration, and normal aeration plus hyperinfl ation were calculated from Hounsfi eld units. Aeration and V/Q were compared (Pearson, ρ). Conclusion The variations of G at diff erent levels of PEEP are consensual with those of Cstat. The study of G during tidal ventilation could help to identify hyperinfl ation in nondependent lung regions and to optimize lung-protective ventilatory strategies. Protective ventilation reduces bacterial growth and lung injury in a porcine pneumonia model J Sperber1, A Nyberg1, M Lipcsey2, A Larsson2, J Sjölin2, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Uppsala University, Uppsala, Sweden Critical Care 2014, 18(Suppl 1):P275 (doi: 10.1186/cc13465) 2 Conclusion In these preliminary cases, changes in shunt and nonaerated tissue correlated well. However, results indicate poor agreement between changes in low and high V/Q and lung morphology. References Introduction Bacterial pneumonia is a common indication for mechanical ventilation in the ICU. Ventilation with high positive end- expiratory pressure (PEEP) and low tidal volume (VT) is recommended in patients with adult respiratory distress syndrome. This improves clinical outcome compared with ventilation with low PEEP and medium-high VT [1]. However, the eff ect of VT and PEEP on bacterial growth in lung tissue is not known. This study contrasted the eff ect of a protective ventilator protocol with a standard medium-high VT and lower PEEP protocol on lung bacterial growth, lung edema formation and lung injury. It was performed in a porcine model of intensive care with the frequently found pathogen Pseudomonas aeruginosa. P277 Gas exchange and CT for patient improving (A) or worsening (B) with PEEP change. 494 ICUs around the world [1]. The Dutch cohort covered 196 patients and the global cohort 7,952. compliance. We present a new graphical user interface (GUI) that supports the clinician to titrate the PEEP level by means of a shape category analysis of the CV curve. Results Vt was 7.6 ml/kg predicted bodyweight in the Dutch cohort versus 8.0 ml/kg and the median applied PEEP was 8 cmH2O versus 5.8 cmH2O (both P <0.01). In the subgroup of patients with ARDS, Vt ml/kg was 7.6 and applied PEEP 8.8 cmH2O in the Netherlands versus 8.6 ml/kg (P = 0.41) and 8.3 cmH2O worldwide. In the Netherlands 7.1% of admitted patients received NIV as fi rst mode of mechanical ventilation versus 15% in the global cohort. In both cohorts 18% of patients were hypercapnic at ICU admission. Fewer patients in the Dutch cohort showed an ICU-acquired pneumonia (4.1 vs. 9.4%, P = 0.007) and sepsis (5.1 vs. 9.0%, P = 0.042), but more patients evolved delirium (16 vs. 5%, P <0.01). Methods A decision support system in the form of a computer-based GUI was developed and tested using respiratory data obtained from patients of the University Medical Center Freiburg. The new clinician- oriented approach provides a breath-by-breath visualization of the patient’s individual intratidal CV curve. The dynamic compliance was analyzed using the gliding-SLICE method [1]. The resulting intratidal CV curve was classifi ed into one of six shape categories according to the defi nition from Mols and colleagues [2]. The actual shape category of the CV curve indicates whether PEEP setting should be changed or whether the volume range of maximal compliance is reached. g p Results The GUI provided a breath-by-breath visualization of the intratidal CV curve and the intuitive individual compliance shape category. Based on the compliance shape category, diff erent guidelines of PEEP titration were applied with the objective of ventilating the patient mechanically within the range of maximal compliance. Conclusion According to our hypothesis, Vt was smaller and applied PEEP was higher in the Dutch cohort. Patients in both cohorts received larger Vt than recommended in prevention of ARDS [2]. Hypercapnia is a main criterion for the use of NIV [3], which suggests that NIV could be used more often in the Netherlands. References 1. Esteban et al.: Am J Respir Crit Care Med 2013, 188:220-230. 1. Esteban et al.: Am J Respir Crit Care Med 2013, 188:220 230. 2. The Acute Respiratory Distress Syndrome Network: N Engl J Med 2000, 342:1301-1308. 3. Brochard: Eur Respir J 2003, 22:31s-37s. p , 2. The Acute Respiratory Distress Syndrome Network: N Engl J Med 2000, p , 2. The Acute Respiratory Distress Syndrome Network: N Engl J Med 2000, 342:1301-1308. P277 The lower incidence of delirium worldwide could be caused by diff erences in sedation or may be due to the used methods of screening. Conclusion The newly developed GUI allows a breath-by-breath visualization of the intratidal CV curve with high reliability. Automated classifi cation of the intratidal CV curve into compliance shape categories provides the rationale basis for patient-individual PEEP titration. References 1. Schumann S, et al.: Physiol Meas 2009, 30:1341-1356. 2 Mols G et al : Intensive Care Med 1999 25:1084 1091 1. Schumann S, et al.: Physiol Meas 2009, 30:1341-1356. 2. Mols G, et al.: Intensive Care Med 1999, 25:1084-1091. P278 Graphical user interface for visualization of a decision support system for PEEP titration S Lozano-Zahonero, S Buehler, S Schumann, J Guttmann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P278 (doi: 10.1186/cc13468) P277 Ventilator settings in ICUs: comparing a Dutch with a global cohort M Van IJzendoorn1, M Koopmans1, U Strauch2, S Heines2, S Den Boer3, B Kors4, P Van der Voort5, P Dennesen6, I Van den Hul7, E Alberts7, P Egbers1, A Esteban8, F Frutos-Vivar8, M Kuiper1 1Medisch Centrum Leeuwarden, the Netherlands; 2Universitair Medisch Centrum Maastricht, the Netherlands; 3Spaarneziekenhuis, Hoofdorp, the Netherlands; 4Kennemer Gasthuis, Haarlem, the Netherlands; 5Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands; 6Medisch Centrum Haaglanden, Den Haag, the Netherlands; 7Vrije Universiteit Medisch Centrum, Amsterdam, the Netherlands; 8Hospital Universitario de Getafe, Madrid, Spain Critical Care 2014, 18(Suppl 1):P277 (doi: 10.1186/cc13467) Methods Sixteen pigs were anesthetized and randomized to mechanical ventilation with two diff erent ventilator settings for 6 hours; Prot-V (PEEP 10 cmH2O, VT 6 ml/kg, n = 8) and Control (PEEP 5 cmH2O, VT 10 ml/kg, n = 8). At 0 hours, 1×1011 colony-forming units (cfu) of P. aeruginosa were instilled intratracheally. At the end of the experiment, six postmortem lung biopsies from predefi ned declivial locations were taken from each animal for cultures and weight measurements. Introduction In data collected during the Third International Study on Mechanical Ventilation, we compared data from the Netherlands with a global cohort. We hypothesized that tidal volumes (Vt) were smaller and applied positive end-expiratory pressure (PEEP) was higher in the Netherlands, compared with the global cohort. We also compared use of non-invasive ventilation (NIV) and outcomes in both cohorts. Methods A post-hoc analysis of a prospective observational study of patients receiving invasive mechanical ventilation was conducted in Results P. aeruginosa growth in lung tissue and wet to dry ratio were lower in the Prot-V group than in the Control group (P <0.05 and P <0.05). PaO2/FiO2 was higher in the Prot-V group than in the Control group (P <0.05) (Table 1). Conclusion Protective ventilation with low VT and higher PEEP reduces P. aeruginosa growth in lung tissue, lung edema formation and lung injury in contrast with medium-high VT and lower PEEP ventilation in this porcine pneumonia model. Methods A post-hoc analysis of a prospective observational study of patients receiving invasive mechanical ventilation was conducted in Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S99 Figure 1 (abstract P276). Gas exchange and CT for patient improving (A) or worsening (B) with PEEP change. Figure 1 (abstract P276). Gas exchange and CT for patient improving (A) or worsening (B) with PEEP change. 6). P279 The aim of the study was to show time- dependent diff erences in the induction of apoptosis due to mechanical overload, by fl uorescence microscopic observations. in SF6 retention at the end of inspiration and a return to baseline during expiration similar to the changes observed in pigs with ALI at PEEP0. In ALI at PEEP15, shunt decreased throughout inspiration and returned to baseline levels during expiration. Conclusion Serial dynamic pulmonary shunt measurements during mechanical ventilation showed distinct variations over the respiratory cycle in both healthy and injured lungs. Increasing PEEP from 0 to 15 cmH2O altered the patterns of dynamic pulmonary shunt before and after ALI. Thus, serial assessment of dynamic pulmonary shunt fraction by SF6 retention with MMIMS-MIGET could prove useful for optimization of mechanical ventilation in healthy and injured lungs. yl Methods Alveolar epithelial cells (A549) were grown on RGD-coated, highly fl exible polydimethyl siloxane membranes. After becoming approximately 100% confl uent, cells were stained with Hoechst 33342/TMRE/caspase-3 and caspase-7/PI and continuously observed by fl uorescence microscopy. The cells were stimulated either with ventilation-analogue or spontaneous-breathing analogue profi le [1]. For both settings the frequency was 0.25 Hz, the surface increase 20% and the cell monolayers were stimulated over a time period of 2 hours in the bioreactor [2].l P281 P281 Eff ect of positive end-expiratory pressure on right ventricle function assessed by speckle tracking echocardiography SR Orde, A Behfar, PG Stalboerger, JK Oh, GC Kane Mayo Clinic, Rochester, MN, USA Critical Care 2014, 18(Suppl 1):P281 (doi: 10.1186/cc13471) Results The analysis of fl uorescence microscopic images showed the fi rst evidence of apoptosis induction after 40 minutes of ventilation-analogue stimulation. In contrast, apoptosis induction after spontaneous-breathing analogue stimulation occurred after 90 minutes. Introduction We sought to determine in a swine model whether a novel echocardiography method, speckle tracking echocardiography (STE), could determine deterioration in right ventricle (RV) function induced by escalating levels of positive end-expiratory pressure (PEEP), and to compare STE with RV fractional area change (FAC). Acute cor pulmonale and hypotension can be induced by high levels of PEEP used in the management of acute respiratory distress syndrome [1]. Quantifying RV function by echocardiography can be challenging due to its shape and position. References 1. Luecke T, et al.: Crit Care 2005, 9:607-621. 2. Huang SJ, et al.: Curr Opin Crit Care 2013, 19:250-257. g p 3. Fine NM, et al.: Circ Cardiovasc Imaging 2013, 6:711-721. 3. Fine NM, et al.: Circ Cardiovasc Imaging 2013, 6:711-721. Methods Pigs (n = 10) were anesthetized and ventilated at a tidal volume of 8 ml/kg and two levels of PEEP (0 and 15 cmH2O – conditions: PEEP0; PEEP15). Hemodynamic, respiratory and multiple inert gas analysis by micropore membrane inlet mass spectrometery (MMIMS- MIGET; Oscillogy, USA) measurements were taken at PEEP0 and PEEP15 before and after saline washout. Retention of SF6, measured fi ve times during one breathing cycle, was taken as a refl ection of shunt fraction. MIGET sample acquisition was synchronized by electrical impedance tomography. P279 STE is a relatively new, feasible, sensitive, angle-independent method for describing cardiac deformation (strain) [2] and is particularly useful in analyzing RV function (RV free wall strain, RVfwS), as has been shown in pulmonary hypertension cohorts [3]. Conclusion The observation of cells during stimulation with continuous fl uorescence microscopic imaging allowed us to analyse the time- dependent induction of apoptosis under the aspects of diff erent stimuli. We could prove our hypothesis that ventilation-analogue stimulation was worse for the cellular viability then spontaneous- breathing analogue stimulation. In future, the direct optical tracking of cell damage should allow one to analyse other stimulation profi les as well, and thereby to improve the stimulation profi les and ultimately the ventilation profi le as well. P279 Time-dependent apoptosis induction after spontaneous-breathing or ventilation-analogue experimental mechanostimulation of monolayer lung cell cultures S Meyer, S Schumann, J Guttmann, K Gamerdinger University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P279 (doi: 10.1186/cc13469) y S Lozano-Zahonero, S Buehler, S Schumann, J Guttmann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P278 (doi: 10.1186/cc13468) y S Lozano-Zahonero, S Buehler, S Schumann, J Guttmann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P278 (doi: 10.1186/cc13468) Introduction The analysis of dynamic volume-dependent compliance provides the rationale basis for PEEP titration during mechanical ventilation. Due to its volume dependence, the compliance of the respiratory system is nonlinear within each single breath (intratidal), which is refl ected in the compliance–volume curve (CV curve). The shape of the CV curve is determined by the PEEP level. The mechanical stress for the mechanically ventilated lung is expected to be minimal when the lung is ventilated within the volume range of maximal Introduction The cyclic strain of lung tissue during mechanical ventilation compared with spontaneous breathing is associated with a largely increased mechanical load due to larger pressure amplitudes and higher acceleration forces. This additional load may change tissue mechanics and may lead to tissue damage. Although experimental mechanostimulation in vitro is a widely used method Introduction The cyclic strain of lung tissue during mechanical ventilation compared with spontaneous breathing is associated with a largely increased mechanical load due to larger pressure amplitudes and higher acceleration forces. This additional load may change tissue mechanics and may lead to tissue damage. Although experimental mechanostimulation in vitro is a widely used method S100 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 to analyse mechanical load on cells or tissue, in vitro experiments with ventilation-analogue stimulation are missing. In this work we compare for the fi rst time the changes of monolayer lung cells after ventilation-analogue stimulation with spontaneous-breathing analogue mechanostimulation. The aim of the study was to show time- dependent diff erences in the induction of apoptosis due to mechanical overload, by fl uorescence microscopic observations. to analyse mechanical load on cells or tissue, in vitro experiments with ventilation-analogue stimulation are missing. In this work we compare for the fi rst time the changes of monolayer lung cells after ventilation-analogue stimulation with spontaneous-breathing analogue mechanostimulation. p References 1. Gamerdinger et al.: Acta Bioeng Biomech 2012, 14:53-62. 2. Schumann et al.: J Biomed Mater Res B Appl Biomater 2013, 101:1164-1171. 1. Gamerdinger et al.: Acta Bioeng Biomech 2012, 14:53-62. 2. Schumann et al.: J Biomed Mater Res B Appl Biomater 2013, 101:1164-1171. 1. Gamerdinger et al.: Acta Bioeng Biomech 2012, 14:53-62. 2. Schumann et al.: J Biomed Mater Res B Appl Biomater 2013, 101:1164-1171. Methods Ten pigs, 40 to 90 kg, anaesthetized, fully mechanically ventilated at 6 to 8 mg/kg were subject to stepwise escalating levels of PEEP at 2-minute intervals (0, 5, 10, 15, 20, 25 and 30 cmH2O). RV images were obtained using intracardiac echocardiography (for optimal frame- rate and endocardial defi nition) and were analyzed offl ine for FAC and RVfwS (using Velocity Vector Imaging; Siemens). Infl uence of positive end-expiratory pressure on cyclic recruitment and derecruitment during one breathing cycle in porcine acute lung injury g y g g Results Escalating levels of PEEP were strongly associated with signifi cant reductions in mean blood pressure (R2 = 0.8, P <0.0001), FAC (R2 = 0.8, P <0.0001) and RVfwS (R2 = 0.9, P <0.001). Paired t tests indicated signifi cant reductions in RVfwS with each step increase in PEEP. FAC only showed signifi cant deterioration at 15 cmH2O PEEP. Signifi cant hypotension (a decrease of more than 20 mmHg) occurred after 10 cmH2O PEEP. RVfwS decreased by a larger extent and earlier than FAC and mean blood pressure with increasing levels of PEEP. j y IS Sulyok1, A Johannes1, S Böhme1, KU Klein1, J Baumgardner2, O Kimberger1, K Markstaller1, R Ullrich1 1Medical University of Vienna, Austria; 2Oscillogy LLC, Folsom, PA, USA Critical Care 2014, 18(Suppl 1):P280 (doi: 10.1186/cc13470) Introduction Cyclic recruitment and derecruitment (R/D) of lung parenchyma during mechanical ventilation are responsible for atelectrauma. Theoretically, cyclic R/D can lead to varying degrees of pulmonary shunt fraction throughout the respiratory cycle. Measurements of dynamic pulmonary shunt fraction could help in assessing the degree of cylic R/D. However, common methods of measuring pulmonary shunt do not allow for dynamic serial measurements during one respiratory cycle. In this study, we measured serial dynamic pulmonary shunt fractions during one breathing cycle and investigated the eff ect of positive end-expiratory pressure (PEEP) on cyclic R/D in artifi cially ventilated pigs before and after saline washout induced acute lung injury. p g Conclusion RVfwS measured by STE is a sensitive method for determining RV dysfunction induced by PEEP. RVfwS displays a stronger association, greater deterioration and earlier reduction than FAC and mean blood pressure with escalating levels of PEEP. This potentially has interesting implications for the role of STE in managing PEEP levels in critically ill patients with acute lung injury. P283 E i P283 Experimental VILI begins with subpleural lung lesions C Chiurazzi, M Gotti, M Amini, C Rovati, M Brioni, G Rossignoli, A Cammaroto, C Bacile, S Luoni, K Nikolla, C Montaruli, T Langer, D Dondossola, M Cressoni, L Gattinoni Fondazione IRCCS Cà Granda, Milan, Italy Critical Care 2014, 18(Suppl 1):P283 (doi: 10.1186/cc13473) Introduction During mechanical ventilation some of the energy delivered to the respiratory system (RS) is dissipated within it, while some is recovered during expiration. The amount of unrecovered energy represents mechanical work done on the RS by the ventilator and may be related to the development of ventilatory-induced lung injury (VILI). The unrecovered energy is measured as the hysteresis area of the pressure–volume (PV) curve in static and dynamic conditions. We explored how and where the energy is dissipated inside the RS.i Introduction We developed an experimental model of VILI, ventilating piglets at a strain (TV/FRC) >2.5. It is possible that lung inhomogeneities act as stress raisers within the lung parenchyma, locally multiplying pressures. In a healthy lung the pleural surface, vessels and bronchi are detected as natural lung inhomogeneities. We studied the development i Introduction We developed an experimental model of VILI, ventilating piglets at a strain (TV/FRC) >2.5. It is possible that lung inhomogeneities act as stress raisers within the lung parenchyma, locally multiplying pressures. In a healthy lung the pleural surface, vessels and bronchi are detected as natural lung inhomogeneities. We studied the development i p gy p Methods In fi ve piglets (weight 21 ± 2 kg) under general anesthesia, we recorded PV curves to quantify dynamic dissipated energy (DE) at increasing tidal volume (TV) (150, 300, 450, 600, 750, 900) and at increasing respiratory rate (RR) (3, 6, 9, 12, 15). We then recorded PV curves for the same TV infl ated with a super-syringe (100 ml), to quantify static DE. We also quantifi ed airway DE connecting the postmortem isolated tracheobronchial tree to the ventilator and recording PV curves at the respiratory setting previously described. of VILI with CT scan to determine where the fi rst lung lesion developed. Methods Piglets were sedated, orotracheally intubated and instrumented with arterial and central venous catheter and urinary catheter. The whole study was performed in the animal CT scan facility, which was equipped as an ICU, and CT scan was performed every 3 hours or if respiratory parameters (plateau/peak pressure) changed. P284 1. Maung AA, Luckianow G, Kaplan LJ: Lessons learned from airway pressure release ventilation. J Trauma 2012, 72:624-628. 1. Maung AA, Luckianow G, Kaplan LJ: Lessons learned from airway pressure release ventilation. J Trauma 2012, 72:624-628. 8 Dissipated energy inside the respiratory system during mechanical ventilation M Gotti, C Chiurazzi, M Amini, C Rovati, M Brioni, G Rossignoli, A Cammaroto, C Bacile di Castiglione, S Luoni, K Nikolla, C Montaruli, T Langer, G Conte, M Cressoni, L Gattinoni Università degli studi di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P274 (doi: 10.1186/cc13474) 2. Kaplan LJ, Bailey H, Formosa V: Airway pressure release ventilation increases cardiac performance in patients with acute lung injury/adult respiratory distress syndrome. Crit Care 2001, 5:221-226. 2. Kaplan LJ, Bailey H, Formosa V: Airway pressure release ventilation increases cardiac performance in patients with acute lung injury/adult respiratory distress syndrome. Crit Care 2001, 5:221-226. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P284 Dissipated energy inside the respiratory system during mechanical ventilation M Gotti, C Chiurazzi, M Amini, C Rovati, M Brioni, G Rossignoli, A Cammaroto, C Bacile di Castiglione, S Luoni, K Nikolla, C Montaruli, T Langer, G Conte, M Cressoni, L Gattinoni Università degli studi di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P274 (doi: 10.1186/cc13474) Figure 1 (abstract P283). improving oxygenation in ventilated patients with cardiogenic shock and severe progressive hypoxemia. improving oxygenation in ventilated patients with cardiogenic shock and severe progressive hypoxemia. Figure 1 (abstract P283). Methods All cardiac and cardiac surgical patients with cardiogenic shock and ALI/ARDS admitted to our ICU were enrolled between January 2012 and September 2013. Data were collected on admission while the patients were on the conventional mode of ventilation and after 48 hours from switching to APRV. All enrolled patients were hemodynamically monitored with a pulmonary artery catheter and frequent echocardiography assessment. A retrospective analysis of these data was performed. Results Completed datasets were obtained from 29 patients. The cardiac index was increased by 30% (P <0.013), serum lactate decreased by 37% (P <0.001), central venous saturation increased by 42% (P <0.001) and peak airway pressure decreased 19% (P <0.001), with 50% increase of mean airway pressure, hypoxemia improved within the fi rst few hours of alveolar recruitment with PaO2/FIO2 increased by 23% (P <0.018), and there was less use of vasopressors, sedation and neuromuscular blockage over the course of APRV application.i g pp Conclusion In our patient series, APRV signifi cantly improved oxygenation and allowed for spontaneous breathing and a reduction in peak airway pressures. Furthermore, this strategy improved hemodynamics and facilitated weaning from MV. Therefore, our data suggest that this ventilation modality has favorable results and appears to be an eff ective alternative for lung recruitment in patients with cardiogenic shock and acute lung injury during their course of stay in cardiac ICU [2]. Figure 1 (abstract P283). Airway pressure release ventilation restores hemodynamic stability in patients with cardiogenic shock: initial experience in cardiac intensive carei A Taha, A Shafi e, M Mostafa, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2014, 18(Suppl 1):P282 (doi: 10.1186/cc13472) Results We observed dynamic changes in pulmonary shunt fraction, expressed as changes in SF6 retention, within all breathing cycles recorded, before and after induction of ALI. In healthy lungs at PEEP0 there was a decrease in SF retention at the end of inspiration and a return to baseline levels during expiration. In contrast, SF6 retention increased at PEEP0 in ALI during inspiration and decreased during expiration. In healthy pigs ventilated with PEEP15 there was an increase Introduction Airway pressure release ventilation (APRV) is a pressure- limited, time-cycled mode of mechanical ventilation. It increases the cardiac index, resulting in improved organ perfusion, which is crucial in cardiogenic shock patients preventing organ failure secondary to inadequate perfusion [1]. The purpose of this study was to evaluate the eff ectiveness of APRV in restoring hemodynamic stability and S101 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets g g Methods LUS and whole-lung CT scan were performed on ARDS sedated, paralyzed, mechanically ventilated patients at PEEP 5 and 15 cmH2O. LUS was performed considering six areas for each lung, with a comprehensive scan of the intercostal spaces in each area. We assigned to each area a score of aeration [1]: 0 (normal lung), 1 (≥3 noncoalescent B-lines), 2 (≥3 coalescent B-lines), 3 (consolidation). A cumulative LUS score (LUSS, ranging from 0 to 36 for the two lungs) was obtained as sum of all areas’ individual scores, each area’s score being the average of all pertaining LUS fi ndings. LUS recruiters upon PEEP increase from 5 to 15 cmH2O were defi ned by the switch of at least three areas to well aerated (area score 0). LUS-based assessment of lung aeration and lung recruitability was compared with qCT fi ndings. Results We enrolled seven patients (six males, age 54.1 ± 22.2 years, BMI 24.2 ± 4.9 kg/m2, PaO2/FiO2 186 ± 78, tidal volume 445 ± 140 ml, RR 14.5 ± 3.4 breaths/minute, PEEP 12.5 ± 3.3 cmH2O). In the 14 conditions evaluated, median LUSS was 19 (IQR 14 to 23); LUSS ≤19 (n = 8) corresponded to 34 ± 13% of nonaerated tissue at qCT; LUSS >20 (n = 6) to 48 ± 18% (P <0.05). A good linear correlation was found between reduction at LUS of consolidated areas (area score 3) versus reduction of qCT nonaerated volume (R2 = 0.66), and between reduction at LUS of y Methods In eight healthy piglets we measured total hysteresis and the peak inspiratory pressure (Ppeak) while randomly increasing VT (with no PEEP) or PEEP (with fi xed VT). P1 was extrapolated from the drop in airway pressure during an end-inspiratory pause [3]. Hysteresis attributable to lung parenchyma was computed as: total hysteresis – ((Ppeak – P1) × VT).i Results The main fi ndings are shown in Figure 1. P values refer to one- way repeated-measures analysis of variance. Conclusion Lung hysteresis increases with VT, but not with PEEP. Further studies are needed to prospectively evaluate the role of lung hysteresis in the pathogenesis of ventilator-induced lung injury. References i Results We enrolled seven patients (six males, age 54.1 ± 22.2 years, BMI 24.2 ± 4.9 kg/m2, PaO2/FiO2 186 ± 78, tidal volume 445 ± 140 ml, RR 14.5 ± 3.4 breaths/minute, PEEP 12.5 ± 3.3 cmH2O). P286f CT scan and ultrasound comparative assessment of PEEP-induced lung aeration changes in ARDS I Algieri1, S Mongodi2, D Chiumello3, F Mojoli2, M Cressoni1, G Via2, S Luoni1, A Colombo1, G Babini1, A Braschi2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 3Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P285 (doi: 10.1186/cc13475) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Static DE(J) had a nonlinear relationship with the TV (ml/kg) applied: static DE = 0.0031TV1.5198, R2 = 0.96. Subtracting from the PV curve hysteresis area of a single breath (dynamic DE) the airway DE, the resulting curve overlapped static DE at every RR considered, suggesting that the amount of energy spent on the RS is equal to the static DE. Static DE can be estimated knowing fl ow (l/minute) and dynamic DE(J) since:ll poorly aerated areas (area score 1 to 2) versus reduction of qCT poorly aerated volume (R2 = 0.74). Change at LUS of at least three areas to well aerated (LUS recruiters, n = 4) corresponded to a qCT increase in well-aerated lung volume of 788 ± 262 g versus 431 ± 35 g in the LUS nonrecruiter group (n = 3) (P <0.05). Conclusion These preliminary data suggest that LUS could be an accurate tool to assess lung aeration and recruitment at the bedside, avoiding the risks and workload related to the use of CT scan. Reference (dynamic–static)DE = 0.0014[fl ow]2 – 0.0173fl ow + 0.1387, R2 = 0.90 (Figure 1). 1. Bouhemad B, et al.: Bedside ultrasound assessment of positive end- expiratory pressure–induced lung recruitment. Am J Respir Crit Care Med 2011, 183:341-347. Conclusion According to our data, the amount of energy that may be related to the development of VILI is static DE; it is a nonlinear function of TV and can be estimated knowing fl ow and dynamic DE. 1. Bouhemad B, et al.: Bedside ultrasound assessment of positive end- expiratory pressure–induced lung recruitment. Am J Respir Crit Care Med 2011, 183:341-347. P283 E i We defi ned as new lesion lung regions the appearance of poorly infl ated/not infl ated lung regions not present in the previous CT scan image. We select the fi rst CT scan in which a relevant number of new lesions appeared (>15) and manually delineated the lesions; the lesions were classifi ed as close/not close to the pleural surface. Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). i Results Six piglets were studied (22 ± 8 kg) that were ventilated with a TV of 750 ± 71 ml (41 ± 1 ml/kg) up to development of VILI, defi ned radiologically as infi ltrates present in all pulmonary fi elds at CT scan plus development of lung edema. In the fi rst CT scan where lesions appeared, a median of 28 lesions (IQ range 22 to 30) were present. Of these lesions, 18 (17 to 22) (72%) were located near the pleura and nine (6 to 11) (28%) near vessels/bronchi. See Figure 1. Conclusion In an experimental model of VILI the fi rst lung lesions appear below the pleural surface. Mutiple nonmutually exclusive possible explanations are possible: the pleural surface acts as a stress raiser; the mechanical friction of the lung with the ribs at very high tidal volume leads to parenchimal injury; and the lung skeleton is a fan-like structure starting from the hilum and going to the pleural surface, leading to increased stress/strain of the subpleural regions. Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). S102 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 CT scan and ultrasound comparative assessment of PEEP-induced lung aeration changes in ARDS Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets DT Andreis1, M Milesi1, P Pugni1, F Nicosia1, GE Iapichino1, M Monti2, B Comini2, E Votta3, A Protti2, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy; 3Politecnico di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P286 (doi: 10.1186/cc13476) Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets I Algieri1, S Mongodi2, D Chiumello3, F Mojoli2, M Cressoni1, G Via2, S Luoni1, A Colombo1, G Babini1, A Braschi2 I Algieri1, S Mongodi2, D Chiumello3, F Mojoli2, M Cressoni1, G Via2, S Luoni1, A Colombo1, G Babini1, A Braschi2 Introduction Growing evidence suggests that, as long as the total lung capacity is not overcome, dynamic (that is, tidal volume, VT) is more injurious than static (that is, positive end-expiratory pressure, PEEP) lung deformation [1]. Because the lung behaves like a viscoelastic body [2], hysteresis may play a role in the development of ventilator- induced lung injury. The aim of the study was to investigate the eff ects of increasing VT or PEEP on lung hysteresis. Introduction Growing evidence suggests that, as long as the total lung capacity is not overcome, dynamic (that is, tidal volume, VT) is more injurious than static (that is, positive end-expiratory pressure, PEEP) lung deformation [1]. Because the lung behaves like a viscoelastic body [2], hysteresis may play a role in the development of ventilator- induced lung injury. The aim of the study was to investigate the eff ects of increasing VT or PEEP on lung hysteresis. Introduction CT-scan quantitative analysis (qCT) represents the gold standard to assess lung aeration and recruitment in ARDS patients. Lung ultrasound (LUS) has been proposed as a bedside nonirradiating alternative to assess lung recruitability, identifying patients who may benefi t from higher PEEP levels. We compared the two methods in the assessment of PEEP-induced lung aeration changes. g g Methods LUS and whole-lung CT scan were performed on ARDS sedated, paralyzed, mechanically ventilated patients at PEEP 5 and 15 cmH2O. LUS was performed considering six areas for each lung, with a comprehensive scan of the intercostal spaces in each area. We assigned to each area a score of aeration [1]: 0 (normal lung), 1 (≥3 noncoalescent B-lines), 2 (≥3 coalescent B-lines), 3 (consolidation). A cumulative LUS score (LUSS, ranging from 0 to 36 for the two lungs) was obtained as sum of all areas’ individual scores, each area’s score being the average of all pertaining LUS fi ndings. LUS recruiters upon PEEP increase from 5 to 15 cmH2O were defi ned by the switch of at least three areas to well aerated (area score 0). LUS-based assessment of lung aeration and lung recruitability was compared with qCT fi ndings. Diaphragm microcirculatory dysfunction and lipid accumulation in endotoxemic rabbits during mechanical ventilation Diaphragm microcirculatory dysfunction and lipid accumulation in endotoxemic rabbits during mechanical ventilation Y Yang1, T Yu1, J Liu1, C Pan1, F Longhini2, L Liu1, Y Huang1, F Guo1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P289 (doi: 10.1186/cc13479) Y Yang1, T Yu1, J Liu1, C Pan1, F Longhini2, L Liu1, Y Huang1, F Guo1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P289 (doi: 10.1186/cc13479) Results The three zones presented increased over-insuffl ated areas and decreased areas with normal insuffl ation with increasing Ppeak from 5 to 15 cmH2O. At 5 cmH2O, the areas of over-insuffl ation and normal insuffl ation in zones I, II and III were 13% and 36%; 4% and 22%; and 0.7% and 15%, respectively. At 15 cmH2O, the areas of over-insuffl ation and normal insuffl ation in zones I, II and III were 74% and 55%; 81% and 57%; and 82% and 71%, respectively. Introduction Sepsis-induced diaphragm dysfunction (SIDD) has been widely described in the literature as a condition aff ecting the diaphragm muscle characterized by contractility loss of function and associated with a high mortality, assessed at around 54% [1]. Previous studies have investigated the pathogenesis of ventilator-induced diaphragmatic dysfunction (VIDD), its lipid metabolic alterations [2] and microcirculatory function processes. This study was designed to investigate on diaphragm muscle the eff ects of LPS-induced endotoxemia in rabbits undergoing two diff erent modes of mechanical ventilation. Conclusion The higher proportion of overly distended pulmonary areas in high Ppeak may increase the risk of lung injury. The lowest Ppeak (5 cmH2O) showed less potential to lung injury as it has higher areas of normal insuffl ation and less areas of over-insuffl ation in all gravitational zones. Methods A prospective randomized animal study in 25 invasively monitored and mechanically ventilated New Zealand White rabbits. The rabbits were randomized to control (n = 5), controlled mechanical ventilation (CMV) (n = 5), pressure support ventilation (PSV) (n = 5), or CMV or PSV with LPS-induced endotoxemia (CMV-LPS and PSV- LPS respectively) (n = 5 for each). The endotoxemia was induced by LPS injection in the CMV-LPS and PSV-LPS groups. Rabbits were anesthetized and ventilated for 24 hours, except for the control (30 minutes). Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets In the 14 conditions evaluated, median LUSS was 19 (IQR 14 to 23); LUSS ≤19 (n = 8) corresponded to 34 ± 13% of nonaerated tissue at qCT; LUSS >20 (n = 6) to 48 ± 18% (P <0.05). A good linear correlation was found between reduction at LUS of consolidated areas (area score 3) versus reduction of qCT nonaerated volume (R2 = 0.66), and between reduction at LUS of 1. Protti A, et al.: Which is the most important strain in the pathogenesis of ventilator-induced lung injury: dynamic or static? Curr Opin Crit Care 2014, 20:33-38. 2. Bayliss LE, et al.: The visco-elastic properties of the lung. Q J Exp Physiol 1939, 29:27-47. 3. Bates JHT, et al.: General method for describing and extrapolating monotonic transients and its application to respiratory mechanics. Med Biol Eng Comput 1987, 25:131-135. Figure 1 (abstract P286). Lung hysteresis as a function of VT (A) and PEEP (B). P <0.05 versus VT 250 ml or PEEP 0 cmH2O (Holm–Sidak method). Figure 1 (abstract P286). Lung hysteresis as a function of VT (A) and PEEP (B). P <0.05 versus VT 250 ml or PEEP 0 cmH2O (Holm–Sidak method). S103 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P287 on the Beckman Coulter Epics XL cytometer (USA). The statistical power of the study was 80% (α ≤0.05). P287 Evaluation and quantifi cation of pulmonary hyperinfl ation in three gravitational zones of domestic felines by computed tomography A Rodrigues de Carvalho Martins1, D Tabacchi Fantoni2, D Aya Otsuki1, A Magalhães Ambrósio2, A Brandão de Campos Fonseca Pinto2, J França dos Santos2, C Rodrigues de Carvalho Martins3, L Sá Malbouisson1 1Faculdade de Medicina da Universidade de São Paulo, Brazil; 2Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Brazil; 3UFAPE, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P287 (doi: 10.1186/cc13477) Results In Group I relative to group II at 3 to 5 days we registered an increase in mature monocytes (CD14) – 23.1 ± 0.8% (P <0.05); reduction in the relative content of CD69 – 3.8 ± 0.21%, lymphocyte of apoptosis: (Annexin V–FITC+PI–) – 7.12 ± 0.46% and (Annexin V–FITC+PI+) – 0.79 ± 0.07% (P <0.001). The duration of mechanical ventilation was 4.1 ± 1.4 days (P <0.05). All patients survived. None of the patients showed clinical or laboratory evidence of adverse eff ects of inhaled nitric oxide. Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets In Group II seven newborns died, and the duration of mechanical ventilation in survivors was 18 ± 3.4 days. Critical Care 2014, 18(Suppl 1):P287 (doi: 10.1186/cc13477) Introduction Mechanical ventilation (MV) aims to enhance blood oxygenation and to remove carbon dioxide. However, excessive hyperinfl ation by MV may cause lung injury. Introduction Mechanical ventilation (MV) aims to enhance blood oxygenation and to remove carbon dioxide. However, excessive hyperinfl ation by MV may cause lung injury. y Conclusion Inhalable of nitric oxide activates monocyte–macrophage immunity, stabilizes the apoptosis of T-lymphocytes, and reduces mortality and duration of mechanical ventilation in newborns with respiratory diseases on mechanical ventilation. References Methods Eighteen cats (4 ± 1 kg) were anesthetized with propofol (loading dose of 6 mg/kg and constant rate infusion of 0.5 mg/kg/ minute) and neuromuscular blockade was achieved with rocuronium at 1 mg/kg/minute. Their lungs were initially mechanically ventilated in FiO2 of 40%, with peak inspiratory pressure (Ppeak) of 5 cmH2O for 20 minutes, and then the Ppeak was increased by 5 cmH2O increments until 15 cmH2O every 5 minutes. Following that, Ppeak was decreased by 2 cmH2O every 5 minutes until reaching Ppeak of 5 cmH2O. The ventilator maintained the respiratory rate and inspiratory time at 15 breaths/minute and 1 second, respectively. Between the Ppeak increments, we applied a 4-second pause for a 5-mm computed tomography (CT) scan of the thorax area. The radiographic attenuation (in Hounsfi eld units, HU) was classifi ed as over-insuffl ation (1,000 to 900 HU), normal insuffl ation (900 to 500 HU) and atelectasic (500 to 100 HU). We split the lungs into three proportional gravitational zones (I, II and III) from apex to base. 1. Dudareva MV, Estrin VV: The state of immunity in newborn infants with respiratory disease. Allergy Immunol 2008, 9:349-350. y gy 2. Puhtinskaya MG, Estrin VV, Dudareva MV, Gulova ES: Method of treatment of RDS in neonates receiving mechanical ventilation. RU Patent No. 2434653; 27 November 2011. 2. Puhtinskaya MG, Estrin VV, Dudareva MV, Gulova ES: Method of treatment of RDS in neonates receiving mechanical ventilation. RU Patent No. 2434653; 27 November 2011. P288f Eff ect of inhaled nitric oxide on apoptosis of lymphocytes in newborns in a critical state M Puhtinskaya Research Institute of Obstetrics and Pediatrics, Rostov-on-Djn, Russia Critical Care 2014, 18(Suppl 1):P288 (doi: 10.1186/cc13478) y Research Institute of Obstetrics and Pediatrics, Rostov-on-Djn, Russia Critical Care 2014, 18(Suppl 1):P288 (doi: 10.1186/cc13478) Introduction Activation of lymphocyte apoptosis while reducing the endogenous nitric oxide is a predictor of adverse outcome in newborns on mechanical ventilation [1]. With the aim to improve the results of treatment we studied the eff ect of inhalable nitric oxide on the immune system in newborns with respiratory diseases on mechanical ventilation [2]. i Results In endotoxemic animals, after 24 hours, the diaphragm contractility and fi ber structure, the microcirculation, mitochondrial membrane potential and lipid accumulation were severely compromised, but not in the CMV and PSV groups. Moreover, a slight but signifi cant increase of lipid accumulation was observed in the CMV and PSV groups in comparison with control (P <0.05). Methods With the permission of the ethics committee in a controlled, randomized, blind clinical trial we included 27 newborns with respiratory diseases on mechanical ventilation. Randomization was performed by the method of envelopes. Group I (n = 17), patients receiving inhalation of nitric oxide at a concentration of 10 ppm for 24 hours controlling the level of methemoglobin (Pulmonox mini; Messer II NO Therapeutics, Austria). Group II (n = 10) did not receive inhaled NO. At admission and at 3 to 5 days we studied subpopulations of lymphocytes by one-parameter immunophenotyping using reagents (Immunotech Beckman Coulter, USA): fi tz-labeled CD3, CD4, CD8, CD14, CD19, CD34, CD56, CD69, CD71, CD95 monoclonal antibody, the relative content of lymphocytes in early and late apoptosis using Annexin V+-labeled FITK and propidium iodide (PL+), labeled with PE (Saltag, USA), with results Conclusion In endotoxemic rabbits, the impaired microcirculation resulted in an increased lipid accumulation and in a disturbance of the mitochondria membrane potential and contractility of the diaphragm. No microvascular alterations have been observed in ventilated non-endotoxemic animals. Moreover, the diaphragm contractility dysfunction was more pronounced in endotoxemic animals. References Diaphragm microcirculatory dysfunction and lipid accumulation in endotoxemic rabbits during mechanical ventilation A catheter able to detect the electrical activity of the diaphragm was placed to evaluate the diaphragm contractility at baseline and after 6, 12 and 24 hours. After 24 hours, we evaluated: the diaphragm microcirculation assessed by a sidestream dark-fi eld videomicroscopy; the mitochondria membrane potential; the lipid accumulation; and the diaphragm muscular fi ber structure. EIT comparison of airway pressure release ventilation and conventional ventilation S Jog, S Sable, D Patel, P Tambur g Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P291 (doi: 10.1186/cc13481) Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P291 (doi: 10.1186/cc13481) g Conclusion EIT may help to identify patients with severe ARDS on VCV with a potential of increasing recruitment by tidal redistribution of ventilation with APRV. Introduction The aim was to study EIT as a monitoring tool for tidal ventilation (TV) redistribution following switching patients from volume-controlled ventilation (VCV) to airway pressure release ventilation (APRV) in patients with severe ARDS. P292 Methods Six patients with severe ARDS having Pplat ≥30 cm were included in the study. Patients ventilated with the ARDSnet strategy were subjected to EIT analysis. Regional TV distribution was monitored by an EIT system (PulmoVista 500®; Dräger Medical GmbH, Lübeck, Germany), dividing the lung fi eld into four same-size regions of interest (ROIs): ventral right (ROI 1) and left (ROI 2) and dorsal right (ROI 3) and left (ROI 4). In step 1, patients ventilated with VCV as per the ARDSnet protocol were subjected to EIT analysis. In step 2, patients were switched to APRV. Ventilation parameters, arterial blood gas analysis Lung-protective ventilation suppresses plasma levels of cell-free DNA in porcine experimental postoperative sepsis A Nyberg1, J Sperber1, M Lipcsey2, J Jylhävä3, M Hurme3, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Eskilstuna, Sweden; 2Uppsala University, Uppsala, Sweden; 3University of Tampere, Finland Critical Care 2014, 18(Suppl 1):P292 (doi: 10.1186/cc13482) High-frequency oscillatory ventilation use in patients with H1N1: a single-centre review Introduction Limited evidence exists as to the value of high-frequency oscillatory ventilation (HFOV) for patients with H1N1 [1,2]. We describe a subgroup of patients, from a single UK centre, who received HFOV and had H1N1-positive virology. gy Methods Local permissions for research were obtained. Patients with confi rmed H1N1 who underwent HFOV between 2008 and 2012 were included. Data collected retrospectively included demographics, diagnosis, illness severity and outcome measures.i g y Results We identifi ed 10 patients (Table 1) who received both HFOV and had confi rmed H1N1. Two patients were transferred to a tertiary centre to receive ECMO and both critical care and 6-month mortality were 40%. Table 1 (abstract P290). Patient characteristics Table 1 (abstract P290). Patient characteristics Male (%) 60 Age (years) 39.4 ± 9.75 Mean APACHE II 19.5 ± 4.88 Pre-HFOV neuromuscular blockade infusion (%) 40 Pre-HFOV vasoactive drug infusion (%) 50 Mean pre-HFOV VT exp (ml/kg) 8.54 ± 2.68 Median P/F ratio (mmHg) Pre HFOV 67.5 0 hours 94.35 4 hours 110.4 Table 1 (abstract P290). Patient characteristics Table 1 (abstract P290). Patient characteristics Conclusion This study adds to the literature on the use of HFOV in H1N1 patients. We managed to replicate some of the existing evidence in respect to the population age [1,2] and the incidence of mortality. Whilst P/F ratios improved on initiation of HFOV, these patients subsequently had long critical care and hospital stays. There is uncertainty regarding the use of HFOV in ARDS, but it may be a valuable treatment for H1N1 patients. The ventilation strategies employed and the subsequent consequences for H1N1 patients require further evaluation. References Figure 1 (abstract P291). EIT comparison of VCV and APRV. and percentage of tidal ventilation distribution in the four ROIs were recorded at steps 1 and 2. Analyses were performed by paired t test. Results Patients on VCV had P/F ratio of 79.5 ± 12.5 with PEEP of 14.16 ± 1.32 There was a signifi cant improvement in P/F ratios on switching to APRV (126.16 ± 23.69, P = 0.002) at 30 minutes of ventilation on APRV. There was a trend to decrease in FiO2 (0.82 ± 0.15 vs. 0.68 ± 0.10, P = 0.068) and PCO2 (52.5 ± 6.15 vs. 45.00 ± 8.67, P = 0.071) and increase in PaO2 (65.83 ± 14.53 vs. 84.83 ± 12.22, P = 0.056) at step 2. High-frequency oscillatory ventilation use in patients with H1N1: a single-centre review The proportional distribution of ventilation in the dorsal ROI 3 and ROI 4 also improved on switching to APRV. TV in ROI 3 during VCV, 12.76 ± 6.76%, improved to 24.58 ± 6.61% (P = 0.067). Similarly TV in ROI 4 during VCV, 24.58 ± 6.61%, improved to 26.6 ± 6.09% (P = 0.068). Due to small sample size, improvement in TV in dorsal ROIs was not statistically signifi cant. Upper panel of Figure 1 shows end-inspiratory and end-expiratory images of EIT on VCV with poor TV in dorsal ROI 3 and ROI 4. Lower panel of fi gure shows two EIT images at Phigh = 30 cm showing improved TV in dorsal ROIs. and percentage of tidal ventilation distribution in the four ROIs were recorded at steps 1 and 2. Analyses were performed by paired t test. Results Patients on VCV had P/F ratio of 79.5 ± 12.5 with PEEP of 14.16 ± 1.32 There was a signifi cant improvement in P/F ratios on switching to APRV (126.16 ± 23.69, P = 0.002) at 30 minutes of ventilation on APRV. There was a trend to decrease in FiO2 (0.82 ± 0.15 vs. 0.68 ± 0.10, P = 0.068) and PCO2 (52.5 ± 6.15 vs. 45.00 ± 8.67, P = 0.071) and increase in PaO2 (65.83 ± 14.53 vs. 84.83 ± 12.22, P = 0.056) at step 2. The proportional distribution of ventilation in the dorsal ROI 3 and ROI 4 also improved on switching to APRV. TV in ROI 3 during VCV, 12.76 ± 6.76%, improved to 24.58 ± 6.61% (P = 0.067). Similarly TV in ROI 4 during VCV, 24.58 ± 6.61%, improved to 26.6 ± 6.09% (P = 0.068). Due to small sample size, improvement in TV in dorsal ROIs was not statistically signifi cant. Upper panel of Figure 1 shows end-inspiratory and end-expiratory images of EIT on VCV with poor TV in dorsal ROI 3 and ROI 4. Lower panel of fi gure shows two EIT images at Phigh = 30 cm showing improved TV in dorsal ROIs. 1. Riscilli BP, et al.: PLOS One 2011. doi: 10.1371/journal.pone.0018166. 2. Boots RJ, et al.: Anaesth Intensive Care 2011, 39:837-846. 1. Riscilli BP, et al.: PLOS One 2011. doi: 10.1371/journal.pone.0018166. 2. Boots RJ, et al.: Anaesth Intensive Care 2011, 39:837-846. dysfunction References Demoule A, et al.: Am J Respir Crit Care Med 2013, 188:213-219. Picard M, et al.: Am J Respir Crit Care Med 2012, 186:1140-1149. Demoule A, et al.: Am J Respir Crit Care Med 2013, 188:213-219. Picard M, et al.: Am J Respir Crit Care Med 2012, 186:1140-1149. Demoule A, et al.: Am J Respir Crit Care Med 2013, 188:213-219. Picard M, et al.: Am J Respir Crit Care Med 2012, 186:1140-1149. S104 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P290 High-frequency oscillatory ventilation use in patients with H1N1: a single-centre review N Smith, N Pathmanathan, V Martinson, J Glazebrook, R Rao, L Sleight, A Gratrix Hull and East Yorkshire NHS Trust, Hull, UK Critical Care 2014, 18(Suppl 1):P290 (doi: 10.1186/cc13480) Figure 1 (abstract P291). EIT comparison of VCV and APRV. Comparison of HFOV and conventional ventilation in H1N1 infl uenza ARDS l S Jog, S Sable, D Patel, N Kapadnis, P Rajhans, P Akole, B Pawar, B Bhurke, M Kothari, S Gururaj D h M hk H i l d R h C P I di j Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P293 (doi: 10.1186/cc13483) Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P293 (doi: 10.1186/cc13483) g y ( g p y ) Results Eighteen patients (male 12, age 56.0 ± 18.6 years, BMI 25.6 ± 5.2 kg/m2, PaO2/FiO2 166 ± 70, tidal volume 496 ± 84 ml, RR 19 ± 7.4 breaths/minute, PEEP 11.4 ± 3.7 cmH2O) were enrolled. The fraction of lung parenchyma that could be recruited at plateau pressure above 30 cmH2O was highly variable with a median of 5% (IQ range 1 to 17%) corresponding to a median 16% (IQ range 10 to 47%) of total lung recruitability. Indeed we observed a statistically relevant diff erence in lung recruitment between airway pressures of 30 and 45 cmH2O (P = 0.016). With PEEP 5 cmH2O, median opening/closing was 129 g (IQ range 124 to 145 g) in the HR group and 50 g (IQ range 24 to 76 g) in LR (P = 0.006), corresponding to 8% of lung parenchyma (IQ range 6 to 8%) in HR and 4% (2 to 7%) in LR (P = 0.053). Increasing the PEEP level to 15 cmH2O, median opening/closing was 67 g (IQ range 24 to 95 g) in the HR group and 45 g (0 to 80 g) in the LR group (P = 0.512), corresponding to 3% (1 to 5%) in HR and 3% (0 to 5%) in LR (P = 0.93). We observed a statistical diff erence between recruitments with PEEP 5 and PEEP 15 in the HR group (P = 0.013) but not in the LR group (P = 0.781).i Introduction HFOV is a promising rescue therapy for refractory hypoxia in severe ARDS. Methods This is a retrospective comparative study. We retrieved data for all patients with H1N1 infl uenza-related severe ARDS treated in the ICU during October 2009 to April 2013. Our ICU had only one HFOV machine during the pandemic. Patients were divided into two groups: HFOV group (received HFOV at fi rst eligibility) and conventional lung protective ventilation (CLPV) group (did not receive HFOV at fi rst eligibility due to nonavailability of HFOV). P294 P294 Opening pressures and intratidal opening and closing in ARDS lung I Algieri1, D Chiumello2, M Cressoni1, A Colombo1, G Babini1, S Luoni1, M Brioni1, F Crimella1, C Chiurazzi1, B Comini2, M Monti2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P294 (doi: 10.1186/cc13484) Introduction Intratidal opening/closing is believed to be one of the main triggers of ventilator-induced lung injury; the application of higher PEEP united with the limitation of plateau pressure at 30 cmH2O may limit this phenomenon. We aim to evaluate the intratidal opening/ closing at diff erent PEEP levels and the amount of lung parenchyma that regains infl ation going from 30 (pressure limit of tidal ventilation) to 45 cmH2O. Introduction Intratidal opening/closing is believed to be one of the main triggers of ventilator-induced lung injury; the application of higher PEEP united with the limitation of plateau pressure at 30 cmH2O may limit this phenomenon. We aim to evaluate the intratidal opening/ closing at diff erent PEEP levels and the amount of lung parenchyma that regains infl ation going from 30 (pressure limit of tidal ventilation) to 45 cmH2O. Conclusion In experimental postoperative sepsis, protective ventila tion suppresses arterial levels of cfDNA. The liver seems to be a signifi cant contributor to systemic cfDNA levels, an eff ect that is suppressed during protective ventilation. References 1. Sperber J, et al.: PLOS One 2013. doi: 10.1371/journal.pone.0083182. 2. Saukkonen K, et al.: Clin Chem 2008, 54:1000-1007. 2 Methods Patients with ARDS underwent whole-lung low-dose (60 mAs, 120 kV) computed tomography at PEEP 5 cmH2O end expiration and end inspiration, PEEP 15 cmH2O end expiration and end inspiration, and airway pressure 30 and 45 cmH2O end inspiration. Quantitative analysis of CT data was performed and recruitability was defi ned as the fraction of lung parenchyma that regains infl ation going from 5 cmH2O end expiration to 45 cmH2O end inspiration. Patients were classifi ed as high recruiters (HR) and low recruiters (LR) according to the median lung recruitability (14% of lung parenchyma). Comparison of HFOV and conventional ventilation in H1N1 infl uenza ARDS Eligibility criteria for rescue therapy by HFOV were: P/F ratio ≤100; PEEP needed above 12 cm; Pplat ≥30 cm on CLPV. There was no selection or omission bias for HFOV application and HFOV was applied to the fi rst eligible patient. Patient demographic data, laboratory parameters, hemodynamic variables, and oxygenation and ventilator settings were recorded while on CLPV at fi rst HFOV eligibility in all patients. i g y Results The total of 43 patients who met the rescue therapy criteria were further grouped into the HFOV group (24 patients) and the CLPV group (19 patients) depending upon modality of ventilation received after satisfying fi rst-time HFOV eligibility criteria. Both groups were comparable for diff erences with Fisher’s t test for qualitative variables and ANOVA for quantitative variables (Table 1), except for higher mortality in the CLPV group (16/19 (84.4%) vs. 12/12 (50%), P = 0.026). On logistic regression analysis to fi nd independent variables diff erentiating the two groups, mortality was higher in the CLPV group Conclusion A highly variable and signifi cant fraction of lung parenchyma is always closed with tidal ventilation at 30 cmH2O plateau pressure, regardless of the PEEP level applied; this implies that sigh or periodic recruitment maneuvers may lead to opening and closing. Intratidal opening/closing was reduced but not abolished in all patients while ventilating at higher PEEP level (15 cmH2O). P295 Lung-protective ventilation suppresses plasma levels of cell-free DNA in porcine experimental postoperative sepsis p p p p p A Nyberg1, J Sperber1, M Lipcsey2, J Jylhävä3, M Hurme3, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Eskilstuna, Sweden; 2Uppsala University, Uppsala, Sweden; 3University of Tampere, Finland Critical Care 2014, 18(Suppl 1):P292 (doi: 10.1186/cc13482) Introduction Mechanical ventilation aff ects systemic infl ammation where protective ventilation attenuates the response [1]. Plasma S105 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 levels of cell-free DNA (cfDNA) increase and have prognostic value in sepsis [2]. In the present study, the eff ect of tidal volume and PEEP on arterial and transorgan levels of cfDNA was investigated in a porcine postoperative sepsis model. (P = 0.02, odds ratio (CI) 71.60 (1.85 to 2,766.59)) compared with the HFOV group. levels of cell-free DNA (cfDNA) increase and have prognostic value in sepsis [2]. In the present study, the eff ect of tidal volume and PEEP on arterial and transorgan levels of cfDNA was investigated in a porcine postoperative sepsis model. Conclusion HFOV when applied as rescue therapy for refractory hypoxia due to severe ARDS caused by H1N1 infl uenza pneumonia is associated with better outcome compared with CLPV. Methods Two groups of anaesthetised pigs were ventilated with either protective ventilation (VT 6 ml/kg, PEEP 10 cmH2O; n = 20) or controls (VT 10 ml/kg, PEEP 5 cmH2O; n = 10) for 7 hours. An artery, the hepatic vein, the portal vein and the jugular bulb were catheterized. Continuous endotoxin infusion at 0.25 μg/kg/hour for 5 hours was started after 2 hours of laparotomy that simulated a surgical procedure. Results The group receiving protective ventilation showed lower levels of cfDNA in arterial blood compared with controls (P = 0.02). Trans- hepatic levels of cfDNA were higher compared with trans-splanchnic levels during the experiment (P = 0.02), but this eff ect was attenuated in the group receiving protective ventilation. No diff erence between the groups was detected in blood samples from the jugular bulb. P294 References References 1. ARDsnet: N Engl J Med 2002, 342:1301-1308. 2. Young D, et al.: N Engl J Med 2013, 368:806-813. 3. The ARDS Defi nition Task Force: JAMA 2012, 307:2526-2533. 1. ARDsnet: N Engl J Med 2002, 342:1301-1308. 2. Young D, et al.: N Engl J Med 2013, 368:806-813. 3. The ARDS Defi nition Task Force: JAMA 2012, 307:2526-2533. 1. ARDsnet: N Engl J Med 2002, 342:1301-1308. 2. Young D, et al.: N Engl J Med 2013, 368:806-813. 3. The ARDS Defi nition Task Force: JAMA 2012, 307:2526-2533. Results The study was completed with 36 patients overall, being 11 patients in failure and 25 patients in the success group. In both groups, areas under the curve (AUCs) were calculated for each minute via ROC analysis using minute SPIF and SPEF values measured during SBT. The maximum AUC was calculated at minute 23 for SPIF (0.564; 95% CI: 0.363 to 0.764) and at minute 9 for SPEF (0.542; 95% CI: 0.316 to 0.376). Cutoff values were determined for the minutes in which maximum AUC values for SPIF and SPEF were detected; and sensitivity and specifi city values were calculated. When the cutoff value for SPIF was accepted as >26.7 l/minute at minute 23, sensitivity and specifi city was calculated as 72.0% and 28.0%, respectively. When the cutoff value for SPEF was accepted as >24.7 l/minute at minute 9, sensitivity and specifi city were calculated as 63.6% and 48.8%, respectively. 1. Boles JM, et al.: Weaning from mechanical ventilation. Eur Respir J 2007, 29:1033-1056. f References 1. Schumann S. et al.: Determination of respiratory system mechanics during inspiration and expiration by FLow-controlled EXpiration (FLEX): a pilot study in anesthetized pigs. Minerva Anestesiol 2014, in press. 2. Goebel U. et al.: Flow-controlled expiration (FLEX): a novel ventilation mode to attenuate experimental porcine lung injury. Br J Anaesth 2014, in press. 1. Schumann S. et al.: Determination of respiratory system mechanics during inspiration and expiration by FLow-controlled EXpiration (FLEX): a pilot study in anesthetized pigs. Minerva Anestesiol 2014, in press. y p g p 2. Goebel U. et al.: Flow-controlled expiration (FLEX): a novel ventilation mode to attenuate experimental porcine lung injury. Br J Anaesth 2014, in press. P298 P298 Lung ultrasound fi ndings predict weaning failure from mechanical ventilation Mechanisms underlying the lung-protective eff ects of FLow- controlled EXpiration p S Schumann, U Goebel, J Haberstroh, J Guttmann p S Schumann, U Goebel, J Haberstroh, J Guttmann University Medical Center Freiburg, Germany y g, y Critical Care 2014, 18(Suppl 1):P296 (doi: 10.1186/cc13486) Introduction During mechanical ventilation the expiration occurs passively and is determined by the recoil forces of the respiratory system. In an experimental study in pigs we could fi nd that linearization of the expiratory fl ow via FLow-controlled EXpiration (FLEX) [1] is lung protective [2]. Utilizing electrical impedance tomography (EIT) we aimed at investigating the mechanisms underlying the lung-protective eff ects of FLEX. p y Conclusion We think that minute SPIF measurement which has better sensitivity and minute SPEF measurement which has better specifi city compared with available traditional predictors [1] may be used as a potential bedside weaning predictor when evaluated in comprehensive studies. f Methods All experiments were approved by the local animal welfare committee. Twelve pigs with oleic acid-induced lung injury were ventilated in the volume-controlled mode (VCV). In six animals, expiratory fl ow was linearized via FLEX. PEEP was set to achieve similar mean airway pressure in the control group (n = 6) and in the FLEX group (n = 6). Using EIT, the local distribution of ventilation was measured and alveolar derecruitment during the no-fl ow phase in late expiration was quantifi ed. Value of peak fl ow rates measured during a spontaneous breathing trial to predict success of weaning from mechanical ventilation K Gundogan, S Baldane, R Coskun, I Bahar, G Altunyurt, H Mumcuoglu, M Guven, M Sungur Value of peak fl ow rates measured during a spontaneous breathing trial to predict success of weaning from mechanical ventilation K Gundogan, S Baldane, R Coskun, I Bahar, G Altunyurt, H Mumcuoglu, M Guven, M Sungur Erciyes University, Kayseri, Turkey Critical Care 2014, 18(Suppl 1):P297 (doi: 10.1186/cc13487) Value of peak fl ow rates measured during a spontaneous breathing trial to predict success of weaning from mechanical ventilation K Gundogan, S Baldane, R Coskun, I Bahar, G Altunyurt, H Mumcuoglu, M Guven, M Sungur Erciyes University, Kayseri, Turkey Critical Care 2014, 18(Suppl 1):P297 (doi: 10.1186/cc13487) Table 1 (abstract P295). Tidal volume With ARDS (%) At risk of ARDS (%) <6 ml/kg 25.3 15.7 6 to 8 ml/kg 31.7 29.3 >8 ml/kg 43 55 Introduction Numerous parameters have been suggested for the prediction of weaning from mechanical ventilation; however, these parameters have limited success in the prediction of weaning outcome. The aim of this study is to assess the success of minute peak fl ow rates (spontaneous peak inspiratory fl ow rate (SPIF) and spontaneous peak expiratory fl ow rate (SPEF)) measured during a spontaneous breathing trial (SBT) in the prediction of weaning outcome. Conclusion Compliance with protective lung ventilation in our ICU is suboptimal. This may be due to the lack of education and guidelines in the unit regarding protective lung ventilation. Moreover, accurate recording of patient height and determination of predicted body weight should be documented to facilitate accurate tidal volume calculation and protective lung ventilation. Methods Patients managed and receiving mechanical ventilation support for at least 24 hours in the medical and surgical ICUs of Erciyes University between March 2011 and May 2012 were included in the present study. Over 30 minutes, SPIF and SPEF values were measured during a SBT in patients spontaneously breathing by T tube. Patients who tolerated 30 minutes of SBT were extubated. Patients who did not need reintubation for 48 hours after extubation were considered successful weaning, while those needing reintubation were considered weaning failure. Reference Reference 1. Boles JM, et al.: Weaning from mechanical ventilation. Eur Respir J 2007, 29:1033-1056. P295 Compliance with protective lung ventilation in an Irish teaching hospital Table 1 (abstract P293). Variable CLPV (n = 19) HFOV (n = 24) P value Age 41.15 32.83 0.07 SOFA 4.95 4.62 0.41 Pplat 29.57 30.46 0.26 O.I. 38.88 35.70 0.54 P/F 70.63 76.79 0.50 PCO2 59.89 59.92 0.99 Death 16 12 0.02 g p Critical Care 2014, 18(Suppl 1):P295 (doi: 10.1186/cc13485 Introduction The importance of protective lung ventilation in reducing mortality in adult respiratory distress syndrome (ARDS) patients is well described [1]. However, suboptimal compliance with the recommended tidal volumes has been reported [2]. Therefore, we wished to assess our compliance in adhering to protective lung ventilation in patients with, or at risk of developing, ARDS. p g Methods A retrospective review was done on all mechanically ventilated patients in the ICU of Tallaght Hospital over a 6-month period (February to July 2013). Hourly tidal volumes were recorded S106 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 automatically in electronic charts. Compliance was assessed by calculating the total time patients with, or at risk of developing, ARDS were ventilated with tidal volumes <6 ml/kg, 6 to 8 ml/kg, and >8 ml/ kg during the fi rst 72 hours on mechanical ventilation. ARDS is defi ned as per the Berlin criteria [3]. Exclusion criteria were patients who did not receive invasive ventilation or who were ventilated for less than 72 hours. We also assessed whether patients’ height was documented. Results A total of 72 patients were ventilated for >72 hours. Of these patients (44 males, 28 females, mean age 65.5 years), ARDS criteria were met in 17 patients and 22 patients were determined to be at risk of developing ARDS. For patients with ARDS, the ventilated time with tidal volumes <6 ml/kg, 6 to 8 ml/kg, and >8 ml/kg was 25.3%, 31.7% and 43% respectively. For patients at risk of developing ARDS, the ventilated time with tidal volumes <6 ml/kg, 6 to 8 ml/kg, and >8 ml/ kg was 15.7%, 29.3%, 55%, respectively (Table 1). A total of 16 patients (nine who had ARDS) had no documentation of their height. distribution of ventilation is more homogeneous. These mechanisms of alveolar recruitment maintenance can explain the lung-protective eff ects of FLEX. Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients AC Pecanha Antonio1, M Basso Gazzana2, P Souza Castro1, M Knorst1 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P299 (doi: 10.1186/cc13489) Introduction The rapid shallow breathing index (RSBI) is considered a good parameter to predict mechanical ventilator liberation. We hypothesized that the RSBI provides no benefi t when clinical readiness criteria are met. AC Pecanha Antonio1, M Basso Gazzana2, P Souza Castro1, M Knorst1 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P299 (doi: 10.1186/cc13489) AC Pecanha Antonio1, M Basso Gazzana2, P Souza Castro1, M Knorst1 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P299 (doi: 10.1186/cc13489) Introduction Fluid balance (FB) has been associated with weaning and extubation failure, particularly of cardiac origin. Also, the increased right ventricular afterload, a feature common in chronic obstructive pulmonary disease (COPD) patients especially during a weaning test, may hinder diastolic fi lling of the left ventricle through a biventricular interdependence mechanism. We aimed to investigate the relationship of the FB in the 48 hours prior to a spontaneous breathing trial (SBT) and weaning outcomes in a subgroup of COPD patients admitted to a medical–surgical ICU. Methods Adults with acute respiratory who required MV for more than 24 hours, excluding COPD, were assessed daily as a liberation protocol (Figure 1). During the RSBI step, RSBI was recorded and blinded to the Figure 1 (abstract P300). Standard mechanical ventilator weaning protocol. Methods We conducted a 2-year prospective, multicenter, obser- vational study in two adult medical–surgical ICUs. All enrolled patients met eligibility criteria for ventilation liberation. Patients with tracheostomy were excluded. We collected demographic, physiologic, 48-hour FB (measured inputs minus outputs) and lung ultrasound fi ndings immediately before a SBT in 29 COPD patients. Our main outcome of interest was weaning failure (WF), defi ned as the inability to tolerate a T-tube trial during 30 to 120 minutes, in which case patients were not extubated. p Results Weaning success (WS) (n = 19) and WF (n = 10) patients were similar in relation to age, sex, APACHE II score, reason for mechanical ventilation (MV) and comorbidities. Mean duration of MV was 11 days. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Therefore, the presence of cardiovascular dysfunction can contribute to weaning failure (WF). A randomized trial concluded that bedside lung ultrasound could predict post-extubation distress through changes in aeration during a T-tube test; however, it could not screen patients before submission to a SBT [1]. We aim to assess the reliability of lung ultrasound as a predictor of weaning outcomes. Figure 1 (abstract P299). Association between positive fl uid balance WF in COPD patients. Methods We conducted a prospective, multicenter, observational study in two adult medical–surgical ICUs. Lung ultrasound was performed immediately before SBT. Three or more B-lines in a single view were called a B-pattern. B-predominance was defi ned as a B-pattern on at least one of the four anterior chest wall zones. All enrolled patients met eligibility criteria for ventilation liberation. Patients with tracheostomy were excluded. Results During 2 years, 250 SBTs were analyzed. WF, defi ned as an inability to tolerate a T-tube trial during 30 to 120 minutes, occurred in 51 (20.4%). There was a higher prevalence of chronic obstructive pulmonary disease in the WF group as well as higher duration of mechanical ventilation. WF patients were also younger. Patients succeed at SBT and were extubated at fi rst time in 75.9% of cases. We observed a signifi cant association between B-predominance prior to submission to SBT and WF (OR = 1.99 (1.04 to 3.84)). For diagnosing WF, B-predominance showed 69% sensitivity, 48% specifi city, 25% positive predictive value, and 86% negative predictive value.i Figure 1 (abstract P299). Association between positive fl uid balance WF in COPD patients. Reference Conclusion The fi nding of B-predominance at bedside lung ultrasound performed before SBT predicts WF, although it shows low accuracy. Reference . Perren A, Brochard L: Managing the apparent and hidden diffi culties of weaning from mechanical ventilation. Intensive Care Med 2013, 39:1885-1895. . Perren A, Brochard L: Managing the apparent and hidden diffi culties of weaning from mechanical ventilation. Intensive Care Med 2013, 39:1885-1895. 1. Soummer A, Perbet S, Brisson H, Arbelot C, Constantin JM, Lu Q, Rouby JJ; Lung Ultrasound Study Group: Ultrasound assessment of lung aeration loss during a successful weaning trial predicts postextubation distress. Crit Care Med 2012, 40:2064-2072. P300 P300 Role of the rapid shallow breathing index to predict the success of mechanical ventilator liberation in acute respiratory failure C Chanpharm, R Bhurayanontachai Prince of Songkla University, Hat Yai, Songkhla, Thailand Critical Care 2014, 18(Suppl 1):P300 (doi: 10.1186/cc13490) Lung ultrasound fi ndings predict weaning failure from mechanical ventilation i Results During ventilation with FLEX the no-fl ow phase in late expiration was reduced by 50% compared with passive expiration. Derecruitment during the no-fl ow phase was clearly reduced by FLEX compared with VCV. Furthermore, intratidal ventilation was more homogeneously distributed during ventilation with FLEX compared with conventional passive expiration. Lung ultrasound fi ndings predict weaning failure from mechanical ventilation AC Pecanha Antonio1, P Souza Castro1, LF Schulz1, J Maccari1, R Oliveira1, C Teixeira1, M Knorst2 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P298 (doi: 10.1186/cc13488) AC Pecanha Antonio1, P Souza Castro1, LF Schulz1, J Maccari1, R Oliveira1, C Teixeira1, M Knorst2 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P298 (doi: 10.1186/cc13488) Conclusion In comparison with conventional VCV with passive expiration, the no-fl ow phase in late expiration is reduced and so is the time the lung persists on the lowest pressure level (PEEP) during the breath. The reduced low-pressure time is associated with reduced end- tidal derecruitment. In a lung mechanically stabilized and recruited by sustained airway pressure throughout the expiration phase, the Introduction Lung ultrasound is increasingly becoming a diagnostic tool in the critical care setting. The B-line is an artifact that correlates with interstitial edema. Decreases in intrathoracic pressure during a spontaneous breathing trial (SBT) will augment venous return and impede left ventricular ejection, increasing intrathoracic blood volume. Introduction Lung ultrasound is increasingly becoming a diagnostic tool in the critical care setting. The B-line is an artifact that correlates with interstitial edema. Decreases in intrathoracic pressure during a spontaneous breathing trial (SBT) will augment venous return and impede left ventricular ejection, increasing intrathoracic blood volume. S107 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P303 i Results Twenty-two (63.04% of all) patients successfully passed the 2-hour SBT. Of them, fi ve failed the subsequent 24-hour SBT. Almost all of the latter (four out of fi ve) were ventilated due to chronic respiratory or cardiac diseases. The following parameters were signifi cantly diff erent between the groups of patients who successfully and unsuccessfully completed 2-hour SBT: pre-inclusion body weight; PEEPi (end-expiratory occlusion method), peak and mean airway pressures during CMV; PaO2, PaO2/FiO2 and oxygenation index during the SBT; respiratory rate, tidal volume, f/Vt, patient work of breathing (modifi ed Campbell method), total body oxygen consumption, respiratory quotient and energy expenditure during ZEEP and the SBT. The logistic regression model devised to predict the outcome of the 2-hour SBT included the following parameters: type of artifi cial airway (tracheotomy vs. translaryngeal), oxygen consumption, f/Vt and energy expenditure during the SBT. P301 g Results A total of 268 patients were enrolled: 187 (69.8%) patients with solid tumors and 81 (30.2%) patients with hematological malignancies. In total, 167 (62.3%) patients had septic shock, and ICU and hospital mortality rates were 45.5% and 67.9%. Microbiological confi rmation was present in 140 (52%) patients with 56% Gram-negative. The most frequent pathogens were methicillin-sensitive S. aureus (36 (26%)), P. aeruginosa (35 (25%)) and S. pneumoniae (16 (12%)). Low incidence of MR (16 (11.4%)) was observed. Adequate antibiotic therapy based on microbiological identifi cation was prescribed in 120 (85.72%) patients. Adherence to ATS/IDSA guidelines was observed in 41 (15.3%) patients. There were no diff erences regarding ATS/IDSA guideline adherence (MR 3 (18.8%) vs. no MR 38 (15.1%), P = 0.719). We observed a trend towards higher hospital mortality in the MR patients (MR 14 (87.5%) vs. no MR 168 (66.7%), P = 0.101). In multivariate analysis, mechanical ventilation (OR 2.52 (1.19 to 5.32)), dialysis (3.86 (1.23 to 12.10)) and higher SAPS2 (OR per point 1.03 (1.01 to 1.05)) were associated with increased hospital mortality whereas successful noninvasive ventilation was associated with lower mortality (0.32 (0.13 to 0.77)). MR were forced into MV analysis but were not associated with outcomes. Conclusion Severe pneumonia in cancer patients presents high hospital mortality, with particular clinical and microbiology features. Despite low adherence to ATS/IDSA guidelines, antibiotic therapy was adequate in most of patients following local guidelines based on local bacterial profi les. Further investigation is needed to clarify the impact of MRs in clinical outcomes of cancer patients with severe pneumonia. P302 Microbiology and outcomes of severe pneumonia in critically ill cancer patients L Rabello1, J Salluh1, M Soares1, M Rosolem1, V Bogado1, I Souza1, J Lapa e Silva2, L Azevedo3 1DOr Institute of Research, Rio de Janeiro, Brazil; 2Postgraduate Program of Internal Medicine – Universidade Federal do Rio de Janeiro, Brazil; 3Hospital Sirio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P302 (doi: 10.1186/cc13492) researcher. The liberation process was continued regardless of the RSBI result. The primary outcome was the success rate of mechanical ventilator liberation with or without RSBI. Introduction Pneumonia is the most frequent types of infection in cancer patients. The presence of multiresistant pathogens (MR) is often associated with inadequate antimicrobial therapy. The aims of this study were to describe the microbiology and outcomes of cancer patients with severe pneumonia requiring ICU admission. Results Analysis of 120 cases with clinical characteristics as presented in Table 1. There was no statistically signifi cant diff erence between using only clinical readiness and using clinical readiness and the RSBI (92% vs. 89%, P = 0.43). Methods A secondary analysis of a prospective cohort study was performed from 2002 to 2011 at Instituto Nacional de Cancer and Hospital Sirio-Libanes, Brazil. Adult patients with a diagnosis of cancer with pneumonia (not acquired in the hospital setting) were evaluated at ICU admission. Demographic, clinical and laboratory data were collected during the fi rst day in the ICU, severity scores, comorbidities, performance status, cancer-related data, microbiologic identifi cation, empiric antibiotics and the adherence to treatment guidelines. Conclusion The inclusion of RSBI in our standard mechanical ventilator liberation protocol for patients who met the clinical readiness criteria did not signifi cantly increase the success rate of mechanical ventilator liberation. Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients FB in the 48 hours prior to the SBT did not diff er between the WS and WF groups (1,091.11 ± 2,195.89 ml and 2,398.80 ± 1,533.15 ml, respectively). Nevertheless, comparing individuals with 48-hour FB above and under the cutoff value of 0 ml according to weaning outcomes resulted in signifi cant association between positive FB and WF in COPD patients (odds ratio = 1.77 (1.24 to 2.53)). That cutoff point was obtained on the ROC curve. See Figure 1. g Conclusion Positive FB in the 48 hours preceding the SBT predicted WF in COPD individuals. We recognized that no intervention was performed in order to accelerate the weaning process. Brain natriuretic peptide levels were not available. Figure 1 (abstract P300). Standard mechanical ventilator weaning protocol. Figure 1 (abstract P300). Standard mechanical ventilator weaning protocol. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S108 Table 1 (abstract P300). Character CR CR + RSBI P value Age 55.79 ± 19.6 56.62 ± 19.4 0.74 Male sex (%) 58.3 59.1 0.90 Success rate (%) 89.2 92.2 0.43 P302 Microbiology and outcomes of severe pneumonia in critically ill cancer patients L Rabello1, J Salluh1, M Soares1, M Rosolem1, V Bogado1, I Souza1, J Lapa e Silva2, L Azevedo3 1DOr Institute of Research, Rio de Janeiro, Brazil; 2Postgraduate Program of Internal Medicine – Universidade Federal do Rio de Janeiro, Brazil; 3Hospital Sirio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P302 (doi: 10.1186/cc13492) P301 Determinants of ventilator weaning outcome in a medical–surgical ICU G Georgiev1, S Milanov1, L Todorova2, M Matveev2 1University Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria; 2Bulgarian Academy of Sciences, Sofi a, Bulgaria i Critical Care 2014, 18(Suppl 1):P301 (doi: 10.1186/cc13491) Introduction The purpose of this study is to prospectively evaluate the determinants of weaning outcome in a selected sample of ventilator- dependent patients.i Introduction The purpose of this study is to prospectively evaluate the determinants of weaning outcome in a selected sample of ventilator- dependent patients.i Methods After fulfi lling a set of inclusion/exclusion criteria, 46 patients treated in a single medical–surgical ICU were prospectively evaluated between October 2011 and January 2013. The study protocol followed the generic course of mechanical ventilatory support and its discontinuation. A period of CMV was followed by stepwise reduction in pressure support. The outcomes were recorded at the second and 24th hour after the beginning of the PSV (8 cmH2O PS) spontaneous breathing trial (SBT). A short period of ZEEP breathing was introduced after the CMV as an additional respiratory stressor. A large number of parameters (including those derived via indirect calorimetry and esophageal balloon catheter) were analyzed. The following statistical methods were used: Student’s t test or its nonparametric alternatives for intergroup comparisons, repeated-measurements ANOVA for intragroup comparisons, cross-tabulation and Fisher’s exact test to compare categorical variables. Logistic regression analysis was conducted with regard to success/failure classifi cation. P304 g Results Median age and SOFA scores on ICU admission were 66 (interquartile range: 50 to 77) years and 6 (4 to 8) years, respectively. Primary diagnosis on ICU admission was coma including post-cardiac arrest syndrome (31%), trauma (29.4%), and sepsis (9.8%). Prediction of MDRPs was signifi cantly higher in the risk group than in the nonrisk group (36.8% vs. 15.2%, P = 0.004). Guideline adherence was lower in the risk group (45.6% vs. 65.2%, P = 0.016). Treatment adequacy was greater with guideline adherence than with nonadherence (75.7% vs. 55.9%, P <0.01). Hospital mortality was not aff ected by guideline adherence (P = 0.70). Multivariate analysis revealed that the independent factors related to hospital mortality were adequate antimicrobial treatment (odds ratio: 0.53, 95% CI: 0.29 to 0.95; P = 0.034), age (1.02, 1.00 to 1.04; P = 0.004), history of malignancy (8.59, 1.48 to 49.6; P = 0.016), trauma (0.41, 0.2 to 0.87; P = 0.02), acute kidney injury (5.54, 2.69 to 11.4: P <0.001), and severe sepsis at VAP onset (2.7, 1.4 to 5.1; P = 0.002). Conclusion The prediction of MDRPs using the 2005 ATS/IDSA guidelines was acceptable. To ensure adequate antimicrobial treatment, strict guideline adherence is required in the Japanese setting. Clinical pulmonary infection score calculator in the early diagnosis and treatment of ventilator-associated pneumonia in the ICU O Celik, N Koltka, S Devrim, B Sen, M Gura Celik Istanbul Medeniyet University, Goztepe Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P304 (doi: 10.1186/cc13494) Introduction Ventilator-associated pneumonia (VAP) is a frequently occurring nosocomial infection in ICU patients and has been associated with increased morbidity, prolonged duration of ventilation and ICU stay and increased costs for healthcare. It was shown that early diagnosis of VAP and immediate initiation of appropriate antibiotics is associated with reduced morbidity and mortality. The aim of this study is to evaluate the potential ability of a screening test based on the clinical pulmonary infection score (CPIS) to identify and treat patients with VAP.i y Methods All fi les belonging to patients between 18 and 80 years old admitted to the ICU and supported by mechanical ventilation for longer than 48 hours were evaluated retrospectively. Demographic data of the patients, the time of mechanical ventilation, duration of the ICU stay and results (survival or death) were recorded. The CPIS was calculated after 48 hours for the diagnosis of VAP. References References 1. Vincent JL, et al.: JAMA 2009, 302:2323-2329. 2. Chastre J, et al.: Am J Respir Crit Care Med 2002, 165:867-903. 3. Conway Morris A, et al.: Thorax 2010, 65:201-207. 4. Wilkinson TS, et al.: Chest 2012, 142:1425-1432. P305 This study aimed to evaluate the eff ectiveness of microbial prediction and validate the adequacy of these guidelines for antibiotic strategy in VAP patients. g p Conclusion This study demonstrates that IL-1β eff ectively excludes VAP when validated in a multicentre study. The performance is further improved by the addition of IL-8, and the combination could form a rapid diagnostic assay to exclude VAP. Biomarker analysis appears to have the potential to improve antibiotic stewardship, and this concept should be formally tested in the setting of a randomised controlled trial. y y Methods We retrospectively analyzed 296 patients who received empiric antimicrobial treatment for VAP between January 2006 and December 2010 in 10 ICUs of Japanese tertiary hospitals. VAP was diagnosed according to CDC criteria. After assigning patients to the ATS/IDSA risk group (n = 250; late onset or risk factors for MDRPs) or non-risk group (n = 46; early onset without risk factors for MDRPs), we determined the accuracy of the guidelines for predicting pathogens and the impact of guideline adherence on patient outcome. P305 non-VAP groups and receiver operating characteristics (ROC) curves were constructed for individual biomarkers and combinations of markers before optimum cutoff points were determined. non-VAP groups and receiver operating characteristics (ROC) curves were constructed for individual biomarkers and combinations of markers before optimum cutoff points were determined. Validation of the 2005 American Thoracic Society/Infectious Diseases Society of America guidelines for ventilator-associated pneumonia: a Japanese multicenter observational study N Saito1, N Shime2, K Sugiyama3, H Yasuda4, K Ishii5, Y Masuda6, T Kanemura7, W Sakamoto8, T Hirayu9 1Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan; 2Kyoto Medical Center, Kyoto, Japan; 3Tokyo Metropolitan Hospital Bokuto, Tokyo, Japan; 4Musashino Red Cross Hospital, Tokyo, Japan; 5Fukuyama City Hospital, Fukuyama, Japan; 6Nagasaki Medical Center, Omura, Japan; 7National Disaster Medical Center, Tokyo, Japan; 8Nippon Medical School Hospital, Tokyo, Japan; 9Kurume University Hospital, Kurume, Japan Critical Care 2014, 18(Suppl 1):P305 (doi: 10.1186/cc13495) f Results A total of 150 patients had paired semi-quantitative culture and biomarker results. Fifty-three (35%) patients had VAP and 97 (65%) patients formed the non-VAP group. All BALF biomarkers were signifi cantly higher in the VAP group (P <0.001). The area under the ROC curve for IL-1β was 0.81, for IL-8 was 0.736, for MMP-8 was 0.758, for MMP-9 was 0.785 and for HNE was 0.777. Using a cutoff value of 17 pg/ ml, IL-1β had a sensitivity of 96.2%, a specifi city of 43.3%, a negative predictive value (NPV) of 95.5% and a post-test probability of 4.5% (95 CI: 1 to 16%). A combination of IL-1β and IL-8 excluded VAP with a sensitivity of 100%, a specifi city of 44.3% and a NPV of 1. There was no signifi cant diff erence in serum biomarkers between the VAP and non- VAP groups. Introduction To select the empirical antimicrobial treatment for patients with ventilator-associated pneumonia (VAP), the 2005 American Thoracic Society/Infectious Disease of America (ATS/IDSA) guidelines classify patients according to time of onset and risk factors for potential multidrug-resistant pathogens (MDRPs). This study aimed to evaluate the eff ectiveness of microbial prediction and validate the adequacy of these guidelines for antibiotic strategy in VAP patients. Introduction To select the empirical antimicrobial treatment for patients with ventilator-associated pneumonia (VAP), the 2005 American Thoracic Society/Infectious Disease of America (ATS/IDSA) guidelines classify patients according to time of onset and risk factors for potential multidrug-resistant pathogens (MDRPs). P304 The patients with CPIS >5 intubated were evaluated VAP(+) and the others with CPIS ≤5 were thought VAP(–). The diagnosis of VAP was bacteriologically confi rmed with the culture of endotracheal aspirate. Statistical evaluations were done according to the results on the day of intubation and the results on days 2, 3, 5, 8 and 10 after intubation. Scores of APACHE II and CRP levels were also recorded on the same days. P306 P306 Biomarker-based exclusion of ventilator-associated pneumonia: a multicentre validation study T Hellyer, J Simpson T Hellyer, J Simpson Newcastle University, Newcastle, UK Critical Care 2014, 18(Suppl 1):P303 (doi: 10.1186/cc13493) T Hellyer, J Simpson Newcastle University, Newcastle, UK Critical Care 2014, 18(Suppl 1):P303 (doi: 10.1186/cc13493) Introduction Ventilator-associated pneumonia (VAP) remains a leading cause of nosocomial infection in the ICU [1]. VAP is confi rmed by positive microbiology in approximately one-third of patients with suspected VAP [2], implying that there is scope to improve antibiotic stewardship. In a single-centre study, bronchoalveolar lavage fl uid (BALF) infl ammatory mediators (in particular interleukin-1 beta (IL-1β)) [3] and neutrophil proteases [4] demonstrated potential as biomarkers to exclude VAP. We aimed to validate these fi ndings in a multicentre study. i Methods We conducted a prospective, multicentre observational study of 167 patients with clinically suspected VAP from 12 ICUs across the UK. VAP was confi rmed by growth of a potential pathogen in BALF at >104 colony-forming units/ml. IL-1β, IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were measured in BALF by cytometric bead array. IL-6, IL-8, MMP-8, MMP-9 and HNE were measured in serum. Patients were dichotomised into VAP and Conclusion The presence of serious respiratory and/or cardiac comorbidities might require longer duration of the SBT. In our study, respiratory load–muscle capacity balance and metabolic activity appear to play a major role in determining the weaning outcome. A short period of ZEEP breathing is safe and might have prognostic utility regarding the outcome of mechanical ventilatory support discontinuation. S109 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Extracorporeal carbon dioxide removal as a bridge to lung transplantation in life-threatening hypercapnia Results Sixteen subjects were recruited for this study. There was a signifi cant increase in paO2 (13.6 ± 2.35 kPa, P = 0.01) and decrease in paCO2 (4.82 ± 1.27 kPa, P = 0.02) in the chair group at 1 hour after sitting out in the chair when compared with baseline (10.9 ± 2.44 kPa; 5.41 ± 1.32 kPa). Also there was a signifi cant increase in tidal volume in the chair group after 1 minute of sitting out (403 ± 118 ml) compared with baseline (314 ± 105 ml). There was no diff erence in the electric bed group for all physiological parameters. The chair group had a better CPAX score on discharge from intensive care (chair group 24; electric bed group 13) and on discharge from the hospital (chair group 39; electric bed group 16). There were no adverse cardiovascular responses to either position. Introduction The introduction of the lung allocation score has resulted in a growing number of patients who are considered for lung transplantation (LTX) while being acutely decompensated. In the sickest of these patients, mechanical ventilation (MV) alone may not be suffi cient to establish adequate gas exchange. Thus, diff erent modes of extracorporeal life support have come to the focus of interest in this setting. Conclusion Sitting suitable critically ill patients out into a chair is safe and can signifi cantly improve the arterial blood gas measurement and the tidal volume when compared with sitting a patient into the sitting position in an electric bed. g Methods A retrospective analysis of 17 patients (male/female ratio: 6/11; median age: 35 (range 16 to 63)) who underwent arteriovenous or venovenous interventional lung assist (iLA; Novalung, Germany) support as bridging to primary LTX (n = 11) or re-LTX (n = 6) between 2005 and 2013. p Reference 1. Zhiqiang L, et al.: Arch Phys Med Rehab 2013, 94:551-561. 1. Zhiqiang L, et al.: Arch Phys Med Rehab 2013, 94:551-561. 1. Zhiqiang L, et al.: Arch Phys Med Rehab 2013, 94:551-561. Results The underlying diagnosis was bronchiolitis obliterans syndrome III in re-LTX patients (n = 6), cystic fi brosis (n = 5), idiopathic pulmonary fi brosis (n = 2), emphysema (n = 1), adult respiratory distress syndrome (n = 1), hemosiderosis (n = 1), and chronic obstructive lung disease (n = 1), respectively. The type of iLA was arteriovenous in 10 and venovenous (iLA active) in seven patients. The median bridging time was 14 (1 to 58) days. The type of transplantation was bilateral LTX (n = 6), size-reduced bilateral LTX (n = 5), lobar bilateral LTX (n = 4), and right single LTX with contralateral pneumonectomy (n = 1), respectively. Hypercapnia was eff ectively corrected in all patients within the fi rst 12 hours of iLA therapy: PaCO2 levels declined from 145 (70 to 198) to 60 (36 to 99) mmHg, P <0.0001. iLA was initiated during non-invasive ventilation in three patients, of whom one was intubated prior to LTX. All other patients (n = 14) were placed on iLA while on invasive MV. Of those, three patients were extubated and remained on iLA until LTX, one patient was weaned from iLA and remained on MV until LTX, and one patient was weaned from iLA and MV prior to LTX. Five patients were switched to extracorporeal membrane oxygenation (venovenous n = 2, venoarterial n = 3) after 5 (1 to 30) days on iLA support. One patient died prior to LTX due to septic multiorgan failure (SMOF). All others (n = 16; 94%) were successfully transplanted. Of these, two patients died in the ICU due to SMOF. The remaining 14 patients (82%) survived to hospital discharge and were alive at a median follow-up of 20 (1 to 63) months. P309 Quantifying sputum production in intensive therapy C Pope, R Spacie, S Reynolds, H Jones Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P309 (doi: 10.1186/cc13499) Reference 1. Magill SS, et al.: Developing a new national approach to surveillance for ventilator-associated events: executive summary. Crit Care Med 2013, 41:2467-2475. 1. Magill SS, et al.: Developing a new national approach to surveillance for ventilator-associated events: executive summary. Crit Care Med 2013, 41:2467-2475. Diff erence in physiological parameters between sitting out of bed into a chair or sitting up on an electric bed in the adult ICU A Fitzgerald, D Breen Diff erence in physiological parameters between sitting out of bed into a chair or sitting up on an electric bed in the adult ICU A Fitzgerald, D Breen Cork University Hospital, Cork, Ireland Critical Care 2014, 18(Suppl 1):P308 (doi: 10.1186/cc13498) Introduction The primary aim of this study was to identify in adult intensive care patients whether there was a diff erence in the acute physiological response when a patient is sat out into a chair compared with when a patient is placed in a chair position using an electric bed. The secondary aim of this study was to observe the functional outcome of these patients [1]. Conclusion While similar numbers of VAEs are triggered using CDC and clinical criteria, we noted a disparity in the VAP incidence. Four clinical VAPs were not triggered due to the hierarchical nature of the CDC criteria because the initial criterion of sustained minimum PEEP or FiO2 rise was not met despite clear clinical criteria based on increased infl ammatory markers, temperature spikes and new microbiology. We are therefore doubtful of the robustness of this tool in its current format as an accurate measure of quality assurance and agree with recent statements from the CDC [1] that modifi cations may be necessary. R f Methods The study was conducted in an adult tertiary referral ICU over a 3-month period. Patients that met predetermined inclusion/exclusion criteria were allocated to either sitting in a chair or sitting up in an electric bed. Heart rate, respiratory rate, tidal volume and mean arterial pressure were obtained for all patients when they were supine in bed, at 1 minute and at 1 hour in the new position. Arterial blood gases were obtained at 1 hour in their new position. A functional outcome measure known as the Chelsea Critical Care Physical Assessment Tool (CPAX) was also taken on the day of admission, on the day of sitting out, on the day of discharge from intensive care and at ward level. All data were analysed using Student’s t test. Surveillance and evaluation of ventilator-associated events as per Centers for Disease Control and Prevention guidelines M Brown, B Rose Surveillance and evaluation of ventilator-associated events as per Centers for Disease Control and Prevention guidelines M Brown, B Rose Lewisham and Greenwich NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P306 (doi: 10.1186/cc13496) Lewisham and Greenwich NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P306 (doi: 10.1186/cc13496) Introduction While ventilator-associated pneumonia (VAP) can aff ect all patients ventilated for >2 days in an ICU, an issue during the last two decades has been identifi cation of a universally accepted defi nition. The Centers for Disease Control and Prevention (CDC) in Atlanta, GA, USA, have designed a new surveillance paradigm aiming to increase the objectivity of VAE diagnosis ranging from ventilator-associated condition (VAC), infective VAC to possible and probable VAP [1]. We assessed the feasibility and accessibility of the audit tool developed from the CDC guidelines as a marker of quality assurance. Results The duration of mechanical ventilation and ratio of death were signifi cantly higher in the patients with VAP(+). CPIS levels in the patients with VAP(+) were signifi cantly higher than the patients with VAP(–) in the days after the diagnosis. CPIS levels were also higher in the patients with VAP(+) on the day of diagnosis. At the same day the parameters, which included the CPIS, body temperature, leukocyte number, tracheal secretions, PaO2/FiO2 levels and the presence of infi ltrates on the chest radiograph, were signifi cantly higher in VAP(+) patients (P <0.05). ROC curves were formed for CPIS scores to be used in diagnosis VAP and the cutoff point had a sensitivity of 97.44% and a specifi city of 100% for diagnosing VAP. Methods A prospective audit of patients admitted to University Hospital Lewisham ICU for intubation and ventilation was performed for September and October 2013. Recording of minimum PEEP and FiO2, minimum and maximum temperatures, WBC count, specimens sent, antibiotics administered and date of organisms found was performed on a daily basis. VAEs were recorded as per modifi ed CDC criteria (absence of quantitative microbiological testing) and compared Conclusion At the end of the study, it was concluded that using the CPIS for early diagnosis and treatment of VAP and thinking that the patients with CPIS >5 were VAP(+) are guiding factors to resolve the problems associated with VAP in ICU patients. S110 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion In patients with life-threatening hypercapnia, bridging to LTX with iLA is feasible, and results in favorable short-term and long- term outcome. Conclusion In patients with life-threatening hypercapnia, bridging to LTX with iLA is feasible, and results in favorable short-term and long- term outcome. against a surrogate marker of clinically diagnosed VAEs based on information available to the clinician. Results Forty patients were admitted with 29 fulfi lling inclusion criteria. Of n = 29, mean age was 58 (SD 21) with a median number of 5 (2 to 29) ventilator-days. We recorded eight VAEs based on CDC criteria, one VAC, four IVAC and three VAPs. Clinical decision-making indicated seven VAEs, of which fi ve were VAPs. CDC and clinical criteria correlated in only fi ve of eight VAPs and clinical criteria alone would have indicated 40% more VAPs than CDC criteria. Median time to VAE trigger was 8 days (4 to 20), median number of ventilator-days if a CDC VAE triggered was 14 compared with 3 if no CDC VAE was triggered (P <0.01), and 11 compared with 3 (P <0.01) for clinical VAEs. P307 Extracorporeal carbon dioxide removal as a bridge to lung transplantation in life-threatening hypercapnia K Riss, G Lang, T Staudinger, R Ullrich, CG Krenn, C Sitzwohl, A Bojic, P Wohlfarth, WR Sperr, W Rabitsch, C Aigner, S Taghavi, P, W Klepetko, P Schellongowski Medical University of Vienna, Austria Critical Care 2014, 18(Suppl 1):P307 (doi: 10.1186/cc13497) Extracorporeal carbon dioxide removal as a bridge to lung transplantation in life-threatening hypercapnia K Riss, G Lang, T Staudinger, R Ullrich, CG Krenn, C Sitzwohl, A Bojic, P Wohlfarth, WR Sperr, W Rabitsch, C Aigner, S Taghavi, P, W Klepetko, P Schellongowski Medical University of Vienna, Austria Critical Care 2014, 18(Suppl 1):P307 (doi: 10.1186/cc13497) Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit MC Faulds, S Lobaz, AJ Glossopfi Introduction Bilevel non-invasive ventilation (NIV) is an established therapy in chronic obstructive pulmonary disease (COPD) and cardiogenic pulmonary oedema but evidence for its use in other acute respiratory conditions is less robust. Reported ICU mortality after NIV treatment of pneumonia ranges between 18 and 33% [1,2], compared with 10% in exacerbations of COPD [3]. We aimed to study the outcomes of patients with acute respiratory failure of mixed aetiology treated with NIV in our critical care unit and compare fi ndings with those already published. Conclusion In this study, NHF gradually improved oxygenation. Additionally, NHF reduces the respiratory rate and the value of PaCO2. This result might suggest that NHF decreased dead-space ventilation. There was no diff erence between the success group and the failure group. So we can use NHF as the fi rst choice for postoperative respiratory failure, but it is diffi cult to predict success or failure. y p Methods Data were collected retrospectively on patients admitted to our critical care unit with acute respiratory failure requiring NIV over a 3-year period using the Metavision electronic patient record system. Patients with a primary surgical problem and those who received continuous positive airway pressure as a primary intervention were excluded. We recorded: primary respiratory diagnosis causing respiratory failure; patient demographics; serial arterial blood gas results; success of NIV as defi ned by the British Thoracic Society (BTS) [4]; and mortality statistics.i Inhalation injury and clinical course in major burned patients S Henrich, TH Rech, IC Warwzeniak, RB Moraes, E Parolo, K Prado, SR Vieira Hospital de Clínicas de Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P312 (doi: 10.1186/cc13502) Results In total, 113 consecutive patients were identifi ed. Mean age was 64 and 50% (56/113) were male. The primary diagnosis was pneumonia in 55 patients and exacerbation of COPD in 40 patients. The overall mortality on critical care, in hospital and at 1 year was 19% (22/113), 34% (38/113) and 41% (46/113) respectively. Success of NIV as defi ned by BTS criteria (pH >7.3 or reduction in PaCO2 by 0.5 kPa) in the fi rst 6 hours was seen in 72% (80/111) of patients. In NIV responders, 1-year mortality was 31% (25/80) compared with 65% (20/31) in nonresponders. Introduction Inhalation injury is the primary determinant of mortality in major burned patients, particularly when associated with pneumonia. Eff ect of nasal high fl ow for postoperative respiratory failure: a prospective observational study Eff ect of nasal high fl ow for postoperative respiratory failure: a prospective observational study p p y S Okahara, K Shimizu, M Hayashi, H Morimatsu Okayama University Hospital, Okayama City, Japan Critical Care 2014, 18(Suppl 1):P311 (doi: 10.1186/cc13501) Introduction We studied the eff ect of nasal high fl ow (NHF) for postoperative respiratory failure after extubation in our general surgical ICU. Recently some studies have reported that NHF improves oxygenation and reduces respiratory rate. However, the usefulness of NHF in the general surgical ICU has not been fully determined. NHF in the general surgical ICU has not been fully determined. Methods A prospective observational study was conducted in our general surgical ICU to investigate the eff ect of NHF on respiratory parameters in patients with postoperative respiratory failure. Patients who were admitted to the ICU for postoperative respiratory failure (defi ned as oxygen saturation as measured by pulse oximetry <96% and/or respiratory rate>24 beats/minute while receiving more than 6 l/ minute oxygen through a facemask) were eligible in this study. Pre and 1 and 6 hours after NHF treatment, we collected PaO2, PaCO2, respiratory rate, heart rate, and blood pressure. Data were presented with means and standard deviations. P <0.05 was considered statistically signifi cant. Results Forty-two patients were treated using NHF in our ICU from February 2013 to November 2013. The mean age of the patients was 62.8 ± 15.8 years, and the male:female ratio was 22:19. PaO2 values after 1 and 6 hours of NHF (104 ± 34 mmHg, 107 ± 26 mmHg, respectively) were signifi cantly higher than that before NHF (89 ± 38 mmHg; P <0.02). The PaO2/FiO2 ratio was increased from 1 hour to 6 hours of NHF (from 218 ± 90 mmHg to 236 ± 86 mmHg, P <0.05). Respiratory rate after NHF (19.6 ± 4.7/minute) was signifi cantly lower than that at baseline (22.3 ± 4.8/minute; P = 0.0006), whereas PaCO2 after NHF was reduced compared with baseline (from 41 ± 7 mmHg to 39 ± 5 mmHg, P <0.02). Thirty-two (76%, success group) patients did not need other positive ventilation. On the other hand, 10 (24%, failure group) patients required non-invasive positive pressure ventilation or intubation. We compared the failure group with the success group. However, there were no signifi cant diff erences in vital signs, total bleeding, operative duration and preoperative respiratory function between the groups. P311 decisions to extubate or decanulate. The quantifi cation of sputum was inconsistent: 39% of respondents counted the frequency of suctioning, 24% measured the quantity of sputum in the suction tubing, whereas 25% used another method. An eff ective cough, consistency and colour were felt to be more important features of sputum than blood staining. Conclusion Our results showed a very high level of agreement on the importance of knowing sputum load for decisions to extubate, decanulate or discharge from the ITU. In contrast, there was little consensus on how we should quantify sputum load in ventilated patients. This lack of standard approach may contribute to uncertainty in the clinical decision-making process. We have developed an objective sputum scoring system. Components identifi ed as important by our survey such as suction frequency, sputum consistency and colour are included. We have recognised the benefi ts of the standardised Bristol stool chart to facilitate communication and believe this can be achieved with sputum load in ventilated patients. R f P310 P310 Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P310 (doi: 10.1186/cc13500) Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit This study sought to describe the association between severity of inhalation injury on bronchoscopic examination and clinical course among fi re victims of Santa Maria, RS, Brazil. Two hundred and forty-two people were killed in the disaster. Methods Eighteen patients with inhalation injury secondary to smoke and fi re exposure in an enclosed space admitted to Hospital de Clínicas de Porto Alegre were divided into groups according to the severity of injury as determined by bronchoscopic criteria: grade 1 (moderate edema and hyperemia), grade 2 (marked edema and hyperemia, with or without carbonaceous debris), or grade 3 (mucosal ulceration or necrosis). Duration of mechanical ventilation (MV), length of ICU stay, overall length of hospital stay, and PaO2/FiO2 ratio on days 1 and 3 were compared among these groups by means of ANOVA with Tukey’s post- hoc correction. Conclusion NIV is used to treat acute respiratory failure due to a wide range of aetiologies in our unit with comparable mortality rates to large published series [1,2,5]. A successful response to NIV in the fi rst 6 hours is associated with a reduction in 1-year mortality when compared with nonresponders. Eff ect of nasal high fl ow for postoperative respiratory failure: a prospective observational study Conclusion In this study, NHF gradually improved oxygenation. Additionally, NHF reduces the respiratory rate and the value of PaCO2. This result might suggest that NHF decreased dead-space ventilation. There was no diff erence between the success group and the failure group. So we can use NHF as the fi rst choice for postoperative respiratory failure, but it is diffi cult to predict success or failure. 1. Fahy et al.: Airway mucus function and dysfunction. N Engl J Med 2010, 363:2233-2247 1. Fahy et al.: Airway mucus function and dysfunction. N Engl J Med 2010, 363:2233-2247 2. Kulkarni et al.: Extubation failure in intensive care: predictors and management. Indian J Crit Care Med 2008, 12:1-9. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Quantifying sputum production in intensive therapy C Pope, R Spacie, S Reynolds, H Jones Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P309 (doi: 10.1186/cc13499) Introduction Sputum is essential for the protection of the respiratory tract but also plays a signifi cant role in the pathophysiology of lung disease [1]. This is evident in critical care where high sputum loads contribute to respiratory failure [2]. The quantity of sputum produced by a patient can impact on key decisions such as weaning, extubation and discharge. We undertook a survey to establish whether there was a consensus on how we quantify sputum on our intensive therapy unit (ITU). Methods We conducted a multidisciplinary team questionnaire of our 28-bed tertiary ITU. Staff were asked how they quantifi ed sputum load in intubated patients. They were also asked to rate statements on a fi ve-point scale pertaining to sputum characteristics. The results were analysed in Excel 2010.f Results One hundred members of staff completed the sputum production in intensive therapy (SPIT) questionnaire (21% doctors, 71% nurses, 8% physiotherapists). Sputum load was deemed to be important or essential by more than 95% of respondents when making S111 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References 1. Carillo et al.: Intensive Care Med 2012, 38:458-466. 2. Jolliet et al.: Intensive Care Med 2001, 27:812-821. 3. Lightowler et al.: BMJ 2003, 326:185-189. 4. 2013 BTS NIV Audit [http://www.brit-thoracic.org.uk/Portals/0/Audit%20 Tools/SummaryReports/NIV%20Audit%202013-FINAL.pdf] 5. Confalonieri et al.: Am J Respir Crit Care Med 1999, 160:1585-1591. yg References 1. Cancio L, et al.: Clin Plastic Surg 2009, 36:555-567. 2. Mlcak RP, et al.: Burns 2007, 33:2-13. Figure 1 (abstract P314). Frequency of interventions in the base hospital (BH) and SARF centre. Nebulized C1-esterase inhibitor treatment does not attenuate pulmonary complement activation in a rat model of severe Streptococcus pneumoniae pneumonia FM De Beer, GJ Glas, CJ Beurskens, J Horn, MJ Schultz, WK Lagrand Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P315 (doi: 10.1186/cc13505) Introduction While complement protein defi ciencies are associated with severe and recurrent pulmonary infections, excessive complement activation plays a role in the pathogenesis of lung injury. We hypo- thesized that inhibition of the complement system by repetitive treatment with nebulized plasma-derived human C1-esterase inhibitor (C1-INH) reduces pulmonary complement activation and subsequently attenuates lung injury and lung infl ammation in a model of severe Streptococcus pneumoniae pneumonia. Conclusion ECMO/iLA therapy can be used as rescue therapy in adult trauma cases with severe hypoxemic respiratory failure, even in the presence of coagulopathy, bleeding and/or brain injury. The benefi ts of oxygenation and circulatory support must be weighed individually against the risk of hemorrhage. Further research should determine whether ECMO therapy also confers survival benefi t. Methods Thirty-two male rats were intratracheally challenged with S. pneumoniae to induce pneumonia. Rats were repeatedly exposed to nebulized C1-INH or saline, 30 minutes before induction of pneumonia and every 6 hours thereafter. Rats were sacrifi ced 20 or 40 hours after inoculation to investigate early and late eff ects. BALF and lung tissue were obtained for measuring levels of complement activation (C4b/c in BALF), lung injury (total protein levels in BALF), and infl ammation (IL-6 levels in lung tissue). p References 1. Carillo et al.: Intensive Care Med 2012, 38:458-466. 2. Jolliet et al.: Intensive Care Med 2001, 27:812-821. 3. Lightowler et al.: BMJ 2003, 326:185-189. 4. 2013 BTS NIV Audit [http://www.brit-thoracic.org.uk/Portals/0/Audit%20 Tools/SummaryReports/NIV%20Audit%202013-FINAL.pdf] 5. Confalonieri et al.: Am J Respir Crit Care Med 1999, 160:1585-1591. 1. Carillo et al.: Intensive Care Med 2012, 38:458-466. 2. Jolliet et al.: Intensive Care Med 2001, 27:812-821 Results Three patients had grade 1 injury, four had grade 2 injury, and 11 had grade 3 injury. Seven patients developed ventilator-associated pneumonia. Mean duration of MV increased progressively in relation S112 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P314). Frequency of interventions in the base hospital (BH) and SARF centre. to injury severity (grade 1: 2.7 ± 0.6 days vs. grade 2: 5.7 ± 2.1 days vs. grade 3: 13.0 ± 5.4 days; P = 0.004), as did length of ICU stay (grade 1: 4 days vs. grade 2: 7.2 ± 2.2 days vs. grade 3: 19.9 ± 7.7 days; P = 0.001) and overall length of hospital stay (grade 1: 5 days vs. grade 2: 14.5 ± 5.0 days vs. grade 3: 63.4 ± 43.6 days; P = 0.025). There were no signifi cant diff erences in PaO2/FiO2 ratio between groups at day 1. However, as expected, diff erences in PaO2/FiO2 ratio were found on day 3 (grade 1: 501 vs. grade 2: 424 ± 115 vs. grade 3: 318 ± 120; P = 0.049). Conclusion In this cohort of patients with major burns, the severity of inhalation injury was associated with prolonged MV, length of ICU stay, and overall length of hospital stay, as well as with deterioration in oxygenation on day 3. Reference 1. Peek GJ, Mugford M, Tiruvoipati R, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351–1363. doi: 10.1016/ S0140-6736(09)61069-2. g Results Thirteen casualties had an average Injury Severity Score of 50.3 ± 10.5 (age 27.7 ± 8.6 years, 69.2% male) and were supported 9.9 ± 4.8 days on ECMO (n = 7) and 7.16 ± 5.9 days on iLA (n = 6). All suff ered severe chest injuries, including one cardiac perforation. Most were coagulopathic prior to initiation of ECMO/iLA support. Among the seven patients with TBI, four had active intracranial hemorrhage. Only 30% of the patients received continuous anticoagulation during the fi rst 24 hours of support without clotting of the system or diagnosis of a thromboembolic event. Complications directly related to support therapy were not lethal; these included hemorrhage from a cannulation site (n = 1), accidental removal of a cannula (n = 1) and pressure sores (n = 3). Deaths occurred due to septic (n = 3) and cardiogenic shock (n = 1). Survival rates were 57 and 83% on ECMO and iLA, respectively. Follow-up of survivors detected no neurological deterioration. Severe respiratory failure in multiple trauma patients: extracorporeal support as a salvage therapy – a single-center experience PB Biderman Rabin Medical Center, Petah Tikva, Israel Critical Care 2014, 18(Suppl 1):P313 (doi: 10.1186/cc13503) Methods All patients transferred to the Severe Adult Respiratory Failure (SARF) unit between April 2012 and April 2013 for advanced respiratory care who did not require ECMO, identifi ed from the ECMO database, underwent retrospective notes’ review to identify the advanced respiratory techniques performed at the SARF unit and the base hospital. Methods All patients transferred to the Severe Adult Respiratory Failure (SARF) unit between April 2012 and April 2013 for advanced respiratory care who did not require ECMO, identifi ed from the ECMO database, underwent retrospective notes’ review to identify the advanced respiratory techniques performed at the SARF unit and the base hospital. PB Biderman Rabin Medical Center, Petah Tikva, Israel Critical Care 2014, 18(Suppl 1):P313 (doi: 10.1186/cc13503) Introduction Use of extracorporeal life support (ECLS) in trauma casualties is limited by concerns regarding hemorrhage, particularly in the presence of traumatic brain injury (TBI). We report usage of ECMO/ interventional lung assist (iLA) as salvage therapy in 13 trauma patients. A high-fl ow technique without anticoagulation was used in cases with coagulopathy or severe TBI. p Results Ten patients were admitted for advanced respiratory care who did not require ECMO. Eight patients had community-acquired pneumonia, one had an inhalational injury and one had a previously undiagnosed cardiac sarcoma. The techniques utilised in the SARF unit and base hospital are shown in Figure 1. Nine patients were discharged from the SARF unit alive (90%). Conclusion A negative fl uid balance and targeting higher haemoglobin are more frequently achieved in a SARF unit. Further studies are required to fully elucidate the advanced respiratory care techniques undertaken at specialist SARF units. g p y Methods Data were collected from all adult trauma cases referred to one center for ECMO/iLA treatment due to severe hypoxemic respiratory failure. Thirteen consecutives cases are reported. The type of assistance was chosen based on a fl owchart. Type of study: therapeutic, level of evidence IV. We analyzed patient data, injury data, blood gases before connection, methods of assistance, coagulation study, complications, survival and neurological outcome. Advanced respiratory care techniques in a severe adult respiratory failure uniti Advanced respiratory care techniques in a severe adult respiratory failure unit D Rafi q, A Needham, R Porter, G Lau Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P314 (doi: 10.1186/cc13504) D Rafi q, A Needham, R Porter, G Lau Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P314 (doi: 10.1186/cc13504) Introduction Outcome is improved when patients with severe adult respiratory distress syndrome are transferred to an extracorporeal membrane oxygenation (ECMO)-capable unit [1]. Not all patients transferred require ECMO, and this service evaluation examines which techniques are utilised in patients who ultimately do not require ECMO. Introduction Outcome is improved when patients with severe adult respiratory distress syndrome are transferred to an extracorporeal membrane oxygenation (ECMO)-capable unit [1]. Not all patients transferred require ECMO, and this service evaluation examines which techniques are utilised in patients who ultimately do not require ECMO. Results Pneumonia was characterized by bilateral macroscopic infi ltrates, bacterial outgrowth in the lung and clinical signs of illness. Pneumonia was associated with pulmonary complement activation. In rats treated with nebulized C1-INH, a functional fraction of C1-INH was detectable in BALF. However, C1-INH treatment did not aff ect S113 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). Conclusion Severe S. pneumoniae pneumonia is associated with pulmonary complement activation in rats. Nebulized C1-INH treat- ment, in an attempt to reduce pulmonary complement activation, neither aff ects pulmonary complement activation or lung injury and infl ammation. P316 Novel carbon dioxide removal device driven by a renal-replacement system without hemofi lter: an experimental approach and validation T Godet1, A Combes2, E Zogheib3, M Jabaudon1, E Futier1, J Constantin1 1CHU Clermont-Ferrand, France; 2CHU Pitié-Salpêtrière, Paris, France; 3CHU Amiens, France Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Introduction Management of acute respiratory diseases must avoid ventilator-induced lung injuries along with the rise of PaCO2 and respiratory acidosis [1]. Eff orts are made to fi nd devices assisting protective ventilation and able to remove arterial CO2 and correct acidosis [2]. An extracorporeal CO2 removal device driven by the widely used Prismafl ex® platform called PrismaLung® was tested in vivo. Methods Five hypercapnic ventilated pigs were equipped with the PrismaLung® system designed to remove CO2 from the bloodstream through a decarboxylation membrane mounted on a renal replacement device without any hemofi lter. P316 P316 Novel carbon dioxide removal device driven by a renal-replacement system without hemofi lter: an experimental approach and validation T Godet1, A Combes2, E Zogheib3, M Jabaudon1, E Futier1, J Constantin1 1CHU Clermont-Ferrand, France; 2CHU Pitié-Salpêtrière, Paris, France; 3CHU Amiens, France Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Advanced respiratory care techniques in a severe adult respiratory failure uniti Experiments examined the potential for blood decarboxylation by gas-exchanger membrane with diff erent sets of parameters (blood fl ow rate: 200, 300 and 400 ml/minute, sweep gas fl ow: 2, 5, 10 and 50 l/minute, FiO2: 21 and 100%). Statistical analysis was performed with the Student t test. Results The extracorporeal device allowed effi cient CO2 removal rates at FiO2 1 (Figure 1) and 0.21 (Figure 2), ranging from 40 to 60 ml/ minute. Effi ciency was increased with blood and sweep gas fl ows. Carbia and pH of animals were signifi cantly modifi ed after 10 minutes Figure 1 (abstract P315). Figure 1 (abstract P316). CO2 removal rates at FiO2 1. pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). Conclusion Severe S. pneumoniae pneumonia is associated with l l t ti ti i t N b li d C1 INH t t Figure 1 (abstract P315). pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or inflammation (Figure 1C) Figure 1 (abstract P315). Figure 1 (abstract P315). Figure 1 (abstract P315). pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). Figure 1 (abstract P316). CO2 removal rates at FiO2 1. l Conclusion Severe S. pneumoniae pneumonia is associated with pulmonary complement activation in rats. Nebulized C1-INH treat- ment, in an attempt to reduce pulmonary complement activation, neither aff ects pulmonary complement activation or lung injury and infl ammation. Novel carbon dioxide removal device driven by a renal-replacement system without hemofi lter: an experimental approach and validation T Godet1, A Combes2, E Zogheib3, M Jabaudon1, E Futier1, J Constantin1 1CHU Clermont-Ferrand, France; 2CHU Pitié-Salpêtrière, Paris, France; 3CHU Amiens, France Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Introduction Management of acute respiratory diseases must avoid ventilator-induced lung injuries along with the rise of PaCO2 and respiratory acidosis [1]. Eff orts are made to fi nd devices assisting protective ventilation and able to remove arterial CO2 and correct acidosis [2]. An extracorporeal CO2 removal device driven by the widely used Prismafl ex® platform called PrismaLung® was tested in vivo. Figure 1 (abstract P316). CO2 removal rates at FiO2 1. of parameters (blood fl ow rate: 200, 300 and 400 ml/minute, sweep gas fl ow: 2, 5, 10 and 50 l/minute, FiO2: 21 and 100%). Statistical analysis was performed with the Student t test.fi Methods Five hypercapnic ventilated pigs were equipped with the PrismaLung® system designed to remove CO2 from the bloodstream through a decarboxylation membrane mounted on a renal replacement device without any hemofi lter. Experiments examined the potential for blood decarboxylation by gas-exchanger membrane with diff erent sets Results The extracorporeal device allowed effi cient CO2 removal rates at FiO2 1 (Figure 1) and 0.21 (Figure 2), ranging from 40 to 60 ml/ minute. Effi ciency was increased with blood and sweep gas fl ows. Carbia and pH of animals were signifi cantly modifi ed after 10 minutes Results The extracorporeal device allowed effi cient CO2 removal rates at FiO2 1 (Figure 1) and 0.21 (Figure 2), ranging from 40 to 60 ml/ minute. Effi ciency was increased with blood and sweep gas fl ows. Carbia and pH of animals were signifi cantly modifi ed after 10 minutes S114 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 2 (abstract P316). CO2 removal rates at FiO2 0.21. centre and mortality among patients with severe 2009 infl uenza A(H1N1). JAMA 2011, 306:1659-1668. 3. Davies A, et al.: Australia and New Zealand Extracorporeal Membrane Oxygenation (ANZ ECMO) Infl uenza investigators. Extracorporeal membrane oxygenation for 2009 infl uenza A(H1N1) acute respiratory distress syndrome. JAMA 2009, 302:1888-1895. 4. NI Stewart, et al.: Regionalisation of intensive care and extra-corporeal membrane oxygenation services in the UK: beliefs about the evidence, benefi t and harm. J Intensive Care Med 2012, 13:244-250. Reference 1. Berra et al.: Crit Care Med 2001, 1:119-124. p p Results See Figure 1. ECMO services have expanded rapidly in the last 5 years in England following the publication of evidence for its effi cacy and concerns regarding an infl uenza pandemic. The referral rates for ECMO for severe hypoxic respiratory failure vary greatly around the country from 88 per 1,000,000 population in Leicester City to no referrals in 32 PCTs. Possible explanations could include: the distributions of swine fl u around the country, referring doctors’ beliefs about the effi cacy of ECMO, local access to high-frequency oscillation ventilation and possible reluctance of teaching hospitals to refer to specialist centres. Further investigation to account for this variation appears indicated. Assessment of an endotracheal tube cleaning closed-suctioning system by micro-computed tomography: preliminary clinical data A Coppadoro1, G Bellani1, A Bronco1, N Eronia1, A Barletta1, M Teggia Droghi1, R Borsa1, M Battistini1, S Bramati2, L Berra3, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy;3Massachusetts General Hospital, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P318 (doi: 10.1186/cc13508) Assessment of an endotracheal tube cleaning closed-suctioning system by micro-computed tomography: preliminary clinical data A Coppadoro1, G Bellani1, A Bronco1, N Eronia1, A Barletta1, M Teggia Droghi1, R Borsa1, M Battistini1, S Bramati2, L Berra3, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy;3Massachusetts General Hospital, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P318 (doi: 10.1186/cc13508) Assessment of an endotracheal tube cleaning closed-suctioning system by micro-computed tomography: preliminary clinical data A Coppadoro1, G Bellani1, A Bronco1, N Eronia1, A Barletta1, M Teggia Droghi1, R Borsa1, M Battistini1, S Bramati2, L Berra3, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy;3Massachusetts General Hospital, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P318 (doi: 10.1186/cc13508) Introduction Using micro-computed tomography (MicroCT), we assessed the eff ectiveness of a cleaning closed-suctioning system (CSS) to remove secretions from the endotracheal tube (ETT) lumen. Biofi lm growing within the ETT, soon after intubation, increases the patient’s risk to develop ventilator-associated pneumonia, and new cleaning devices have been designed to keep the ETT clean from secretions [1]. Methods In a bench test, we injected a water-based gel into unused ETTs to evaluate MicroCT scan (SkyScan 1172; Bruker, Belgium) eff ectiveness to measure secretions. In six critically ill patients, a cleaning CSS (Airway Medix Closed Suction system; Biovo, Tel Aviv) was used three times a day to keep the ETT clean. After extubation, we measured ETT secretions volume by MicroCT scanning over a length of 20 cm from the ETT tip. We also collected ETTs from 11 patients treated with a standard CSS as controls, and evaluated ETT microbial colonization. of treatment. No signifi cant modifi cation of blood oxygenation was obtained with pure oxygen, allowing the use of ambient air as the sweep gas through the membrane. No side eff ects or fi lter clotting occurred during experiments.lfi Conclusion The device based on the Prismafl ex® platform effi ciently removed CO2 from blood and decreased PaCO2 and acidosis of hypercapnic pigs. Benefi ts for patients with acute to chronic respiratory diseases need evaluation. References 1. Slutsky AS, et al.: N Engl J Med 2013, 369:2126-2136. 1. Slutsky AS, et al.: N Engl J Med 2013, 369:2126-2136. 1. Slutsky AS, et al.: N Engl J Med 2013, 369:2126-2136. 2. Pesenti A, et al.: Crit Care Med 2010, 38:S549-S554. 1. Slutsky AS, et al.: N Engl J Med 2013, 369:2126 2136. 2. Pesenti A, et al.: Crit Care Med 2010, 38:S549-S554. 2. Pesenti A, et al.: Crit Care Med 2010, 38:S549-S554. P317 Results The volume of gel measured by MicroCT strongly correlated with the volume of injected gel (P <0.001, R2 = 0.99). At extubation, a lower amount of secretions was measured in the ETTs treated with the cleaning CSS as compared with controls (0.031 ± 0.029 vs. 0.350 ± 0.417 mm3, P = 0.028), corresponding to a smaller occupation of the cross-sectional area (average 0.3 ± 0.4 vs. 3.8 ± 4.5% respectively, P = 0.030). Microbial colonization tended to be reduced in the ETTs treated with the cleaning CSS (total bacterial charge 1.3 ± 1.7 vs. 3.6 ± 2.7 log(CFU/ml), P = 0.08). Does geography aff ect referral rates for extracorporeal membr oxygenation in England? J Barnett Royal United Hospital, Bath, UK Critical Care 2014, 18(Suppl 1):P317 (doi: 10.1186/cc13507) Royal United Hospital, Bath, UK Introduction Referral for ECMO has been demonstrated to reduce mortality in severe hypoxic respiratory failure [1-3]. The numbers of patients that undergo ECMO is still small and the service depends on timely referral from regional ICUs. There is evidence that intensivists’ views on the role of ECMO are mixed [4]. The purpose of this study is to determine whether there are variations in the geographical distribution of patients that receive ECMO. Conclusion MicroCT scan showed high precision and accuracy in measuring the volume of secretions in bench tests and can thus be used to evaluate the eff ectiveness of actions or devices studied to reduce ETT biofi lm accumulation. In a small nonrandomized population of critically ill patients, the use of an ETT cleaning device appeared eff ective to reduce the volume of secretions present in the ETT at extubation. p Methods NHS England provided the home primary care trust (PCT) of all adult patients referred for ECMO for potentially reversible respiratory failure from 2008 to 2012. The referrals from each PCT were indexed to the population of each area to produce a referral rate per 1,000,000 people. Reference Does cost aff ect endotracheal tube performance? P Lichtenthal, UB Borg University of Arizona, Tucson, AZ, USA Critical Care 2014, 18(Suppl 1):P319 (doi: 10.1186/cc13509) Does cost aff ect endotracheal tube performance? P Lichtenthal, UB Borg University of Arizona, Tucson, AZ, USA Critical Care 2014, 18(Suppl 1):P319 (doi: 10.1186/cc13509) Introduction Today’s healthcare environment has forced providers to constantly evaluate the materials used, and to fi nd less expensive alternatives. Recently, low-cost endotracheal tubes (ETTs) have been introduced to the market. The aim of this study was to test these tubes (Portex AirCare and Cardinal Health ETT) compared with endotracheal tubes with known performance (Hi-Lo Covidient pre ISO standard and Taper-Guard Covidient, post ISO standard). Conclusion The referral rates for ECMO vary greatly around the country. References Conclusion The referral rates for ECMO vary greatly around the country. References 1. Peek GJ, et al.; CESAR Trial Collaboration: Effi cacy and economic assessment of Conventional Ventilatory Support Versus Extra-Corporeal Membrane Oxygenation for Severe Adult Respiratory Failure (CESAR): a multicentre randomised controlled trail. Lancet 2009, 374:1351-1363. 1. Peek GJ, et al.; CESAR Trial Collaboration: Effi cacy and economic assessment of Conventional Ventilatory Support Versus Extra-Corporeal Membrane Oxygenation for Severe Adult Respiratory Failure (CESAR): a multicentre randomised controlled trail. Lancet 2009, 374:1351-1363. Methods We used the required test setups according to the ISO standard for cuff sealing performance. The tubes were tested versus each other in size 7.0, 7.5 and 8.0 mm. Kink performance was done in the routine manner. 2. Noah MA, et al.: Referral to an extracorporeal membrane oxygenation S115 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P317). Figure 1 (abstract P317). Figure 1 (abstract P317). Results The leak test showed that the Portex AirCare ETT leaked signifi cantly more compared with both the Hi-Lo (P <0.05) or Taper- Guard (P <0.001) ETTs. The standard deviation leak rate for AirCare was large, suggesting varying seal performance between tubes from various lots. The kink test showed no diff erence among tubes.f Results The leak test showed that the Portex AirCare ETT leaked signifi cantly more compared with both the Hi-Lo (P <0.05) or Taper- Guard (P <0.001) ETTs. The standard deviation leak rate for AirCare was large, suggesting varying seal performance between tubes from various lots. The kink test showed no diff erence among tubes.f price alone but should take into account documented performance in standardized tests. price alone but should take into account documented performance in standardized tests. P320 P320 Tracheostomy in obese patients: the best tube choice issue L Marullo, G Izzo, A Torino, A D’Elia, L Vessicchio, F Ferraro Second University of Naples, Italy Critical Care 2014, 18(Suppl 1):P320 (doi: 10.1186/cc13510) Conclusion Although tubes look the same there may be diff erences in performance. This study demonstrated that the new cheaper ETTs had greater cuff leak compared with the two tubes used for comparison. We can only speculate whether diff erences found in this study is a result of cost cutting. However, the great variability in sealing performance between ETTs of the same size from diff erent lots would indicate that manufacturing controls may be less stringent. Although we cannot demonstrate that the higher incidence of leak will result in adverse patient outcomes, one can surmise that the possibility exists. Thus, selection of endotracheal tubes should not be based on purchase Introduction Obesity is not an absolute contraindication for percutaneous tracheostomy (PDT). Video-endoscopy (video-FBS) and ultrasound (US) facilitate PDT techniques and reduce complications in obese patients (OPs) [1,2]. OPs may have a higher trachea–skin distance that makes it diffi cult to place or to manage a tracheostomy tube (TT). S116 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A retrospective review was performed using data in the last 5 years. All OPs were from the ICU of our university hospital. Only OPs who underwent a PDT were selected with BMI >30 kg/m2. All OPs needed prolonged mechanical ventilation. A total of 67 OPs were identifi ed, with 60 PDTs placed using the Ciaglia Blue Rhino (CBR) Introducer Kit and seven PDTs with the UniPerc PDT Kit. All PDTs were performed by dedicated staff including residents. Valuation of clinical anatomical and physio-pathological features of the OPs and US scan of the neck came before the procedure. At the beginning of the procedure we placed a 5 mm ID orotracheal tube by tube exchange with video-FBS assistance, as already described [3]. An 8 to 9 mm ID wire-reinforced silicone tracheostomy tube (rTT) with adjustable fl ange was chosen instead of a standard PVC or silastic TT (sTT) in all OPs treated with CBR because of the anatomical particularities of OPs and because of external traction by the weight of the tubing attached to the TT. P322 Development of the novel Tracoe Twist Plus tracheostomy tube L Barrass, M Healy, D Kennedy, H Drewery Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P322 (doi: 10.1186/cc13512) y p , , Critical Care 2014, 18(Suppl 1):P322 (doi: 10.1186/cc13512 Introduction The 4th National Audit project, conducted by the Royal College of Anaesthetists in 2011, reviewed major airway complications in the UK [1]. It highlighted tracheostomy tube displacement as a signifi cant risk in critical care patients. Prior to 2011, at the Royal London Hospital ICU, the Tracoe Twist (marketed by Kapitex) was primarily used as the default choice of tracheostomy tube. However, over a 5-year period, there were a signifi cant number of critical incidents related to tracheostomy tube displacement or blockage. On investigation of these incidents, root-cause analysis identifi ed inadequate length of the Tracoe Twist as a major contributing factor. As a consequence of this, there was also an increasing requirement for the use of adjustable fl ange tubes. This was particularly apparent in obese patients. Our objective was to develop a new tracheostomy tube that would reduce the number of incidents related to inadequate length. An additional aim was to maximise the inner diameter for a given external diameter, thus reducing airway resistance and potentially aiding weaning. Results No major complications (aborting procedure, >50 ml bleed- ing, TT misplacement, death) were observed. We had only minor complications (<50 ml bleeding: 3%; ring fracture: 2%; diffi cult insertion: 21% only with CBR rTT because of the step between the tip of the rTT and its introducer). UniPerc eTT placement has always been easy. Conclusion In our experience, the data do not support what previous studies have shown suggesting increased risk of complication in OPs [1,2]. We know that the sTT could not be eff ective in OPs. The use of US, video-FBS assistance [3], and rTT with an adjustable fl ange allows a safe and eff ective adjustment to anatomical OP particularities, avoiding collected risks. Impact of high-fl ow oxygen therapy delivered through a tracheostomy on arterial blood gases and endotracheal pressure D Natalini, FA Idone, DL Grieco, L Spaziani, MT Santantonio, F Toni, M Antonelli, SM Maggiore A Gemelli Hospital, University of the Sacred Heart, Rome, Italy Critical Care 2014, 18(Suppl 1):P321 (doi: 10.1186/cc13511) i Results We developed a prototype tracheostomy tube that had an increased length for a given internal diameter. We also increased the mobility to fl ange, in order to reduce pressure areas and assist with fi xation. A third modifi cation was to ensure a maximal internal diameter for a given external diameter. We piloted its use in 20 patients. No adverse events were observed and the clinical impression was that the increased length was benefi cial to the patients. Introduction High-fl ow oxygen therapy (HFOT) delivered through nasal cannulas can improve oxygenation as compared with low-fl ow oxygen devices. It has been shown that nasal HFOT can generate a positive airway pressure, which increases linearly with the gas fl ow rate. Data on the use of HFOT delivered through a tracheostomy are scarce. The aim of the present randomized, controlled, cross-over trial was to assess the eff ects of HFOT delivered through a tracheostomy on arterial blood gases and endotracheal pressure in critically ill patients. i Conclusion Following the success of the pilot study, this new tracheostomy tube was developed and then marketed by Kapitex. This tube was named the Tracoe Twist Plus. We have now used this tube for 2 years. We have since performed an extensive retrospective audit of tracheostomy usage and the eff ect of the introduction of the novel Tracoe Twist Plus, and the complication rate was reduced in terms of displacement and obstruction. y Methods Tracheostomized patients underwent HFOT with three gas fl ow rates (10 l/minute, 30 l/minute and 50 l/minute), randomly applied for 20-minute periods. At the end of each period, arterial blood gases, respiratory rate, and endotracheal pressure (Ptrach) were measured. Ptrach was recorded over the last 3 minutes of each study period: the maximum expiratory pressure (MEPtrach) and mean expiratory pressure were measured and averaged for all respiratory cycles during 1-minute recording with stable breathing. FiO2 was kept constant during the whole study. P320 An extralong TT (eTT) was chosen because the pretracheal tissue was too thick for a regular-sized TT in 16 OPs with BMI >40 kg/m2 treated with CBR. For three OPs treated with CBR we needed to change rTT to eTT because of tube dislodgement and subocclusion X-ray and video-FBS diagnosis. The UniPerc technique was chosen for OPs with BMI >40 kg/m2. 1.32 ± 0.4 cmH2O, and 1.89 ± 0.5 cmH2O at 10 l/minute, 30 l/minute and 50 l/minute, respectively, P <0.01) and mean expiratory pressure (0.54 ± 0.27 cmH2O, 0.91 ± 0.29 cmH2O, and 1.36 ± 0.35 cmH2O, at 10 l/ minute, 30 l/minute and 50 l/minute, respectively, P <0.01) increased with fl ow. Changes in PaO2/FiO2 were not correlated with changes in expiratory pressures. Conclusion When HFOT is used through a tracheostomy at increasing gas fl ow rate, oxygenation increases up to 30 l/minute while CO2 clearance and the respiratory rate do not vary. Tracheal expiratory pressure increases with fl ow, but changes are small and probably of limited clinical relevance. Changes in oxygenation are not related to the variations of tracheal expiratory pressure. HFOT through a tracheostomy has diff erent eff ects from when a nasal interface is used. Reference 1. Cook TM, Woodall N, Harper J, Benger J; Fourth National Audit Project: Major complications of airway management in the UK: results of the Fourth National Audit Project of the Royal College of Anaesthetists and the Diffi cult Airway Society. Part 2: intensive care and emergency departments. Br J Anaesth 2011, 106:632-642. Results Seventeen tracheostomized patients were enrolled (SAPS II 52 ± 10, PaO2 96 ± 27 mmHg, PaCO2 33 ± 10 mmHg). Increasing the gas fl ow rate from 10 l/minute to 30 l/minute was associated with an increase in PaO2/FiO2 that did not improve further when 50 l/minute was used (259 ± 66, 317 ± 79, and 325 ± 76, respectively, P <0.001). The same trend was observed with PaO2 (89 ± 19 mmHg, 109 ± 26 mmHg, and 113 ± 29 mmHg, respectively, P <0.001) and SaO2 (96 ± 3%, 98 ± 2%, 98 ± 2%, respectively, P <0.001). PaCO2 (32 ± 8 mmHg on average) and respiratory rate (27 ± 7 breaths/minute on average) did not change with diff erent gas fl ow rates. MEPtrach (0.96 ± 0.43 cmH2O, References 1. McCague A, et al.: Laryngoscope 2012, 122:1031-1034. 2. Guinot PG, et al.: Crit Care 2012, 16:R40. 3. Ferraro et al.: Chest 2004, 126:159-164. 1. McCague A, et al.: Laryngoscope 2012, 122:1031-1034. 2. Guinot PG, et al.: Crit Care 2012, 16:R40. 3. Ferraro et al.: Chest 2004, 126:159-164. Methods We formed a consultation committee, consisting of consultants in critical care, a consultant surgeon and a consultant anaesthetist with a specialist interest in head and neck anaesthesia and tracheostomy care. We collaborated with the Kapitex design team to develop a new tracheostomy tube which addressed some of the perceived defi cits of existing devices. P321 Impact of high-fl ow oxygen therapy delivered through a tracheostomy on arterial blood gases and endotracheal pressure D Natalini, FA Idone, DL Grieco, L Spaziani, MT Santantonio, F Toni, M Antonelli, SM Maggiore A Gemelli Hospital, University of the Sacred Heart, Rome, Italy Critical Care 2014, 18(Suppl 1):P321 (doi: 10.1186/cc13511) Ability to speak in ventilator-dependent tracheostomized ICU patients Methods The nonlinear constant of the Rohrer equation (K2) was calculated as resistive properties, during a continuous fl ow of 10 to 90 l/minute, for a conventional endotracheal tube (ETT) with and without FOB (ETT size 7, 7.5, 8, 7f, 7.5f, 8f), ventilation tube of TLT (F4 and F5) and DLET. The variation of gas exchange (Δ) was measured with arterial blood gas samples obtained before and after the PT. During PT, all patients received sedation, analgesia, neuromuscular blocking and volume-controlled ventilation set with FiO2 100%, TV 500 ml, RR 15 breaths/minute, PEEP 5 cmH2O. Methods The aim of this study was to compare weaning from MV by gradually decreasing the level of support in cuff -defl ated ventilation with use of a BiPAP Vision® and a Passy Muir® speaking valve, or by trials of spontaneous breathing with use of a speaking valve, both for progressively longer periods of time. We examined the diff erences in the ability to speak, the duration of the weaning period, the occurrence of delirium and the frequency of tracheal suctioning. We performed a single-centre retrospective and prospective observational study in a 22-bed mixed ICU during 1 year. Data were collected using the patient data management system. Baseline criteria were age, gender, APACHE IV score, ICU length of stay and duration of MV before placement of the tracheostomy.ii 2 Results In vitro evaluation showed that the DLET had the lowest K2 (7 = 11.33; 7.5 = 8.74; 8 = 7.57; 7f = 6.13; 7.5f = 10.52; 8f = 12.28; F4 = 130.0; F5 = 11.12; DLET = 5.25 cmH2O/l/minute). During in vivo evaluation, PT was performed with the conventional ETT with FOB and DLET for fi ve patients in each group (age 69 ± 13 vs. 71 ± 16; SAPS II: 56 ± 14 vs. 52 ± 20; GCS 3 vs. 4). Gas exchange before and after the procedure did not diff er between the groups, but the Δ values of pH, PaO2 and PaCO2 measured before and after the procedure were, ETT+FOB versus DLET: ΔpH: –0.05 ± 0.05 versus 0.01 ± 0.02, P = 0.04; ΔPaO2: –112.6 ± 112.6 versus 41.6 ± 25.3, P = 0.01; ΔPaCO2: 14.5 ± 10.8 versus 0.6 ± 1.1, P = 0.02; ΔHCO3: 0.5 ± 2 versus –0.04 ± 0.3, P = 0.6. Ability to speak in ventilator-dependent tracheostomized ICU patients p R Bultsma, M Koopmans, M Kuiper, P Egbers Medisch Centrum Leeuwarden, the Netherlands Critical Care 2014, 18(Suppl 1):P323 (doi: 10.1186/cc13513) p R Bultsma, M Koopmans, M Kuiper, P Egbers Medisch Centrum Leeuwarden, the Netherlands Critical Care 2014, 18(Suppl 1):P323 (doi: 10.1186/cc13513) Introduction Mechanical ventilation (MV) and inability to speak increases psycho-emotional distress [1]. Although not commonly used S117 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P323). Number of patients able to speak. Figure 1 (abstract P324). Point 1 just before vocal cord, point 2 asymmetric elliptical cuff just before the carena. Figure 1 (abstract P324). Point 1 just before vocal cord, point 2 asymmetric elliptical cuff just before the carena. Figure 1 (abstract P323). Number of patients able to speak. Figure 1 (abstract P323). Number of patients able to speak. in ICU patients, MV with a defl ated cuff in patients with a tracheostomy can be provided safely and comfortably, by use of a BiPAP Vision®. Air leakage to the upper airway enables speech [2]. By adding a Passy- Muir® speaking valve as second step, the quality of speech and cough will improve. The ability to speak provides an important improvement in communication. (FOB), and a lower channel exclusively dedicated to the patient’s ventilation. The aim of this study is to achieve an in vitro and an in vivo evaluation of PT performed with the DLET. (FOB), and a lower channel exclusively dedicated to the patient’s ventilation. The aim of this study is to achieve an in vitro and an in vivo evaluation of PT performed with the DLET. Methods The nonlinear constant of the Rohrer equation (K2) was calculated as resistive properties, during a continuous fl ow of 10 to 90 l/minute, for a conventional endotracheal tube (ETT) with and without FOB (ETT size 7, 7.5, 8, 7f, 7.5f, 8f), ventilation tube of TLT (F4 and F5) and DLET. The variation of gas exchange (Δ) was measured with arterial blood gas samples obtained before and after the PT. During PT, all patients received sedation, analgesia, neuromuscular blocking and volume-controlled ventilation set with FiO2 100%, TV 500 ml, RR 15 breaths/minute, PEEP 5 cmH2O. Ability to speak in ventilator-dependent tracheostomized ICU patients Results Ten patients were included, fi ve in the BiPAP group and fi ve in the spontaneous group. There were no signifi cant diff erences in the baseline criteria. On the second day after tracheostomy, three out of fi ve patients in the BiPAP group were able to speak compared with one in the spontaneous method group. A diff erence in speaking ability remained until day 9 (see Figure 1). At fi rst time of speaking, the BiPAP group had higher PEEP level (10 vs. 7.5 cmH2O) and higher SOFA score (6.2 vs. 4.6) compared with the spontaneous group. There was no signifi cant diff erence in delirium, duration of weaning and tracheal suctioning between both groups.fl 3 Conclusion The DLET resulted in adequate airway patency and minimal obstruction due to the lower channel exclusively dedicated to patient’s ventilation. Gas exchange in PT with the DLET remained stable without any variation in oxygenation and carbon dioxide levels, although the same settings of mechanical ventilation. Conclusion Cuff -defl ated MV in ICU patients enables speaking during ventilator dependence. With this technique the ability to speak started in an earlier phase of weaning compared with weaning with spontaneous breathing trials and a speaking valve. References 1. Engström A, et al.: People’s experiences of being mechanically ventilated in an ICU: a qualitative study. Intensive Crit Care Nurs 2013, 29:88-95. 1. Engström A, et al.: People’s experiences of being mechanically ventilated in an ICU: a qualitative study. Intensive Crit Care Nurs 2013, 29:88-95. A Garrett, C Strauss, S Saha Barking, Havering and Redbridge University Hospitals NHS Trust, Romford, UK Critical Care 2014, 18(Suppl 1):P325 (doi: 10.1186/cc13515) A Garrett, C Strauss, S Saha Barking, Havering and Redbridge University Hospitals NHS Trust, Romford, UK Critical Care 2014, 18(Suppl 1):P325 (doi: 10.1186/cc13515) 2. Hess DR: Facilitating speech in the patient with a tracheostomy. Respir Care 2005, 50:519-525. 2. Hess DR: Facilitating speech in the patient with a tracheostomy. Respir Care 2005, 50:519-525. Introduction Respiratory weaning in ICUs can be a lengthy and expensive process [1], but may be facilitated by the use of tracheostomies. Discharging patients with tracheostomies to general wards improves ICU bed availability but raises potential patient safety issues. This is demonstrated by the increased mortality compared with patients decannulated before discharge from the ICU [2]. We investigated how often ICUs in the UK discharge patients with tracheostomies to wards, which wards these are and whether systems are in place to ensure adequate safety on discharge.f Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A retrospective review of all repeat bedside percutaneous dilatational tracheostomies (Ciaglia technique with direct broncho- scopic guidance) performed on our cardiothoracic critical care unit from January 2004 to February 2013 was conducted. Methods A retrospective review of all repeat bedside percutaneous dilatational tracheostomies (Ciaglia technique with direct broncho- scopic guidance) performed on our cardiothoracic critical care unit from January 2004 to February 2013 was conducted.i 39 did occasionally. Five discharged to the high dependency unit only, 60 to respiratory wards only, 70 to specialist wards and 15 to any or most wards. Eighty-fi ve out of 190 units discharged patients with tracheostomy cuff s both up and down, 72 discharged with the cuff down or cuffl ess and 16 with the cuff ‘usually down’. A total of 141 hospitals had routine follow-up for tracheostomy patients from critical care outreach or other services. Critical care outreach was available 24 hours a day in 65 hospitals. y y Results From a total of 1,001 patients undergoing PDT, we identifi ed 36 patients with repeat PDT. Patients’ previous tracheostomies dated back between 5 days and 2.7 years (mean 122 days). Mean age was 60.3 ± 14.3 years, 42% of patients were female. The mean time from intubation to PDT was 3.7 ± 3.9 days. There were no deaths associated with PDT but one major procedure-related complication: one patient suff ered from a periprocedural laceration of the brachiocephalic trunk. After emergency surgery and surgical tracheotomy (ST), the patient recovered completely. In all other patients, no conversion to ST, no loss of airway, no paratracheal insertion, and no accidental tracheal extubation was observed. No pneumothorax, pneumomediastinum, hypotension, hypoxemia, or arrhythmias were recorded. A mild bleeding was observed in 13 patients (36%). A moderate but not signifi cant bleeding was observed in only one patient (2.8%). A tracheal ring fracture occurred in six patients (16.7%). Fourteen patients (38.9%) could be weaned successfully from the respirator and the tracheostomy could be removed. Thirteen patients (36.1%) were transferred to another ICU with the tracheostomy in place. The functional and cosmetic outcomes of PDT were excellent. y p Conclusion The vast majority of ICUs in the UK perform tracheostomies for respiratory weaning and many routinely discharge patients to the wards prior to decannulation. Routine follow-up is usually available, but cover may only be available during the day. P328 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) P328 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) y p itical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) Introduction Percutaneous dilatational tracheostomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. However, patients with severe coagulopathy or thrombocytopenia might have an increased risk of periprocedural bleeding. Introduction The tracheostomy is an ancient technique that more recently has developed a percutaneous technique. Percutaneous tracheostomies (PCT) have been shown to be safer and reduce infection, cost and other complications over surgical techniques [1-3]. Ventilator- associated pneumonia (VAP) is a serious complication resulting from the use of endotracheal tubes (ETT) and tracheostomies. Changes in design of these tubes by the addition of a subglottic suction port have been shown to improve VAP rates in mechanically ventilated patients [4,5]. A large meta-analysis review showed that subglottic drainage reduced the number of days of mechanical ventilation required and reduced the number of days stayed on the ICU [4]. Methods We retrospectively reviewed the records of all patients who underwent PDT (using the Ciaglia technique with bronchoscopic guidance) on our cardiothoracic ICU between January 2004 and February 2013. Patients were stratifi ed into two groups: no coagulopathy (group 1), and coagulopathy/thrombocytopenia defi ned as international normalized ratio >1.5, partial thromboplastin time >50 seconds and/or platelet count <50 × 109/l (group 2). p g p Results From a total of 1,001 patients (46% male, mean age 68.1 years) that underwent PDT, we identifi ed 441 patients (44.1%) with a severe coagulopathy (group 2). There were no procedure-related deaths. Major procedure-related complications included a severe bleeding (requiring transfusion and/or surgery) in two patients in each group (one laceration of the brachiocephalic trunk, one venous bleeding, two bleedings from a thyroid vessel), injury of the membranous wall of the trachea in two patients in group 2 as well as a pneumothorax and a device failure in group 1. P326 P Percutaneous dilatational tracheostomy in patients with severe coagulopathy or thrombocytopenia K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Patients may go to a specialist ward with trained nurses but this is not always the case. Patients are often discharged to wards with their tracheostomy cuff up, raising major safety issues if their tracheostomy tubes block and nurses are not trained for such emergencies. Twenty-four hours a day critical care outreach cover may improve patient safety, but further research and the production of guidelines is needed to facilitate the safe discharge of patients with tracheostomies from ICU to the wards. References 1. Vitacca M, et al.: [Experience of an intermediate respiratory intensive therapy in the treatment of prolonged weaning from mechanical ventilation] [Review in Italian]. Anestesiology 1996, 62:57-64. 1. Vitacca M, et al.: [Experience of an intermediate respiratory intensive therapy in the treatment of prolonged weaning from mechanical ventilation] [Review in Italian]. Anestesiology 1996, 62:57-64. 2. Martinez GH, et al.: Tracheostomy tube in place at intensive care unit discharge is associated with increased ward mortality. Respir Care 2009, 54:1644-1652. Conclusion Repeat PDT is a safe procedure in experienced hands and should not be generally considered a contraindication. However, special attention should be paid to the anatomical situation with an increased risk of vascular complications. P328 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) Of the group of ICUs that did not use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 10. Conclusion Signifi cant diff erences in practise exist with PCT and subglottic suction ports on tubes. The size of the ICUs in these groups is variable. The larger units are more likely to use PCT over the smaller units. Regarding subglottic suction ports on ETT and tracheostomy tubes, the size of the ICU does not necessarily dictate their use. We propose that all ICUs review their policy on the use of PCT and subglottic suction-assisted tubes to help improve surgical complications, cost, VAP rates and ICU stays. Conclusion Periprocedural bleeding complications during and after PDT are rare, even in patients with a severe coagulopathy, and thus PDT can be safely performed in these patients. Double-lumen endotracheal tube for percutaneous tracheostomy: in vitro and in vivo preliminary data Double-lumen endotracheal tube for percutaneous tracheostomy: in vitro and in vivo preliminary data M Vargas1, G Servillo1, A Marra1, D Salami2, P Pelosi3 1University of Naples ‘Federico II’, Naples, Italy; 2AOU San Martino IST, Genoa, Italy; 3University of Genoa, Italy Critical Care 2014, 18(Suppl 1):P324 (doi: 10.1186/cc13514) p y M Vargas1, G Servillo1, A Marra1, D Salami2, P Pelosi3 1University of Naples ‘Federico II’, Naples, Italy; 2AOU San Martino IST, Genoa, Italy; 3University of Genoa, Italy Critical Care 2014, 18(Suppl 1):P324 (doi: 10.1186/cc13514) Methods We telephoned 217 ICUs in the UK. Nursing staff answered a series of questions regarding the discharge of patients with tracheostomies to the wards and their follow-up. Introduction A double-lumen endotracheal tube (DLET; bilumen ventilation tube, PCT/IT2012/000154; Deas S.r.l., Italy) (Figure 1) has been developed to improve the safety of patients and procedural comfort during percutaneous tracheostomy (PT). The DLET is divided into an upper channel, for placement of a fi beroptic bronchoscope Introduction A double-lumen endotracheal tube (DLET; bilumen ventilation tube, PCT/IT2012/000154; Deas S.r.l., Italy) (Figure 1) has been developed to improve the safety of patients and procedural comfort during percutaneous tracheostomy (PT). The DLET is divided into an upper channel, for placement of a fi beroptic bronchoscope Results We obtained information from 203 ICUs. A total of 201 units used tracheostomies for respiratory weaning. In total, 151 routinely discharged patients to wards with tracheostomies, 11 never did and S118 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P327 P327 Repeat bedside percutaneous tracheostomy: still a contraindication? K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P327 (doi: 10.1186/cc13517) P327 Repeat bedside percutaneous tracheostomy: still a contraindication? K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P327 (doi: 10.1186/cc13517) P328 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) The incidence of moderate periprocedural bleeding was comparable between the two groups (n = 43 (9.75%) vs. n = 41 (7.3%), P = NS). y y Methods We contacted all ICUs in the UK by telephone and spoke to the nurse-in-charge to ascertain their normal practice with regards to PCT and subglottic suction use. Results We contacted a total of 246 general ICUs, 72% of which we received a response. The average number of beds per ICU from all units who responded was 11. Ninety-eight per cent of ICUs that we questioned did use PCT. For three units, the average bed number per unit was 11 and the other 2% of ICUs who did not use PCT had fi ve beds per unit on average. The proportion of ICUs that employed subglottic suction ports on their ETTs was 43% having on average 11 beds per unit, whilst the proportion of ICUs that did not employ subglottic suction ports was 57%, also with 11 beds per unit on average. Regarding PCT subglottic suction ports, 38% of ICUs did utilise these tubes whilst 62% did not. Of the group of ICUs that did use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 12. Of the group of ICUs that did not use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 10.if Results We contacted a total of 246 general ICUs, 72% of which we received a response. The average number of beds per ICU from all units who responded was 11. Ninety-eight per cent of ICUs that we questioned did use PCT. For three units, the average bed number per unit was 11 and the other 2% of ICUs who did not use PCT had fi ve beds per unit on average. The proportion of ICUs that employed subglottic suction ports on their ETTs was 43% having on average 11 beds per unit, whilst the proportion of ICUs that did not employ subglottic suction ports was 57%, also with 11 beds per unit on average. Regarding PCT subglottic suction ports, 38% of ICUs did utilise these tubes whilst 62% did not. Of the group of ICUs that did use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 12. Eff ect of subglottic secretion drainage for preventing ventilator-associated pneumonia Eff ect of subglottic secretion drainage for preventing ventilator-associated pneumonia Conclusion There is a wide variation in post-ICU/HDU management of tracheostomy patients throughout the UK. Although there are well established UK national guidelines for the management of tracheostomy patients, outside the ICU/HDU environment there is a lack of full implementation of the NTSP recommendations, increasing the risk of tracheostomy-related morbidity and mortality. References A Koker1, F Gok2, I Erayman2, A Yosunkaya2 1Konya Education and Research Hospital, Konya, Turkey; 2Necmettin Erbakan University, Meram Faculty of Medicine, Konya, Turkey Critical Care 2014, 18(Suppl 1):P331 (doi: 10.1186/cc13521) A Koker1, F Gok2, I Erayman2, A Yosunkaya2 1Konya Education and Research Hospital, Konya, Turkey; 2Necmettin Erbakan University, Meram Faculty of Medicine, Konya, Turkey Critical Care 2014, 18(Suppl 1):P331 (doi: 10.1186/cc13521) y y y y Critical Care 2014, 18(Suppl 1):P331 (doi: 10.1186/cc13521) Introduction We aimed to assess the eff ect of continuous drainage of subglottic secretion in the prevention of ventilator-associated pneumonia (VAP) in patients requiring prolonged mechanical ventilation for more than 48 hours in the ICU as a prospective, randomized, controlled study. 1. Cook TM, Woodall N, Frerk C (Eds): The Fourth National Audit Project of the Royal College of Anaesthetists and the Diffi cult Airway Society: Major Complications of Airway Management in the UK. Report and Findings. London: Royal College of Anaesthetists; 2011. 2. National Tracheostomy Safety Project Manual 2013 [http://www. tracheostomy.org.uk] Methods Our study was performed with a document from the ethics committee and written informed consent from the relatives of patients between April 2011 and February 2012 in our 14-bed ICU. Fifty-four patients whose mechanical ventilation requirements were expected to be longer than 72 hours were included in our study. Patients were randomly divided into two groups. These were formed as the group using a conventional intubation tube (Group C) and the group using an intubation tube allowing aspiration of subglottic secretions (Group S). In Group S, continuous subglottic aspiration occurred under constant pressure with a special device. In both groups, the cuff pressure was maintained at a constant pressure of 20 to 30 using a digital cuff pressure device [1]. P329 Rello J, et al.: Chest J 2002, 122:2115-2121. 2. Shi Z, et al.: Cochrane Database Syst Rev 2013, 8:CD008367, 3. Barkvoll et al.: J Clin Periodontol 1989, 16:593-595. 4. Kolahi J, et al.: Quintessence Int 2006, 37:605-612. P327 Repeat bedside percutaneous tracheostomy: still a contraindication? K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P327 (doi: 10.1186/cc13517) Conclusion Signifi cant diff erences in practise exist with PCT and subglottic suction ports on tubes. The size of the ICUs in these groups is variable. The larger units are more likely to use PCT over the smaller units. Regarding subglottic suction ports on ETT and tracheostomy tubes, the size of the ICU does not necessarily dictate their use. We propose that all ICUs review their policy on the use of PCT and subglottic suction-assisted tubes to help improve surgical complications, cost, VAP rates and ICU stays. Introduction Percutaneous tracheostomy has become an established procedure in airway management of critically ill patients. Repeat PDT is considered a (relative) contraindication as a result of distorted anatomy. S119 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References References 1. Baumann HJ, et al.: Pneumologie 2010, 64:769-776. 2. Engels PT, et al.: Can J Surg 2009, 52:427-433. 3. Muscedere J, et al.: Crit Care Med 2011, 39:1985-1991. 4. Bouza E, et al.: Chest 2008, 134:938-946. 5. Smulders K, et al.: Chest 2002, 121:858-862 Table 1 (abstract P330). Timing diff erence between toothpaste and chlorhexidine (minutes) Minutes Number of ICUs (%) >30 74 (50.3%) <30 32 (21.8%) Nil 38 (25.9%) Variable 3 (2.0%) Table 1 (abstract P330). Timing diff erence between toothpaste and chlorhexidine (minutes) P329 The frequency of chlorhexidine application varied between ICUs; 15 (9.9%) applied 4-hourly, 91 (59.9%) 6-hourly, 20 (13.2%) 8-hourly, 19 (12.5%) 12-hourly and seven (4.6%) applied at variable times. Ninety-seven per cent (n = 152) brushed patient’s teeth; 86% (n = 130) used toothpaste, 3% (n = 5) used chlorhexidine gel and 11% (n = 17) used both. Ninety-seven per cent (n = 147) of ICUs using chlorhexidine also brushed patient’s teeth with toothpaste. Forty-eight per cent (n = 70) administered chlorhexidine within 30 minutes of toothpaste application (Table 1). Introduction Over 5,000 tracheostomies are performed in the UK per year [1]. The 4th National Audit Project identifi ed signifi cant morbidity and mortality associated with tracheostomy care [1]. The National Tracheostomy Safety Project (NTSP) 2013 manual highlighted the need for: local policy; an appropriate care environment; immediate availability of emergency equipment; trained staff and local training programmes; and bed-head sign and emergency algorithms for tracheostomy patients [2]. Following these guidelines, we asked: how are adult tracheostomy patients managed post discharge from the intensive care/high dependence unit (ICU/HDU) throughout the UK? Introduction Over 5,000 tracheostomies are performed in the UK per year [1]. The 4th National Audit Project identifi ed signifi cant morbidity and mortality associated with tracheostomy care [1]. The National Tracheostomy Safety Project (NTSP) 2013 manual highlighted the need for: local policy; an appropriate care environment; immediate availability of emergency equipment; trained staff and local training programmes; and bed-head sign and emergency algorithms for tracheostomy patients [2]. Following these guidelines, we asked: how are adult tracheostomy patients managed post discharge from the intensive care/high dependence unit (ICU/HDU) throughout the UK? Methods In November 2013, 200 adult ICU/HDUs throughout the UK were contacted to take part in a telephone survey. Data were collected on tracheostomy weaning, post-ICU/HDU care, safety guidelines, emergency protocols and training for clinicians and nurses. g g p Results Sixty-fi ve per cent of ICUs in the UK responded to our survey (n = 157). Ninety-seven per cent (n = 152) used chlorhexidine and 96% (n = 150) used it as part of a ventilator care bundle. Forty-six per cent (n = 70) used a gel, 32% (n = 48) used a mouthwash and 23% (n = 34) used both preparations. P329 The frequency of chlorhexidine application varied between ICUs; 15 (9.9%) applied 4-hourly, 91 (59.9%) 6-hourly, 20 (13.2%) 8-hourly, 19 (12.5%) 12-hourly and seven (4.6%) applied at variable times. Ninety-seven per cent (n = 152) brushed patient’s teeth; 86% (n = 130) used toothpaste, 3% (n = 5) used chlorhexidine gel and 11% (n = 17) used both. Ninety-seven per cent (n = 147) of ICUs using chlorhexidine also brushed patient’s teeth with toothpaste. Forty-eight per cent (n = 70) administered chlorhexidine within 30 minutes of toothpaste application (Table 1). intensive care/high dependence unit (ICU/HDU) throughout the UK? Methods In November 2013, 200 adult ICU/HDUs throughout the UK were contacted to take part in a telephone survey. Data were collected on tracheostomy weaning, post-ICU/HDU care, safety guidelines, emergency protocols and training for clinicians and nurses. g y p g Results Out of the 200 adult ICU/HDUs contacted, 134 took part in the survey. Out of these, 44% have a tracheostomy weaning protocol, 69% initiate weaning whilst the patient is mechanically ventilated, and 92% use a speaking valve in their weaning process. Also, 87% allow tracheostomy patients to have oral nutritional intake and in 59% of these the decision involves speech and language therapy. Post ICU/ HDU, 67% of units discharge to specialised wards, 22% to nonspecialised wards, 4% to dedicated step-down units and 6% do not step down their tracheostomy patients. A critical care outreach team reviews the patients in 73% of the hospitals surveyed. Furthermore, only 11% of the hospitals have a consultant lead tracheostomy ward round and 17% have a tracheostomy multidisciplinary team (MDT). Also within these hospitals, 57% have their own tracheostomy safety guidelines and 70% have emergency tracheostomy management protocols. On the wards: 34% have tracheostomy bed-head information signs, 93% have emergency bed-side tracheostomy equipment, 89% have a tracheostomy training programme, and 50% have a MDT approach to decannulation. Conclusion Chlorhexidine is being used too soon after the application of toothpaste in 48% of ICUs in the UK. This results in attenuation of its eff ect and may remove its benefi cial risk reduction in VAP. Awareness of this interaction should be emphasised. References 1. Rello J, et al.: Chest J 2002, 122:2115-2121. 2. Shi Z, et al.: Cochrane Database Syst Rev 2013, 8:CD008367, 3. Barkvoll et al.: J Clin Periodontol 1989, 16:593-595. 4. Kolahi J, et al.: Quintessence Int 2006, 37:605-612. 1. P329 Is the post-critical care environment safe for tracheostomy patients? J Siah, S Begum, S Wijayatilake, G De La Cerda, T Jovaisa Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P329 (doi: 10.1186/cc13519) ill patients, as studies suggest a risk reduction in VAP [2]. Chlorhexidine reacts with soaps in toothpaste to form inactive insoluble salts [3]. A minimum delay of 30 minutes between tooth brushing and the sub- sequent application of chlorhexidine is therefore recommended [4]. Methods A telephone questionnaire was conducted on all ICUs in the UK to assess current oral decontamination procedures with regards to chlorhexidine use and the timing of tooth brushing with toothpaste. Results Sixty-fi ve per cent of ICUs in the UK responded to our survey (n = 157). Ninety-seven per cent (n = 152) used chlorhexidine and 96% (n = 150) used it as part of a ventilator care bundle. Forty-six per cent (n = 70) used a gel, 32% (n = 48) used a mouthwash and 23% (n = 34) used both preparations. The frequency of chlorhexidine application varied between ICUs; 15 (9.9%) applied 4-hourly, 91 (59.9%) 6-hourly, 20 (13.2%) 8-hourly, 19 (12.5%) 12-hourly and seven (4.6%) applied at variable times. Ninety-seven per cent (n = 152) brushed patient’s teeth; 86% (n = 130) used toothpaste, 3% (n = 5) used chlorhexidine gel and 11% (n = 17) used both. Ninety-seven per cent (n = 147) of ICUs using chlorhexidine also brushed patient’s teeth with toothpaste. Forty-eight per cent (n = 70) administered chlorhexidine within 30 minutes of toothpaste application (Table 1). ill patients, as studies suggest a risk reduction in VAP [2]. Chlorhexidine reacts with soaps in toothpaste to form inactive insoluble salts [3]. A minimum delay of 30 minutes between tooth brushing and the sub- sequent application of chlorhexidine is therefore recommended [4]. Methods A telephone questionnaire was conducted on all ICUs in the UK to assess current oral decontamination procedures with regards to chlorhexidine use and the timing of tooth brushing with toothpaste. Results Sixty-fi ve per cent of ICUs in the UK responded to our survey (n = 157). Ninety-seven per cent (n = 152) used chlorhexidine and 96% (n = 150) used it as part of a ventilator care bundle. Forty-six per cent (n = 70) used a gel, 32% (n = 48) used a mouthwash and 23% (n = 34) used both preparations. Survey of the use and practicalities of subglottic suction drainage in the UK Methods Anesthetized Yorkshire swine were randomized (n = 6/ group) to receive a bolus of the PFC Oxycyte™ either 45 minutes before (PFC-B) or after (PFC-A) induction of ARDS or nothing as a control (NON). ARDS was induced via intravenous oleic acid infusion (time 0 (T0)) over 30 minutes. Animals were monitored for physiological and hematological parameters. They were euthanized at T180 minutes and a full necropsy and histopathological analysis was performed. F Baldwin, R Gray, A Chequers y y p g Critical Care 2014, 18(Suppl 1):P332 (doi: 10.1186/cc13522) Introduction Subglottic secretion drainage (SSD) has been shown to reduce the incidence of ventilator-associated pneumonia (VAP) [1]. We reviewed current UK practice and practicalities surrounding implementation of SSD using a survey. p y p g y p Results Survival was 100% in the NON group, 80% in the PFC-A group and 20% in the PFC-B group. Mean arterial pressure (MAP) and mean pulmonary artery pressure (MPAP) were signifi cantly increased during infusion of PFC and during ARDS in the PFC-B group, while cardiac output (CO) was signifi cantly reduced. In the PFC-A group it was observed that MAP and MPAP increased and CO decreased during ARDS induction, but not during PFC infusion. Those changes were signifi cant in comparison with the NON group. Oxygen delivery and consumption in the PFC-A group were signifi cantly increased. Histopathological analysis is currently being performed. Interim analysis showed a trend to reduced alveolar damage in PFC-A animals. y Methods We constructed a survey of 10 questions using SurveyMonkey and circulated it via an email link to the Linkmen of the UK Intensive Care Society. Responses were received between August and November 2013. Results We had 77 responses. The majority were from doctors (88%) and the rest from nurses. Of respondents, 63% worked in district general hospitals, 28% in teaching hospitals and the rest in specialist units. Overall, 54% of respondents worked in units using SSD. From these responses, the types of patients receiving SSD are summarised in Table 1. One hundred per cent of units used intermittent SSD. Seventy- one per cent of respondents reported that SSD tubes were stored only on their ICU, with 26% reporting availability in acute areas and the rest hospital wide. Twenty-eight per cent of respondents indicated it was unit policy to reintubate patients to facilitate SSD. P333 statistically signifi cant diff erences were detected (P = 0.276). However, in the fi rst 5 days the development of VAP was signifi cantly higher in Group C (respectively, 4.3% vs. 25%, P = 0.046). The growth rate of VAP (VAP number/ventilator-day × 1,000) in Group C was 17.48, while in Group S it was 11.62. Between the groups there were no statistically signifi cant diff erence according to ICU mortality, length of ICU stay and length of hospital stay (P ≥0.05). Intravenous perfl uorocarbons increased oxygen delivery/ consumption in ARDS in swine Intravenous perfl uorocarbons increased oxygen delivery/ consumption in ARDS in swine A Scultetus1, A Haque1, F Arnaud1, G McNamee2, L Dickson1, C Auker1, R McCarron1, R Mahon1 A Scultetus1, A Haque1, F Arnaud1, G McNamee2, L Dickson1, C Auker1, R McCarron1, R Mahon1 1Naval Medical Research Center, Silver Spring, MD, USA; 2Uniformed Services University of the Health Sciences, Bethesda, MD, USA Critical Care 2014, 18(Suppl 1):P333 (doi: 10.1186/cc13523) Conclusion This prospective randomized controlled study demonstrated that the incidence of VAP (especially early development of the VAP) with continuous aspiration of subglottic secretions without creating any undesirable clinical damage in the airways was signifi cantly reduced. Introduction Emulsifi ed perfl uorocarbons (PFC) are synthetic hydrocarbons that can carry 50 times more oxygen than human plasma. Their properties may be advantageous in applications requiring preservation of tissue viability in oxygen-deprived states, which makes them a potential candidate for combat and civilian pre- hospital resuscitation. Our hypothesis was that an intravenous dose of PFC increases vital organ tissue oxygenation, improves survival and reduces or prevents the development of ventilator-associated ARDS. Here we report data from the second part (ARDS only) of a multiphase swine study to investigate the benefi ts of PFC in treating hemorrhagic shock and preventing ARDS. Reference 1. Bouza E, Perez MJ, Munoz P, Rincon C, et al.: Continuous aspiration of subglottic secretions in the prevention of ventilator-associated pneumonia in the postoperative period of major heart surgery. Chest 2008, 134:938-946. P334 Prevention of pneumothorax using venovenous ECMO in acute respiratory distress syndrome with emphysematous/cystic changes in the lung T Otani, S Ohshimo, K Ota, Y Kida, T Inagawa, J Itai, S Yamaga, K Une, Y Iwasaki, N Hirohashi, N Kohno, K Tanigawa Hiroshima University, Hiroshima, Japan Critical Care 2014, 18(Suppl 1):P334 (doi: 10.1186/cc13524) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1. Muscedere J, et al.: Crit Care Med 2011, 39:1985-1991. Survey of the use and practicalities of subglottic suction drainage in the UK More than 90% of units had a ventilator care bundle and regularly measured cuff pressures. Overall, 83% of those surveyed thought SSD was benefi cial in the prevention of VAP. Conclusion Administration of PFC before induction of ARDS was detrimental, while giving PFC after ARDS improved oxygen delivery and increased oxygen consumption. Although survival in this group was lower than in the NON control group (80% vs. 100%, not signifi cant), a reduction in alveolar damage was observed. This might improve long- term outcome after ARDS. Based on these data we will continue to the fi nal phase of this project and evaluate the capacity of PFC to prevent ARDS in combination with HS. Table 1 (abstract P332). SSD in specifi c patient groups (more than one per responder) Patients Number All 23 Tube >72 hours 6 Tracheostomy 13 Selected 7 Table 1 (abstract P332). SSD in specifi c patient groups (more than one per responder) 2. High Impact Intervention [http://nhschoicestraining.spinningclock.com/ Documents/HII_-_Ventilator_associated_pneumonia.pdf] Survey on the use of chlorhexidine and toothpaste as part of oral care in UK ICUs I Kangesan1, F Millinchamp1, JV Williams1, LA Burrows2 1Royal United Hospital, Bath, UK; 2Frenchay Hospital, Bristol, UK Critical Care 2014, 18(Suppl 1):P330 (doi: 10.1186/cc13520) Introduction Ventilator-associated pneumonia (VAP) is the most common nosocomial infection among ventilated patients and is associated with increased mortality and morbidity [1]. Oral chlorhexidine has been used to decontaminate the airway in critically Results In Group C, VAP was developed in 10 (35.7%) of 28 patients. In group S, VAP was developed in fi ve (21.7%) of 23 patients. In both groups when compared according to the development of VAP, no S120 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Forty-one patients were included in this study (ECMO and non- ECMO group, 21 and 20 patients, respectively). There were no signifi cant diff erences between ECMO and non-ECMO groups as regards age, sex, underlying disease, PaO2/FIO2 ratio, duration of ICU stay, and survival. In the ECMO group, the mean duration of ECMO use was 17 ± 13 days, and bleeding due to anticoagulation was observed in fi ve patients. The mean airway pressure in the ECMO group was signifi cantly lower than in the non-ECMO group (12 ± 6 cmH2O, 22 ± 6 cmH2O, respectively; P <0.0001). The incidence of pneumothorax was also signifi cantly lower in the ECMO group than the non-ECMO group (10%, 45%, respectively; P = 0.015). In Kaplan–Meier analysis, the proportion of pneumothorax- free patients was signifi cantly higher in the ECMO group (P = 0.014). In multivariate analysis, conventional ventilator management, presence of interstitial pneumonia and the duration of intubation were the independent risk factors of pneumothorax (hazard ratio (HR), 18.0, P = 0.010; HR 33.3, P = 0.025; HR 1.05, P = 0.041, respectively).f secretion of Th17 (IL-6, IL-8, IL-9, IL-17) and Th1 (TNFα, IL-15, IL-12p70) cytokines [1,2]. However, the exact role of T-helper cells (Th) in the ALI model remains unknown. We hypothesized that there might be Th imbalance within the lung in the early phase of LPS-induced ALI. This study was to assess the role of lung Th polarization response in ALI mice. Methods C57BL/6 mice were randomly divided into two groups: control group and ALI group. ALI animals received 2 mg/kg LPS. Lung wet weight/body weight (LW/BW) was recorded to assess lung injury. The pathological changes were examined under an optical microscope. The mRNA expression levels of T-bet, GATA-3 and RORγt were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. Meanwhile, levels of IL-6, IFNγ, IL-4 and IL-17 in lung homogenates were assessed by enzyme-linked immunosorbent assay. Results The increase in LW/BW was induced in ALI mice. Histologically, widespread alveolar wall thickening caused by edema, severe hemorrhage in the interstitium and alveolus, and marked and diff use interstitial infi ltration with infl ammatory cells were observed in the ALI group. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. References 1. Bermejo-Martin JF, et al.: Crit Care 2009, 13:R201. 2. Hagau N, et al.: Crit Care 2010, 14:R203. 1. Bermejo-Martin JF, et al.: Crit Care 2009, 13:R201. 2. Hagau N, et al.: Crit Care 2010, 14:R203. 1. Bermejo-Martin JF, et al.: Crit Care 2009, 13:R201. 2. Hagau N, et al.: Crit Care 2010, 14:R203. g j y Methods Infant (~1/2 month), juvenile (~1 month), adult (~4 months) and older (~19 months) Wistar rats were challenged with intratracheal LPS and injurious ventilation using tidal volumes of 15 ml/kg for 4 hours. Lung injury was assessed by wet-to-dry ratio and changes in P/F ratio. Levels of infl ammatory mediators were measured in bronchoalveolar lavage fl uid; mRNA expression of key genes of the pulmonary RAS was determined in lung homogenates. P335 Injurious ventilation has an age-dependent aff ect on the pulmonary renin–angiotensin system in LPS-challenged rats LR Schouten1, HJ Helmerhorst1, GT Wagenaar2, AP Bos1, MJ Schultz1, RM Wösten-van-Asperen1 1Academic Medical Center, Amsterdam, the Netherlands; 2Leiden University Medical Center, Leiden, the Netherlands Critical Care 2014, 18(Suppl 1):P335 (doi: 10.1186/cc13525) Introduction The underlying molecular mechanisms for the association between aging and a higher susceptibility to develop ARDS are poorly understood. The pulmonary renin–angiotensin system (RAS), with a lung-protective (angiotensin-converting enzyme (ACE)2–angiotensin (Ang)-1,7–Mas receptor) axis and a lung-injurious (ACE–Ang II–Ang II receptor (AT1)) axis, has been implicated in the pathogenesis of ARDS and changes with age. We hypothesized that injurious ventilation has an age-dependent eff ect on the pulmonary RAS in LPS-challenged rats that is associated with increased lung injury. Conclusion ALI provokes Th1 and Th17 polarization response. Th1 and Th17 may participate in the early infl ammatory response to ALI. Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. References Conclusion ALI provokes Th1 and Th17 polarization response. Th1 and Th17 may participate in the early infl ammatory response to ALI. y p p yl y p Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. References Comparison of CD80 level on dendritic cells in acute lung injury mice FCM analysis showed that the level of CD80 on circulating DC in control group was (3.3 ± 1.5)%, CD80 expression on lung DC was (3.6 ± 1.2)%, and expression of CD80 on splenic DC was (9.0 ± 3.6)%, which was Methods Twelve C57BL/6 mice were randomly divided into two groups: control group and ALI group. Blood, lungs and spleens were harvested at 6 hours after LPS or PBS administration. The level of CD80 on DC was assessed by fl ow cytometry (FCM). The IL-6 level in the lung was measured by enzyme-linked immunosorbent assay. Lung wet weight/ body weight (LW/BW) was recorded to assess lung injury. Meanwhile, pathological changes were examined under an optical microscope.i j g g Conclusion The eff ects of injurious ventilation on lung injury are age dependent in a model of LPS-challenged rats, which is associated with a more pronounced imbalance of the pulmonary RAS at the expense of the lung-protective axis with increasing age. Comparison of CD80 level on dendritic cells in acute lung injury mice 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Results LPS-challenged and ventilated older rats showed higher mortality, larger change in wet-to-dry ratio and larger decline in P/F ratio, compared with other age groups. Increases in neutrophil infl ux and infl ammatory mediators were age dependent, with higher levels with increasing age. Compared with controls, ventilated LPS- challenged rats showed a decrease in mRNA expression of ACE, ACE2, AT-1 and Mas receptor in all age groups, except for infants. The relative decrease in the mRNA expression of the Mas receptor was most extensive in older rats, thereby shifting the balance towards the lung injurious axis in this age group.f Critical Care 2014, 18(Suppl 1):P337 (doi: 10.1186/cc13527) Critical Care 2014, 18(Suppl 1):P337 (doi: 10.1186/cc13527) Introduction Dendritic cells (DC) may play an important role in acute lung injury (ALI) [1]. CD80 is the crucial co-stimulatory molecule that is expressed on the surface of DCs. However, little is known about the expression of CD80. The purpose of this study was to observe the expression of CD80 on circulating, lung and splenic dendritic cells (DC) in ALI mice. Methods Twelve C57BL/6 mice were randomly divided into two groups: control group and ALI group. Blood, lungs and spleens were harvested at 6 hours after LPS or PBS administration. The level of CD80 on DC was assessed by fl ow cytometry (FCM). The IL-6 level in the lung was measured by enzyme-linked immunosorbent assay. Lung wet weight/ body weight (LW/BW) was recorded to assess lung injury. Meanwhile, pathological changes were examined under an optical microscope. Results LPS-ALI resulted in a signifi cant increase in lung W/D ratio. Histologically, widespread alveolar wall thickening caused by edema, severe hemorrhage in the interstitium and alveolus, and marked and diff use interstitial infi ltration with infl ammatory cells were observed in the ALI group. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 The mRNA expression of T-bet and RORγt was upregulated in ALI mice at 24 hours and 48 hours relative to normal mice (P <0.05 vs. Con). There was no signifi cant diff erence in the expression of GATA-3 among groups at 24 hours and 48 hours. Meanwhile, the levels of IFNγ, IL-17 and IL-6 in lung tissue were signifi cantly enhanced at 24 hours and 48 hours in the LPS-induced ALI mice. In addition, the levels of IL-4 in lung tissue were signifi cantly enhanced at 48 hours in the LPS-induced ALI mice. The expression of T-bet mRNA and RORγt mRNA had a strong correlation with the IL-6 concentration. However, there was no signifi cant correlation of GATA- 3 with the IL-6 concentration. In addition, there was a signifi cant correlation of IFNγ, IL-4 and IL-17 with the IL-6 level in LPS-induced ALI at 24 hours and 48 hours. y Conclusion Although the survival rate was not statistically diff erent, the use of ECMO for ARDS patients complicated with emphysematous/ cystic changes in the lung markedly reduced the incidence of pneumothorax. Prevention of pneumothorax using venovenous ECMO in acute respiratory distress syndrome with emphysematous/cystic changes in the lung Introduction Venovenous extracorporeal membrane oxygenation (VV ECMO) is a treatment option for acute respiratory distress syndrome (ARDS) to minimize ventilator-induced lung injury including life- threatening pneumothorax. The purpose of our study was to investigate the safety and effi cacy of VV ECMO for preventing pneumothorax in ARDS patients who were complicated with emphysematous/cystic changes in the lung. Conclusion Despite specifi c recommendations from the UK Department of Health [2], only about one-half of respondents work in ICUs where SSD has been adopted. Most studies show benefi t in patients expected to be ventilated for greater than 72 hours [1], but most units used SSD in all intubated patients. The reintubation rate to facilitate SSD was also reasonably high, despite a lack of evidence to support this practice. In the vast majority of hospitals, SSD endotracheal tubes are stored only on the ICU and so the need for reintubation may result from a lack of available appropriate tubes at the point of fi rst intubation. References Methods We have retrospectively analyzed data of ARDS patients complicated with emphysematous/cystic changes in the lung who were admitted to our ICU from 2006 through 2012. We divided the subjects into two groups, patients treated with VV ECMO (ECMO group), and those treated only by conventional ventilator management (non- ECMO group). Correlations between age, sex, underlying disease, PaO2/ FIO2 ratio on admission, duration of ICU stay, survival and incidence of pneumothorax were evaluated. S121 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P338 Five-year single-centre review of ARDS patients receiving high-frequency oscillatory ventilation N Pathmanathan1, N Smith1, V Martinson1, V Allgar2, R Rao1, J Glazebrook1, L Gray-Nicholson1, A Gratrix1 1Hull and East Yorkshire Hospitals, Hull, UK; 2Hull and York Medical School, York, UK Critical Care 2014, 18(Suppl 1):P338 (doi: 10.1186/cc13528) Results The increase in LW/BW induced by LPS was partly prevented by pretreated with losartan. Histologically, losartan eff ectively attenuated the LPS-induced lung hemorrhage, and leukocyte cell infi ltration in the interstitium and alveolus. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. With pretreatment of ALI mice with losartan, the level of IL-6 in lungs markedly decreased. The mRNA expression of T-bet and RORγt was upregulated in ALI mice at 24 hours and 48 hours relative to normal mice (P <0.05 vs. Con). There was no signifi cant diff erence in the expression of GATA-3 among groups at 24 hours and 48 hours. Of note, pretreatment of ALI mice with losartan resulted in signifi cantly reduced mRNA expression of T-bet at 24 hours and 48 hours and RORγt mRNA expression at 48 hours (P <0.05 vs. ALI). Meanwhile, the levels of IFNγ, IL-4, IL-17 and IL-6 in lung tissue were signifi cantly enhanced at 24 hours and 48 hours in the LPS-induced ALI mice. In addition, both IFNγ and IL-17 in lung tissue at 24 hours and 48 hours decreased signifi cantly in losartan-pretreated mice compared with the ALI mice. With pretreatment of ALI mice with losartan, the level of IL-4 in lungs was not changed. Critical Care 2014, 18(Suppl 1):P338 (doi: 10.1186/cc13528) Introduction Two recent RCTs (OSCAR and OSCILLATE [1,2]) showed that high-frequency oscillatory ventilation (HFOV) had no positive impact on mortality. We present our experience over 5 years. p y p p y Methods Adult ARDS patients who received HFOV from 2008 to 2012 were included. Demographics, illness severity and outcomes were collected retrospectively. Results A total of 118 patients were included; 56.8% were male, mean age was 54.8 years. RRT use was 45% during admission. Vasoactive agent use and neuromuscular blockade infusion rate was 81.9 and 29.7% pre HFOV respectively. The 28-day and 6-month mortality was 61.9 and 70.3%. A total of 60.1% had less than 48 hours conventional ventilation (CV) pre HFOV. The 6-month mortality was 64.8% for this group. Patients who had over 48 hours CV pre HFOV had a 6-month mortality of 76.6%. See Table 1. y Conclusion Mortality rates were higher than in recent trials [1,2]. Our patients represent a more critically unwell group with lower PF ratios pre HFOV and high vasoactive and RRT use. P339 signifi cantly higher than that on circulating DC and lung DC (P <0.05). In the ALI mouse, the level of CD80 on peripheral blood DC was (5.1 ± 2.1)%; the CD80 level on lung DC was (9.6 ± 2.50)%, which was signifi cantly higher than that on peripheral blood DC (P <0.05); and the level of CD80 on splenic DC was (25.2 ± 4.7)%, which was signifi cantly higher than CD80 levels on the peripheral blood and lung DC (P <0.05). The CD80 level on lung and splenic DC in ALI mice was signifi cantly higher than that on lung and splenic DC in control mice (P <0.05 vs. Con). Blocking angiotensin type 1 receptor modulates Th1-mediated and Th17-mediated responses in lipopolysaccharide-induced acute lung injury mice J Liu1 W Li2 J Liu , W Li 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Research Institute of General Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Critical Care 2014, 18(Suppl 1):P339 (doi: 10.1186/cc13529) Conclusion There is a dynamic characteristic in the expression of CD80 on DC populations in normal and ALI mice. Elevated expression of CD80 on DC seems to play an important role in the pathogenesis of ALI. Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. Reference Introduction Losartan, an antagonist of angiotensin II (Ang II) type 1 receptor, is a potential therapeutic drug for acute lung injury (ALI). Recent reports suggest that losartan inhibits T-helper (Th)-1 immune response and ultimately attenuates infl ammation in several angiotensin II-mediated infl ammatory diseases [1,2]. However, the possible protective mechanisms of losartan in ALI remain poorly understood. This study was to assess the eff ect of losartan on the lung Th polarization response in ALI. 1. Venet F, et al.: Am J Pathol 2010, 176:764-773. Methods C57BL/6 mice were randomly divided into three groups: control group, ALI group and ALI + losartan group. ALI animals received 2 mg/kg LPS; ALI + losartan animals received 2 mg/kg LPS and 15 mg/kg losartan 30 minute before intratracheal injection of LPS. The pathological changes were examined under an optical microscope. The mRNA expression levels of T-bet, GATA-3 and RORγt were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. P338 Five-year single-centre review of ARDS patients receiving high-frequency oscillatory ventilation N Pathmanathan1, N Smith1, V Martinson1, V Allgar2, R Rao1, J Glazebrook1, L Gray-Nicholson1, A Gratrix1 1Hull and East Yorkshire Hospitals, Hull, UK; 2Hull and York Medical School, York, UK Critical Care 2014, 18(Suppl 1):P338 (doi: 10.1186/cc13528) HFOV may still have a role in the treatment of these very sick patients with treatment refractory to conventional ventilation. g Conclusion Ang II-induced Th1 and Th17 polarization response could upregulate infl ammatory response and induce lung injury, and losartan may be a promising substance for clinical use in LPS-induced ALI. Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. References 1. Hoch NE, et al.: Am J Physiol Regul Integr Comp Physiol 2009, 296:R208-R216. 2. Platten M, et al.: Proc Natl Acad Sci U S A 2009, 106:14948-14953. P336 Role of Th1 and Th17 imbalance in acute lung injury mice J Liu1 W Li2 p g g p p Results LPS-ALI resulted in a signifi cant increase in lung W/D ratio. Histologically, widespread alveolar wall thickening caused by edema, severe hemorrhage in the interstitium and alveolus, and marked and diff use interstitial infi ltration with infl ammatory cells were observed in the ALI group. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. FCM analysis showed that the level of CD80 on circulating DC in control group was (3.3 ± 1.5)%, CD80 expression on lung DC was (3.6 ± 1.2)%, and expression of CD80 on splenic DC was (9.0 ± 3.6)%, which was , 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China , 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Critical Care 2014, 18(Suppl 1):P336 (doi: 10.1186/cc13526) Critical Care 2014, 18(Suppl 1):P336 (doi: 10.1186/cc13526) Introduction Acute lung injury (ALI) is characterized by an excessive infl ammatory response. Several recent clinical observations have shown that severe H1N1 infl uenza with ARDS is characterized by early S122 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Risk factors for multi-resistant organisms in sepsis L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P342 (doi: 10.1186/cc13532) Introduction The increasing prevalence of infections by multi-resistant organisms (MDR) has increased over the last decades, with implications not only in the overall level of therapeutic success, but also in the selective pressure exerted by the use of broad-spectrum antibiotics to defeat increasingly resistant agents, thereby creating a vicious cycle [1,2]. The aim of this study is to describe risk factors associated with infection by MDR organisms among septic patients. g p Methods We report a retrospective analysis of out-of-center ECMO implantation in patients with severe ARDS. Our center provides a 24/7 service for out-of-hospital ECMO implantation with a team consisting of two intensive care physicians and a perfusionist. In all patients, ECMO therapy was initiated in a non-ECMO center prior to transportation to our hospital. Implantation of a dual-lumen catheter was fi rst choice in all patients and was guided echocardiographically. y g g p p Methods A retrospective cohort study including all adult patients with microbiological documented sepsis, admitted to the emergency room of a tertiary care, university hospital between 1 July 2011 and 30 June 2012. g g y Results Between January 2011 and November 2013, a total of 56 patients (average age 53.3 years) underwent out-of-center venovenous ECMO implantation. In 52 cases (94.6%), a dual-lumen catheter could be implanted successfully using echocardiographic guidance. Either 31 Fr (65.0%) or 27 Fr (35.0%) Avalon Elite catheters were employed. No patient developed any major or even fatal complications related to ECMO implantation. In three out of 56 patients, poor imaging quality or technical issues hampered the implantation of a dual-lumen catheter and a femoral approach had to be made. Concerning short-term survival, 81.0% of all patients could be dismissed from the ICU. Results During the study period, 162 patients were admitted to the emergency room with severe sepsis; 79 (49%) had microbiological documentation, and were included in this study. The mean (SD) age was 71 (15) years; 62% were men. Forty patients (51%) had an infection by a MDR organism. References 1. Young D, et al.: High-frequency oscillation for ARDS. N Engl J Med 2013, 368:806-813. 1. Hoch NE, et al.: Am J Physiol Regul Integr Comp Physiol 2009, 296:R208-R216. 2. Platten M, et al.: Proc Natl Acad Sci U S A 2009, 106:14948-14953. 2. Ferguson N, et al.: High-frequency oscillation in early ARDS. N Engl J Med 2013, 368:795-805. 1. Hoch NE, et al.: Am J Physiol Regul Integr Comp Physiol 2009, 296:R208-R216. 2. Platten M, et al.: Proc Natl Acad Sci U S A 2009, 106:14948-14953. Table 1 (abstract P338). OSCAR OSCILLATE Hull HFOV Control HFOV Control Mean APACHE II 22.0 21.8 21.7 29 29 Mean Vt (ml/kg predicted) 8.6 8.7 8.3 7.2 7.1 PaO2:FiO2 (mmHg) 81.5 113 113 121 114 Vasoactive agents (%) 81.9 43.5 (day 1) 44.6 (day 1) 67 63 Hospital mortality (%) 66.1 50.1 48.4 46.9 35.2 Table 1 (abstract P338). S123 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 loads were higher in TA compared with NP, up to 2 log viral copies. Duration of positivity of infl uenza in daily samples was up to two times longer in TA samples than in NP samples. Of all 35 virus-positive patients, 10 had received viral diagnostics via routine care. loads were higher in TA compared with NP, up to 2 log viral copies. Duration of positivity of infl uenza in daily samples was up to two times longer in TA samples than in NP samples. Of all 35 virus-positive patients, 10 had received viral diagnostics via routine care. P340 Echocardiographic guidance for Avalon Elite dual-lumen catheter implantation D Staudacher1, A Schmutz2, P Biever1, J Kalbhenn2, C Bode1, T Wengenmayer1 1University Heart Center Freiburg, Germany; 2University Clinic Freiburg, Germany Critical Care 2014, 18(Suppl 1):P340 (doi: 10.1186/cc13530) Conclusion The prevalence of respiratory viruses in this unselected patient population is high. Forty percent would have been missed if only NP was sampled. Thereby, TA sampling adds to the detection of respiratory viruses in critically ill patients. Accuracy and implications of these results needs to be investigated further. Introduction Venovenous ECMO is a therapeutic option in patients with severe ARDS. The dual-lumen catheter (Avalon Elite; Maquet Medical) off ers excellent oxygenation and decarboxylation through a single insertion site and facilitates patient mobilization as it avoids femoral access. Lower airway sampling greatly increases detection of respiratory viruses in critically ill patients: the COURSE studyf j Conclusion The presence of diabetes and decreased functional capacity should be considered risk factors for infection by a MDR organism and should be taken into consideration in the empirical therapy prescription. SP Rebers1, KD Verheul1, R Molenkamp1, AM Spoelstra-de Man2, P Spronk3, MD De Jong1, MJ Schultz1 g 1Academic Medical Center, Amsterdam, the Netherlands; 2VU Medical Center, Amsterdam, the Netherlands; 3Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2014, 18(Suppl 1):P341 (doi: 10.1186/cc13531) P341 Lower airway sampling greatly increases detection of respiratory viruses in critically ill patients: the COURSE study F Van Someren Gréve1, KF Van der Sluijs1, NP Juff ermans1, T Winters1, SP Rebers1, KD Verheul1, R Molenkamp1, AM Spoelstra-de Man2, P Spronk3, MD De Jong1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU Medical Center, Amsterdam, the Netherlands; 3Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2014, 18(Suppl 1):P341 (doi: 10.1186/cc13531) Lower airway sampling greatly increases detection of respiratory viruses in critically ill patients: the COURSE studyf P342 Risk factors for multi-resistant organisms in sepsis L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P342 (doi: 10.1186/cc13532) Risk factors for multi-resistant organisms in sepsis L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P342 (doi: 10.1186/cc13532) Risk factors associated with infection by a MDR organism were the presence of any comorbidity (OR = 3.542, P = 0.022), diabetes mellitus (OR = 4.500, P = 0.006), Karnofsky performance status (KPS) <70% (OR = 3.882, P = 0.005), the presence of modifi ers of etiology (OR = 5,040, P = 0.010), chronic wounds (OR = 5.371, P = 0.040), healthcare-associated infections (OR = 3.325, P = 0.026) and hospital- acquired infections (OR = 5.225, P = 0.016). The multivariate model retained as independent variables associated with infection by a MDR organism: the presence of diabetes mellitus (adjusted OR = 4.1,95% CI: 1.4 to 12.6) and the need for assistance in daily activities (KPS <70%, adjusted OR = 3.6, 95% CI: 1.3 to 9.7). Conclusion Echocardiographic guidance for out-of-center Avalon Elite catheter implantation is a safe and effi cient option when performed by an experienced team. References As incorrect placement of the catheter might result in perforation and cardiac tamponade, fl uoroscopy guidance is advocated in the literature. Transporting an unstable patient for fl uoroscopy, however, can endanger the patient. y References 1. Siegel JD, et al.: Am J Infect Control 2007, 35(10 Suppl 2):S165-S193. 2 Wright SW et al : Am J Emerg Med 2000 18:143 146 1. Siegel JD, et al.: Am J Infect Control 2007, 35(10 Suppl 2):S165-S193. 1. Siegel JD, et al.: Am J Infect Control 2007, 35(10 Suppl 2):S165-S193. 2. Wright SW, et al.: Am J Emerg Med 2000, 18:143-146. Introduction The prevalence of viral respiratory infections in critically ill patients on the ICU and the diagnostic potential of tracheal aspirate sampling are unknown. For this study, the prevalence of respiratory viruses was investigated in intubated patients by simultaneous sampling of nasopharynx and tracheal aspirate. 2. Wright SW, et al.: Am J Emerg Med 2000, 18:143-146. Retrospective observational analysis of the infective risk of arteri lines in a general ICU M Wylie, M Clark Victoria Hospital, Kirkcaldy, UK Critical Care 2014, 18(Suppl 1):P345 (doi: 10.1186/cc13535) g M Wylie, M Clark y Victoria Hospital, Kirkcaldy, UK P344 Clostridium diffi cile infection in ICU patients E Belesiotou, C Routsi, M Nepka, E Magira, P Kaltsas, Z Psaroudaki, E Kraniotaki, A Argyropoulou, T Pittaras, S Zakynthinos Evangelismos Hospital, Athens, Greece Critical Care 2014, 18(Suppl 1):P344 (doi: 10.1186/cc13534) P343 Infections from MDR strains (K. pneumoniae, P. aeruginosa, A. Baumannii complex) in a multivalent ICU K Kontopoulou, E Antypa, I Sgouropoulos, G Voloudakis, P Tasioudis, E Antoniadou G. Genimatas General Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P343 (doi: 10.1186/cc13533) Methods During March and April 2013, consecutive acutely admitted, intubated patients were included in three ICUs in the Netherlands, regardless of diagnosis at admission. Daily sampling of the nasopharynx (NP) with fl ocked swab and tracheal aspirate (TA) was performed until successful weaning from mechanical ventilation or death. Admission samples were tested via multiplex RT-PCR for infl uenza A and B, parainfl uenza, RSV, human metapneumovirus, bocavirus, coronavirus, rhinovirus, enterovirus, parechovirus and adenoviruses. Of the infl uenza-positive patients, subsequent daily samples were tested for infl uenza. Results of viral diagnostics performed by routine care were collected, and compared with results found in this study. G. Genimatas General Hospital, Thessaloniki, Greece p Critical Care 2014, 18(Suppl 1):P343 (doi: 10.1186/cc13533) Introduction The infection incidences from MDR strains in our ICU are in accordance with the results of respective studies in other ICUs of our country. Our aim is to identify potential risk factors for the development of MDR infection in ICU patients. Methods The sample consisted of 882 patients, admitted to our ICU from 1 January 2010 until 30 June 2013. The factors studied were: age, gender, length of stay in the ICU, origin of patient (for example, home or transferred from another institution), APACHE II score, adjusted mortality score, previous colonization with MDR, prior use of antimicrobial agents, duration of ventilator use, immunosuppression, diabetes, hypoproteinemia and presence of central venous catheter (CVC). Bivariable analyses were conducted to determine the association p y Results As part of an ongoing observational study (COURSE study), 128 patients were included, of which 35 were virus-positive (27%). Of these, 13 patients were positive in both NP and TA, eight only in NP, and 14 only in TA. Thereby, 40% of the viruses would have been missed if only NP was performed in this study group. In eight out of 12 coronavirus- positive patients, only the TA sample was virus-positive, with negative NP. In subsequent daily samples of infl uenza-positive patients, viral S124 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 between potential risk factors and development of MDR infection. Categorical variables were compared using the Fisher test. Odds ratios (ORs) and 95% confi dence intervals (CIs) were calculated to evaluate the strength of any association. Continuous variables were compared using the Student t test or the Wilcoxon test where appropriate. Multivariable analysis was performed using multiple logistic regression. Variables with P <0.20 in bivariable analyses were considered for inclusion in a multivariable model. Methods During the last year, all stool specimens received in the microbiology department from patients hospitalized in the 30-bed, multidisciplinary ICU of a tertiary-care hospital were evaluated. Specimens were ordered by physicians in the presence of clinical features compatible with C. diffi cile-associated infection. Each specimen was subjected to diagnostic tests for C. diffi cile infection including toxin enzyme immunoassays for C. diffi cile toxins A and B detection (DUO Toxin A&B; VEDA.LAB, France), and glutamate dehydrogenase (GDH) for cell wall antigen detection (C. DIFF Quik Chek Complete®; USA). Results Out of 882 patients, 135 (15.3%) developed MDR infection. The following factors showed statistical signifi cant diff erence (P <<0.01): length of stay in the ICU, origin of patient, APACHE II score, previous colonization, previous use of carbapenems, duration of mechanical ventilation, immunosuppression, diabetes mellitus, hypoproteinemia and the presence of CVC. The multivariant analysis showed statistically signifi cant correlations for the following independent risk factors: previous use of carbapenems (OR = 632.64, P <<0.01), origin of patient (OR = 19.60, P = 0.014), presence of CVC (OR = 14.19, P = 0.015), previous colonization (OR = 4.71, P = 0.037) and duration of ventilator use (OR = 1.10, P = 0.029). g p Results During the study period, 335 stool specimens were evaluated. Results obtained with the two-stage immunoassay tests are shown in Figure 1. All infected patients were treated with metronidazole or vancomycin. Following a course of therapy, 2% of the infected patients had recurrence or relapse. The crude mortality rate was 17%. Conclusion GDH antigen was positive in 12% of the stool specimens received from ICU patients with suspected C. diffi cile-associated infections. The majority of these specimens (51.4%) produce both C. diffi cile toxins A and B, whereas toxin B is produced in 31.4% and toxin A in the remaining 17.2%. References References Conclusion The analysis showed a very strong correlation between prior use of carbapenems and development of MDR infection. Other signifi cant factors are presence of CVC, origin of patient, previous colonization and duration of ventilator use. The identifi cation of the above factors and the eff ort for their restriction redound to the reduction of the MDR infections and, consequently, to the reduction of the morbidity and the mortality of the patients hospitalized in ICUs. 1. Bobo LB, et al.: Chest 2011, 140:1643. 1. Bobo LB, et al.: Chest 2011, 140:1643. 2. Cohen SH, et al.: Infect Control Clin Epidemiol 2010, 315:431. 2. Cohen SH, et al.: Infect Control Clin Epidemiol 2010, 315:431. P345 Reducing CR-BSI in a general ICU Results Out of 588 patients, 30 (5.1%) developed candidemia (frequency: 12 per 1,000 days of hospitalization). The mortality of patients with candidemia was 66.7% (20/30). The monovariant analysis showed statistical signifi cant diff erence in the following factors: immunosuppression (P = 0.0005), diabetes mellitus (P = 0.0005), hypoproteinemia (P = 0.0005), the presence of central venous catheter (P = 0.0005), the coexistence of bacteremia and the prior use of antimicrobial agents (P = 0.0005), length of stay in the ICU >5 days (P = 0.004) and the type of intervention (P = 0.001). As independent risk factors from the multivariant analysis were found the following: immunosuppression (exp(B) = 218.37, 95% CI = 11.76 to 4,053.72, P = 0.0005), hypoproteinemia (exp(B) = 25.69, 95% CI = 2.82 to 296.8, P = 0.009), presence of central venous catheter (exp(B) = 13.79, 95% CI = 1.86 to 102.51, P = 0.01) and the coexistence of bacteremia in combination with prior use of antimicrobial agents (exp(B) = 404.94, 95% CI = 11.61 to 14190.41, P = 0.001). D Dawson, P Riley, A Rhodes , y, St George’s Healthcare NHS Trust, London, UK St George’s Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P346 (doi: 10.1186/cc13536) St George’s Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P346 (doi: 10.1186/cc13536) g , , Critical Care 2014, 18(Suppl 1):P346 (doi: 10.1186/cc13536) Introduction Central venous catheters (CVCs) are essential for the delivery of medications and fl uids in the ICU patient; however, they carry a substantial infection risk. Evidence from a collaborative, cohort study suggests bundled interventions can provide a sustained decrease in infection [1]. Methods Since December 2009, data have been collected daily on the number of patients with one or more CVC. All positive blood cultures are reviewed monthly against predefi ned criteria to judge whether these are genuine bacteraemic episodes and thus classifi ed as laboratory-confi rmed bloodstream infections. A monthly rate for CR-BSI per 1,000 dwell-days is calculated from these data. Since July 2010, a number of interventions aimed at reducing CR-BSI have been introduced to the ICU. Conclusion Early candidemia identifi cation, prompt collaboration of intensivists with biopathologists and immediate initiation of the proper antifungal treatment is of great signifi cance. The concurrent understanding of the predisposing risk factors constitutes a signifi cant supportive tool for the prediction of such infections. P347 Thirty-seven arterial lines returned no growth (77.08%). Seven cultures grew organisms likely to be contaminants (14.58%). Four cultures grew signifi cant organisms (yield of 8.33%). There were two cases with documented clinical signs of catheter-related local infection (CRLI) at the arterial line puncture site. In one case of CRLI the primary source of infection was felt to be remote from the arterial line. The second represented a local infection with organisms that are typically skin commensals. Of the four cultures likely to represent invasive pathogens, three had clinical suspicion that the primary source was a site remote from the arterial line. In two of these cases this was confi rmed by growing the same organism at an alternative site more likely to be the source of infection. Risk factors of candidemia in postoperative ICU patients: a prospective study K Kontopoulou, E Antypa, I Sgouropoulos, N Voloudakis, E Chassou E Antoniadou G. Genimatas General Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P347 (doi: 10.1186/cc13537) K Kontopoulou, E Antypa, I Sgouropoulos, N Voloudakis, E Chassou, E Antoniadou l l h l k K Kontopoulou, E Antypa, I Sgouropoulos, N Voloudakis, E Chassou, E Antoniadou G. Genimatas General Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P347 (doi: 10.1186/cc13537) Introduction The prediction for and the mortality of patients with candidemia are highly adverse. The aim of this study was to identify the risk factors for the development of candidemia in postoperative patients. Introduction The prediction for and the mortality of patients with candidemia are highly adverse. The aim of this study was to identify the risk factors for the development of candidemia in postoperative patients. Conclusion These results suggest that CRLI rates for arterial lines are low at 1.003 per 1,000 arterial line-days. However, there is a signifi cant bacterial colonisation rate of the arterial lines sampled. Three arterial lines (6.25%) grew organisms that could represent an important potential source of ongoing bacteraemia. There is evidence to suggest that the risk of infection and colonisation of arterial lines may be similar to that of central lines [1]. Further prospective work is needed to assess the impact of catheter care bundles on colonisation and infection rates of arterial lines in the ICU. Methods From 1 July 2010 to 30 June 2013 all postoperative patients (n = 588) admitted to the multivalent ICU of our hospital were enrolled in this study. We recorded the age, sex, length of stay in the ICU, APACHE II score upon admission to the ICU, adjusted mortality score, underlying conditions, recent operations, invasive therapeutic procedures and prior usage of antimicrobial agents. Initial bivariable statistical comparisons were conducted using the χ2 test for categorical data and the Student t test or Wilcoxon test for continuous data. Relative risks (RRs) and their 95% confi dence intervals (CIs) were calculated. Statistical signifi cance was set at P <0.05. To identify patient characteristics associated with candidemia we used multivariable logistic regression. In the multivariant analysis we also included the independent risk factors reported in recent medical literature. Results of the logistic regression analysis are reported as adjusted odds ratio (OR) with 95% CI. P346 P346 Reducing CR-BSI in a general ICU D Dawson, P Riley, A Rhodes St George’s Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P346 (doi: 10.1186/cc13536) Reducing CR-BSI in a general ICU Reducing CR-BSI in a general ICU Results Data are presented on 15,644 CVC dwell-days over 47 months. Table 1 presents data for three full years and one part year (January to November 2013). Despite an increase in bed-days per year, there has been a sustained reduction in infection rates and a reduction in dwell-days. Table 1 (abstract P346). Catheter infection rates, APACHE score and bed-days 2010 to 2014 P348 P348 Escherichia coli infection in Polish neonatology ICUs in 2009 to 2012 J Wójkowska-Mach1, A Chmielarczyk1, D Romaniszyn1, E Helwich2, R Lauterbach2, M Pobiega1, M Borszewska-Kornacka2, E Gulczyńska2, A Kordek2 1Jagiellonian University Medical School, Krakow, Poland; 2Polish Neonatal Surveillance Network, Warsaw, Poland Critical Care 2014, 18(Suppl 1):P348 (doi: 10.1186/cc13538) Escherichia coli infection in Polish neonatology ICUs in 2009 to 2012 J Wójkowska-Mach1, A Chmielarczyk1, D Romaniszyn1, E Helwich2, R Lauterbach2, M Pobiega1, M Borszewska-Kornacka2, E Gulczyńska2, A Kordek2 1Jagiellonian University Medical School, Krakow, Poland; 2Polish Neonatal Surveillance Network, Warsaw, Poland Critical Care 2014, 18(Suppl 1):P348 (doi: 10.1186/cc13538) y Total CR-BSI/ CVC CR-BSI 1,000 APACHE II Bed- Dwell-days/ Year dwell-days annually dwell-days (mean) days bed-days 2010 4,037 6 1.5 15.8 5,870 0.69 2011 3,942 2 0.5 16.3 5,967 0.66 2012 3,822 3 0.8 15.9 6,117 0.62 2013* 3,863 1 0.3 16.0 6,059 0.64 Introduction The aim of our study was the analysis of the epidemiology of infections caused by ECO in Polish NICUs and their resistance to antibiotics, with particular reference to their impact on the safety of infants with very low birth weight, and their relationship to the care of women who are pregnant and in labour. The routes of transmission of ECO strains were also evaluated. Conclusion CR-BSI rates of 1.5/1,000 dwell-days in the fi rst year were similar to the post-intervention rate of 1.4 in the Pronovost study [1]. Subsequent rates have reduced, suggesting we are outperforming the secular trend [2]. The proportion of dwell-days to bed-days was reduced, which may suggest a reduction in duration and/or quantity of CVC placements. Methods Continuous prospective infection surveillance was conducted in 2009 to 2012 in fi ve NICUs and included 1,768 newborns whose birth weight was lower than 1,500 g. Results The incidence of ECO infections was 5.4% and 2.0/1,000 patient- days. The occurrence of ECO infections depended signifi cantly on the NICU and ranged from 3.9 to 17.9%. Multivariate analysis that took into account the combined eff ect of demographic data (gender, gestational Clostridium diffi cile infection in ICU patients p y Critical Care 2014, 18(Suppl 1):P345 (doi: 10.1186/cc13535) p y Critical Care 2014, 18(Suppl 1):P345 (doi: 10.1186/cc13535) Introduction This study aims to describe the infective morbidity of arterial line catheters in a general ICU. Introduction This study aims to describe the infective morbidity of arterial line catheters in a general ICU. Methods All ICU admissions for the year 2012 were listed on the WardWatcher database. All cases in which arterial line tip samples returned any growth were reviewed in conjunction with the medical, nursing and daily chart recordings. Data were collected on the organisms grown and antimicrobial sensitivities. A qualitative decision of clinical relevance was established based on necessitated change in patient management, signs of local site infection or evidence of bacteraemia with the same organism. Introduction Clostridium diffi cile infection is becoming more common worldwide. Critically ill patients are at particularly high risk for this disease due to multiple risk factors in this population. Accurate diagnosis is essential for patient management, infection control and epidemiology. There are a variety of methods to detect the presence of toxigenic C. diffi cile in stools [1,2]. The aim of this study was to evaluate the incidence of C. diffi cile infection in our ICU. Laboratory results of C. diffi cile toxin detection performed by the methods available in our institution are presented. Results During 2012 there were 416 ICU admissions, representing a total of 2,440 ITU-days and 1,994 arterial line-days. A total of 48 arterial line tips suspected of being infected were sent for culture. Figure 1 (abstract P344). Illustration of the tests performed for detection of C. diffi cile. Figure 1 (abstract P344). Illustration of the tests performed for detection of C. diffi cile. S125 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 2. Dixon-Woods et al.: Implement Sci 2013, 8:702. Reference 1. Lucet JC, et al.: Infectious risk associated with arterial catheters compared with central venous catheters. Crit Care Med 2010, 38:1030-1035. 1. Pronovost et al.: N Engl J Med 2006, 355:26. Surveillance for nosocomial pathogens in our ICU Y Kawano, H Ishikura, T Nishida, N Matusmoto, H Ohya, M Mizunuma, Y Nakamura, T Umemura Fukuoka University Hospital, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P350 (doi: 10.1186/cc13540) Surveillance for nosocomial pathogens in our ICU Y Kawano, H Ishikura, T Nishida, N Matusmoto, H Ohya, M Mizunuma, Y Nakamura, T Umemura Fukuoka University Hospital, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P350 (doi: 10.1186/cc13540) Introduction The use of contact precautions is recommended to reduce the transmission of these pathogens. However, there is little research regarding the relationship between the rate of patients with culture- positive fi ndings for Acinetobacter baumannii and the consumption of hand disinfectant. The objective of this study was therefore to evaluate trends in nosocomial bacterial detection, including A. baumannii, and the use of hand disinfectant in our ICU. y Conclusion Unfortunately, the presented data indicate that antibiotic prophylaxis in the presence of symptoms such as chorioamnionitis and PROM did not help to reduce the risk of ECO infection in the group of examined infants. In addition, multivariate analysis demonstrated only one signifi cant risk factor for ECO infection among infants with a birth weight <1,500 g; that is, the impact of the NICU. Epidemiology of ECO infections clearly indicated that observed cases of infections have no connection with the horizontal transmission (thus no proof of a link between the observed ECO infections with possible negligence in hand hygiene or excessive congestion on NICUs). This is confi rmed by the fact that no epidemic clones were observed (DEC-2011/01/D/ N27/00104). Methods A single-center, retrospective, observational study was carried out. The results of all cultures (sputum, urine, blood, and so forth) were used to examine trends in the detection of microbiology in our ICU over a 4-year period from April 2009 through March 2013. The rate of culture-positive patients, frequency of use of hand disinfectant and the antimicrobial use density (AUD) values were investigated for the described periods and compared between the early period group (April 2009 to March 2011) and the late period group (April 2011 to March 2013). Results We encountered 3,302 patients in our ICU during this period. No patients with culture-positive fi ndings for multidrug-resistant A. baumannii were identifi ed in this study. The rates of patients with culture-positive fi ndings for multidrug-resistant Pseudomonas aeruginosa (0‰ vs. 0.05‰ per 1,000 patient-days, P = 0.99) and P. aeruginosa (6.96‰ vs. 7.21‰ per 1,000 patient-days, P = 0.7) were not signifi cantly diff erent between the early period group and the late period group. The rates of patients with culture-positive fi ndings for MRSA (6.96‰ vs. 1. Nalapko Y, Peicheva O, Iegorov O, Nekrylov A: The dependence of infectious complications from the trauma severity. Intensive Care Med 2013, 39(Suppl. 2):P.0791. P350 age and birth weight) and place of birth (NICUs, where the baby was born) showed that only the place of hospitalisation had a signifi cant eff ect on the ECO infection risk. The highest levels of resistance among all ECO isolates were observed against ampicillin (88.8%) and amoxicillin/clavulanic acid (62.2%). ECO isolates showed very diff erent pulsotypes and dominant epidemic clones were not detected. Cluster analysis based on PFGE of the 90 isolates showed 71 unique types, some of which were less than 70% similar, suggesting a genotypically variable population. Isolates that have identical pulsotypes usually were derived from the same patient (as in the case of 11 isolates) or were isolated from diff erent patients of the same NICU in the same period of time (in the case of seven isolates). The location of the NICU and the site of the isolation did not appear to have a correlation in the cluster analysis P351 Comparison of multidrug-resistant Acinetobacter and non-Acinetobacter infections in terms of outcome in critically ill patients A Madaan1, V Singh1, P Shastri2, C Sharma3 1Patel Cancer and Superspeciality Hospital, Jalandhar, India; 2Sir Gangaram Hospital, New Delhi, India; 4Lovely Professional University, Jalandhar, India Critical Care 2014, 18(Suppl 1):P351 (doi: 10.1186/cc13541) Surveillance for nosocomial pathogens in our ICU Y Kawano, H Ishikura, T Nishida, N Matusmoto, H Ohya, M Mizunuma, Y Nakamura, T Umemura Fukuoka University Hospital, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P350 (doi: 10.1186/cc13540) 8.94‰ per 1,000 patient-days, P <0.05) and A. baumannii (4.98‰ vs. 6.51‰ per 1,000 patient-days, P <0.05) were signifi cantly higher in the late period group than in the early period group. The frequency of use of hand disinfectant (99.5 ml/patient- day vs. 85 ml/patient-day, P <0.05) was signifi cantly lower in the late period group. The AUD values for fl uoroquinolones (34 vs. 31.1 defi ned daily doses/1,000 bed-days, P = 0.69), third-generation cephalosporins (23.5 vs. 38.4 defi ned daily doses/1,000 bed-days, P = 0.55) and glycopeptides (34.9 vs. 35.9 defi ned daily doses/1,000 bed-days, P = 0.66) were not signifi cantly diff erent between the early period group and the late period group. However, the AUD values for carbapenems were signifi cantly higher in the early period group (40.6 vs. 71.8 defi ned daily doses/1,000 bed-days, P < 0.05). P349 Infection control as a nonpharmacologic strategy for the prevention of healthcare-associated infections in a Ukrainian hospital Y Nalapko Lugansk State Medical University, Lugansk, Ukraine Critical Care 2014, 18(Suppl 1):P349 (doi: 10.1186/cc13539) P349 Infection control as a nonpharmacologic strategy for the prevention of healthcare-associated infections in a Ukrainian hospital Y Nalapko Lugansk State Medical University, Lugansk, Ukraine Critical Care 2014, 18(Suppl 1):P349 (doi: 10.1186/cc13539) p Lugansk State Medical University, Lugansk, Ukraine Introduction Healthcare-associated infections (HAI) in ICU patients are related to intubation, mechanical ventilation, and central venous and/ or urinary catheters. The incidence of HAI is too high, and antibiotic strategies suff er from resistance to the common classes of antibacterial agents [1]. Methods A complex program including staff teaching on the basic approaches of hand hygiene, microbiological passport of the ICU departments, and detection of the sources of the pathogens polluting the treatment area of the ICU was implemented at the Lugansk Regional Clinical Hospital. The incidence of the HAI was studied in polytrauma patients in 1999 to 2003 (before the implementation of the program) and in 2008 to 2012 (after its implementation). Conclusion Improving compliance with hand hygiene and appropriate use of carbapenems is important for decreasing the rate of patients with culture-positive fi ndings for MRSA and A. baumannii. p Results Before the implementation of the infection control program, the incidence of respiratory tract infections in polytrauma patients staying in ICUs was 57.4% of patients. Urinary infections occurred in 51.9% of patients. Surgical site infections were found in 32.8% of the patients. Combination of the infections was detected in 33.8% of patients. The incidence of the multiple-drug-resistant bacterial colonies was 5.8%. In contrast, recent data obtained after the implementation of the program of the infection control showed that the respiratory infections occurred in 29.3% of the patients, catheter-related urinary tract infections were detected in 32.8%, and surgical site infections were detected in 8.6% of the polytrauma patients. On the other hand, the incidence of the multiple-drug-resistant bacterial colonies increased signifi cantly up to 14.2% (P <0.001). Other types of HAI changed nonsignifi cantly. Results Before the implementation of the infection control program, the incidence of respiratory tract infections in polytrauma patients staying in ICUs was 57.4% of patients. Urinary infections occurred in 51.9% of patients. Surgical site infections were found in 32.8% of the patients. Combination of the infections was detected in 33.8% of patients. The incidence of the multiple-drug-resistant bacterial colonies was 5.8%. p References 1. Pronovost et al.: N Engl J Med 2006, 355:26. 2. Dixon-Woods et al.: Implement Sci 2013, 8:702. S126 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P352 Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) M Albert1, DR Willamson1, J Muscedere2, F Lauzier3, C Rostein4, S Kanji5, X Jiang2, M Hall6, DK Heyland2 1Hopital du Sacré-Coeur de Montréal, Canada; 2Kingston General Hospital, Kingston, Canada; 3Centre hospitalier affi lié universitaire de Québec, Canada; 4Toronto General Hospital, Toronto, Canada; 5The Ottawa Hospital Research Institute, Ottawa, Canada; 6Nationwide Children’s Hospital, Colombus, OH, USA Critical Care 2014, 18(Suppl 1):P352 (doi: 10.1186/cc13542) References 1. Mahgoub S, et al.: Infection Control Hosp Epidemiol 2002, 23:477-479. 2. Hsueh PR, et al.: Emerg Infect Dis 2002, 8:827-832. Retrospective analysis of respiratory isolates post out-of-hospital cardiac arrest to establish choices in empirical antibiotic cover Introduction We analysed positive respiratory isolates for antibiotic resistance in our out-of-hospital cardiac arrest (OOHCA) population to establish adequacy of current empirical regimes. Pneumonia commonly complicates OOHCA, with studies suggesting a prevalence up to 48% [1]. Results MDR AB-infected patients (n = 45, 43%) had greater number of organ dysfunctions, and higher mean lengths of hospital stay (8.4 days vs. 6.1 days) and ICU stay (5.3 days vs. 0.8 days) after the index day than MDR non-AB infection (n = 59, 57%) (Table 1). In-hospital mortality rates for patients with MDR AB infections (42%) were higher than for MDR non-AB infection (10%). F critical = 3.934253. Methods Patients admitted to the ICU between May 2007 and September 2013 who underwent therapeutic hypothermia post OOHCA were included in this study. Demographics and antibiotic resistance were collected from an electronic database. Conclusion We demonstrated in patients infected with MDR strains of any organism that patients infected with Acinetobacter have greater number of organ dysfunction, longer lengths of stay in both the hospital and the ICU as well as increased mortality than patients infected with non-Acinetobacter infection when we controlled for severity of illness. Results A total of 160 patients were admitted to the ICU post OOHCA. In total, 37.5% (60/160) had no respiratory sample sent within 72 hours of admission and were excluded. Forty per cent (40/100) grew a clinically important isolate. Gram-negative bacteria (GNB) were most frequently isolated (42.5%, 17/40) followed by Gram-positive bacteria (32.5%, 13/40) and mixed bacterial growth (25%, 10/40). S. aureus (n = 14) isolates were often resistant to penicillin (92.8%, 13/14 isolates tested) but maintained macrolide (erythromycin 92.8%, 13/14), clindamycin (92.8%, 13/14) and vancomycin (100%, 13/13) sensitivity, if tested. S. pneumoniae isolates (n = 8) maintained penicillin (87.5%, 7/8), levofl oxacin (100%, 6/6), erythromycin (87.5%, 7/8) and vancomycin (100%, 6/6) sensitivity. The most commonly isolated GNB, H. infl uenzae, maintained high-level sensitivity to amoxicillin (81.8%, 9/11) and co- amoxiclav (90.9%, 10/11). Other isolated GNB, however, demonstrated variable resistance to co-amoxiclav (69.2%, 9/13). Isolates were 100% sensitive to pipericillin–tazobactam (17/17), amikacin (12/12) and meropenem (16/16). The British Thoracic Society suggests co-amoxiclav and clarithromycin for fi rst-line treatment of severe community- acquired pneumonia [2]. Based on this, 77.5% (31/40) of our patients would have received adequate cover. If pipericillin–tazobactam replaced co-amoxiclav, 95% (38/40) of our patients would have been treated with appropriate antibiotics. 1. Delisle MS, Williamson DR, Perreault MM, Albert M, Jiang X, Heyland DK: The clinical signifi cance of candida colonization of respiratory tract secretions in critically ill patients. J Crit Care 2008, 23:11-17. References 1. Nielsen N, et al.: Adverse events and their relation to mortality in out-of- hospital cardiac arrest patients treated with therapeutic hypothermia. Crit Care Med 2011, 39:57-64. 1. Nielsen N, et al.: Adverse events and their relation to mortality in out-of- hospital cardiac arrest patients treated with therapeutic hypothermia. Crit Care Med 2011, 39:57-64. hospital cardiac arrest patients treated with therapeutic hypothermia. Crit Care Med 2011, 39:57-64. p Methods We conducted a double-blind, placebo-controlled, multi- center, pilot randomized trial of antifungal therapy in critically ill patients with a clinical suspicion of ventilator-associated pneumonia with positive airway secretion specimens for Candida spp. We also included an observational group without Candida spp. in their airway secretions. We measured the recruitment rate, infl ammatory profi les over time and clinical outcomes. 2. Lim WS, et al.: BTS guidelines for the management of community acquired pneumonia in adults: update 2009. Thorax 2009, 64:1-55. 2. Lim WS, et al.: BTS guidelines for the management of community acquired pneumonia in adults: update 2009. Thorax 2009, 64:1-55. Pharmacokinetics of antituberculosis drugs in critically ill patients with tuberculosis and acute respiratory failure CH H d C d GL L G ti Results We recruited 60 patients into the randomized trial; 29 patients into the observational study. Recruitment was halted before the end of the study because of diffi culty in recruiting patients. Markers of infl ammation and all clinical outcomes were comparable between placebo and antifungal treatment groups at baseline and overtime. At baseline, TNFα levels were higher in the VAP with Candida compared with the observational group (mean ± SD) (21.8 ± 23.1 vs. 12.4 ± 9.3 pg/ ml, P = 0.02) and these patients had lower response to the LPS stimulation test (854.8 ± 855.2 vs. 1,559.4 ± 1,290.6 pg/ml, P = 0.01).fi Instituo Nacional de Enfermedades Respiratorias, Mexico City, DF, Mexico Critical Care 2014, 18(Suppl 1):P354 (doi: 10.1186/cc13544) Introduction The purpose of this study was to describe the pharmacokinetics of antituberculosis drugs, isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA), in eight critically ill patients with tuberculosis and acute respiratory failure. Methods We analyzed plasma concentrations of RIF, INH and PZA. Blood samples were obtained at steady state. Plasma concentrations were determined with HPLC. A population pharmacokinetic approach using a nonlinear mixed-eff ect model was implemented [1]. Interindividual variability in PK parameters was ascribed to an exponential model according to the equation: θj = θp × exp(nj), where θj is the estimate for a pharmacokinetic parameter in the jth patient, θp is the typical population PK parameter value (ka, CL/F, V/F), and n is a random variable from a normal distribution with zero mean and variance ω2. Residual variability was estimated using additive and additive-proportional error models; Cij = Cj + εadd and Cij = Cj(1 + εp) + εadd, where Cij and Cj are observed–predicted concentrations for the pg Conclusion Given the diffi culty recruiting patients and the lack of signal in clinical or infl ammatory outcomes, this study does not provide evidence to support a larger trial examining the effi cacy of empiric antifungal treatment in patients with a clinical suspicion of ventilator- associated pneumonia and Candida in the endotracheal secretions. The presence of Candida in the lung may be associated with persistent infl ammation and immunosuppression. P354 Pharmacokinetics of antituberculosis drugs in critically ill patients with tuberculosis and acute respiratory failure CH Hernandez-Cardenas, GL Lugo-Goytia Instituo Nacional de Enfermedades Respiratorias, Mexico City, DF, Mexico Critical Care 2014, 18(Suppl 1):P354 (doi: 10.1186/cc13544) Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) M Albert1, DR Willamson1, J Muscedere2, F Lauzier3, C Rostein4, S Kanji5, X Jiang2, M Hall6, DK Heyland2 1Hopital du Sacré-Coeur de Montréal, Canada; 2Kingston General Hospital, Kingston, Canada; 3Centre hospitalier affi lié universitaire de Québec, Canada; 4Toronto General Hospital, Toronto, Canada; 5The Ottawa Hospital Research Institute, Ottawa, Canada; 6Nationwide Children’s Hospital, Colombus, OH, USA Critical Care 2014, 18(Suppl 1):P352 (doi: 10.1186/cc13542) X Jiang2, M Hall6, DK Heyland2 1Hopital du Sacré-Coeur de Montréal, Canada; 2Kingston General Hospital, Kingston, Canada; 3Centre hospitalier affi lié universitaire de Québec, Canada; 4Toronto General Hospital, Toronto, Canada; 5The Ottawa Hospital Research Institute, Ottawa, Canada; 6Nationwide Children’s Hospital, Colombus, OH, USA Critical Care 2014, 18(Suppl 1):P352 (doi: 10.1186/cc13542) Introduction Candida spp. are frequently recovered from endotracheal secretions in critically ill patients suspected of having ventilator- associated pneumonia. Observational studies reported an association with worse clinical outcomes [1] but the eff ect of antifungal therapy in these patients remains unclear. We designed this pilot study to assess the feasibility of a larger trial and to evaluate infl ammatory profi les and clinical outcomes in these patients. Introduction Candida spp. are frequently recovered from endotracheal secretions in critically ill patients suspected of having ventilator- associated pneumonia. Observational studies reported an association with worse clinical outcomes [1] but the eff ect of antifungal therapy in these patients remains unclear. We designed this pilot study to assess the feasibility of a larger trial and to evaluate infl ammatory profi les and clinical outcomes in these patients. Conclusion Respiratory samples post OOHCA frequently grow potentially pathogenic bacteria but current antibiotic guidelines fail to provide adequate cover in this population. f Comparison of multidrug-resistant Acinetobacter and non-Acinetobacter infections in terms of outcome in critically ill patients Comparison of multidrug-resistant Acinetobacter and non-Acinetobacter infections in terms of outcome in critically ill patients g In contrast, recent data obtained after the implementation of the program of the infection control showed that the respiratory infections occurred in 29.3% of the patients, catheter-related urinary tract infections were detected in 32.8%, and surgical site infections were detected in 8.6% of the polytrauma patients. On the other hand, the incidence of the multiple-drug-resistant bacterial colonies increased signifi cantly up to 14.2% (P <0.001). Other types of HAI changed nonsignifi cantly. p A Madaan1, V Singh1, P Shastri2, C Sharma3 1Patel Cancer and Superspeciality Hospital, Jalandhar, India; 2Sir Gangaram Hospital, New Delhi, India; 4Lovely Professional University, Jalandhar, India Critical Care 2014, 18(Suppl 1):P351 (doi: 10.1186/cc13541) Introduction Acinetobacter infections have increased and gained attention because of the organism’s prolonged environmental survival and propensity to develop antimicrobial drug resistance [1,2]. We performed a retrospective, matched cohort investigation at a tertiary care hospital in a tier II city hospital of India to examine morbidity and mortality outcome of patients with multidrug-resistant (MDR) Acinetobacter (AB) infection compared with patients with MDR non- Acinetobacter (non-AB) infections. i y Conclusion In the era of the total antibacterial resistance, the education strategies and organization approaches (for example, infection control, antibiotic susceptibility, hand hygiene, and so forth) become more potent and eff ective than pharmacology innovations. Polytrauma patients, as one of the most severe categories suff ering from the HAI, demand a high level of compliance of the infection control approaches from the side of doctors as well as staff of and visitors to ICUs. Methods Patient records from the last 3 years from 2011 through 2013 were studied (n = 104) to examine outcomes of hospitalized patients in terms of number of organ dysfunctions, ICU length of stay, hospital length of stay and mortality in patients infected with MDR AB infection compared with the patients with MDR non-AB infection. 1. Nalapko Y, Peicheva O, Iegorov O, Nekrylov A: The dependence of infectious complications from the trauma severity. Intensive Care Med 2013, 39(Suppl. 2):P.0791. S127 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P351). Outcome evaluated MDR AB MDR non-AB P value F value Mean length of stay 8.488889 6.101695 0.029299 4.886684 after index day Mean ICU stay after 5.311111 0.864407 1.73×10–11 57.30083 index day P355 Eight-year study of the Staphylococcus epidermidis resistance profi le against glycopeptides, oxazolidinones and glycylcyclines in an ICU of a Greek tertiary hospital A Stylianakis, V Kaldis, D Argyris, D Chatzilia, V Kasouris, F Kalogeropoylos, S Kamariotis, K Daskalakis, S Stratoyli, I Alamanos 1General Hospital Kat-Eka, Kifi sia Athens, Greece Critical Care 2014, 18(Suppl 1):P355 (doi: 10.1186/cc13545) Methods The faecal vancomycin concentration was measured in eight patients who suff ered a Clostridium diffi cile infection and were treated with 4×500 mg vancomycin orally per day. In vitro, a (1:2) dilution series of vancomycin (range 6,250 to 0.4 μg/ml) was created and 1 ml vancomycin solution was then added to 1 ml standardized inoculum. One vancomycin-susceptible enterococcus isolate (VSE, MIC = 3 μg/ ml) and two VRE isolates (MIC = 16 μg/ml) were studied. After 1, 7 and 14 days incubation at 35°C, growth was defi ned as macroscopic visible turbidity. To test for surviving bacteria, all inocula were cultured to sheep blood agar plates, which were read after 24-hour incubation at 35°C. E-tests to measure MIC were performed on relevant samples. The in vitro experiment was performed twice. Introduction Staphylococcus epidermidis (SE) is the most often isolated species of coagulase-negative staphylococci, which are recognized as one of the main causes of ICU infections [1]. In this study we aimed to study the resistance profi le of SE clinical isolates against last-line antibiotics (vancomycin (VA), teicoplanin (TEC), linezolid (LZ) and daptomycin (DA)) for treating CNS infections, during an 8-year period. p p Results The faecal vancomycin concentration in patients treated orally with vancomycin was 8,000 μg/ml on average. VSE growth at day 14 was detected at up to 1.5 μg/ml vancomycin, whereas VRE growth was detected at up to 98 μg/ml. The MIC of these VRE species growing at 98 μg/ml vancomycin was increased (≥256 μg/ml). For both VSE and VRE, surviving bacteria were detected at very high concentrations of vancomycin (>98 μg/ml): the MIC of these survivors was not increased. Conclusion Oral treatment with vancomycin results in extremely high faecal concentrations. At these high concentrations, VRE bacteria are killed in vitro; however, a minority of the VRE is able to survive. Vancomycin thus seems unsuitable for eradication. However, high concentrations of vancomycin dramatically reduce the bacterial VRE load. P355 Therefore oral treatment with vancomycin may help to terminate VRE outbreaks: a dramatic reduction in bacterial load of the colonised patient will minimise the risk of patient-to-patient transmission. daptomycin (DA)) for treating CNS infections, during an 8 year period. Methods From January 2005 until December 2012 we examined 518 nonduplicated SE isolates recovered from blood cultures of 421 patients hospitalized in a surgical ICU of our hospital. Species identifi cation and susceptibility testing were performed using the automated VITEK II system (Biomerieux). Additionally we used the E-test method (Biomerieux, ABI-Biodisk) in order to confi rm some isolation resistances against TEC and LZ found by the VITEK II system and to estimate the MIC levels of DA and VA. Mueller–Hinton agar adjusted to contain physiologic levels of free calcium ions (50 μg/ml) was used when testing DA susceptibility. Isolates with MIC >4 mg/l were considered resistant to TEC and LZ and those with MIC <1 mg/l and MIC <4 mg/l susceptible to DA and VA, respectively. Results The percentage resistance rate of the examined SE isolates is shown in Table 1. Methicillin resistance was observed with an overall prevalence of approximately 84.6%. All of the resistant isolates to TEC and LZ were also resistant to methicillin. The MIC values of VA were lower than 2 mg/l (Table 1). P356 P356 Vancomycin-resistant enterococci: eradication using vancomycin? J Van Prehn1, AM Stemerding2, HK Van Saene3, PE Spronk2 1VU University Medical Center, Amsterdam, the Netherlands; 2Gelre Hospitals, Apeldoorn, the Netherlands; 3University of Liverpool, UK Critical Care 2014, 18(Suppl 1):P356 (doi: 10.1186/cc13546) P356 Vancomycin-resistant enterococci: eradication using vancomycin? J Van Prehn1, AM Stemerding2, HK Van Saene3, PE Spronk2 1VU University Medical Center, Amsterdam, the Netherlands; 2Gelre Hospitals, Apeldoorn, the Netherlands; 3University of Liverpool, UK Critical Care 2014, 18(Suppl 1):P356 (doi: 10.1186/cc13546) P356 Vancomycin-resistant enterococci: eradication using vancomycin? J Van Prehn1, AM Stemerding2, HK Van Saene3, PE Spronk2 1VU University Medical Center, Amsterdam, the Netherlands; 2Gelre Hospitals, Apeldoorn, the Netherlands; 3University of Liverpool, UK Critical Care 2014, 18(Suppl 1):P356 (doi: 10.1186/cc13546) Results Cmax of PZA was above the recommended concentration (>20 mg/l). For RIF the Cmax was below the recommended level (>8 mg/l), and the Cmax of INH was below the recommended levels (>3 mg/l). See Table 1. g Conclusion Large interindividual pharmacokinetic variability and concentrations below the recommended levels for RIF and ISO. We need to monitor drugs and to re-evaluate the doses. Reference Introduction Patient-to-patient transmission enables vancomycin- resistant enterococci (VRE) outbreaks. Outbreak management is expensive and time consuming, and therefore the possibility of VRE eradication is desirable. Since vancomycin is scarcely absorbed in the gastrointestinal tract, treatment with vancomycin per os may result in very high gastrointestinal concentrations (many times the minimum inhibiting concentration (MIC)). The purpose of this study is to measure in vivo gastrointestinal concentrations of vancomycin in patients that are treated with a standard dose orally, and to investigate in vitro whether vancomycin is able to kill VRE at concentrations up to 2,000 times the MIC. Introduction Patient-to-patient transmission enables vancomycin- resistant enterococci (VRE) outbreaks. Outbreak management is expensive and time consuming, and therefore the possibility of VRE eradication is desirable. Since vancomycin is scarcely absorbed in the gastrointestinal tract, treatment with vancomycin per os may result in very high gastrointestinal concentrations (many times the minimum inhibiting concentration (MIC)). The purpose of this study is to measure in vivo gastrointestinal concentrations of vancomycin in patients that are treated with a standard dose orally, and to investigate in vitro whether vancomycin is able to kill VRE at concentrations up to 2,000 times the MIC. 1. Immanuel CP: Indian J Med Res 2003, 118:109-114. l Reference 1. Delisle MS, Williamson DR, Perreault MM, Albert M, Jiang X, Heyland DK: The clinical signifi cance of candida colonization of respiratory tract secretions in critically ill patients. J Crit Care 2008, 23:11-17. S128 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P354). Mean population parameters of antituberculosis drugs Parameter RIF INH PZA ka 0.459 3.58 1.41 CL/F 6.69 2.74 1.56 V/F 105 42.3 33.4 ωka 0.645 38.7 0.98 ωCL/F 0.391 65.6 0.23 ωV/F 0.469 46.8 0.38 ε 0.441 0.235 0.089 Table 1 (abstract P354). Mean population parameters of antituberculosis drugs Table 1 (abstract P354). Mean population parameters of antituberculosis drugs Reference 1. Zieburhr W, et al.: Nosocomial infections by Staphylococcus epidermidis. Int J Antimicrob Agents 2006, 28(Suppl 1):S14-S20. Table 1 (abstract P355). 2005 2006 2007 2008 2009 2010 2011 2012 (n = 77) (n = 76) (n = 79) (n = 89) (n = 92) (n = 93) (n = 46) (n = 59) VA 0 0 0 0 0 0 0 0 TEC 1.3 2.6 0 0 0 1.1 0 0 LZ 0 0 1.4 1.2 6.8 12.6 23.1 27.1 DA Not Not Not 0 0 0 0 0 tested tested tested Table 1 (abstract P355). jth patient at time i, respectively, and ε is the error, a random variable with a normal distribution with zero mean and variance σ2. Bayesian estimates were obtained and the pharmacokinetic parameters Cmax, Tmax and AUC0–24 hours were calculated. P357 We aimed to determine the impact of this strategy on antibiotic resistance patterns and patient d i h i il i d d i 2000 [1] outcomes compared with a similar exercise we conducted in 2000 [1]. Methods We conducted a retrospective study of all patients with bacteraemia or fungaemia (community-acquired, hospital-acquired, and ICU-acquired) treated in our university hospital ICU over a 6-month period (December 2012 to May 2013). We compared this against data from blood culture-positive patients admitted between February and July 2000. Information was collected on bacteraemia episodes, causative pathogens, antimicrobial resistance patterns, antibiotic use and duration, and patient outcomes. Notably, our ICU admits many immunosuppressed patients (for example, haemoncology). Results Table 1 presents demographics and incidence of bacteraemia. Antimicrobial resistance remained low in the 2013 cohort with few multi-resistant Gram-negative organisms, few fungaemia episodes and a marked decrease in methicillin-resistant Staphylococcus aureus (MRSA) (Figure 1). The number of relapses and breakthrough bacteraemias remained low. Conclusion A strategy of short-course antibiotic monotherapy is associated with low breakthrough and relapse rates and a low rate of antibiotic resistance. P357 Audit of bacteraemia management in a university hospital ICU V De Santis, MG Gresoiu, A Peter, R Wilson, M Singer University College London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P357 (doi: 10.1186/cc13547) Conclusion The examined SE isolates present a scattered resistance to TEC and they show a remarkable continuing increase of resistance to LZ. These fi ndings enforced the necessity to take the appropriate measures in the ICU environment and during the clinical practice to limit the dissemination and the amplifi cation of these resistances. DA and VA possess an excellent in vitro activity against SE isolates and they could be very good alternative solutions for treating ICU infections caused by this species. Introduction The optimal duration of antibiotic treatment in critically ill patients remains a subject of debate. In our multidisciplinary ICU, a short course of antibiotic monotherapy (5 to 7 days) is generally used as S129 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P357). Microorganisms isolated in each bacteraemia group. Left panel, 2000; right panel, 2013. Top panel, community-acquired bacteraemia; Middle panel, hospital-acquired bacteraemia; lower panel, ICU-acquired bacteraemia. Shaded areas represent numbers of methicillin-resistant strains for S. aureus and coagulase-negative staphylococci; vancomycin-resistant strains for Enterococcus spp.; multidrug-resistant strains for Gram-negative pathogens; and fl uconazole-resistant strains for Candida spp. Figure 1 (abstract P357). Microorganisms isolated in each bacteraemia group. Left panel, 2000; right panel, 2013. Top panel, community-acquired bacteraemia; Middle panel, hospital-acquired bacteraemia; lower panel, ICU-acquired bacteraemia. Shaded areas represent numbers of methicillin-resistant strains for S. aureus and coagulase-negative staphylococci; vancomycin-resistant strains for Enterococcus spp.; multidrug-resistant strains for Gram-negative pathogens; and fl uconazole-resistant strains for Candida spp. Table 1 (abstract P357). Demographics and incidence of bacteraemia Variable 2000 cohort 2013 cohort Total ICU population 713 1,318 Patients with bacteraemia 91 87 Episodes of bacteraemia 102 113 Community-acquired bacteraemia 13 37 Hospital-acquired bacteraemia 28 39 ICU-acquired bacteraemia 60 37 Hospital mortality 45% 32% Monotherapy treatment (%); duration median (IQR) Community-acquired bacteraemia 57%; 6 (5 to 6) 65% 5 (3 to 5) Hospital-acquired bacteraemia 78%; 6 (5 to 8) 63%; 5 (4 to 7) ICU-acquired bacteraemia 80%; 5 (5 to 7) 62%; 4 (3 to 6) Table 1 (abstract P357). Demographics and incidence of bacteraemia a strategy to treat bacteraemia, unless specifi cally indicated otherwise (for example, endocarditis, osteomyelitis). P359 Safety and effi cacy of amphotericin B inhalation for Candida spp. in the respiratory tract of critically ill patients Introduction Nosocomial pneumonia (NP) is frequently caused by multiresistant Gram-negative bacteria. The inhaled antibiotics provide us with a new treatment modality for NP in sepsis. The aim of this study was to estimate the effi cacy of inhaled tobramycin (IT) as an adjunct to systemic antibiotics in the treatment of NP in sepsis. P Van der Geest, E Dieters, B Rijnders, J Groeneveld P Van der Geest, E Dieters, B Rijnders, J Groeneveld Erasmus Medical Center, Rotterdam, the Netherlands , , j , Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P359 (doi: 10.1186/cc13549) y p Methods Fifty ICU ventilated septic patients with NP were enrolled in the study (all male, 55.3 ± 7.3 years old; primary reason for ICU stay – intraabdominal infections (78%), mediastinitis (13%), other (9%)). Diagnosis of NP was made according to the standard clinical and CPIS criteria. Associations of multiresistant Gram-negative bacteria were detected in bronchoalveolar lavage (BAL) of all patients. Seventy- two percent of bacteria were sensitive to tobramycin. Patients were randomized into two groups: IT (group 1, n = 25), addition of IT to systemic antibiotics (carbapenems, aminoglycosides, protected penicillins); and no IT (group 2, n = 25), shift of systemic antibiotics according to sensitivity. Groups were comparable in APACHE II and CPIS scores. IT (Bramitob) was administered 300 mg twice daily via nebulizer. The data were statistically analyzed by STATISTICA 7.0 (M, σ, Newman– Keuls test; P <0.05). Introduction Candida spp. are increasingly isolated in the critically ill, but the clinical signifi cance hereof is hard to establish [1]. Candida spp. colonization has been suggested as a risk factor for ventilator-associated pneumonia (VAP) [2]. The effi cacy and safety of inhalational amphotericin B (AB) is unknown [3]. The hypothesis was that inhalational AB deoxycholate is a safe and eff ective treatment for Candida spp. colonization of the respiratory tract and thereby prevents VAP and prolonged need for mechanical ventilation. p g Methods All patients admitted to the ICU from December 2010 to 2011 with positive Candida spp. cultures of the respiratory tract and requiring mechanical ventilation >48 hours were included. AB treatment was decided by attending intensivists. The colonization index was calculated to determine the eff ect of AB. The clinical pulmonary infection score (CPIS) and the lung injury score (LIS) were calculated to determine pulmonary eff ects of AB. Reference Corona A, et al.: J Antimicrob Chemother 2004, 54:809-881. S130 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P359). Duration of mechanical ventilation grouped by amphotericin B. P358 P358 Sepsis: impact of timely and appropriate empirical antibiotic therapy on mortality L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P358 (doi: 10.1186/cc13548) P360 Inhaled tobramycin for the treatment of nosocomial pneumonia in sepsis 1. Dellinger RP, et al.: Crit Care Med 2008, 36:296-327. 2. Levy MM, et al.: Intensive Care Med 2010, 36:222-231. 3. Kumar A, et al.: Crit Care Med 2006, 34:1589-1596. 4. Kumar A, et al.: Chest 2009, 136:1237-1248. 1. Dellinger RP, et al.: Crit Care Med 2008, 36:296-327. 2. Levy MM, et al.: Intensive Care Med 2010, 36:222-231. 3. Kumar A, et al.: Crit Care Med 2006, 34:1589-1596. 4. Kumar A, et al.: Chest 2009, 136:1237-1248. Critical Care 2014, 18(Suppl 1):P360 (doi: 10.1186/cc13550 P358 Sepsis: impact of timely and appropriate empirical antibiotic therapy on mortality Introduction The administration of timely and appropriate antibiotic therapy is a well-known prognostic factor among severe sepsis patients [1-4]. The purpose of this study is to describe the magnitude of the impact of early and appropriate empirical antibiotic therapy on hospital mortality. y Methods A retrospective cohort study including all adult patients with sepsis admitted to the emergency room of a tertiary care, university hospital between 1 July 2011 and 30 June 2012. p y Results A total of 1,219 patients were admitted to the emergency room during the study period, of which 162 (13%) had severe sepsis. Forty (25%) patients had withheld therapeutic decisions and were excluded from the current analysis; 20 additional patients transferred from other acute healthcare facilities were excluded due to missing or inaccurate data, leaving 102 patients to be included with a hospital mortality rate of 45%. The median time to antibiotics administration was 36 minutes (IQR 0 to 174), 59 (58%) patients had antibiotic administered within the fi rst hour after sepsis recognition; 60 (59%) had positive microbiology, 74% with appropriate empiric antibiotic therapy. An association was found for hospital mortality with: heart failure (OR = 4.297; P = 0.037), decreased functional status (Karnofsky performance status <70%) (OR = 2.368; P = 0.034) and SOFA score (OR per point = 1.415; P <0.001). Two multivariate models with hospital mortality as the dependent variable were built using alternative severity scores: one with SAPS II that was retained in the fi nal model (adjusted OR = 1.068, 95% CI = 1.020 to 1.119) along with heart failure (adjusted OR = 5.859, 95% CI = 0.996 to 34.474); and another with SOFA score that was also retained in the fi nal model (adjusted OR = 1.659, 95% CI = 1.227 to 2.242) along with heart failure (adjusted OR = 12.636, 95% CI = 1.423 to 112.229). Figure 1 (abstract P359). Duration of mechanical ventilation grouped by amphotericin B. associated with a higher CPIS and LIS, even in those with similar degree and duration of colonization and thus probably Candida spp. load at baseline (P <0.001) (Figure 1). There was no diff erence in occurrence of VAP or mortality.f Conclusion AB deoxycholate treatment is eff ective for the treatment of Candida spp. colonization of the respiratory tract in critically ill patients. However, patients are mechanically ventilated for longer. References 1. P358 Sepsis: impact of timely and appropriate empirical antibiotic therapy on mortality El-Ebiary et al.: Am J Respir Crit Care Med 1997, 156:583-590. 2. Azoulay E, et al.: Chest 2006, 129:110-117. 3. Knechtel SA, et al.: Expert Opin Drug Saf 2007, 6:523-532. Conclusion Contrarily to what has been described previously, early and appropriate empirical antibiotic therapy was not associated with better prognosis. The most probable explanation is the higher compliance found with the current recommendations, reinforcing the need for period audits and feedback to the team. 3. Knechtel SA, et al.: Expert Opin Drug Saf 2007, 6:523-532. 3. Knechtel SA, et al.: Expert Opin Drug Saf 2007, 6:523-532. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 of resources and false results is well established and unnecessary cultures can cause patient harm. was reliable in 80% of the patients of group 1 (P <0.02). It is noteworthy that 21% of group 1 patients were in vitro resistant to tobramycin, but it was clinically eff ective, probably due to a local superconcentration. Treatment with IT was associated with an increase of sensitivity of microbes to antibiotics they were prior resistant to (32% of patients). This is probably due to IT eff ects on biolayers. De-escalation of antibiotic therapy was possible in group 1 by day 5 in 42% of patients. The treatment with IT made it possible to wean 40% of patients by day 5.3 ± 1.8 after treatment cessation (vs. 35% and 11.2 ± 1.3 days in group 2, P = 0.02). Hearing loss and tinnitus was detected only in three patients of group 1. There were no cases of bronchospasm. The mortality was 12% (n = 3) in group 1 and 16% (n = 4) in group 2 (P >0.05), and was not related to a progression of NP [1]. Methods All ICU admissions in 2011 were listed retrospectively. Notes were reviewed for those with blood cultures taken during, or within 24 hours prior to or following, ICU admission. Data collected for positive blood cultures included organism, antimicrobial sensitivity, culture timing with respect to ICU admission and antibiotic therapy before and after positive blood culture. Qualitative decisions were made regarding clinical utility of each positive blood culture. Results were deemed useful if management changed – starting, changing or stopping antibiotics or altering antibiotic duration. Confi rmatory results or those not altering treatment were not deemed useful. Statistical analysis was performed using the chi-squared test. p g q Results During 2011 and 2012, there were 450 ICU admissions. In total, 698 blood cultures were taken during, or within 24 hours prior to or following, ICU admission. A total of 135 blood cultures were taken in the 24 hours prior to ICU admission. Of these, 26 grew signifi cant organisms (19.3%). Nine of these cultures were deemed clinically useful (6.7%). A total of 542 blood cultures were taken during ICU admission. Thirty-three of these yielded signifi cant results (6.1%) but only nine (1.7%) were deemed clinically useful. Sternal wound infections in cardiac surgery: eff ects of vancomycin prophylaxis y F Ampatzidou, M Sileli, C Koutsogiannidis, A Vlachou, A Baddour, G Drossos F Ampatzidou, M Sileli, C Koutsogiannidis, A Vlachou, A Baddour, G Drossos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P361 (doi: 10.1186/cc13551) Introduction Appropriate antibiotic prophylaxis plays a crucial role in preventing sternal wound infection after cardiac surgery [1]. In institutions with high prevalence of methicillin-resistant Staphylococci species, vancomycin prophylaxis is recommended either as a monotherapy or as an adjuvant agent [2]. In our study, we assessed sternal wound infection rates before and after the introduction of a vancomycin prophylaxis protocol. y y Methods Twenty-six of a total 227 consecutive cardiac surgical patients, between July and December 2012, developed sternal wound infection (Group A). All of the patients received a standard empirical antibiotic prophylaxis. From January to July 2013, 308 patients underwent cardiac surgery (Group B). In this group, we applied a more restricted antibiotic protocol, considering the resistance patterns and the results of microbiological tests of group A. We also evaluated the results of MIC susceptibility testing of fi ve antibiotics: oxacillin, linezolide, daptomycin, teicoplanin and vancomycin. In the new protocol the fi rst vancomycin dose was given 1 hour before sternal incision followed by three additional doses (48 hours duration). 1. Coburn B, et al.: Does this adult patient with suspected bacteraemia require blood cultures? JAMA 2012, 308:502-510. Employing quality improvement methodology in sepsis: an electronic sepsis order set further improves compliance with the Surviving Sepsis Campaign 3-hour bundle S Rossi, A Shields, N Gauge, M Kinirons, A Hopper, G Glover, R Beale Guys and St Thomas NHS Foundation Trust, London, UK Critical Care 2014, 18(Suppl 1):P363 (doi: 10.1186/cc13553) Employing quality improvement methodology in sepsis: an electronic sepsis order set further improves compliance with the Surviving Sepsis Campaign 3-hour bundle S Rossi, A Shields, N Gauge, M Kinirons, A Hopper, G Glover, R Beale Guys and St Thomas NHS Foundation Trust, London, UK Critical Care 2014, 18(Suppl 1):P363 (doi: 10.1186/cc13553) Results In group A, 26 patients (11.45%) developed sternal wound infection. Twenty out of 26 patients had staphylococcal infections characterized by high prevalence of oxacillin resistance while six patients had Gram-negative infections. In the vancomycin prophylaxis group (group B) we observed a signifi cant reduction in the incidence of sternal wound infection rate (P <0.01). Among 308 patients, six patients (1.94%) developed sternal wound infections, two of them caused by coagulase-negative oxacillin-resistant staphylococci while in four patients Gram-negative microorganisms were cultured. Mortality in infected patients was 0% in both groups. Introduction The Surviving Sepsis Campaign (SSC) has developed guidelines to promote evidence-based management for patients with severe sepsis [1]. Improvements in bundle compliance have been demonstrated over time, but compliance remains below 40%. Sepsis has been studied in acute and critical care environments, but little research has focused on the management in level 1 wards. An initial audit of patients with sepsis who were referred to the GSTT critical care outreach team revealed very low overall compliance to the SSC 3-hour bundle. A novel quality improvement campaign was instituted with the aim of improving bundle compliance. g Conclusion Appropriate antibiotic prophylaxis in cardiac surgery patients is of paramount importance in preventing sternal wound infections, resulting in dramatic reduction of postoperative morbidity and in signifi cant economic benefi ts. i References References 1. Kappeler R, et al.: Antimicrob Chemother 2012, 67:521-522. 2. Garey KW, et al.: Antimicrob Agents Chemother 2008, 52:446-451. References 1. Kappeler R, et al.: Antimicrob Chemother 2012, 67:521-522. 2. Garey KW, et al.: Antimicrob Agents Chemother 2008, 52:446-451. References 1. Kappeler R, et al.: Antimicrob Chemother 2012, 67:521-522. 2. Garey KW, et al.: Antimicrob Agents Chemother 2008, 52:446-451. Methods A retrospective cohort study in a university hospital was performed. Patients on level 1 wards with severe sepsis registered in the adult critical care response team (CCRT) database in November 2012 were identifi ed (Cohort A). Physiological observation, track and trigger scores, compliance with the 3-hour bundle elements (measured lactate, blood cultures before antibiotics, fl uid challenge, early antibiotics), antimicrobial stewardship and 28-day mortality were recorded. Following this, a quality improvement project was initiated: central to this was an electronic ‘SEPSIS’ order set, containing appropriate investigations and a step-by-step management guide for use on level 1 wards. A ‘viral’ print and social media campaign were also undertaken. Compliance to the SSC early care bundle was re-examined Reference 1. Moroz VV, et al.: Gen Reanimatol 2012, VIII:5-10. P359 Safety and effi cacy of amphotericin B inhalation for Candida spp. in the respiratory tract of critically ill patients Results Administration of IT as an adjunct to systemic antibiotics was associated with a decrease of systemic infl ammation and acute respiratory insuffi ciency signs 2.3 ± 1.2 days after the treatment onset (vs. 6.3 ± 1.5 days in group 2, P = 0.03). The decrease of microbial titer to 103 to 104 CFU/ml was detected in both groups by days 5 to 7, but it f Results Fifty-fi ve of 181 patients had been treated with AB. The AB decreased indicators of Candida spp. load but increased the duration of mechanical ventilation as compared with nontreated patients, S131 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 A total of 102 cultures were taken in the fi rst 24 hours of the ICU, of which 17 were positive (16.7%) and six were useful (5.9%). Fifty-three were taken over the next 24 hours, of which two were positive and two were useful (3.8%). Forty-four cultures were taken over the following 24 hours, of which two were positive (4.5%) and one was useful (2.3%). A total of 343 cultures were taken subsequent to this in the ICU, of which 12 were positive (3.5%) and three were deemed useful (0.9%). Of 22 blood cultures taken in the 24 hours post ICU, one was positive but deemed useful (4.5% yield). Conclusion These results demonstrate overall low clinical utility of blood cultures but specifi cally that utility of ICU cultures is signifi cantly lower than pre ICU (1.7% vs. 6.7%; P = 0.0001). The yield of positive blood cultures and their clinical utility also decrease during ICU stay. This may refl ect appropriate empirical antibiotics and lower bacteraemia burden in later illness. Given the low clinical yield and a lack of established sensitive or specifi c triggers [1], we suggest further work in an ICU setting to maximise utility whilst minimising harm. Reference Conclusion Administration of IT as an adjunct to systemic antibiotics is effi cient in treatment of NP caused by multiresistant Gram-negative bacteria in sepsis. Reference Reference 1. Kappeler R, et al.: Antimicrob Chemother 2012, 67:521-522. 2. Garey KW, et al.: Antimicrob Agents Chemother 2008, 52:446-451. Early detection of postoperative acute kidney injury by Doppler renal resistive index in major lung and cardiac operations M Arslantas, I Cinel, A Kararmaz Marmara University Pendik Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P365 (doi: 10.1186/cc13555) Early detection of postoperative acute kidney injury by Doppler renal resistive index in major lung and cardiac operations M Arslantas, I Cinel, A Kararmaz Marmara University Pendik Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P365 (doi: 10.1186/cc13555) Results The mean age of all patients studied (n = 79) was 66.5 years. Fifty-three per cent of the patients were male. Thirty-one per cent were in septic shock at the time of sepsis identifi cation. Overall SSC bundle compliance was 6.60% (Cohort A), 24% (Cohort B) and 45.5% (Cohort C). Improvements in other bundle parameters were also seen, including blood culture (54%, 72%, 91%), antibiotic administration (50%, 69%,76%) and fl uid administration in septic shock (50%, 42%, 75%) in Cohort A, Cohort B and Cohort C respectively. Introduction Lung and cardiac operations cause signifi cant changes in the fl uid balance, and thus have a high incidence of development of postoperative acute kidney injury (AKI). The renal resistive index (RRI) calculated by the pattern of the renal artery fl ow is an indicator of renal artery fl ow. In this research work, we aimed to evaluate the effi ciency of the RRI on the early prediction of postoperative kidney injury in major lung and cardiac operations. p y Conclusion Baseline compliance with the SSC 3-hour bundle on level 1 wards was very low. An electronic sepsis order set was associated with marked improvement. Novel quality improvement methodology may be important to achieve optimal compliance with evidence-based guidelines and an electronic sepsis order set is recommended. Reference g p Methods Twenty-two patients who have undergone lung or cardiac surgery were included in the study. After the kidneys were localized by ultrasonography, the best regions of blood fl ow were detected using color Doppler and then the arterial waveforms of these regions were obtained and optimized by Doppler. The measurements taken from three diff erent regions were averaged. The RRI was calculated at the preoperative and postoperative fi rst and 24th hours respectively.i 1. Dellinger RP, et al.: Crit Care Med 2013, 41:580-637. Results A signifi cant correlation was established between RRI and postoperative creatinine levels (P <0.01). RRI values reached their highest point at the postoperative fi rst day whereas the creatinine levels reached their highest level at the postoperative third day. P365 in two cohorts of patients in July 2013; patients that were referred to the CCRT as before (Cohort B) and also patients who had the electronic order set activated (Cohort C). Early detection of postoperative acute kidney injury by Doppler renal resistive index in major lung and cardiac operations M Arslantas, I Cinel, A Kararmaz Marmara University Pendik Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P365 (doi: 10.1186/cc13555) Although there was no correlation between postoperative creatinine level and duration of staying in hospital, a signifi cant relationship was detected between duration of staying in ICU and the creatinine levels (P <0.01). When the cases were divided into two groups as RRI is less (n = 13) and larger (n = 9) than 0.7, signifi cant diff erences were present with regard to age and creatinine levels. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References Results Our literature search yielded 316 potential papers, of which 57 were included (20 papers on AHF, 15 ACS and 22 CS). A risk of bias analysis showed a low risk for selection bias in 55% of the studies and prospective data collection in 45%. AKIRIFLE was used in 33 studies (RIFLE in 22, AKIN in 14, KDIGO in four), AKIWRF, with six variants, in 24 studies and use of RRT (AKIRRT) in 20 studies. The incidence of AKI in CRS-1 patients defi ned by AKIRIFLE and AKIWRF was similar (22.5%, respectively 22.4%, P = 0.401), and greater than AKIRRT (2.6%, both P <0.001). AKIRIFLE occurred more frequently in AHF patients compared with ACS and CS patients (55.0% vs. 14.9% vs. 19.3%; P = 0.009 respectively P = 0.001, P = NS for ACS vs. CS). This was similar when defi ned by AKIWRF. AKIRRT was evenly distributed among CRS- 1 subtypes (AHF 4.3%, ACS, 1.7%, and CS 3.1%, P = 0.611). Despite predominant low severity of AKIRIFLE (stage 1: 16.9%, stage 2: 3.7%, and stage 3: 3.6%), AKIRIFLE was associated with increased mortality (RR = 5.4), LOSICU (MD 1.7 days), and LOShosp (MD 4.4 days), and increasing AKIRIFLE severity was associated with increase in these three outcomes in all CRS-1 patients as well as in the three subgroups. The impact of AKIRIFLE on mortality was greatest in CS patients (AHF RR = 2.8, ACS RR = 3.5, and CS RR = 9.1). Not surprisingly, AKIWRF had similar impact on outcomes, but AKIRRT had greater impact compared with AKIRIFLE (mortality RR = 9.16, LOSICU MD = 10.6 days, and LOShosp, MD = 20.2 days). P364 P364 Acute kidney injury in cardiorenal syndrome type 1: a meta-analysis W Vandenberghe, S Gevaert, H Peperstraete, I Herck, J Decruyenaere, E Hoste University Hospital Ghent, Belgium Critical Care 2014, 18(Suppl 1):P364 (doi: 10.1186/cc13554) Retrospective analysis of the clinical utility of blood cultures taken surrounding intensive care admission V Humphrey, M Clark V Humphrey, M Clark p y Victoria Hospital, Kirkcaldy, UK Victoria Hospital, Kirkcaldy, UK p y Critical Care 2014, 18(Suppl 1):P362 (doi: 10.1186/cc13552) Introduction This study aimed to establish clinical utility of blood cultures taken surrounding ICU admission. Blood culture cost in terms S132 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P364 Acute kidney injury in cardiorenal syndrome type 1: a meta-analysis W Vandenberghe, S Gevaert, H Peperstraete, I Herck, J Decruyenaere, E Hoste Introduction Cardiorenal syndrome type 1 (CRS-1) refl ects an abrupt worsening in cardiac function leading to acute kidney injury (AKI). Acute cardiac conditions contributing to CRS-1 include acute heart failure (AHF), acute coronary syndrome (ACS) and cardiac surgery (CS). The objective of this study was to evaluate the epidemiology of AKI in CRS-1. Conclusion The RRI, which is used to evaluate renal arterial fl ow, is directly related with increasing renal vascular resistance in the case of AKI. The usefulness of RRI for prediction of AKI was shown both clinically following a renal allograft and experimentally in the acute tubular necrosis modeling [1,2]. Also in septic patients this was asserted, as RRI is a better marker than cystatin C, which is one of the popular markers of recent times for prediction of development of AKI [3]. Our results show that RRI could be a simple, non-invasive and useful technique for early diagnosis of AKI in patients undergoing major operations such as lung and cardiac surgeries. Methods This is a systematic review and meta-analysis. AKI defi ned by the RIFLE defi nition and its modifi cations AKIN and KDIGO is grouped as AKIRIFLE. Similarly, AKI defi ned by variations of worsening renal failure is grouped as AKIWRF. Incidence of AKI is reported by the diff erent defi nitions of AKI. In addition, we report on mortality and length of intensive care and hospital stay (LOSICU and LOShosp) for AKIRIFLE. Data are reported as percentage, risk ratio (RR), and mean diff erence (MD). References References 1. Tranquart F, et al.: Transpl Int 1993, 6:14-17. 2. Yoon DY, et al.: Invest Radiol 1995, 30:168-172. 3. Schnell D, et al.: Shock 2012, 38:592-597. References 1. Tranquart F, et al.: Transpl Int 1993, 6:14-17. 2. Yoon DY, et al.: Invest Radiol 1995, 30:168-172. 3. Schnell D, et al.: Shock 2012, 38:592-597. Acute kidney injury and cardiac surgery: impact of fl uid balance on AKI classifi cation and prognosis Figure 1 (abstract P368). Cox-hazard prediction of AKI class on hospitalization (ICU LOS fi xed at median). Critical Care 2014, 18(Suppl 1):P367 (doi: 10.1186/cc13557) Introduction We assessed the eff ect of fl uid balance (FB) on acute kidney injury (AKI) classifi cation/prognosis in cardiac surgical patients by comparing patients classifi ed with AKI, before and after adjusting the creatinine (used to classify AKI) for FB. Fluid accumulation is associated with negative outcomes including development of AKI in critically ill patients [1]. Cardiac surgical patients commonly receive large volumes of fl uid postoperatively and could be at risk for the harmful eff ects of fl uid accumulation. Furthermore, fl uid accumulation may infl uence serum creatinine concentration and mask AKI [2]. Methods We performed a retrospective analysis of prospectively collected data on all cardiac surgical patients admitted to St Vincent’s Hospital ICU, Melbourne, Australia from 1 July 2004 to 30 June 2012. AKI Network creatinine criteria were used to classify AKI in the usual method and then using FB-adjusted creatinine (FB at 18 hours and an assumption that total body water is 60% of weight involved). FB (total i.v. input minus (total urine output + chest drain losses)) was calculated for 18 hours post surgery as most patients were in the ICU for this period. p Results Patients classifi ed with AKI increased from 27.7% to 37.2% (n = 2,171) after adjusting creatinine for FB. Patients were categorised into four groups based on presence or absence of AKI before and after adjustment for FB: group A, no AKI before or after adjustment for FB; group B, no AKI before/AKI after; group C, AKI before/no AKI after; and group D, AKI before and after. Group B (n = 209) had an in-hospital mortality rate similar to patients in group D (n = 599) (3.4% vs. 4.3%, P = 0.53) and greater than those in group A (n = 1,333) (3.4% vs. 1.6%, P = 0.07). Group B also had an ICU mortality rate similar to patients in group D (2.9% vs. 2.7%, P = 0.88) and signifi cantly greater than those in group A (2.9% vs. 0.7%, P = 0.003). The need for renal replacement therapy (RRT) in group B was also high as for patients in group D (7.7% vs. 12.4%, P = 0.06) and was signifi cantly greater than those in group A (7.7% vs. 1.6%, P <0.001). 1. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. 2. Liu KD, et al.: Crit Care Med 2011, 39:2665-2671. 1. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. 2 Liu KD et al : Crit Care Med 2011 39:2665-2671 Acute kidney injury of all severity is associated with extended hospitalization after critical illness Acute kidney injury of all severity is associated with extended hospitalization after critical illness R Prowle, I Kolic, J Purdell-Lewis, CJ Kirwan Introduction Acute kidney injury (AKI) complicates over 50% of ICU admissions and is associated with signifi cantly increased mortality, length of stay, and costs across a broad spectrum of conditions [1]. Methods We performed a single-centre, retrospective analysis of AKI diagnosis in patients with ICU admissions of 5 days or more who survived to hospital discharge between 2009 and 2011. We examined the relationship between hospital length of stay, AKI diagnosis, demographics and clinical characteristics in a multivariable Cox-hazard analysis. p Conclusion The renal RI measured at admission and its dynamics after the fi rst 24 ICU hours are predictive of the development of AKI and ICU mortality. Further studies are needed to confi rm these results. y Results We identifi ed 700 cases, with a 66% incidence of AKI. The AKI was associated with older age, greater initial illness severity and longer ICU and hospital length of stay in univariate analysis (Table 1). In Cox-hazard analysis, only AKI category and ICU length of stay were signifi cantly associated with lower probability of discharge over time (Figure 1). AKI-1 was associated with a hazard ratio for hospital discharge of 0.66 (0.55 to 0.79), AKI-2 with 0.55 (0.42 to 0.71) and AKI-3 with 0.54 (0.44 to 0.66). 1. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 on day 3. Within the fi rst 5 days a total of 17 patients had AKI stage 2 or 3. The RI at admission was associated with APACHE II score, hypotension at admission, baseline serum lactate and history of type II diabetes treated with insulin. Patients who developed AKI stage 2 or 3 had signifi cantly higher RI on admission (0.79 vs. 0.67, P = 0.021). A cutoff value of 0.72 best diff erentiated patients with AKI 2 or 3. Raise of RI by ≥0.05 during the fi rst 24 hours was associated with the development of AKI stage 2 or 3 (P = 0.034). Patients with persistent AKI on day 3 had higher RI values on admission, and those who recovered lowered their RIs by >0.05. In multivariate analysis, admission RI and raise of RI by ≥0.05 were independently associated with development of AKI stage 2 or 3 and ICU mortality. Best sensitivity for AKI was achieved when both criteria were used: elevated RI at admission (>0.72) and raise of RI by ≥0.05 at 24 hours post admission. 1. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966 973. 2. Liu KD, et al.: Crit Care Med 2011, 39:2665-2671. P366 Renal resistive index at ICU admission and its change after 24 hours predict acute kidney injury in sepsis I Gornik, A Godan, V Gašparović University Hospital Centre Zagreb, Croatia Critical Care 2014, 18(Suppl 1):P366 (doi: 10.1186/cc13556) y p g Critical Care 2014, 18(Suppl 1):P366 (doi: 10.1186/cc13556) Introduction The renal resistive index (RI) measured by Doppler ultrasound refl ects the changes in renal microvascular resistance. It was recently shown that the RI measured at ICU admission was associated with development of AKI and with persistent AKI on day 3 after admission. The aim of this study was to investigate whether change of RI after the fi rst 24 ICU hours has an additional predictive value for the development of AKI. Methods This non-interventional study included adult patients with sepsis admitted to a medical ICU. Patients with renal transplant and those in terminal renal failure were excluded. The RI was measured within 2 hours from admission and 24 ± 2 hours after ICU admission. Occurrence of AKI within the fi rst 5 days was classifi ed according to the AKI Network criteria. There was no intervention guide by RI measurements, nor were the results known to the attending physicians. Results There were 52 patients included in the study. At admission, eight patients had AKI stage 2 or 3 and it was persistent in fi ve patients Conclusion Almost one-quarter of patients with an acute cardiac condition had AKI, and RRT was used in approximately 3%. AKI was associated with signifi cant worse outcomes. AHF patients experienced the highest incidence of AKI, but the impact on mortality was greatest in CS patients. S133 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P367 P367 Acute kidney injury and cardiac surgery: impact of fl uid balance on AKI classifi cation and prognosis EM Moore1, A Tobin2, D Reid2, J Santamaria2, R Bellomo1 1Monash University, Melbourne, Australia; 2St Vincent’s Hospital, Melbourne, Australia Critical Care 2014, 18(Suppl 1):P367 (doi: 10.1186/cc13557) Acute kidney injury and cardiac surgery: impact of fl uid balance on AKI classifi cation and prognosis Thus, hospital and ICU mortality and use of RRT in patients classifi ed with AKI only after adjustment for FB were similar to patients with AKI before and after adjustment for FB and were notably higher than those of patients without AKI. Table 1 (abstract P368). Patient characteristics by AKI No AKI AKI P value Age 46 (32 to 60) 51 (37 to 64) <0.001 SAPS-2 35 (27 to 42) 41 (32 to 49) <0.001 ICU LOS 8 (6 to 12) 12 (7 to 18) <0.001 Hospital LOS 27 (17 to 42) 41 (28 to 72) <0.001 Data presented as median (IQR). Table 1 (abstract P368). Patient characteristics by AKI No AKI AKI P value Table 1 (abstract P368). Patient characteristics by AKI Conclusion AKI was a signifi cant predictor of remaining in hospital at all levels of AKI severity even after allowing for longer ICU stay. Even mild AKI is associated with extended recovery from critical illness and healthcare costs even after ICU discharge. Reference Conclusion Lack of adjustment for FB post cardiac surgery may mask the presence of AKI that is associated with increased risk for death and RRT, which could hinder optimal treatment. References Conclusion Lack of adjustment for FB post cardiac surgery may mask the presence of AKI that is associated with increased risk for death and RRT, which could hinder optimal treatment. Reference S134 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P369 using CG, MDRD and MCQE equations. The predictive capacity of these formulae was tested and compared in relation to AKI and RD incidence by constructing receiver operating characteristic curves for each of the models. Reference Reference 1. Macedo E, et al.: Crit Care 2010, 14:R82. Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery M Sileli, F Ampatzidou, S Tsagkaropoulos, K Diplaris, C Koutsogiannidis, G Drosos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P370 (doi: 10.1186/cc13560) Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery M Sileli, F Ampatzidou, S Tsagkaropoulos, K Diplaris, C Koutsogiannidis, G Drosos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P370 (doi: 10.1186/cc13560) Fluid accumulation increases the risk of AKI progression and death in critically ill patients with early AKI MR Raimundo1, S Crichton2, Y Syed2, J Martin2, M Ostermann2 1Hospital Beatriz Ângelo, Loures, Portugal; 2Guy’s & St Thomas Hospital, London, UK Critical Care 2014, 18(Suppl 1):P371 (doi: 10.1186/cc13561) Fluid accumulation increases the risk of AKI progression and death in critically ill patients with early AKI MR Raimundo1, S Crichton2, Y Syed2, J Martin2, M Ostermann2 1Hospital Beatriz Ângelo, Loures, Portugal; 2Guy’s & St Thomas Hospital, London, UK Critical Care 2014, 18(Suppl 1):P371 (doi: 10.1186/cc13561) References Results In total, 41.7% of the study patients developed AKI (AKIN-I 31.0%, AKIN-II 10%, AKIN-III 4.8%) and 4.8% received RRT. The following factors were diff erent between AKI and non-AKI patients: baseline expected GFR <60 ml/minute/1.73 m2, baseline serum Cr and baseline serum albumin, major abdominal surgery, vascular surgery, combined regional and general anesthesia, APACHE II score, receiving 6% 130/0.4 hydroxyethyl starch (HES) >20 ml/kg/day, 4% gelatin >20 ml/kg/day, crystalloid >30 ml/kg/day and positive fl uid balance in the fi rst 48 hours. Multiple logistic regression showed that independent risk factors of AKI included: baseline eGFR <60 ml/minute/1.73 m2 (OR = 1.53; 95% CI, 1.08 to 1.27, P = 0.02), baseline serum albumin <2 mg/dl (OR = 1.75; 95% CI, 1.01 to 3.06, P = 0.049), admitting hemoglobin <8 g/dl (OR = 2.41; 95% CI, 1.14 to 5.11, P = 0.02), receiving 6% 130/0.4 HES >20 ml/kg/day (OR = 2.02; 95% CI, 1.09 to 3.76, P = 0.03), receiving crystalloid >30 ml/ kg/day (OR = 3.15; 95% CI, 1.53 to 6.48, P = 0.01), vascular surgery (OR = 1.67; 95% CI, 1.05 to 2.64, P = 0.03), and major abdominal surgery (OR = 1.68; 95% CI, 1.11 to 2.55, P = 0.01). AKI patients had higher ventilator- hours (P = 0.02) and ICU length of stay (P = 0.04). 1. Tomaszuk-Kazberuk A, et al.: Ren Fail 2011, 33:983-899. 2. Ekmekci A, et al.: Angiology 2013. [Epub ahead of print] 1. Tomaszuk-Kazberuk A, et al.: Ren Fail 2011, 33:983-899. 2 Ekmekci A et al Angiolog 2013 [Ep b ahead of print] 1. Tomaszuk-Kazberuk A, et al.: Ren Fail 2011, 33:983-899. 2. Ekmekci A, et al.: Angiology 2013. [Epub ahead of print] Fluid accumulation increases the risk of AKI progression and death in critically ill patients with early AKI MR Raimundo1, S Crichton2, Y Syed2, J Martin2, M Ostermann2 1Hospital Beatriz Ângelo, Loures, Portugal; 2Guy’s & St Thomas Hospital, London, UK Critical Care 2014, 18(Suppl 1):P371 (doi: 10.1186/cc13561 Introduction Fluid therapy is a cornerstone in the management of patients with evolving acute kidney injury (AKI). Despite the wide availability of advanced hemodynamic monitoring to guide therapy, there are few data on how to manage hemodynamics optimally once early AKI has occurred. Our aim was to investigate the association between cumulative fl uid balance (FB)/fl uid administration and outcome (progression to AKI III and hospital mortality) in critically ill patients with early AKI, and its interaction with other hemodynamic parameters. y Conclusion The prevalence of early AKI in nonsepsis, noncardiac patients admitted to the general surgical ICU was 41.7%. Surgical patients with risk factors should be carefully cared for to prevent the development of AKI. Receiving 6% 130/0.4 HES >20 ml/kg/day and crystalloid >30 ml/kg/day increased the risk of early AKI in nonsepsis, noncardiac surgical patients. p Methods A retrospective analysis (2-year period) of all patients admitted to an adult ICU with AKI (defi ned by KDIGO criteria) who had hemodynamic monitoring within 12 hours of AKI I. We recorded FB, urinary output (UO), fl uid administered and hemodynamic parameters including mean arterial pressure (MAP) on the day of AKI I and in the following 72 hours. Logistic regression was employed to determine independent predictors of outcome. Reference P369 Early acute kidney injury in nonsepsis, noncardiac surgical patients admitted to a general surgical ICU Results The mean age of the cohort was 64.7 ± 0.45 years and the mean value of estimated kidney function from MCQE, MRDR and CG formulae was 83.7 ± 1.03 ml/minute/1.73 m2, 69.03 ± 0.82 ml/minute/1.73 m2 and 75.3 ± 1.18 ml/minute/1.73 m2 respectively. AKI was identifi ed in 75 (14.2%) patients, whereas early RD was necessary in 16 (3%) patients. All of the three variables showed a good predictive value for estimating AKI and RD after cardiac surgery. The area under the curve values for the early RD group was 0.887, 0.867 and 0.804, respectively and for the AKI group was 0.701, 0.651 and 0.691, respectively. j y g Critical Care 2014, 18(Suppl 1):P369 (doi: 10.1186/cc13559) Introduction Perioperative AKI is a signifi cant factor determining morbidity/mortality in surgical patients. We sought to determine the prevalence and risk factors of early AKI (within 72 hours of ICU admission) in nonsepsis, noncardiac surgical patients admitted to the general surgical ICU. Methods This prospective observational study was done in 600 nonsepsis, noncardiac surgical patients admitted to the 14-bed general surgical ICU of Siriraj Hospital. The following data were collected: patient demographic data, ASA, comorbidity, type and urgency of surgery, type of anesthesia, preoperative and the fi rst 72 hours laboratory data, amount of bleeding, type/amount of fl uid and blood replacement, average intraoperative and the fi rst 72 hours MAP, and severity score. Outcome as ventilator-hours, ICU length of stay and ICU mortality were also determined. Risk factors were identifi ed by multiple logistic regression. AKI was defi ned and classifi ed according to the AKIN criteria using adjusted serum creatinine (Cr) [1]. g p p y Conclusion On the basis of our fi ndings, all of the above algorithms seem to be accurate in predicting AKI and RD incidence in the early ICU postoperative period after elective cardiac surgery. Nevertheless, the MCQE equation more accurately classifi ed individuals compared with MDRD and CG formulae. Our results extend knowledge from previous studies [1,2]. Further investigations should be performed to determine whether these costless formulae could be used as an additional validated predictor in this group of patients and hence whether they could be incorporated into clinical practice. Incidence and outcomes of contrast-induced nephropathy in adult ICU patients p S Alderson1, S Pillai2, A Manoras1, J Papanikitas1, D Lewis1, S McKechnie1 1John Radcliff e Hospital, Oxford, UK; 2Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P374 (doi: 10.1186/cc13564) S Alderson1, S Pillai2, A Manoras1, J Papanikitas1, D Lewis1, S McKechnie1 1John Radcliff e Hospital, Oxford, UK; 2Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P374 (doi: 10.1186/cc13564) Introduction Critically ill patients cared for in ICUs often require radiological investigation using iodinated contrast agents. Contrast- induced nephropathy (CIN) – a form of acute kidney injury (AKI) – is a complication following the use of such contrast. Although CIN has been thoroughly studied in some populations (for example, those undergoing coronary angiography), it has not been investigated in large numbers of ICU patients [1]. p Conclusion Our results demonstrate that AKI occurs in 8.8% of the patients following surgery for gynecologic malignancies. Patients who had AKI were older, had higher body mass index with higher preoperative CRP levels, more frequent distant organ metastasis and longer operation times and higher amounts of blood transfused intraoperatively. Methods We conducted a single-centre retrospective review of the electronic patient records of general adult ICU patients who received iodinated contrast over a 3-year period (2009 to 2011). Our review identifi ed evidence of CIN or AKI post scan (assessed using CIN and KDIGO criteria); and clinical outcomes post scan (renal replacement therapy (RRT), ICU length of stay, mortality). Patients were excluded if: they had received pre-scan RRT; they did not have pre/post-scan creatinine measurements. Univariate analyses investigated the relationship between CIN or AKI and ICU outcomes (use of RRT post scan, ICU mortality and ICU length of stay), using chi-squared tests for categorical outcomes and nonparametric tests for continuous outcomes. Results A total of 479 scans involving 331 patients were included. In total, 303 (63%) scans involved males, median age 61 (IQR 46 to 71) years, 119 (25%) diabetic, with median pre-scan eGFR of 85 (38 to 113) ml/minute/1.73 m2. Scans occurred a median of 2.9 (0.8 to 8.8) days from admission. A total of 266 (56%) scans were associated with CIN (grade 0, n = 167; grade 1, n = 39; grade 2, n = 60). Clinical outcomes were signifi cantly worse in patients developing higher grades of CIN (increased use of RRT post scan, P = 0.02; increased ICU length of stay, P = 0.04; increased ICU mortality, P = 0.01). Reference Methods A total of 1,000 patients were enrolled retrospectively from January 2007 through March 2013. Patients under 18 years of age, those with chronic kidney disease and patients who died within the fi rst week after surgery were excluded. AKI was defi ned according to the KDIGO 2012 Clinical Practice Guideline for Acute Kidney Injury. Perioperative variables of patients were collected from medical charts. Results The mean age was 55.4 ± 12.4 years. The incidence of postoperative AKI was 8.8%, stage 1 occurred in 5.9%, stage 2 in 2.4% and stage 3 in 0.5% of the patients. Patients who had AKI were signifi cantly older (57.9 ± 12.8 vs. 55.1 ± 12.3 years, P = 0.046), had higher body mass index (30.1 ± 7.1 vs. 28.6 ± 6.4, P = 0.031), higher preoperative C-reactive protein (CRP) levels (157.8 ± 121.5 vs. 76.6 ± 81.1 mg/dl, P = 0.037) and more frequently had history of distant organ metastasis (13.3 vs. 7.8%, P = 0.022) when compared with those who did not have AKI. When compared with patients who did not develop AKI postoperatively, longer operation times (149.1 ± 62.5 vs. 123.8 ± 56.6 minutes, P = 0.001), intraoperative usage of higher amounts of erythrocyte suspension (279.5 ± 616.7 vs. 128.3 ± 296.1 ml, P = 0.001) and fresh frozen plasma (165.9 ± 284.8 vs. 94.1 ± 185.9 ml, P = 0.001) were seen in those who developed AKI. 1. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group: KDIGO Clinical Practice Guideline for Acute Kidney Injury. Kidney Int 2012, 2(Suppl):1-138. P373 Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) Postoperative acute kidney injury in patients with gynecologic malignancies haematocrit and low preoperative glomerular fi ltration rate (GFR) were signifi cant risk factors (P = 0.03, P = 0.04 and P <0.01 respectively) for developing postoperative AKI following cardiac surgery. See Table 1. Introduction Postoperative acute kidney injury (AKI) is an important cause of mortality and morbidity among surgical patients. Less is known about the occurrence of AKI after operations for gynecologic malignancies. The aim of this study was to determine the incidence of AKI in patients who underwent surgery for gynecologic malignancies and to compare patients with and without postoperative AKI. p g p p g g y Conclusion Our results suggest that we should focus on LV function and GFR as predictors for developing AKI following cardiac surgery. Strategies to increase preoperative haematocrit should be investigated to reduce in the incidence and severity of postoperative AKI. R f P372 Postoperative acute kidney injury in patients with gynecologic malignancies M Didik, P Zeyneloglu, A Pirat, A Ayhan, Z Kayhan Baskent University Faculty of Medicine, Ankara, Turkey Critical Care 2014, 18(Suppl 1):P372 (doi: 10.1186/cc13562) Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery Results A total of 210 patients (median age 70 years; 138 male) had hemodynamic monitoring within 12 hours of AKI I. In total, 41.5% progressed to AKI III and 43.3% died. Patients with fl uid overload after the diagnosis of AKI I (FB >1 l/day; n = 85) had a higher rate of progression to AKI III (63.5 vs. 23.3%, respectively; P <0.001) and in- hospital mortality (43.5 vs. 24.8%, respectively; P = 0.004) compared with patients with FB <1 l/day. There was no diff erence in the other parameters (demographics, comorbidities, severity scores, hemodynamic parameters on day of AKI I, vasopressor use), with the exception of MAP on the day of AKI I (71 vs. 74 mmHg, respectively; P = 0.01). In multivariate analysis after adjustment for demographics, severity scores, comorbidities and hemodynamic parameters on the day of AKI I, a higher FB was associated with an increased risk of progression to AKI III and death (odds ratio (OR) per each 1 l/day increase 2.8; P <0.001) and death (OR 1.6; P = 0.001). Similarly, a higher amount of fl uid administered was associated with an increased risk of AKI progression (OR per each 1 l/day increase 1.8; P = 0.011), even with adjustment for UO. There were no signifi cant interactions between the risk of progression/death and other hemodynamic parameters. With the exception of MAP, improvements in other hemodynamic General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P370 (doi: 10.1186/cc13560) Introduction The Cockcroft–Gault (CG) equation and the four-variable Modifi cation of Diet in Renal Disease (MDRD) formula are the most commonly used methods to provide estimation of kidney function. The newly developed Mayo Clinic quadratic equation (MCQE) is another alternative. The scope of this study is to investigate the prognostic value of the above algorithms as prediction models for development of acute renal injury (AKI) and early renal dialysis (RD) in cardiac surgery patients during the postoperative ICU stay. Methods A retrospective single-centre study of 528 consecutive patients admitted to the ICU, who underwent elective cardiac surgery under extracorporeal circulation from July 2012 to November 2013. Patients undergoing urgent or emergent surgery were excluded prior to the study. Preoperative estimation of renal function was obtained S135 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P373). Incidence and outcomes of contrast-induced nephropathy in adult ICU patients Ninety-fi ve (20%) scans were associated with AKI (stage 1, n = 45; stage 2, n = 13; stage 3, n = 37). Clinical outcomes were again signifi cantly worse in patients developing AKI (increased use of RRT post scan, P <0.001; increased ICU mortality P = 0.01). Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery Results Age (years) 66 (13.8) Male 69 Female 34 No AKI 69 KDIGO 1 22 KDIGO 2 6 KDIGO 3 6 Table 1 (abstract P373). Results parameters in the days after AKI I diagnosis did not have a signifi cant impact on outcome.l Conclusion In critically ill patients with early AKI, a positive fl uid balance, induced by excessive fl uid administration, is associated with an increased risk of AKI progression and death. Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) g p Results A total of 479 scans involving 331 patients were included. In total, 303 (63%) scans involved males, median age 61 (IQR 46 to 71) years, 119 (25%) diabetic, with median pre-scan eGFR of 85 (38 to 113) ml/minute/1.73 m2. Scans occurred a median of 2.9 (0.8 to 8.8) days from admission. A total of 266 (56%) scans were associated with CIN (grade 0, n = 167; grade 1, n = 39; grade 2, n = 60). Clinical outcomes were signifi cantly worse in patients developing higher grades of CIN (increased use of RRT post scan, P = 0.02; increased ICU length of stay, P = 0.04; increased ICU mortality, P = 0.01). Ninety-fi ve (20%) scans were associated with AKI (stage 1, n = 45; stage 2, n = 13; stage 3, n = 37). Clinical outcomes were again signifi cantly worse in patients developing AKI (increased use of RRT post scan, P <0.001; increased ICU mortality P = 0.01). Introduction Acute kidney injury (AKI) is a signifi cant complication following cardiac surgery associated with an increase in morbidity, hospital stay and mortality. Although the estimated incidence of AKI following cardiac surgery is 30%, few studies have identifi ed the incidence of AKI following cardiac surgery as defi ned by the Kidney Disease: Improving Global Outcomes (KDIGO) group [1]. We conducted a prospective observational study at our institution to identify the incidence and staging of AKI according to the KDIGO defi nition. We also aim to identify the factors that predispose adult patients to developing AKI post cardiac surgery. Methods A prospective analysis was performed on 103 adult patients admitted to ICU post-cardiac surgery from September to October 2013. Data for perioperative risk factors and renal biochemical markers were collected up to the sixth postoperative day and are expressed as mean (SD). Conclusion Renal impairment/injury is common in adult ICU patients undergoing investigations using iodinated contrast and the incidence varies depending upon the classifi cation used. Whichever classifi cation is used, patients developing CIN/AKI following contrast administration have poorer clinical outcomes than patients who do not. Reference Results Ordered logistic regression was used to analyse the data. Thirty- three per cent of cardiac surgery patients at our institution developed AKI. Factors such as poor left ventricular (LV) function, low preoperative 1. Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) Lameire et al.: Contrast-induced acute kidney injury and renal support for acute kidney injury: a KDIGO summary (Part 2). Crit Care 2013, 17:205. . Lameire et al.: Contrast-induced acute kidney injury and renal support for acute kidney injury: a KDIGO summary (Part 2). Crit Care 2013, 17:205. S136 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 of AKI. Signifi cant risk factors for AKI again included male gender (P = 0.009), reduced pre-scan eGFR (P <0.001), and decreasing time from admission to scan (P = 0.003), but also included emergency admission to ICU (P = 0.03), pre-scan shock (P <0.001), and pre-scan oliguria (P <0.001). P375 Human acute kidney injury is associated with a proinfl ammatory phenotype N Gauge, M Varrier, D Boardman, M Hernandez-Fuentes, M Ostermann Guy’s and St Thomas’ Hospital, London, UK Critical Care 2014, 18(Suppl 1):P375 (doi: 10.1186/cc13565) Conclusion Male gender, reduced pre-scan eGFR and decreasing time from admission to scan are risk factors for the development of both CIN and AKI. The association with time from admission to scan may refl ect inadequate patient optimisation prior to contrast administration; this hypothesis is supported by the risk factors signifi cantly associated with AKI alone (emergency admission to ICU and pre-scan shock). Given the adverse clinical outcomes associated with the development of CIN/AKI, these fi ndings necessitate a review of current practice. Reference Introduction Animal research suggests that acute kidney injury (AKI) is an infl ammatory condition [1]. Our aim was to describe the immune phenotype in human AKI. Introduction Animal research suggests that acute kidney injury (AKI) is an infl ammatory condition [1]. Our aim was to describe the immune phenotype in human AKI. y Methods We enrolled patients with: AKI grade II/III (defi ned by KDIGO criteria) and systemic infl ammatory response syndrome (SIRS) without sepsis; SIRS without AKI; and AKI II/III without SIRS. A healthy control population was used for baseline comparison. Serial blood samples were taken on days 0, 2 and 7. Cells were separated using Percoll gradients and phenotyped using fl ow cytometry.i 1. Lameire et al.: Contrast-induced acute kidney injury and renal support for acute kidney injury: a KDIGO summary (Part 2). Crit Care 2013, 17:205. References 1. Martensson J, et al.: Intensive Care Med 2010, 36:1333-1340. 2. Nejat et al.: Crit Care 2010, 14:R85. 2. Nejat et al.: Crit Care 2010, 14:R85. Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) l Results The results from 24 day 0 samples identifi ed statistically signifi cant diff erences between SIRS, AKI, AKI + SIRS and healthy controls amongst: CD8+ cytotoxic T cells, CD45–CD25+++ regulatory T cells, and CD45–CD25++ cytokine secreting non-T-regulatory cells (Table 1). The percentage of CD69-positive neutrophils was signifi cantly increased across all three groups relative to controls, with little variation between AKI, SIRS and AKI + SIRS patients. Test characteristics of acute kidney injury biomarkers in animal models of sepsis p Z Peng, J Zhang, F Zhou, J Kellum g g University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P377 (doi: 10.1186/cc13567) Table 1 (abstract P375). AKI + SIRS AKI no SIRS SIRS alone Control % CD8+ cytotoxic T cells 23.3 16.7 11.6 31.1 % Fr. II T-regulatory cells 4.1 2.7 1.6 1.2 % Fr. III cytokine T cell 11.5 21.2 13.9 8.0 % CD69+ neutrophils 83.9 69.7 65.5 7.35 Conclusion Human AKI is associated with a proinfl ammatory phenotype. Reference 1. Akcay A, et al.: Mediators of infl ammation in acute kidney injury. Mediators Infl amm 2009, 2009:137072. Table 1 (abstract P375). Introduction Approximately 50% of acute kidney injury (AKI) is associated with sepsis. Neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C are the two most widely used biomarkers for AKI. However, these two markers are also aff ected by the systemic infl ammatory response, and their diagnostic value in sepsis-induced AKI is disputed [1,2]. Unlike clinical AKI, animal models can be used to explore single etiology. The purpose of this study is to examine the relationship between infl ammatory mediators and biomarkers for AKI in a sepsis model in rats. Conclusion Human AKI is associated with a proinfl ammatory phenotype. Reference p Methods Sepsis was induced by cecal ligation and puncture (CLP) in 60 adult SD rats and then observed for AKI and survival. Blood and urine samples were collected at baseline, and 18, 22, and 48 hours after CLP. AKI severity was assessed by RIFLE criteria (creatinine only). The associations between plasma IL-6 and plasma NGAL, plasma cystatin C, urine NGAL and urine cystatin C were analyzed. The area under the receiver-operator characteristic curves (AUC) was used to evaluate the diagnostic capability between severe AKI (RIFLE-I or RIFLE-F) and no AKI (includes RIFLE-R) for diff erent biomarkers. 1. Akcay A, et al.: Mediators of infl ammation in acute kidney injury. Mediators Infl amm 2009, 2009:137072. P376 Risk factors for the development of contrast-induced nephropathy in ICU patients f Results The changes of plasma NGAL, plasma cystatin C, urine NGAL and urine cystatin C with time were similar to the changes of plasma IL-6. However, only plasma NGAL levels were closely correlated with levels of plasma IL-6 (R2 = 0.36, P <0.05). The analysis for plasma cystatin C, urine NGAL and urine cystatin C at 22 hours for severe AKI showed AUCs of 0.78, 0.71 and 0.75 respectively (all P <0.05), and the AUC for plasma NGAL was 0.62 (P = 0.11). There were no signifi cant diff erences in plasma NGAL at 22 hours between severe AKI and no AKI (2,143.32 vs. 2,077.02 U/ml, P = 0.21). Introduction Critically ill patients cared for in ICUs often require radiological investigation using iodinated contrast agents. Contrast- induced nephropathy (CIN) – a form of acute kidney injury (AKI) – is a complication following the use of such contrast. Although CIN has been thoroughly studied in some populations (for example, those undergoing coronary angiography), it has not been investigated in large numbers of ICU patients [1]. Conclusion In this animal model of CLP sepsis, plasma NGAL levels were aff ected by the systemic infl ammatory response, and did not discriminate for AKI. Urine NGAL, plasma cystatin C and urine cystatin C were able to diff erentiate severe AKI from no AKI in CLP sepsis. References g Methods We conducted a single-centre retrospective review of the electronic patient records of general adult ICU patients who received iodinated contrast over a 3-year period (2009 to 2011). Our review identifi ed: patient demographics; ICU admission details (specialty, time of admission, elective/emergency admission); physiological status pre scan (evidence of shock, eGFR, urine output); volume of iodinated contrast; and evidence of CIN or AKI post scan (assessed using CIN and KDIGO criteria). Patients were excluded if they had pre-scan renal replacement therapy. Analyses investigated the risk factors for CIN or AKI using chi-squared tests for categorical variables and nonparametric tests for continuous variables (as no continuous variable was normally distributed, even with log transformation). P378 Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? L Desteghe1, W Boer2, Q Swennen3, V Pennemans3, M Vander Laenen2, K Engelen2, C De Deyne2, F Jans2 1Hasselt University, Diepenbeek, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium; 3Biomedical Research Institute, Diepenbeek, Belgium Critical Care 2014, 18(Suppl 1):P378 (doi: 10.1186/cc13568) Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? L Desteghe1, W Boer2, Q Swennen3, V Pennemans3, M Vander Laenen2, K Engelen2, C De Deyne2, F Jans2 1Hasselt University, Diepenbeek, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium; 3Biomedical Research Institute, Diepenbeek, Belgium Critical Care 2014, 18(Suppl 1):P378 (doi: 10.1186/cc13568) Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? L Desteghe1, W Boer2, Q Swennen3, V Pennemans3, M Vander Laenen2, K Engelen2, C De Deyne2, F Jans2 1Hasselt University, Diepenbeek, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium; 3Biomedical Research Institute, Diepenbeek, Belgium Critical Care 2014, 18(Suppl 1):P378 (doi: 10.1186/cc13568) P376 P376 Risk factors for the development of contrast-induced nephropathy in ICU patients S Pillai1, S Alderson2, A Manoras2, J Papanikitas2, D Lewis2, S McKechnie2 1Morriston Hospital, Swansea, UK; 2John Radcliff e Hospital, Oxford, UK Critical Care 2014, 18(Suppl 1):P376 (doi: 10.1186/cc13566) Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? Results In total, 479 scans involving 331 patients were included. A total of 266 (56%) scans were associated with CIN. Signifi cant risk factors for the development of CIN included male gender (P = 0.02), reduced pre- scan GFR (P <0.001), and decreasing time from admission to scan (P = 0.03). Ninety-fi ve (20%) scans were associated with the development Introduction Acute kidney injury (AKI) remains a common complication after cardiopulmonary bypass (CPB) and can be diagnosed by S137 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 serum creatinine [1]. However, serum creatinine is an insensitive and nonspecifi c biomarker [2]. This study was designed to investigate whether a correlation exists between urinary oxygen tension (UOT) and early markers of AKI. The aim was to evaluate whether UOT could provide warning signs of an insuffi cient renal oxygen supply, which can lead to postoperative AKI. serum creatinine [1]. However, serum creatinine is an insensitive and nonspecifi c biomarker [2]. This study was designed to investigate whether a correlation exists between urinary oxygen tension (UOT) and early markers of AKI. The aim was to evaluate whether UOT could provide warning signs of an insuffi cient renal oxygen supply, which can lead to postoperative AKI. dose, mean arterial pressure, fl uid balance and urine output in the fi rst 6 hours after admission were signifi cantly diff erent for patients with low and high risk for AKI. The negative predictive value (NPV) of 0.41 for urine output <1 ml/kg/hour over the fi rst 6 hours after admission was signifi cantly lower if compared with NPV of 0.86 for GFBP7/TIMP-2 ≤0.3 for exclusion of AKI. Similarly NPV of urine output <1 ml/kg/hour of 0.89 for moderate and severe AKI (stage 2 and 3) was lower than the NPV of 1.00 for GFBP7/TIMP-2 ≤0.3. Methods Fourteen subjects undergoing cardiac surgery with CPB were included in this prospective clinical pilot study. UOT was measured perioperatively in all patients, both in the operating room (before, during and after CPB) and in the ICU. Biomarkers of AKI in blood and urine were measured preoperatively and postoperatively at 3, 6, 12 and 24 hours after the initiation of CPB. These included serum creatinine and the early urinary biomarkers kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C. P380 P380 Urine TIMP2 × IGFBP7 increases 24 hours before severe AKI M Ostermann1, L Chawla2, L Forni3, J Kellum, on behalf of Sapphire Investigators4 1Guys & St Thomas Foundation Hospital, London, UK; 2George Washington University, Washington, DC, USA; 3Western Sussex Hospital, Worthing, UK; 4University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P380 (doi: 10.1186/cc13570) Urine TIMP2 × IGFBP7 increases 24 hours before severe AKI M Ostermann1, L Chawla2, L Forni3, J Kellum, on behalf of Sapphire Investigators4 Results There was a signifi cant decrease in UOT between the start of CPB (138.44 ± 22.19 mmHg) and the lowest UOT during CPB (107.70 ± 23.28 mmHg) (P = 0.001). Dividing the subjects into two groups according to the Acute Kidney Injury Network (AKIN) classifi cation, no signifi cant diff erences were found in mean UOTs between the group of patients with a normal kidney function (n = 7) and the group with AKIN stage 1 or 2 (n = 7). For KIM-1, a signifi cant diff erence between the two groups was found at 3 hours (P = 0.041) after the initiation of CPB. Further, for NGAL a signifi cant increase in biomarker concentrations compared with the preoperative value was observed in the group with an AKIN stage 1 or 2 at all diff erent postoperative time points (3 hours (P = 0.013), 6 hours (P = 0.003), 12 hours (P = 0.009) and 24 hours (P = 0.003)). On the contrary, there were no signifi cant increased urinary NGAL levels measured in the group with the normal kidney function. 1Guys & St Thomas Foundation Hospital, London, UK; 2George Washington University, Washington, DC, USA; 3Western Sussex Hospital, Worthing, UK; 4University of Pittsburgh, PA, USA Introduction We recently reported a 728-patient multicenter study (Sapphire) where a biomarker combination of tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) were validated for risk stratifi cation for moderate or severe acute kidney injury (AKI) KDIGO stage 2 and 3 [1].f Methods We subsequently selected two clinical cutoff values for the TIMP2 × IGFBP7 combination from the Sapphire dataset, one (0.3) with high sensitivity (89%) (specifi city = 50%) and one (2.0) with high specifi city (95%) (sensitivity = 42%) for the development of AKI KDIGO stage 2 and 3 within 12 hours of study enrolment. P379 Postoperative acute kidney injury can be predicted by the novel biomarkers insulin-like growth factor-binding protein 7/tissue inhibitor of metalloproteinases-2 as early as 6 hours after surgery I Göcze, R Herzog, M Koch, P Renner, F Zeman, BM Graf, HJ Schlitt, T Bein University Medical Center Regensburg, Germany Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) Postoperative acute kidney injury can be predicted by the novel biomarkers insulin-like growth factor-binding protein 7/tissue inhibitor of metalloproteinases-2 as early as 6 hours after surgery I Göcze, R Herzog, M Koch, P Renner, F Zeman, BM Graf, HJ Schlitt, T Bein University Medical Center Regensburg, Germany Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) Postoperative acute kidney injury can be predicted by the novel biomarkers insulin-like growth factor-binding protein 7/tissue inhibitor of metalloproteinases-2 as early as 6 hours after surgery I Göcze, R Herzog, M Koch, P Renner, F Zeman, BM Graf, HJ Schlitt, T Bein University Medical Center Regensburg, Germany Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) Conclusion The TIMP2 × IGFBP7 biomarker combination identifi es patients who ultimately develop moderate or severe AKI 24 hours earlier than serum creatinine. P380 We examined the timing of change in TIMP2 × IGFBP7 relative to change in creatinine using the sign test. g y Conclusion This pilot study was not able to demonstrate any association between perioperatively measured UOT and markers of postoperative AKI. Additional laboratory and clinical studies will be necessary to further defi ne the relationship between the UOT and new biomarkers of AKI. References References References 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. g g Results TIMP2 × IGFBP7 results were available for 178 patients who developed AKI stage 2 or 3. The median TIMP2 × IGFBP7 result was signifi cantly greater than the cutoff value of 0.3 from 24 hours before to 24 hours after AKI 2 or 3 (P <0.01) (Figure 1). Conversely, median serum creatinine was not diff erent from baseline prior to development of AKI 2 or 3.i 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2 Vaidya VS et al : Annu Rev Pharmacol Toxicol 2008 48:463-493 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? Student’s t tests and Mann–Whitney tests were used to compare continuous variables.i Conclusion The novel urine biomarkers IGFBP7 and TIMP-2 enable early risk stratifi cation of surgical ICU patients at risk for development of AKI. The predictive values of biomarkers were better for early prediction than clinical parameters such as urine output within the fi rst 6 hours after admission to ICU. P382 P382 Urine microscopy score combined with albumin creatinine ratio score improves prediction of future acute kidney injury (AKI) and worsening AKI JJ Dixon1, K Lane1, W Fleming-Nouri2, H Cheema2, P Walker2, I MacPhee3, B Philips1 1St George’s Hospital and University of London, UK; 2St George’s, University of London, UK; 3St George’s Hospital, London, UK Critical Care 2014, 18(Suppl 1):P382 (doi: 10.1186/cc13572) Reference Reference y g g y Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) 1. Kashani K, et al.: Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury. Crit Care 2013, 17:R25. Introduction Acute kidney injury (AKI) in surgical critically ill patients is an independent risk factor for early mortality. Two novel urine biomarkers, insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), may help to detect clinically silent episodes of AKI in the golden hours prior to irreversible damage of the kidney. We evaluated the early predictive value of these biomarkers for AKI, moderate and severe AKI, early requirement of renal replacement therapy (RRT), and ICU mortality, with a cutoff value of IGFBP7/TIMP-2 >0.3. Figure 1 (abstract P380). NC, Nephrocheck (TIMP2 × IGFBP7); Cr, creatinine. Methods Four to six hours after admission to the surgical ICU, urine biomarkers were prospectively evaluated in all patients with present exposures and susceptibilities for AKI according to the KDIGO recommendation. The incidence and severity of AKI (KDIGO 2012) and requirement of RRT were assessed over 48 hours after admission. In addition, ICU mortality and variables such a norepinephrine dose, mean arterial pressure, hemoglobin level, cumulative fl uid balance and urine production were noted at the time of biomarker evaluation (4 to 6 hours) and for the fi rst 24 hours after admission. i Results A total of 120 patients were included in the study. The area under the curve (AUC) for IGFBP7/TIMP2 >0.3 was 0.83 for early detection of AKI, 0.86 for moderate and severe AKI, 0.86 for RRT in the fi rst 48 hours after admission and 0.86 for ICU mortality. Patients with IGFBP7/ TIMP-2 >0.3 had a higher risk (odds ratio 11.7) for development of AKI compared with patients with IGFBP7/TIMP-2 ≤0.3. The norepinephrine Figure 1 (abstract P380). NC, Nephrocheck (TIMP2 × IGFBP7); Cr, creatinine. S138 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P381 who developed AKI or worsening AKI (n = 58) had a mean score of 2.79 ± 1.21 versus 2.30 ± 1.14 in those who never developed AKI or improved (n = 150), P = 0.006. UMS-ACRS score >2 on admission had a sensitivity of 0.89 for identifying progressive AKI. Reference UMS-ACRS score of 5 to 8 on admission had a positive predictive value for worsening AKI of 60%, a negative predictive value of 69% and a likelihood ratio 3.1 for developing AKI or worsening AKI. who developed AKI or worsening AKI (n = 58) had a mean score of 2.79 ± 1.21 versus 2.30 ± 1.14 in those who never developed AKI or improved (n = 150), P = 0.006. UMS-ACRS score >2 on admission had a sensitivity of 0.89 for identifying progressive AKI. UMS-ACRS score of 5 to 8 on admission had a positive predictive value for worsening AKI of 60%, a negative predictive value of 69% and a likelihood ratio 3.1 for developing AKI or worsening AKI. P381 Urinary tissue inhibitor of metalloproteinases-2 and insulin-like growth factor-binding protein 7 as early biomarkers of acute kidney injury and renal recovery following cardiac surgery A Zarbock1, M Meersch1, C Schmidt1, S Martens1, J Rossaint1, K Singbartl2, D Görlich1, J Kellum3, H Van Aken1 1University of Münster, Germany; 2Penn State College of Medicine Hershey, PA, USA; 3University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P381 (doi: 10.1186/cc13571) Conclusion The mean UMS-ACRS is higher in patients with AKI. Combining UMS with ACRS improves prediction and stratifi cation of which patients will develop AKI, or progressive AKI, after ICU admission. Clinical implications are that urine microscopy and ACR calculation are safe, inexpensive, non-invasive and may improve prediction of AKI in critically ill patients, potentially leading to earlier intervention and improved outcomes. Introduction Diffi culties in prediction and early identifi cation of acute kidney injury (AKI) have hindered the ability to develop preventive and therapeutic measures for this syndrome. We tested the hypothesis that a urine test measuring insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), both inducers of G1 cell cycle arrest, a key mechanism implicated in AKI, could predict AKI in cardiac surgery patients. P383 Resveratrol ameliorates apoptosis induced by contrast medium ioxitalamate in HK-2 human renal proximal tubule cells in vitro YY Chen, CC Cheng, TC Lin, Huang Buddhist Tzu Chi General Hospital, Hualien, Taiwan Critical Care 2014, 18(Suppl 1):P383 (doi: 10.1186/cc13573) y Methods We studied 50 patients at high risk for AKI undergoing cardiac surgery with cardiopulmonary bypass. Serial urine samples were analyzed for [TIMP-2] × [IGFBP7] concentrations. The primary outcome measure was AKI as defi ned by international consensus criteria following surgery. Furthermore, we investigated whether urine [TIMP- 2] × [IGFBP7] could predict renal recovery from AKI prior to hospital discharge. Introduction Computed tomography with contrast medium is a common diagnostic tool in emergency and critical medicine. Contrast- induced nephropathy (CIN) is one of the leading causes of hospital- acquired acute kidney injury. Focus on renal tubule protection may be a hope to improve the eff ectiveness of current strategies. Results Twenty-six patients (52%) developed AKI. Diagnosis based on serum creatinine and/or oliguria did not occur until 1 to 3 days after cardiopulmonary bypass. In contrast, urine concentration of [TIMP-2] × [IGFBP7] rose from a mean of 0.49 (0.24) at baseline to 1.51 (0.57) 4 hours after cardiopulmonary bypass in patients who developed AKI. The maximum urinary [TIMP-2] × [IGFBP7] concentration achieved in the fi rst 24 hours following surgery (composite time point) demonstrated an area under the receiver-operating characteristic curve of 0.84. Sensitivity was 0.92, and specifi city was 0.81 for a cutoff value of 0.50. The decline in urinary [TIMP-2] × [IGFBP7] values was the strongest predictor for renal recovery. f Methods We used HK-2 human renal proximal tubule cells to evaluate the therapeutic potential of resveratrol, a polyphenol phytoalexin Figure 1 (abstract P383). Immunostaining of cytoplasmic DNA fragmentation in HK-2 cells. Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. Figure 1 (abstract P383). Immunostaining of cytoplasmic DNA fragmentation in HK-2 cells. p y Conclusion Urinary [TIMP-2] × [IGFBP7] serves as a sensitive and specifi c biomarker to predict AKI early after cardiac surgery and to predict renal recovery. Recovery from AKI by KDIGO criteria Recovery from AKI by KDIGO criteria D S h J G G d B h p p p Methods This single-centre, randomized, double-blind, placebo- controlled study included 75 patients scheduled for CABG with preexisting renal impairment (estimated glomerular fi ltration rate based on p-cystatin C <60 ml/minute and >15 ml/minute). The patients either received a single high dose of EPO (400 IU/kg) or placebo preoperatively. The primary endpoint was renal protection evaluated by p-cystatin C at the third postoperative day compared with the preoperative values. Incidence of acute kidney injury and other renal biomarker changes were among secondary endpoints.if y p g Critical Care 2014, 18(Suppl 1):P386 (doi: 10.1186/cc13576) Introduction Data on recovery of AKI are mainly limited to persistent dialysis dependency in patients with dialysis-requiring AKI. The aim of this analysis is to evaluate recovery from diff erent stages of AKI. y yf g Methods In a large database (n = 4,640) of a previous RCT [1] we estimated renal recovery from AKI defi ned by KDIGO criteria (without urine output criteria). Patients with end-stage renal disease (n = 56), kidney transplantation (n = 15) or incomplete data (n = 9) were excluded. Patients were classifi ed according to their maximal AKI stage (AKImax) during the ICU stay. Recovery was evaluated by AKI stage at hospital discharge. Complete recovery was defi ned as the absence of AKI, partial recovery as persistent AKI with a decrease in AKI stage compared with AKImax and no recovery as persistence of AKImax or worsening of AKI after ICU discharge. A persistent 0.3 mg/dl increase of Screat was also considered as no or partial recovery. Results There was no signifi cant diff erence on the third postoperative day for p-cystatin C levels (2.1 ± 0.8 mg/l for the study group and 1.9 ± 0.5 mg/l for the control group, P = 0.51). There were no signifi cant diff erences in other renal biomarkers or measures between the groups (p-NGAL, p-creatinine, p-urea, and estimated glomerular fi ltration rate). There were no other diff erences in outcome variables between the groups. g p Conclusion Intravenous administration of a single high dose (400 IU/ kg) of EPO did not have a renal protective eff ect in patients with reduced kidney function undergoing coronary artery bypass surgery. References Results A total of 1,296 patients (28%) developed AKI. Recovery from AKI by KDIGO criteria AKImax was stage 1 in 580 (45%) (416 with >50% increase of Screat), stage 2 in 207 (16%) and stage 3 in 509 (39%) (348 needing RRT). Mortality increased from 12 to 42% (P <0.0001), hospital stay increased from 21 (14 to 37) to 34 (17 to 63) days (P <0.0001) and complete recovery in survivors decreased from 82 to 53% (P <0.0001) with increasing severity of AKI. In patients requiring RRT, 51% of survivors left the hospital without AKI whereas 16% remained dialysis dependent. Within the AKI 3 group, the need for RRT signifi cantly increased mortality (P = 0.0002), but did not aff ect complete recovery in survivors (51% vs. 58%, P = 0.16). Patients with a ‘0.3 mg/dl increase of serum creatinine only’ had a signifi cantly higher mortality than patients without AKI in the ICU (P = 0.0004). They also had a worse kidney outcome at hospital discharge (P = 0.006). 1. Bahlmann FH, Fliser D: Erythropoietin and renoprotection. Curr Opin Nephrol Hypertens 2009, 18:15-20. 2. Moore E, Bellomo R: Erythropoietin (EPO) in acute kidney injury. Ann Intensive Care 2011, 1:3. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ICU nonsurvivors (n = 229), patients on dialysis at ICU discharge (n = 77) and patients for whom Clcr on the last day of ICU was not available (n = 206), 784 patients were included in this analysis. We compared eGFR (MDRD equation) with measured Clcr (based on 24-hour urine collection and corrected for BSA) at ICU discharge for patient groups with diff erent ICU stays. We also evaluated the impact of using the two GFR measurements on the estimation of complete recovery relative to baseline eGFR. Parameters were compared with the paired t test and McNemar’s test. produced naturally by several plants, for contrast ioxitalamate-induced toxicity in vitro. Cytotoxicity was determined by MTT assay. Patterns of cell death were observed by fl ow cytometry. Cytoplasmic DNA fragmentation was examined by ELISA. Western blots were used to analyze the expression of related proteins. y p p Results In a 48-hour administration, ioxitalamate elicited cytotoxicity on HK-2 cells. Annexin V+ cells were signifi cantly increased after 30 mg/ ml ioxitalamate exposure. A decrease in bcl-2 expression explained the ioxitalamate-induced apoptosis. Co-treatment with resveratrol ameliorated the cytotoxicity induced by ioxitalamate. Resveratrol at 12.5 μM decreased ioxitalamate-induced DNA fragmentation through upregulated expression of survivin. See Figures 1 and 2. Results Amongst the 784 patients with AKI, 456 (58%) reached stage 1, 143 (18%) stage 2 and 185 (24%) stage 3. Mean ± SD Clcr and eGFR at ICU discharge were respectively 54.5 ± 28 and 76 ± 55 ml/minute/1.73 m2 (P <0.0001). eGFR was not signifi cantly diff erent from Clcr in patients with ICU stay <7 days. In patients with ICU stay between 8 and 14 days, eGFR was signifi cantly higher than Clcr (79 ± 51 vs. 48.5 ± 20, P <0.0001) and the diff erence increased even further in patients with ICU stay over 14 days (102 ± 70 vs. 42.6 ± 20, P <0.0001). The percentage of patients with complete recovery diff ered signifi cantly when evaluated by eGFR (35.3%) or Clcr (28.7%) (P = 0.007). In patients with ICU stay >14 days, this diff erence increased to 56.4% by eGFR versus 14.1% by Clcr (P <0.0001). Conclusion Resveratrol ameliorated apoptosis induced by contrast medium ioxitalamate in human renal proximal tubule cells. Investigations using animal models will be conducted in the future. Estimated GFR versus creatinine clearance for evaluation of recovery from acute kidney injury y y j y P Timmermans, J Gunst, G Van den Berghe, M Schetz KU Leuven University Hospital, Leuven, Belgium y p g Critical Care 2014, 18(Suppl 1):P385 (doi: 10.1186/cc13575) Conclusion Increasing severity of AKI according to the KDIGO criteria is associated with increased mortality and decreased recovery of kidney function. The need for RRT signifi cantly increases mortality but complete recovery in survivors of AKI 3 is not diff erent with or without RRT. The 0.3 mg/dl criterion proves valid with regard to mortality and kidney outcome. Introduction The aim of this study is to quantify the impact of using eGFR instead of measured creatinine clearance (Clcr) on the evaluation of recovery from acute kidney injury (AKI). Introduction The aim of this study is to quantify the impact of using eGFR instead of measured creatinine clearance (Clcr) on the evaluation of recovery from acute kidney injury (AKI). Methods From a large RCT’s database [1] we excluded patients with end-stage renal disease and kidney transplants. In the remaining 4,560 patients, 1,296 (28%) developed AKI (KDIGO criteria). After exclusion of Casaer et al.: N Engl J Med 2011, 365:506-517. P386 R P386 Recovery from AKI by KDIGO criteria D Schrijvers, J Gunst, G van den Berghe, M Schetz KU Leuven University Hospital, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P386 (doi: 10.1186/cc13576) Urine microscopy score combined with albumin creatinine ratio score improves prediction of future acute kidney injury (AKI) and worsening AKI Urine microscopy score combined with albumin creatinine ratio score improves prediction of future acute kidney injury (AKI) and worsening AKI g JJ Dixon1, K Lane1, W Fleming-Nouri2, H Cheema2, P Walker2, I MacPhee3, B Philips1 p 1St George’s Hospital and University of London, UK; 2St George’s, University of London, UK; 3St George’s Hospital, London, UK Critical Care 2014, 18(Suppl 1):P382 (doi: 10.1186/cc13572) Introduction Patients with AKI have a high morbidity and mortality. Diagnosis of AKI may be improved by examination of the urinary sediment with microscopy and by measurement of urine albumin. A urine microscopy score (UMS; 0 to 4 points) of renal tubular epithelial cells and granular casts has previously been developed to aid diagnosis. We have devised a urine albumin:creatinine ratio score (ACRS; 0 to 4 points) and have combined this with the UMS with the aim of stratifying the risk of developing future AKI or worsening AKI (UMS- ACRS; 0 to 8 points). The aims were to compare UMS-ACRS in critically ill patients with and without AKI at ICU admission, and to determine whether a high UMS-ACRS can predict if critically ill patients develop AKI or worsening AKI. Figure 1 (abstract P383). Immunostaining of cytoplasmic DNA fragmentation in HK-2 cells. Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. g Methods Investigators were blinded to diagnosis prior to urine collection. Microscopy was performed on centrifuged urine obtained from 227 consecutive critically ill patients in a general ICU on day 1 of admission. Five photographs were taken and the mean UMS was calculated. An independent reviewer scored the photographs. The urine albumin:creatinine ratio was calculated and the ACRS determined. The UMS was then combined with the ACRS. Results Mean UMS-ACRS ± SD was higher (2.66 ± 1.57) in patients with AKI on ICU admission (n = 106) compared with those without AKI (mean UMS-ACRS = 2.40 ± 1.03; n = 120), unpaired t test P = 0.14. Patients Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. S139 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P384 P384 Erythropoietin and Protection of Renal function in Cardiac Surgery (EPRICS) trial A Dardashti Lund University and Skane University Hospital, Lund, Sweden Critical Care 2014, 18(Suppl 1):P384 (doi: 10.1186/cc13574) Conclusion Estimated GFR at ICU discharge is signifi cantly higher than the measured Clcr in patients with prolonged ICU stay. This diff erence can be explained by loss of muscle mass with decreased creatinine production and results in an important overestimation of recovery. Reference Introduction To date, there are no known methods for preventing acute kidney injury after cardiac surgery. Increasing evidence suggests that erythropoietin (EPO) has renal anti-apoptotic and tissue protective eff ects [1], and in animal models a single high dose of EPO has been shown to ameliorate reperfusion injury after ischemia [2]. However, recent human studies have shown confl icting results. We aimed to study the eff ect of a single high dose of EPO preoperatively on renal function after coronary artery bypass grafting (CABG) in patients with preoperative impaired renal function. 1. Casaer et al.: N Engl J Med 2011, 365:506-517. 1. Casaer et al.: N Engl J Med 2011, 365:506-517. Recovery of renal function after acute kid continuous renal replacement therapy p py HY Jung, KH Kim, SC Park, JY Choi, SH Park, CD Kim, YL Kim, JH Cho Kyungpook National University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P389 (doi: 10.1186/cc13579) y HY Jung, KH Kim, SC Park, JY Choi, SH Park, CD Kim, YL Kim, JH Cho Kyungpook National University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P389 (doi: 10.1186/cc13579) Kyungpook National University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P389 (doi: 10.1186/cc13579) Introduction Acute kidney injury (AKI) is increasingly common in critically ill patients and many patients with severe kidney injury require continuous renal replacement therapy (CRRT). However, little is known regarding the incidence rate and associated factors for developing chronic kidney disease after CRRT in AKI patients. This study aimed to investigate renal outcome and the factors associated with incomplete renal recovery in AKI patients who received CRRT. Conclusion By the KDIGO defi nition, AKI occurred in two-thirds of patients following LTx. AKI portended greater risk of death and loss of kidney function. Methods Between January 2011 and August 2013, 397 patients received CRRT in our ICU. Among them, patients who had normal renal function before AKI and were discharged without maintenance renal replacement therapy (RRT) were included in this study. We examined the incidence of incomplete renal recovery with estimated glomerular fi ltration rate (eGFR) <60 ml/minute/1.73 m2 during follow-up. Factors that increased risk of incomplete renal recovery after AKI were investigated with multiple logistic regression. y References 1. Arnaoutakis GJ, George TJ, Robinson CW, et al.: Severe acute kidney injury according to the RIFLE (risk, injury, failure, loss, end stage) criteria aff ects mortality in lung transplantation. J Heart Lung Transplant 2011, 30:1161-1168. 2. Wehbe E, Brock R, Budev M, et al.: Short-term and long-term outcomes of acute kidney injury after lung transplantation. J Heart Lung Transplant 2012, 31:244-251. 2. Wehbe E, Brock R, Budev M, et al.: Short-term and long-term outcomes of acute kidney injury after lung transplantation. J Heart Lung Transplant 2012, 31:244-251. Results Forty-one AKI patients were discharged without further RRT and followed up for a mean of 7 months. Sixteen (39.0%) of 41 patients incompletely recovered their renal function. Patients with incomplete renal recovery showed older age and longer duration of anuria compared with complete renal recovery patients (69.7 ± 7.0 vs. 54.8 ± 16.9 years, P = 0.002; 128.6 ± 192.1 vs. 26.9 ± 66.6 hours, P = 0.019). Multivariate analysis adjusting for sex, initial eGFR, hemoglobin level, diabetes mellitus and hypertension showed that old age and long duration of anuria were independent risk factors for incomplete renal recovery (OR = 1.143, 95% CI = 1.020 to 1.281, P = 0.021 and OR = 1.011, 95% CI = 1.001 to 1.032, P = 0.038, respectively). 3. Paradela de la Morena M, De La Torre Bravos M, Prado RF, et al.: Chronic kidney disease after lung transplantation: incidence, risk factors, and treatment. Transplant Proc 2010, 42:3217-3219. 3. Paradela de la Morena M, De La Torre Bravos M, Prado RF, et al.: Chronic kidney disease after lung transplantation: incidence, risk factors, and treatment. Transplant Proc 2010, 42:3217-3219. y Reference Casaer et al.: N Engl J Med 2011, 365:506-517. S140 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P387 Incidence and outcomes of acute kidney injury following orthotopic lung transplant: a population-based cohort study P Fidalgo1, M Ahmed1, SR Meyer1, D Lien1, J Weinkauf1, FS Cardoso1, K Jackson2, SM Bagshaw1 1University of Alberta, Edmonton, Canada; 2Alberta Health Services, Edmonton, Canada Critical Care 2014, 18(Suppl 1):P387 (doi: 10.1186/cc13577) P387 criteria were used to classify AKI using creatinine adjusted for fl uid balance. Results Renal replacement therapy was performed on 136 patients in this group (4.2%). The fl uid accumulation percentage was associated with an 8% increase in odds for AKI (OR (CI), 1.08 (1.04 to 1.12)), and a 13% increase in odds for requiring renal replacement therapy (1.13 (1.05 to 1.21)) for each percent increase in fl uid accumulation (l/kg%) after cardiac surgery, after adjusting for variables including APACHE score, cardiac failure, type of surgery, and inotrope use in multivariate analysis. Introduction Acute kidney injury (AKI) is a serious complication following lung transplantation (LTx) [1-3]. We aimed to describe the incidence and outcomes associated with AKI following LTx. Introduction Acute kidney injury (AKI) is a serious complication following lung transplantation (LTx) [1-3]. We aimed to describe the incidence and outcomes associated with AKI following LTx. y Conclusion In this relatively homogeneous patient group undergoing cardiac surgery, postoperative percent fl uid accumulation at 18 hours was associated with AKI and need for renal replacement therapy. Whether there is residual confounding due to indication for fl uid use or unmeasured risk factors requires further investigation in controlled trials. g Methods A retrospective population-based cohort study of all adult recipients of LTx at the University of Alberta between 1990 and 2011 was performed. The primary outcome was AKI, defi ned and classifi ed according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria, in the fi rst seven postoperative days. Secondary outcomes included risk factors, utilization of renal replacement therapy (RRT), occurrence of postoperative complications, mortality and kidney recovery. Fluid accumulation post cardiac surgery and risk for renal replacement therapy p py EM Moore1, A Tobin2, D Reid2, J Santamaria2, R Bellomo1 EM Moore , A Tobin , D Reid , J Santamaria , R Bellomo 1Monash University, Melbourne, Australia; 2St Vincent’s Hospital, Melbourne, Australia Conclusion The renal outcome of severe AKI requiring CRRT was poor even in patients with previous normal renal function. Long-term monitoring of renal function is needed especially in severe AKI patients with old age and long duration of anuria. Critical Care 2014, 18(Suppl 1):P388 (doi: 10.1186/cc13578) Critical Care 2014, 18(Suppl 1):P388 (doi: 10.1186/cc13578) Introduction We assessed the impact of fl uid accumulation on the development of acute kidney injury (AKI) and need for continuous renal replacement therapy in cardiac surgical patients. Fluid accumulation has been associated with negative outcomes including development of AKI in critically ill patients [1-3]. As cardiac surgical patients commonly receive large volumes of i.v. fl uid within 24 hours of surgery, they could be at risk of the harmful eff ects of fl uid accumulation. References 1. Bouchard J, et al.: Kidney Int 2009, 76:422-427. 1. Bouchard J, et al.: Kidney Int 2009, 76:422 427. 2 Payen D, et al.: Crit Care (London) 2008, 12:R74. 3. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. 2 Payen D, et al.: Crit Care (London) 2008, 12:R74. y Results Of 445 LTx recipients included, AKI occurred in 306 (68.8%), with severity classifi ed as stage I in 38.9% (n = 173), stage II in 17.5% (n = 78) and stage III in 12.4% (n = 55). RRT was received by 36 (8.1%). Independent risk factors associated with AKI included longer duration of cardiopulmonary bypass (per minute, odds ratio (OR) 1.003; 95% CI, 1.001 to 1.006; P = 0.02), and mechanical ventilation (per hour (log-transformed), OR 5.30; 95% CI, 3.04 to 9.24; P <0.001), and use of cyclosporine (OR 2.03; 95% CI, 1.13 to 3.64; P = 0.02). In-hospital and 1-year mortality were signifi cantly higher in those with AKI compared with no AKI (7.2% vs. 0%, adjusted P = 0.001; 14.4% vs. 5.0%, adjusted P = 0.02, respectively). At 3 months, those with AKI had greater sustained loss of kidney function compared with no AKI (estimated glomerular fi ltration rate (mean (SD)) 68.9 (25.7) vs. 75.3 (22.1) ml/minute/1.73 m2, P = 0.01).i Renal replacement therapy in very elderly critical care patients K Brown, HG Jones Renal replacement therapy in very elderly critical care patients K Brown, HG Jones Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P392 (doi: 10.1186/cc13582) Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P392 (doi: 10.1186/cc13582) Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P392 (doi: 10.1186/cc13582) Introduction The very elderly (>80 years) UK population is increasing and along with it there is an increased utilisation of critical care resources [1]. The use of chronological age as a bar to treatment is unethical, yet a discrepancy of opinion remains between the disinclined and the elderly advocate intensivist [2]. Acute kidney injury requiring dialysis is a frequent complication of critical illness and is associated with high morbidity and mortality. An increased risk is seen with age due to the high prevalence of risk factors and chronic kidney disease in the elderly [3]. g p Results The total cohort was composed of 7,276 patients with 63.91 ± 12.45 years and 61.1% male gender. Elective scheduled surgery was done in 85.9% of patients. Surgical risk assessed by the additive EuroSCORE was 5.86 ± 3.14 points and predicted mortality by the Logistic EuroSCORE was 8.10%. Mortality in the ICU was 7.6% and in- hospital mortality was 10.1% (8.1% missing data). Prior to surgery, 10.4% of patients had a creatinine level between 1.2 and 2 mg/dl, 1.1% between 2 and 2.3 mg/dl, 0.8% between 2.3 and 3.5 mg/dl and 0.3% above 3.5 mg/dl. In the nonmatched cohort, 180 patients (2.5%) needed RRT. RRT was needed in 3.5% of 3,771 patients with diuretics and in 1.4% of 3,505 patients not treated with them (P <0.001); 2.61 (1.87 to 3.65). After adjusting with logistic regression by additive EuroSCORE, SAPS 3, bypass time exceeding 120 minutes and prior renal dysfunction, the OR was 1.67 (1.15 to 2.45). When we analysed 3,426 matched patients according to the propensity score, RRT was needed in 3% of 1,713 patients with diuretics and in 1.6% of 1,713 not treated with them (P <0.009), 1.85 (1.16 to 2.94). Methods Very elderly patients admitted to our tertiary referral ITU were included in a 2-year (December 2011 to November 2013) retrospective cohort analysis. The outcome of those receiving RRT was reviewed. Results There were 2,297 admissions to the ITU, of which 323 (14%) were classifi ed as very elderly with a mean APACHE II score of 17.3. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A prospective cohort of adult patients who underwent cardiac surgery in 11 institutions all over the Andalusia community from March 2008 to July 2012 was included in the ARIAM adult cardiac surgery project. Diuretic users prior to the intervention were pair-matched to nonusers on the basis of a propensity score based on demographics, comorbidities, medication and surgical data. We analysed diff erences in RRT in both groups. Continuous renal replacement therapy (CVVHD) for acute kidney injury in critical care: incidence and outcome across South West Wales K Brown, M Challis, A Mikhail K Brown, M Challis, A Mikhail Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P391 (doi: 10.1186/cc13581) Critical Care 2014, 18(Suppl 1):P391 (doi: 10.1186/cc13581) Introduction Renal replacement therapy in critical care is associated with increased mortality. It is not known for which patients RRT confers the most benefi t, or who will recover function and remain dialysis independent on a long-term basis.f References 1. Bagshaw SM, et al.: Crit Care 2009, 13:R45. 2. Nguyen YL, Et al.: Ann of Intensive Care 2011, 1:29. 3. Stevens LA, et al.: Adv Chronic Kidney Dis 2010, 17:293-301. 4. Cracknell R: The Ageing Population. London: House of Commons Library Research; 2010. 5. Ansell D, et al., eds: UK Renal Registry Report: 2002. 6. Wight JP, et al.: Qual Health Care. 1998, 7:209-221. 7. McDermid RC, et al.: Crit Care 2011, 15:125. Methods All ICU patients receiving CVVHD (diff usive haemodialysis) for AKI were analysed in a retrospective cohort study of mixed non/surgical patients at a tertiary renal centre over a 2-year period (December 2011 to November 2013). Children <18 years, patients on intermittent haemodialysis or patients requiring plasma exchange were excluded from analysis. y Results A total of 2,297 patients were admitted to the ICU, of which 14% (319) required CRRT. Thirteen patients were excluded from analysis; n = 306, of which 58% were male. Causes of AKI included sepsis (37%), surgery (13%, of which 82% were emergency procedures) and vascular events (9%). Mean values for patients requiring RRT versus all ICU patients: age (65 vs. 62.5 years), APACHE II score (21.2 vs. 14.8), length of stay (13 vs. 8.2 days). Forty-seven per cent of patients received incident dialysis <24 hours of admission, with mean fl ow rates of 31 ml/kg/hour (2 l) in 39%. Mortality at hospital discharge was 56% for RRT versus 20% in all ICU patients admitted over the same time period. P393 Preventing continuous renal replacement therapies (CRRT)-induced hypophosphatemia using a phosphate-containing CRRT solution in the setting of regional citrate anticoagulation V Pistolesi1, S Morabito1, L Tritapepe1, E Vitaliano2, L Zeppilli1, F Polistena1, MI Sacco1, E Fiaccadori3, A Pierucci1 1Umberto I, Policlinico di Roma, Rome, Italy; 2Pertini Hospital, Rome, Italy; 3University of Parma Medical School, Parma, Italy Critical Care 2014, 18(Suppl 1):P393 (doi: 10.1186/cc13583) Preventing continuous renal replacement therapies (CRRT)-induced hypophosphatemia using a phosphate-containing CRRT solution in the setting of regional citrate anticoagulation V Pistolesi1, S Morabito1, L Tritapepe1, E Vitaliano2, L Zeppilli1, F Polistena1, MI Sacco1, E Fiaccadori3, A Pierucci1 1Umberto I, Policlinico di Roma, Rome, Italy; 2Pertini Hospital, Rome, Italy; 3University of Parma Medical School, Parma, Italy Critical Care 2014, 18(Suppl 1):P393 (doi: 10.1186/cc13583) Preventing continuous renal replacement therapies (CRRT)-induced hypophosphatemia using a phosphate-containing CRRT solution in the setting of regional citrate anticoagulation Conclusion The reported incidence of AKI in critical care ranges from 20 to 50%, with the highest rates seen in sepsis [1]. Utilisation of CRRT for AKI is higher at our centre than the described 5% [2], potentially due to close collaboration between critical care and nephrology [3] or relatively lenient ICU admission criteria. Increasing mortality was seen with age, APACHE II score and delay in initiation of RRT. Prospective analysis is required to look at determining biomarkers for AKI and risk factors for mortality; dynamic monitoring of haemodynamic responders (MAP, vasopressor requirement <24 hours), percentage creatinine decrease [4], severity-of-illness scores and urine output. Results of current RCTs are awaited, which may provide more information on mode of clearance, fl ow rates and early versus standard initiation of RRT to more accurately prognose patients’ outcomes. Introduction Phosphate depletion is a known issue during continuous renal replacement therapies (CRRT) with an incidence of hypophosphatemia up to 80% when standard CRRT solutions are used. The aim was to evaluate the eff ects on serum phosphate and phosphorus supplementation needs of a regional citrate anticoagulation (RCA) protocol for CRRT combining the use of citrate with a phosphate-containing CRRT solution. Renal replacement therapy in very elderly critical care patients K Brown, HG Jones RRT was utilised in 12.4% (n = 40) of the very elderly patients with a mean APACHE II score of 25. Forty-fi ve per cent of AKI was due to sepsis and 25% due to emergency surgery. ITU very elderly patient mortality was 30.1%, and in those who received RRT was 42% and 67% at ITU and hospital discharge respectively. Recovery of renal function was seen in 19 patients at ITU discharge, of these 11 survived to hospital discharge. Two patients required ongoing IHD in the community. Conclusion Preoperative use of diuretics is associated with an increased risk of need for RRT. P391 y Conclusion Current predictions estimate that the very elderly UK population will almost double by 2030 [4]. The biggest uptake in acceptance of chronic RRT has been in the elderly [5], where no diff erence has been shown in health-related quality of life from the general dialysis population [6]. Our experience suggests that RRT has a role in the critically ill elderly patient, with 33% surviving to hospital discharge. The challenge is identifying those most likely to benefi t from a cohort with multiple comorbidities, against the available resources, rising demand for critical care in an aging population and increasing expectations [7]. Continuous renal replacement therapy (CVVHD) for acute kidney injury in critical care: incidence and outcome across South West Wales K Brown, M Challis, A Mikhail Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P391 (doi: 10.1186/cc13581) Relation between preoperative use of diuretics and renal replacement therapy after cardiac surgery: a propensity score analysis E Curiel-Balsera1, M Delange van der Kroft2, I Macias-Guarasa1, R Hinojosa Perez1, J Ravina-Sanz1, M Garcia-Delgado1, R Rivera-Fernandez1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain E Curiel-Balsera1, M Delange van der Kroft2, I Macias-Guarasa1, R Hinojosa Perez1, J Ravina-Sanz1, M Garcia-Delgado1, R Rivera-Fernandez1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain fl Methods We performed a retrospective analysis of prospectively collected data on all patients admitted after cardiac surgery to St Vincent’s Hospital ICU, Melbourne, Australia from 1 July 2004 to 30 June 2012 (n = 3,207). The fl uid accumulation percentage (total urine and chest drain losses subtracted from total i.v. intake (l) /weight (kg) × 100) was calculated for 18 hours post surgery as most patients were in the ICU for this period. Acute Kidney Injury Network (AKIN) creatinine q g p Critical Care 2014, 18(Suppl 1):P390 (doi: 10.1186/cc13580) Introduction The aim was to evaluate the relation between preoperative use of diuretics and renal replacement therapy (RRT) in postoperative patients of cardiac surgery. Introduction The aim was to evaluate the relation between preoperative use of diuretics and renal replacement therapy (RRT) in postoperative patients of cardiac surgery. S141 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients We found no statistical signifi cant diff erences for: age (P = 0.06); SAPS II (P = 0.28); SOFA (P = 0.19); the timing of beginning of the technique (P = 0.61), with 34.8% versus 47.5% of patients in R (RIFLE), 27.8% versus 18% in I (RIFLE) and 16% versus 21% in F (RIFLE); duration of the technique (P = 0.74) and length of stay. Although we noticed a greater loss of dose and absolute creatinine clearance in the citrate group, this had no statistical signifi cance (P = 0.18 and P = 0.13). The mortality found for citrate and heparin groups was 60.4% and 39.4% respectively. The diff erences with statistical signifi cance related to dialitrauma emerged in K+ (P = 0.03), Ca2+ (P = 0.02), Na+ (P = 0.004), platelets (P = 0.002), pH (P = 0.02) and bicarbonate (P = 0.0001). Logistic regression for mortality in the citrate versus heparin groups showed the following values: eff ective dose (ROC 0.435 vs. ROC 0.663), clearance (ROC 0.606 vs. ROC 0.663), SAPS II (ROC 0.482 vs. ROC 0.696), SOFA (ROC 0.713 vs. ROC 0.696) and RIFLE (ROC 0.695 vs. ROC 0.636). Conclusion We may say that there are functional differences that must Methods We performed a retrospective and analytical study including patients exclusively submitted to citrate or heparin through years 2011 and 2012. Included were demographic data with SAPS II, SOFA, RIFLE scores and mortality rate. For functional analysis we consider the timing of the beginning, duration, loss of dose and loss of creatinine clearance. We analyzed dialitrauma, considering the variation of: potassium, total and ionized calcium, magnesium, sodium, phosphorus, pH, lactates, bicarbonate, platelets, albumin, creatinine and urea. Data are presented as the average and standard deviations. To access the infl uence of dose and clearance losses on mortality, we used logistic regression test. yp p p Conclusion The use of a phosphate-containing CRRT solution, accounting for about 50 to 60% of the CRRT dose in the setting of RCA-CVVH or RCA-CVVHDF, allowed one to prevent CRRT-induced phosphate depletion in most of the patients, minimizing the need for phosphate supplementation and maintaining phosphorus levels in a near-normal range throughout CRRT days. Results The study included 44 patients in the citrate group versus 61 in the heparin group. P395 corrected for predilution, was at least 25 ml/kg/hour with about 50 to 60% of dialysis dose given as phosphate-containing solution. By convention, hypophosphatemia was defi ned as follows: mild (<0.81 mmol/l), moderate (<0.61 mmol/l) and severe (<0.32 mmol/l). Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients We found no statistical signifi cant diff erences for: age (P = 0.06); SAPS II (P = 0.28); SOFA (P = 0.19); the timing of beginning of the technique (P = 0.61), with 34.8% versus 47.5% of patients in R (RIFLE), 27.8% versus 18% in I (RIFLE) and 16% versus 21% in F (RIFLE); duration of the technique (P = 0.74) and length of stay. Although we noticed a greater loss of dose and absolute creatinine clearance in the citrate group, this had no statistical signifi cance (P = 0.18 and P = 0.13). The mortality found for citrate and heparin groups was 60.4% and 39.4% respectively. The diff erences with statistical signifi cance related to dialitrauma emerged in K+ (P = 0.03), Ca2+ (P = 0.02), Na+ (P = 0.004), platelets (P = 0.002), pH (P = 0.02) and bicarbonate (P = 0.0001). Logistic regression for mortality in the citrate versus heparin groups showed the following values: eff ective dose (ROC 0.435 vs. ROC 0.663), clearance (ROC 0.606 vs. ROC 0.663), SAPS II (ROC 0.482 vs. ROC 0.696), SOFA (ROC 0.713 vs. ROC 0.696) and RIFLE (ROC 0.695 vs. ROC 0.636).f Elimination rates of electrolytes, vitamins and trace elements during continuous renal replacement therapy with citrate CVVHD: infl uence of treatment dose l P Von Freyberg1, W Stahl2, H Reinelt2, K Träge Conclusion We may say that there are functional diff erences that must be taken into account. Despite not having statistical signifi cance on this sample, losses of dose and creatinine clearance showed a direct relation with mortality. Introduction During continuous renal replacement therapy (CRRT), relevant losses of nutritional substrates, vitamins and trace elements due to diff usive transport and elimination via the fi lter may occur. We investigated the amount of these losses with regard to treatment dose during a 72-hour treatment period. Reference Hetzel GR, Schmitz, Wissing H, et al.: Regional citrate versus systemic heparin for anticoagulation in critically ill patients on continuous venovenous haemofi ltration: a prospective randomized multicentre trial. Nephrol Dial Transplant 2011, 26:232-239. Methods Forty-one patients with CRRT were investigated. Patients were treated with a citrate CVVHD with diff erent standard doses resulting in a treatment dose range from 14 to 53 (median 27) ml/ kg/hour. Losses were calculated from substrate concentrations in the dialysate × (dialysate + ultrafi ltration) fl ow. During a 72-hour treatment period, each 24 hours were determined separately. Regression analysis was performed for the respective per-day losses related to treatment doses. P394 Elimination rates of electrolytes, vitamins and trace elements during continuous renal replacement therapy with citrate CVVHD: infl uence of treatment dose P Von Freyberg1, W Stahl2, H Reinelt2, K Träger2 1Klinikum Heidenheim, Germany; 2University Hospital Ulm, Germany Critical Care 2014, 18(Suppl 1):P394 (doi: 10.1186/cc13584) y References Methods In critically ill heart surgery patients undergoing CRRT for acute kidney injury, we adopted RCA in a CVVH or CVVHDF modality combining a commercially available citrate solution (18 mmol/l) with a phosphate-containing CRRT solution as dialysate and/or replacement fl uid (HCO3 – 30 mmol/l, phosphate 1.2). The prescribed CRRT dose, 1. Case J, et al.: Crit Care Res Pract 2013, 2013:479730 4. Herrera-Gutiérrez ME, et al.: ASAIO J 2006, 52:670-676. S142 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P396 Results Regression analyses of ER for Ca2+, Mg2+, PO4 2–, zinc, folic acid and vitamin B12 are shown in Table 1 as the median and interquartile range. Development of key performance indicators for renal replacement therapy in adult intensive care to guide safe and cost-eff ective therapy therapy Table 1 (abstract P394). ER elimination rates Table 1 (abstract P394). ER elimination rates Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients Results Forty-eight patients were treated with RCA-CRRT for at least 72 hours (total running time 12,502 hours). Two-hundred and nineteen RCA-CVVH circuits were used with a mean fi lter life of 57.1 ± 41.7 hours (median 47, IQR 24 to 83). Acid–base status was adequately maintained without the need for additional interventions on RCA-CRRT parameters (pH 7.43 (7.40 to 7.47), bicarbonate 25.3 mmol/l (23.8 to 26.6), BE 0.9 (–0.7 to 2.4); median (IQR)). Serum phosphate was steadily maintained in a narrow range throughout RCA-CRRT days (1.2 mmol/l (0.97 to 1.45); median (IQR)). At some times during CRRT, only 10 out of 48 patients (20.8%) received a low amount of phosphate supplementation (D-fructose-1,6-diphosphate 1.05 ± 2.04 g/day) for mild (n = 7) to moderate (n = 3) hypophosphatemia. In particular, considering all patients, only 33 out of 513 serum phosphorus determinations met the criteria for mild (n = 24) to moderate (n = 9) hypophosphatemia. Severe hypophosphatemia was never observed. p V Goulão, E Lafuente, J Silva, M Fernandes, F Santos, A Ferreira CHTS, Penafi el, Portugal Critical Care 2014, 18(Suppl 1):P395 (doi: 10.1186/cc13585) V Goulão, E Lafuente, J Silva, M Fernandes, F Santos, A Ferreira CHTS, Penafi el, Portugal Critical Care 2014, 18(Suppl 1):P395 (doi: 10.1186/cc13585) Introduction The aim of this study is to analyze the functional alterations that may infl uence the fi nal result when using citrate versus heparin anticoagulation in critically ill patients [1]. p g y p Methods We performed a retrospective and analytical study including patients exclusively submitted to citrate or heparin through years 2011 and 2012. Included were demographic data with SAPS II, SOFA, RIFLE scores and mortality rate. For functional analysis we consider the timing of the beginning, duration, loss of dose and loss of creatinine clearance. We analyzed dialitrauma, considering the variation of: potassium, total and ionized calcium, magnesium, sodium, phosphorus, pH, lactates, bicarbonate, platelets, albumin, creatinine and urea. Data are presented as the average and standard deviations. To access the infl uence of dose and clearance losses on mortality, we used logistic regression test. Results The study included 44 patients in the citrate group versus 61 in the heparin group. Table 1 (abstract P394). ER elimination rates y A Fischer, S Finney A Fischer, S Finney Table 1 (abstract P394). ER elimination rates Parameter Average daily ER Day 1 R2 Day 2 R2 Day 3 R2 Ca2+ (mmol/day) 97 (83 to 107) 0.2125 0.2448 0.2315 Mg2+ (mmol/day) 6 (3 to 9) 0.0247 0.0708 0.0733 PO4 2– (mmol/day) 53 (40 to 69) 0.003 0.0556 0.0012 Zinc (μmol/day) 46 (27 to 73) 0.005 0.0527 0.0502 Folic acid (nmol/day) 268 (180 to 388) 0.0935 0.0031 0.0353 Vitamin B12 (pmol/day) 2,060 (1,690 to 2,558) 0.0009 0.0092 0.0152 Conclusion Only Ca2+ showed a correlation of ER and treatment dose. Mg2+, PO4 2– zinc, folic acid and vitamin B12 were eliminated without a correlation to treatment dose. ( ) Parameter Average daily ER Day 1 R2 Day 2 R2 Day 3 R2 Ca2+ (mmol/day) 97 (83 to 107) 0.2125 0.2448 0.2315 Mg2+ (mmol/day) 6 (3 to 9) 0.0247 0.0708 0.0733 PO4 2– (mmol/day) 53 (40 to 69) 0.003 0.0556 0.0012 Zinc (μmol/day) 46 (27 to 73) 0.005 0.0527 0.0502 Folic acid (nmol/day) 268 (180 to 388) 0.0935 0.0031 0.0353 Vitamin B12 (pmol/day) 2,060 (1,690 to 2,558) 0.0009 0.0092 0.0152 Royal Brompton Hospital, London, UK y p p Critical Care 2014, 18(Suppl 1):P396 (doi: 10.1186/cc13586) Introduction Renal replacement therapy (RRT) is common. We undertook to develop and report some key performance indicators (KPIs) to monitor our provision of this costly therapy. We utilised data already collected in an electronic clinical information system that records the care received by our patients. We reduced our prescribed RRT dose to 25 ml/kg/hour in December 2011 following an appraisal of the literature [1]. We assessed whether the KPIs informed us if our practice changed and such changes were sustained.fl Methods We calculate the hourly effl uent rate corrected for a patient’s predicted body weight, and the lifespan of haemofi lters. This takes less than 30 minutes each month. Statistical process control charts (SPCs) are used to assimilate the indicators over time. S143 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P396). KPI for renal replacement therapy 2013 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 AICU-RBH no. of patients on CVVHD 8 8 17 15 6 7 13 12 9 15 21 AICU-RBH no. A Fischer, S Finney We assessed the eff ectiveness of EMIC2 with citrate anticoagulation in AKI prevention of post-cardiac surgical patients.i g Conclusion The KPIs could be produced quickly and allowed monitoring of the reduction in RRT dosing, assuring us that it is in excess of 20 ml/ kg/hour. The KPIs did not require additional data collection processes. We are developing similar indicators for other organ systems, therapies and processes. A Fischer, S Finney 602 763 689 636 695 294 629 9 5,526 7,004 6,325 5,838 6,380 2,699 5,774 9, 28.17 29.10 30.53 33.93 31.52 31.25 29.90 3 23.44 22.23 23.60 29.32 24.43 21.34 23.93 2 0.84 0.75 0.67 0.84 0.77 0.66 0.77 0 48.77 34.02 32.60 38.74 31.30 26.65 23.70 3 2,322 4,025 3,638 2,632 3,870 2,012 4,644 5, 753 954 861 795 869 368 786 1, 3,075 4,979 4,499 3,427 4,739 2,380 5,430 6, 8,601 11,983 10,824 9,265 11,119 5,079 11,204 15 1,075 705 722 1,544 1,588 391 934 1, 118 122 126 143 131 115 146 1 Results A total of 736 patients received RRT during the study. Prior to the dose change, the mean set and delivered doses were 39 and 31 ml/ kg/hour respectively. Thereafter the mean set and delivered doses were 33 and 26 ml/kg/hour respectively. Whilst higher than our guideline dose, they are signifi cantly less than the doses prior to the change in practice (both P <0.001). The SPC indicates that the change in practice has been sustained. See Figure 1 and Table 1. P397 Eff ectiveness of sub-albumin protein leakage membrane EMIC2 in post-cardiac surgery rhabdomyolysis G Paternoster, A Covino, R Mercorella, C Di Leo, M Calabrese, G Pittella San Carlo Hospital, Potenza, Italy Critical Care 2014, 18(Suppl 1):P397 (doi: 10.1186/cc13587) P397 Eff ectiveness of sub-albumin protein leakage membrane EMIC2 in post-cardiac surgery rhabdomyolysis G Paternoster, A Covino, R Mercorella, C Di Leo, M Calabrese, G Pittella San Carlo Hospital, Potenza, Italy Critical Care 2014, 18(Suppl 1):P397 (doi: 10.1186/cc13587) Introduction A high postoperative serum myoglobin (MyG) concentration predicts the incidence of acute kidney injury (AKI) and need for renal replacement therapy (RRT), as reported in several surgical settings. The incidence of such events in the ICU is reported to be 2 to 5% of all causes of AKI [1], but can even worsen in the case of cardiac surgery (40.3%) [1]. MyG is a small protein (17.8 kDa) that can be removed with RRT, typically in convection cases. New-generation membranes, removing sub-albumin protein molecular weight solutes, can be used in diff usive treatments (CVVHD) with the advantage of limiting albumin loss and easily combining with citrate anticoagulation, pivotal for cardio-surgical settings. A Fischer, S Finney of patient-days on CVVHD 61 73 98 86 65 85 44 77 112 119 137 AICU-RBH fi lter started 32 30 52 47 34 50 26 60 71 62 74 AICU-RBH hours of CVVHD 1,154 1,463 1,769 1,532 1,317 1,565 693 1,422 2,215 2,157 2,393 AICU-RBH estimated no bags 544 602 763 689 636 695 294 629 995 952 1,054 AICU-RBH fl uid cost (£) 4,994 5,526 7,004 6,325 5,838 6,380 2,699 5,774 9,134 8,739 9,676 AICU-RBH ml/kg/hour actual 39.90 28.17 29.10 30.53 33.93 31.52 31.25 29.90 30.40 32.55 32.36 AICU-RBH ml/kg/hour 24 hours 32.46 23.44 22.23 23.60 29.32 24.43 21.34 23.93 25.25 25.38 24.01 AICU-RBH percent on CVVHDF 0.79 0.84 0.75 0.67 0.84 0.77 0.66 0.77 0.82 0.76 0.73 AICU-RBH average fi lter life (hours) 36.06 48.77 34.02 32.60 38.74 31.30 26.65 23.70 31.20 34.79 32.34 AICU-RBH fi lter cost (£) 2,477 2,322 4,025 3,638 2,632 3,870 2,012 4,644 5,495 4,799 5,728 AICU-RBH effl uent bags cost (£) 680 753 954 861 795 869 368 786 1,244 1,190 1,318 AICU-RBH total consumable (£) 3,157 3,075 4,979 4,499 3,427 4,739 2,380 5,430 6,739 5,989 7,045 AICU-RBH total cost CVVHD (£) 8,151 8,601 11,983 10,824 9,265 11,119 5,079 11,204 15,873 14,728 16,721 AICU-RBH average cost per patient (£) 1,019 1,075 705 722 1,544 1,588 391 934 1,764 982 796 AICU-RBH average cost per patient day (£) 134 118 122 126 143 131 115 146 142 124 122 Table 1 (abstract P396). KPI for renal replacement therapy 2013 during the study. Prior to doses were 39 and 31 ml/ and delivered doses were igher than our guideline es prior to the change in hat the change in practice y and allowed monitoring hat it is in excess of 20 ml/ data collection processes. r organ systems, therapies nal-replacement therapy in 7. P397 Eff ectiveness of sub-albumin protei post-cardiac surgery rhabdomyolys G Paternoster, A Covino, R Mercorella, C San Carlo Hospital, Potenza, Italy Critical Care 2014, 18(Suppl 1):P397 (do Introduction A high postoperat concentration predicts the inciden and need for renal replacement ther surgical settings. The incidence of suc be 2 to 5% of all causes of AKI [1], bu cardiac surgery (40.3%) [1]. MyG is a be removed with RRT, typically in co membranes, removing sub-albumin can be used in diff usive treatments limiting albumin loss and easily comb pivotal for cardio-surgical settings. W EMIC2 with citrate anticoagulation i surgical patients. Plasma fi ltration with dialysis (plasma diafi ltration) in critically ill patients with acute liver failure Plasma fi ltration with dialysis (plasma diafi ltration) in critically ill patients with acute liver failure Y Eguchi1, H Nakae2, T Furuya3, M Isono4, Y Kishi5, T Yoshioka6 1Shiga University of Medical Science, Shiga, Japan; 2Akita University School of Medicine, Akita, Japan; 3Akita Red Cross Hospital, Akita, Japan; 4Otsu Municipal Hospital, Shiga, Japan; 5Hikone Municipal Hospital, Hikone, Japan; 6Nishi-Kyoto Hospital, Kyoto, Japan Critical Care 2014, 18(Suppl 1):P399 (doi: 10.1186/cc13589) Y Eguchi1, H Nakae2, T Furuya3, M Isono4, Y Kishi5, T Yoshioka6 1Shiga University of Medical Science, Shiga, Japan; 2Akita University School of Medicine, Akita, Japan; 3Akita Red Cross Hospital, Akita, Japan; 4Otsu Municipal Hospital, Shiga, Japan; 5Hikone Municipal Hospital, Hikone, Japan; 6Nishi-Kyoto Hospital, Kyoto, Japan Critical Care 2014, 18(Suppl 1):P399 (doi: 10.1186/cc13589) y Results The pretreatment MyG median value was 10,789 ng/ml; it signifi cantly reduced on average 92.8% during CVVHD (see Figure 1) and it remained low at ICU discharge (median value 114 ng/ml). sCr remained stable (average time value equal to 0.94 mg/dl) during CVVHD; PCT also decreased over time with a reduction rate equal to 78% (from 5.35 ± 4.39 mg/dl to 1.23 ± 1.09 mg/dl at the end of CVVHD). Finally, six patients survived at 90 days. Introduction Removing the middle molecular weight substances including cytokines and albumin-bound toxin could be eff ective for patients with acute liver failure (ALF). We have developed a new system, plasma fi ltration with dialysis (plasma diafi ltration (PDF)) [1,2], and assessed its effi cacy in multicenter analysis. y y Conclusion This small experience confi rms that serum MyG is likely to increase in post-cardiac surgical high-risk patients and suggests a benefi cial eff ect of CRRT treatments with EMIC2 membranes and citrate on serum MyG, potentially preventing AKI. Further larger assessment can be advised for confi rmation. R f fi y y Methods A subgroup analysis of an observational study conducted in the ICUs of six hospitals. In PDF, simple plasma exchange is performed using a selective membrane plasma separator (Evaxclio EC-2A; Kawasumi Chemical Inc., Tokyo Japan), which has a sieving coeffi cient of 0.3 for albumin, while the dialysate fl ows outside the hollow fi bers. The fl ow rate of the blood, dialysate, substitute and additional substitute was 80 to 100 ml/minute, 600 ml/hour, and 0 to 450 ml/hour according to the rate of water elimination and 150 ml/ hour, respectively. 1. Keil R, et al.: Comp Met Progr Biomed 2013, S0169:2607. P399 and mean aortic cross-clamping of 98 minutes (range 25 to 190). We measured MyG, procalcitonin (PCT), and creatinin (sCr) at ICU admission and, if serum MyG was higher than 600 mg/dl, the patient was treated with CVVHD-EMIC2–citrate anticoagulant within 12 hours of ICU admission for 72 hours and a dose of 2,000 ml/hour. Biochemical assays were obtained at 12, 24, and 72 hours and at ICU discharge. Myoglobin removal of small-protein leakage membrane (EMIC2) in patients in the ICU: a case series P Fabbrini1, R Rona1, M Migliari1, M Viganò1, A Pesenti2, A Stella2 1AO San Gerardo, Monza, Italy; 2Università degli Studi Milano-Bicocca, Milan, Italy Critical Care 2014, 18(Suppl 1):P398 (doi: 10.1186/cc13588) y p g Results A multicenter study was underway from October 2005 to August 2011. We performed PDF on 65 patients with ALF (severe sepsis, 22; post operation, 15; fulminant hepatitis, 11; alcohol hepatitis, 3; graft versus host disease, 4; and others, 10). The serum total bilirubin, plasma PT-INR and the model for end-stage liver disease (MELD) score before the PDF procedure were 15.0 ± 8.15 mg/dl (average ± SD), 2.3 ± 1.5 and 35.8 ± 9.3, respectively. PDF was performed as 9.2 ± 13.2 sessions per patient and the overall 28-day survival rate was 68.5%. According to the severity of the MELD score, we stratifi ed patients into three categories defi ned by the MELD score. The numbers of patients were 15 (23%) in score 20 to 29, 30 (46%) in score 30 to 39 and 19 (29%) in score over 40. The 28-day survival rates were 73.3%, 40% and 16%, respectively. Introduction Rhabdomyolysis is characterized by breakdown of striated muscle due to a great number of causes. Acute kidney injury (AKI) is a common complication as a consequence of high concentrations of circulating myoglobin (Mb). The AKI degree can vary but often requires dialysis, a condition which drastically worsens the ICU stay and prognosis. Since Mb overconcentration represents the cause of AKI, one of the therapy’s aims should be its removal to prevent further kidney damage and to allow faster renal recovery. Both intermittent hemodialysis and high-volume CVVHF are poorly eff ective in removing Mb, while small- protein leakage membranes seem to be promising in this setting. The aim of our study was therefore to measure effi cacy of Mb removal of a new high cutoff membrane (EMIC2; Fresenius, cutoff value 40 kDa) for continuous renal replacement therapies (CRRT) in the ICU setting. Conclusion PDF may be a useful blood purifi cation therapy for ALF, but PDF should be performed below a MELD score of 30. P400fi Effi cacy of continuous plasma diafi ltration therapy T Komura, T Taniguchi, T Noda, M Okajima, S Kaneko Kanazawa University, Kanazawa, Japan Critical Care 2014, 18(Suppl 1):P400 (doi: 10.1186/cc13590) g p p Results The median Mb value at CRRT start was 6,971 ng/ml (range 4,679 to 48,011 ng/ml). CRRT were delivered with an average blood fl ow rate of 143 ± 45 ml/minute and a dialysate fl ow rate of 2,134 ± 1,334 ml/hour. These operating conditions allowed one to stop treatment on average after 75 ± 47 hours (median 54 hours) with a Mb reduction of 82.2% (range 99.4 to 44.4%). Overall median Mb removal per treatment was 59 mg (range 33 to 279 mg) mainly due to the fi rst 24 hours of treatment (54 mg, range 20 to 187 mg). Only two patients had residual renal function that was in one case measured to account for only 7.45 mg Mb removal during the entire treatment. Six patients survived and recovered renal function with no dialysis need at present follow-up. One patient died during the ICU stay. Introduction Acute liver failure (ALF) is a critical illness with high mortality. Plasma diafi ltration (PDF) is a blood purifi cation therapy in which simple plasma exchange is performed using a selective membrane plasma separator while the dialysate fl ows outside the hollow fi bers. While several studies demonstrated that PDF therapy is a useful blood purifi cation therapy for patients with ALF, PDF therapy is often diffi cult to employ in ALF patients complicated with multiple organ failure, especially in those with unstable hemodynamics. Furthermore, it is likely to re-occur immediately after PDF therapy. We developed continuous PDF (CPDF) as a new concept in PDF therapy, and assessed its effi cacy and safety in ALF patients compared with conventional plasma exchange (PE) plus continuous hemodiafi ltration (CHDF) therapy in this study. Conclusion Our data measured high performance of the EMIC2 membrane in Mb removal and confi rm theoretical models indicating that CRRT with a high cutoff membrane can achieve major Mb removal within 24 hours with great superiority in comparison with all other available techniques. Methods Ten ALF patients (gender: male/female = 6/4, age: 47 ± 14) employed CPDF therapy. The primary outcomes were altered liver function, measured by the model for end-stage liver disease (MELD) score, and total bilirubin and prothrombin time International References Methods We report results of EMIC2-based treatments in seven patients (four male/three female) with diff erent causes of rhabdomyolysis (trauma, sepsis, limb ischemia). Five patients had classic dialysis indications (persistent anuria) while in two patients treatment was prophylactically started. CRRT were delivered in CVVHD mode with the EMIC2 dialyzer and with loco-regional trisodium–citrate anticoagulation. Mb plasma levels were assessed each 12 hours while the removal rate, total body and dialyzer clearances were estimated by kinetic modeling as previously described [1]. Clinical data were also collected and both global and renal patient survival was reported. 1. Mori T, et al.: Ther Apher 2002, 6:463-466. 2. Nakae H, et al, Ther Apher Dial 2010, 14:444-450. 1. Mori T, et al.: Ther Apher 2002, 6:463-466. 2. Nakae H, et al, Ther Apher Dial 2010, 14:444-450. Plasma fi ltration with dialysis (plasma diafi ltration) in critically ill patients with acute liver failure As the substitute from the additional fl uid line, we added 1,200 ml (150 ml/hour) of fresh frozen plasma followed by 50 ml of 25% albumin considering the loss of albumin by diff usion. As an anticoagulant, nafamostat mesilate (Torii Pharmaceutical Co. Ltd, Tokyo, Japan) was used at a rate of 15 to 25 mg/hour. 1. Benedetto U, et al.: J Thorac Cardiovasc Surg 2010, 140:646-670. 1. Benedetto U, et al.: J Thorac Cardiovasc Surg 2010, 140:646-670. Reference 1. RENAL Investigators: Intensity of continuous renal-replacement therapy in critically ill patients. N Engl J Med 2009, 361:1627. EMIC2 with citrate anticoagulation in AKI prevention of post-cardiac surgical patients. Methods This is a case series of eight patients (mean age 62.7 years, fi ve male, EuroSCORE log 15.61) in cardiac surgery on CPBP for 150 minutes Figure 1 (abstract P396). Delivered dose of RRT each month. Figure 1 (abstract P397). Figure 1 (abstract P396). Delivered dose of RRT each month. Figure 1 (abstract P396). Delivered dose of RRT each month. g p Methods This is a case series of eight patients (mean age 62.7 years, fi ve male, EuroSCORE log 15.61) in cardiac surgery on CPBP for 150 minutes Figure 1 (abstract P397). Figure 1 (abstract P397). Figure 1 (abstract P397). S144 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P403 Pharmacokinetics of meropenem during continuous renal replacement therapy in critically ill patients J Solé Violán1, J Ferrer Agüero1, B Sádaba2, S Sancho3, R Zaragoza3, P Luque4, M Nieto4, M López5, F García6, C Hernández6, J Azanza2 1HUGC Dr Negrín, Las Palmas de GC, Spain; 2Universidad de Navarra, Pamplona, Spain; 3Hospital Dr Peset, Valencia, Spain; 4Hospital Clínico, Zaragoza, Spain; 5Hospital General Yagüe, Burgos, Spain; 6Hospital Morales Meseguer, Murcia, Spain Critical Care 2014, 18(Suppl 1):P403 (doi: 10.1186/cc13593) p p g Methods Patients with microbiologically confi rmed severe sepsis/ septic shock and AKI (RIFLE criteria F or more) treated with HCO- CVVHD, started within 12 hours from the diagnosis, were prospectively included in the study. The cumulative vasopressor index (CVI), C-reactive protein levels (CRP), serum lactate concentration (Lac) and central venous oxygen saturation (ScvO2) were measured before (T0h) and at 24 hours and 48 hours after HCO-CVVHD initiation. Data are expressed as the median (range). The Mann–Whitney U test was applied to detect diff erences in CVI, CRP, Lac and ScvO2 at the three time points (statistical signifi cance for P <0.05). Introduction Antibiotic dosing for patients with acute renal failure receiving continuous renal replacement therapy (CRRT) is a clinical challenge. The aim of this study was to investigate the pharmacokinetics of meropenem (M) during CRRT. Methods A prospective and multicenter study was conducted at seven hospitals. Fifteen critically ill patients undergoing either continuous venovenous hemofi ltration (CVVHF) or hemodiafi ltration (CVVHDF) were included. Serum and ultrafi ltrate (UF) levels of M were determined by liquid chromatography. Blood samples were drawn 24 hours after starting CRRT at 08:00 a.m., 09:00 a.m., 10:00 a.m., 01:00 p.m., 06:00 p.m., 20:00 p.m. and 08:00 a.m. of the following day. CRRT clearance (Cl), total amount of M in the UF (MUF), percentage of the dose extracted by CRRT (EF) and the AUC (ng/hour/ml) were calculated. i Results In 16 ICUs, a total of 16 patients (six cardiac surgery, four abdominal surgery and six medical) met the inclusion criteria and were enrolled in the study. A signifi cant reduction in CRP levels was observed over time: 263 (216 to 358) mg/dl at T0h to 153 (56 to 186) mg/dl at T48h (P <0.05). ScvO2 signifi cantly increased from 45 (40 to 55)% at T0h to 75 (68 to 77)% at T48h (P <0.05). Pharmacodynamics and pharmacokinetics of ciprofl oxacin during sustained low-effi ciency dialysis Pharmacodynamics and pharmacokinetics of ciprofl oxacin during sustained low-effi ciency dialysis fi K Olsen, T Plumb, N Reardon, K Bogard, A Branch-Woods, G Peitz University of Nebraska Medical Center, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P402 (doi: 10.1186/cc13592) Results The MELD score (34.5 to 28.0; P = 0.005), total bilirubin (10.9 to 7.25 mg/dl; P = 0.048), PT-INR (1.89 to 1.31; P = 0.084), and SOFA score (10.0 to 7.5; P <0.039) were improved 5 days after CPDF therapy. Nine patients were alive and one patient died due to acute pancreatitis, complicated by ALF. The effi cacy of CPDF therapy for maintaining liver function and renal function was not inferior to PE plus CHDF therapy. Parameters of renal function such as the creatinine value were also improved 5 days after CPDF therapy. Circulation parameters such as mean arterial pressure and heart rate were maintained without inotropic and vasopressor support during the CPDF treatment period. The oxygenation index (PaO2/FiO2) as a measure of pulmonary function tended to increase after this treatment. We could employ this treatment without any adverse events, such as infections and unstable hemodynamics. Introduction Little information is available regarding ciprofl oxacin pharmacokinetics and pharmacodynamics in sepsis patients receiving sustained low-effi ciency dialysis (SLED). This study determined the pharmacokinetics and simulated pharmacodynamics of ciprofl oxacin in ICU patients during SLED. Methods This study was a prospective evaluation of ciprofl oxacin pharmacokinetics in patients with sepsis and >18 years of age, urine output <200 ml/day and receiving SLED for at least 8 hours. Following informed consent, plasma samples were collected at baseline and 1, 2, 4, and 8 hours after a ciprofl oxacin 400 mg dose i.v. during SLED and post-SLED therapy at the same times. Dialysate samples were collected at 4-hour intervals during SLED. Pharmacokinetic parameters were determined using WinNonlin and compared between the two periods. Simulated pharmacodynamic parameters were determined for Pseudomonas aeruginosa using MIC = 2. y Conclusion In the present study, CPDF therapy safely supported liver function and generally improved the condition of critically ill patients with ALF. Results A total of seven patients (four male, three female, age 56.9 ± 7.6, APACHE II 26.8 ± 2.4) were enrolled. Ciprofl oxacin was cleared relatively rapidly with a half-life of 6.9 hours and a Ke of 0.108/hour during SLED compared with 11.9 hours and 0.057/hour post-SLED (P <0.05). Simulated pharmacodynamics demonstrated inadequate coverage for P. 1. Morgera S: Crit Care Med 2006, 34:2099-2104. P403 Finally, serum lactate decreased from 5.1 (3.0 to 9.5) mmol/l at T0h to 1.6 (1.0 to 4.6) mmol/l at T48h (P <0.05). Conversely, CVI did not signifi cantly reduce over time (8.2 (4 to 9) at T0h vs. 4.5 (4 to 8) at T48h, P >0.05). g Results Nine patients were treated with CVVHDF and six with CVVHF. M (0.5 to 2 g) was administered every 6 to 12 h by i.v. infusion over 15 minutes. Data (mean and SD) concerning the dialysate fl ow rate (DF; ml/hour), blood fl ow rate (ml/minute) and the average UF rate (ml/ kg/hour) for CRRT techniques are shown in Table 1. Pharmacokinetic Conclusion Our preliminary data show that patients with sepsis- related AKI may benefi t from early treatment with HCO-CVVHD. The modulation of proinfl ammatory and anti-infl ammatory mediators, as previously demonstrated [1], may improve microcirculation, tissue perfusion and cellular oxygenation. Although promising, our results must be confi rmed at the end of the study with larger observations. Finally, a subgroup analysis is absolutely mandatory in order to explore diff erent behaviors of tissue perfusion indexes in diff erent populations of patients. Table 1 (abstract P403). Parameters of CRRT techniques CVVHF (n = 6) CVVHDF (n = 9) DF rate 964/94 Blood fl ow rate 190/33 187/29 UF rate 37.7/5.7 29.8/15.6 Table 1 (abstract P403). Parameters of CRRT techniques Reference S145 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Normalized Ratio (PT-INR), 5 days after CPDF therapy. Secondary outcomes included Sequential Organ Failure Assessment (SOFA) scores, 5 days after CPDF therapy, and the survival rate 14 days after this therapy. Pharmacodynamics and pharmacokinetics of ciprofl oxacin during sustained low-effi ciency dialysis aeruginosa during SLED with Cmax/MIC ratio 5.7 ± 1.2 and AUC/MIC 77.5 ± 22.3.l P401 P401 Hemodialysis with high cutoff membranes improves tissue perfusion in severe sepsis: preliminary data of the Sepsis in Florence sTudy (SIFT) G Villa, C Chelazzi, S Valente, AR De Gaudio, S Romagnoli University of Florence, Italy Critical Care 2014, 18(Suppl 1):P401 (doi: 10.1186/cc13591) Conclusion Ciprofl oxacin is rapidly cleared during SLED similar to clearance during normal renal function, which may result in adequate pharmacodynamic coverage for some pathogens. Introduction It has been demonstrated that blood purifi cation therapy performed by means of venovenous hemodialysis with high cutoff membranes (HCO-CVVHD) may modulate the host infl ammatory response in septic patients with acute kidney injury (AKI), potentially limiting organ dysfunction. Improvement in hemodynamics and respiratory function has been described during HCO-CVVHD treatment [1]. The Sepsis in Florence sTudy (SIFT) has been designed to evaluate changes in infl ammatory biomarkers and tissue oxygenation/perfusion indexes in septic ICU patients with AKI during HCO-CVVHD.i P406 Long-term outcomes in acute kidney injury patients treated with renal replacement therapy who were alive at hospital discharge V Sergoyne, W De Corte, J Vanhalst, A Dhondt, S Claus, S Oeyen, J Decruyenaere, E Hoste University of Ghent, Belgium Critical Care 2014, 18(Suppl 1):P406 (doi: 10.1186/cc13596) Results Data from 33 patients receiving renal replacement therapy were analysed. Mean time (± SD) of treatment interruption was 1.4 ± 2.7 hours/day due to fi lter changes, transfers for CT scans and surgical procedures. Therefore, 94% of the prescribed dose was delivered. There was a mean of 2.3 fi ltration dose prescriptions per patient over the observation period (due to changing clinical conditions), with a total of 77 dose adaptations. Mean E/ABW was signifi cantly higher than calculated IBW (76.3 kg vs. 61.4 kg), and thus a diff erence of 14.9 kg (95% CI = 9.2 to 20.6 kg). This resulted in a signifi cant diff erence in mean fi ltration dose delivered of 33.1 ml/kg/ hour for E/ABW versus 39.7 ml/kg/hour for IBW respectively (P <0.001). As a consequence, in 29.6% (8/27) of cases where HVHDF (>35 ml/kg/ hour) was prescribed, standard volume haemodiafi ltration (SVHDF) (≤35 ml/kg/hour) was delivered. In 10% (5/50) of cases where SVHDF was prescribed, HVHDF was delivered (P <0.001). Introduction Acute kidney injury (AKI) treated with renal replacement therapy (RRT) in ICU patients is associated with high mortality, chronic kidney disease (CKD) (eGFR <60 ml/minute/1.73 m2, or CKD stage ≥3) and end-stage kidney disease (ESKD). Data on long-term outcomes vary among studies due to diff erences in age, CKD, severity of illness, RRT modality and timing of initiation of RRT. Long-term patient and kidney outcomes in AKI-RRT patients were evaluated in this study. y p y Methods A retrospective study of all consecutive treated AKI-RRT patients in a 50-bed ICU academic hospital from August 2004 to December 2012. Data were retrieved from the electronic ICU, RRT and hospital patient fi les. Long-term outcomes data were obtained by a telephone survey. p Conclusion We conclude that the delivered UF rate in our cohort of patients diff ered signifi cantly depending on measured or calculated body weight. In almost one-third of cases where HVHDF was prescribed, SVHDF was delivered. As many interventions in the ICU are based on IBW and daily weighing of patients is not uniformly practiced, this makes comparison of data diffi cult. Impact of ideal versus estimated body weight on haemofi ltration dosing in critically ill patients with AKI y p y Results During the 9-year study period, a total of 1,291 AKI-RRT patients were included. Short-term mortality at day 30 was 47.2%; mortality at 1 year was 64.3%. Compared with nonsurvivors, 1-year survivors had similar age (65 vs. 67 years, P = 0.077), worse kidney function at baseline (eGFR 46 vs. 52 ml/minute/1.73 m2, P = 0.001; CKD stage ≥3 65% vs. 58%, P = 0.019), a greater proportion was male (69.0% vs. 63.2%, P = 0.048), and more were admitted to the cardiac surgery ICU (39% vs. 46%, P = 0.012). They were less severely ill as illustrated by lower SAPS 2 score at ICU admission (52 vs. 69, P <0.001), and at the time of initiation of RRT, and a lower SOFA score (9 vs. 11; P <0.001). A smaller proportion was on mechanical ventilation (85.1% vs. 89.7%, P = 0.042) and on vasoactive drugs (45.7% vs. 63.9%, P <0.001). Survivors had earlier initiation of RRT (2 vs. 3 days, P = 0.034), and more frequently intermittent RRT was used (84.8% vs. 63.2%; P <0.001). When corrected for gender, age, severity of illness, and modality and timing of RRT, worse baseline kidney function, defi ned as CKD stage ≥3, remained associated with better 1-year survival (odds ratio 1.6, 95% CI: 1.1 to 2.2, P = 0.011). Introduction Acute kidney injury (AKI) in the critically ill is an independent risk factor for adverse outcome [1]. Previously, it was suggested that high-volume haemofi ltration (HVHF) may confer a mortality benefi t and lead to a reduction in organ failure compared with standard ultrafi ltration rates (UF) [2]. This was not confi rmed by a recent investigation [3]. It has also not been determined whether ideal (IBW) or estimated/actual body weight (E/ABW) was used to calculate UF rates. We sought to determine what impact diff erent weight calculations have on delivered UF rates. Methods A retrospective single-centre study in a tertiary referral institution. Continuous venovenous haemodiafi ltration (CVVHDF) was administered according to the patient’s IBW. The delivered UF rate was then calculated both for IBW and E/ABW. The latter was based on measurements obtained at the time of ICU admission. We compared the highest predilution, postdilution and dialysate volume administered each day according to the diff erent weight estimate measurements respectively. Student’s t tests and chi-square tests were used for statistical analysis (P <0.05). 1. Chertow GM, et al.: J Am Soc Nephrol 2005, 16:3365-3370. 2. Ronco C, et al.: Lancet 2000, 355:26-30. 3. Bellomo R, et al.: N Engl J Med 2009, 361:1627-1638. Reference Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S146 Table 2 (abstract P403). Pharmacokinetic parameters for meropenem CVVHF CVVHDF AUC 357/188 433/369 M dose (g/day) 1.7/0.4 2.2/1.4 MUF (mg) 338/104 625/499 EF (%) 28/13 30/26 M Cl (l/hour) 1.2/0.7 1.7/1.5 P405 ICU patients treated with RRT for AKI who have chronic kidney disease have better 1-year outcome compared with patients with better kidney function W De Corte1, V Sergoyne2, J Vanhalst3, A Dhondt4, S Claus4, S Oeyen4, J Decruyenaere4, E Hoste4 1AZ Groeninge, Kortrijk, Belgium; 2Stedelijk Ziekenhuis, Aalst, Belgium; 3Sint- Jozefziekenhuis, Izegem, Belgium; 4Ghent University Hospital, Ghent, Belgium Critical Care 2014, 18(Suppl 1):P405 (doi: 10.1186/cc13595) parameters for M are depicted in Table 2. No diff erences in either EF or M Cl between the two CRRT techniques were observed. Conclusion A signifi cant removal of M by CVVHF/CVVHDF was observed. Dose adjustment is necessary in critically ill patients receiving CRRT. Acknowledgement Supported by EC 81/00469. Table 2 (abstract P403). Pharmacokinetic parameters for meropenem CVVHF CVVHDF AUC 357/188 433/369 M dose (g/day) 1.7/0.4 2.2/1.4 MUF (mg) 338/104 625/499 EF (%) 28/13 30/26 M Cl (l/hour) 1.2/0.7 1.7/1.5 Introduction Chronic kidney disease (CKD) is a risk factor for developing acute kidney injury (AKI) with need for renal replacement therapy (RRT). AKI-RRT is associated with important short-term mortality, and recent data showed there is also important increased risk for 1-year mortality. The aim of this study is to evaluate variables associated with 1-year survival, and in particular the impact of baseline CKD in a cohort of AKI-RRT patients. parameters for M are depicted in Table 2. No diff erences in either EF or M Cl between the two CRRT techniques were observed.i q Conclusion A signifi cant removal of M by CVVHF/CVVHDF was observed. Dose adjustment is necessary in critically ill patients receiving CRRT. g Acknowledgement Supported by EC 81/00469. Methods A single-center observational study in a 50-bed ICU tertiary care hospital. During the study period August 2004 to December 2012, all consecutive adult AKI-RRT patients were included. Data were retrieved from the electronic ICU patient fi le, the electronic hospital patient fi le and the electronic ICU-RRT database. Long-term outcome data were collected by a telephone survey. Impact of ideal versus estimated body weight on haemofi ltration dosing in critically ill patients with AKI Conclusion In ICU patients who had AKI-RRT, 1-year survival was associated with lower severity of illness. Surprisingly, worse kidney function or CKD stage ≥3 was also associated with long-term survival. This eff ect remained present after adjudication for relevant covariates. P406 References Results During the study period 1,291 ICU patients were treated with RRT for AKI. Mortality was 47.2% at day 30 and 57.2% at hospital discharge. Mortality in hospital survivors showed an important increase until 3-year follow-up, and a moderate increase later (1 year: 14.4%, 2 years: 20%, 3 years: 35.7%, and 7 years: 39%). In-hospital survivors’ Scr and eGFR at baseline were comparable to 1-year follow-up (1.4 vs. P409 P409 Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility bundle in adult ICU patients: a preliminary cost analysis G Peitz1, K Dvoracek2, J Sankaranarayanan3, M Balas4, K Olsen1 1University of Nebraska Medical Center, Omaha, NE, USA; 2Avera McKennan Hospital and University Health Center, Sioux Falls, SD, USA; 3University of Conneticut/Hartford School of Pharmacy, Storrs, CT, USA; 4The Ohio State University, Columbus, OH, USA Critical Care 2014, 18(Suppl 1):P409 (doi: 10.1186/cc13599) Polymyxin B-immobilized fi ber hemoperfusion therapy improves sepsis-related immunosuppression Cruz DN, Antonelli M, et al.: Early use of polymyxin B hemoperfusion in abdominal septic shock. The EUPHAS randomized controlled trial. JAMA 2009, 301:2445-2452. P407 Polymyxin B-immobilized fi ber hemoperfusion therapy improves sepsis-related immunosuppression A Kimura, S Ono, S Hiraki, R Takahata, H Tsujimoto, M Kinoshita, S Aosasa, K Hatsuse, D Saitoh, K Hase, J Yamamoto National Defense Medical College, Tokorozawa, Japan Critical Care 2014, 18(Suppl 1):P407 (doi: 10.1186/cc13597) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1.4 mg/dl, P = 0.162, respectively 46 vs. 51 ml/minute/1.73 m2) and we observed an increase of CKD stage in 36.0% of patients, a decrease in 36.3%, and stable CKD stage in 27.7%. A total 43.1% of patients with CKD stage <3 at baseline had an increase to CKD stage ≥3 at 1 year. A total 26.6% of patients with CKD stage ≥3 at baseline decreased to CKD stage <3. A total of 8.3% of hospital survivors developed ESKD. Patients with increase of CKD stage had similar age (67 vs. 64 years, P = 0.145), SAPS 2 (57 vs. 51, P = 0.858), and SOFA score (9 vs. 9, P = 0.275) compared with patients with stable or decreased CKD stage. There were no diff erences in type of ICU, modality of RRT, or number of patients treated with vasopressors (44% vs. 47%, P = 0.617) or invasive ventilation (84% vs. 81%, P = 0.496). However, these patients were more probably male (76.9% vs. 65.6%, P = 0.042), had lower Scr at baseline (1.1 vs. 1.7 mg/dl, P <0.001) and at ICU admission (1.7 vs. 2.4 mg/dl, P <0.001), and were started on RRT later (3 vs. 2 days, P = 0.008). Endotoxin activity assay and polymyxin B hemoperfusion use in a cohort of critically ill patients Endotoxin activity assay and polymyxin B hemoperfusion use in a cohort of critically ill patients y SL Cutuli, G De Pascale, V Alicino, S Cicconi, V Di Gravio, D Silvestri, D Giacobelli, E Gasperin, S Marsili, MS Vallecoccia, M Antonelli Sacro Cuore Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P408 (doi: 10.1186/cc13598) Introduction Endotoxin plays a crucial role in the pathogenesis of severe sepsis and septic shock (SS&SSh) [1]. The aim of this study is to analyze the impact of extracorporeal endotoxin removal with polymyxin B hemoperfusion (PMX-DHP) (Toraymyxin®). y y y y Methods All patients admitted to our ICU between 1 April 2011 and 30 June 2013 who developed SS&SSh and underwent endotoxin activity assay (EAA) measurement were retrospectively evaluated. y Conclusion The annual mortality in AKI-RRT hospital survivors is approximately 10% per year during the fi rst 3 years of follow-up. One- third of patients had an increase of CKD stage at 1 year of follow-up and almost one-half of patients who had eGFR >60 ml/minute/1.73 m2 developed CKD. Patients who had an increase of CKD had similar severity of illness, but lower Scr at baseline and at ICU admission, and had later initiation of RRT compared with patients who had stable or decreased CKD stage. Results During the study period, EAA was dosed in 100 patients. Eighty- one patients were aff ected by septic shock. The source of infection was identifi ed in 70.4% of cases (45% abdominal) and the percentage of microbiologically confi rmed episodes was 77% (81% Gram-negatives). The mortality rate was 49%. The mean EAA level was 0.66 ± 0.2, and in 66% of patients the value was higher than 0.6. No signifi cant diff erences were found in terms of SAPS II (P = 0.32) and SOFA score (P = 0.67), according to EAA level (>/≤0.6). Patients with levels >0.6 presented a higher percentage of microbiologically confi rmed infections (84% vs. 66%; P = 0.09). Thirty-two of 66 patients with EAA >0.6 were treated with Toraymyxin®. No complications leading to treatment interruption were recorded and a relevant decrease of cardiovascular SOFA score and lactate levels were observed 72 hours after treatment (P = 0.05 and P = 0.06, respectively). Source control and Toraymyxin® treatment resulted as the only modifi able factors improving the ICU survival rate (Table 1). Polymyxin B-immobilized fi ber hemoperfusion therapy improves sepsis-related immunosuppression p pp A Kimura, S Ono, S Hiraki, R Takahata, H Tsujimoto, M Kinoshita, S Aosasa, K Hatsuse, D Saitoh, K Hase, J Yamamoto National Defense Medical College, Tokorozawa, Japan Critical Care 2014, 18(Suppl 1):P407 (doi: 10.1186/cc13597) Table 1 (abstract P408). Multivariate analysis for ICU mortality risk factors P value OR (95% CI) SAPS II score 0.04 1.1 (1.01 to 1.1) Septic shock 0.02 10.4 (1.5 to 71) PMX-DHP 0.04 0.2 (0.1 to 0.9) Source control 0.01 0.1 (0.03 to 0.5) Table 1 (abstract P408). Multivariate analysis for ICU mortality risk factors Introduction Sepsis-induced immunosuppression has long been considered a factor in the late mortality of sepsis patients, but little is known about the immunity of immunocompetent cells and the eff ect of polymyxin B-immobilized fi ber hemoperfusion therapy (PMX- DHP) on sepsis-induced immunosuppression. The present study was designed to evaluate the eff ect of PMX-DHP on recovery from sepsis- related immunodefi ciency. i Methods Patients with septic shock who were treated with PMX- DHP were enrolled in this study. Study 1: (1) numbers of peripheral lymphocytes and CD4+ T cells, especially regulatory T cells (Tregs), and serum cytokine levels were examined to evaluate the eff ects of PMX- DHP in septic shock patients. (2) Peripheral blood mononuclear cells (PBMCs) in these patients were examined to evaluate infl ammatory cytokine production before and after PMX-DHP. The obtained PBMCs were stimulated with interleukin (IL)-2 and IL-12, anti-CD3 antibody, or lipopolysaccharide for 24 hours, and tumor necrosis factor alpha and interferon-gamma (IFNγ) production in the culture supernatants was measured using enzyme-linked immunosorbent assay. Study 2: whole blood from patients with sepsis was incubated with a polymyxin B-immobilized fi lter (cut into small sizes) for small animals for 2 hours (PMX group), or were treated with 200 μg polymyxin B for 2 hours (PLB group), or were not treated (sepsis group). IFNγ production by PBMCs was compared among the three groups. Conclusion EAA is a rapid and reliable method to identify patients who may be treated with polymyxin B hemoperfusion. Source control and extracorporeal endotoxin removal have appeared as two eff ective interventions that should be implemented in the early management of patients with SS&SSh. 1. Cruz DN, Antonelli M, et al.: Early use of polymyxin B hemoperfusion in abdominal septic shock. The EUPHAS randomized controlled trial. JAMA 2009, 301:2445-2452. 1. P408 Endotoxin activity assay and polymyxin B hemoperfusion use in a cohort of critically ill patients SL Cutuli, G De Pascale, V Alicino, S Cicconi, V Di Gravio, D Silvestri, D Giacobelli, E Gasperin, S Marsili, MS Vallecoccia, M Antonelli Sacro Cuore Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P408 (doi: 10.1186/cc13598) References S147 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 An assessment of long-term sleep quality using actigraphy in survivors of critical illness Methods Fifty-nine patients consented to complete the RCSQ for each night of their ICU admission. Alongside this, the nurses who had cared for these patients were asked to assess their patients’ sleep using the RCSQ. These were then matched with their patients’ responses. The Bland–Altman method was applied to assess for agreement between patient and nurse SEIs in order to reveal whether nurses could accurately estimate their patients’ night sleep. Additionally, Cronbach’s alpha was derived to assess for internal consistency. Ethical approval was gained prior to the start of the study.f K Solverson, C Doig University of Calgary, Canada y y Critical Care 2014, 18(Suppl 1):P410 (doi: 10.1186/cc13600) Critical Care 2014, 18(Suppl 1):P410 (doi: 10.1186/cc13600) Introduction Recent studies have suggested that critical illness survivors experience long-term sleep disruption [1,2]. The Actigraph device is a sleep watch that has been shown to have equivalent accuracy to polysomnography and previously used during critical illness to show sleep disruption [3]. Our objective was to assess patients long-term sleep quality using the Actigraph device. Results A total of 126 pairs or RCSQs were gathered. The mean diff erence between nurse and patient SEIs was –0.9, suggesting there is no signifi cant trend regarding nurses overestimating or underestimating patients’ SEIs. In total, 94.2% of nurses’ estimations fell within the limits of agreement. The variability of the diff erences was consistent across the range of averages. Cronbach’s alpha was 0.63 between nurse and patient scores, suggesting questionable reliability between the RCSQ pairs. See Figure 1. g y g g Methods Study patients were selected from a 24-bed multidisciplinary ICU. Thirteen patients who were ≥18 years old, stayed longer than 4 days in the ICU and did not have and acute brain injury were followed up at 2 months post hospital discharge. The Actigraph device was given to patients to take home and worn for 72 hours. Previously validated algorithms were used to analyze sleep and wake cycles [4]. Additionally, patient completed the Pittsburgh Sleep Quality Index (PQSI), as a measure of subjective sleep quality. p g Conclusion The data gathered here demonstrate that nurses are not able to accurately estimate their patients sleep using the RCSQ, and hence alternative methods of sleep monitoring should be developed for routine use. Results Sixty-two percent of patients at 2 months post hospital discharge reported poor sleep quality as per the PSQI. P411 P411 Study to assess whether staff are able to accurately assess sleep quality and quantity in intensive care patients J Patel, J Baldwin, P Bunting, S Laha Royal Preston Hospital, Manchester, UK Critical Care 2014, 18(Suppl 1):P411 (doi: 10.1186/cc13601) P411 Study to assess whether staff are able to accurately assess sleep quality and quantity in intensive care patients J Patel, J Baldwin, P Bunting, S Laha Royal Preston Hospital, Manchester, UK Critical Care 2014, 18(Suppl 1):P411 (doi: 10.1186/cc13601) yi Results Data were analyzed from 146 and 150 patients in the pre- ABCDE and post-ABCDE groups respectively. There were mean decreases of 0.59 MV days and 0.83 delirium days between the pre and post bundle implementation. The mean costs (per patient per ICU stay) of implementing the bundle components were signifi cantly higher in the post-ABCDE cohort versus the pre-ABCDE cohort ($191 vs. $92, P <0.0001), with an additional increase in total costs ($8,930 vs. $8,624, P = 0.0233). Costs to prevent 1 day of MV and to prevent 1 day of delirium were $552 and $368 respectively. In the non-MV patients, the post-ABCDE bundle demonstrated fewer days of delirium than the pre- ABCDE cohort (0.8 vs. 1.13 days, P <0.05) but had higher overall costs ($5,417 vs. $4,546, P <0.05), resulting in a cost of $2,639 to prevent 1 day of delirium in a non-MV patient. Introduction Sleep deprivation is recognised as an important cause of morbidity after ICU admission, but most centres do not routinely assess their patients’ sleep. Considering the invasive nature and costs associated with objective sleep measurements, they are unsuitable for routine use. Subjective measurements off er an easy-to-use and economical alternative, the most well validated of which is the Richards–Campbell Sleep Questionnaire (RCSQ). This can be used to derive an accurate estimation of the sleep effi ciency index (SEI), a well-validated measure of sleep. However, there are several concerns regarding patients reporting their sleep quality and quantity using these questionnaires [1]. Additionally, they cannot be used to assess sleep in sedated or delirious patients. It has been suggested that one way to bypass the drawbacks of patients assessing their own sleep would be to utilise nursing staff [1]. Previous smaller scare studies have since agreed with this suggestion [2]. This study aimed to assess whether staff were able to use the RCSQ to accurately assess their patients’ sleep. P411 Conclusion Implementing the ABCDE bundle in adult ICU patients appears to be a feasible cost-eff ective strategy when considering costs of mechanical ventilation and ICU delirium. References consisted of the following components: daily spontaneous awakening trials (SATs); daily spontaneous breathing trials coordinated with the SATs; delirium monitoring; and early mobility. The study’s primary endpoint was the cost-eff ectiveness of the ABCDE bundle in terms of the bundle’s cost to prevent 1 day of mechanical ventilation (MV) or 1 day of delirium for all patients, as well as for a subgroup of non-MV patients. All cost-eff ectiveness ratios (CERs) were constructed from the hospital’s cost perspective. The economic analyses were carried out in two steps. First, the mean cost per patient per hospital stay and total costs in the pre-ABCDE and post-ABCDE bundle periods were compared. Next, CERs of each respective primary endpoint were computed as incremental costs of implementing the bundle to prevent 1 day of MV and to prevent 1 day of delirium. P <0.05 was considered statistically signifi cant. References 1. Orwelius L, et al.: Crit Care 2008, 12:R97. 2. Lee C, et al.: Intensive Care Med 2009, 35:314-320. 3. Beecroft J, et al.: Intensive Care Med 2008, 34:2076-2083. 4. Hedner J, et al.: Sleep 2004, 27:1560-1566. 5. Buysse D, et al.: J Clin Sleep Med 2008, 4:563-571. References 1. Orwelius L, et al.: Crit Care 2008, 12:R97. 2. Lee C, et al.: Intensive Care Med 2009, 35:314-320. 3. Beecroft J, et al.: Intensive Care Med 2008, 34:2076-2083. 4. Hedner J, et al.: Sleep 2004, 27:1560-1566. 5. Buysse D, et al.: J Clin Sleep Med 2008, 4:563-571. Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility bundle in adult ICU patients: a preliminary cost analysis Results Study 1: (1) the number of CD4+ T cells was lower and the percentage of Tregs in CD4+ T cells was higher in septic shock patients compared with those without shock. A signifi cant increase in the number of CD4+ T cells, a signifi cant decrease in the percentage of Tregs in the CD4+ T-cell population, and a signifi cant decrease in serum IL-10 levels were observed 24 hours after PMX-DHP in septic shock patients who survived compared with those who did not. (2) IFNγ production by PBMCs was signifi cantly lower in patients with sepsis than in healthy volunteers. IFNγ production by IL-2-stimulated and IL-12-stimulated PBMCs signifi cantly increased after PMX-DHP therapy. Study 2: IFNγ production by PBMCs in patients with sepsis increased signifi cantly in the PMX and PLB groups compared with that in the sepsis group. y G Peitz1, K Dvoracek2, J Sankaranarayanan3, M Balas4, K Olsen1 1University of Nebraska Medical Center, Omaha, NE, USA; 2Avera McKennan Hospital and University Health Center, Sioux Falls, SD, USA; 3University of Conneticut/Hartford School of Pharmacy, Storrs, CT, USA; 4The Ohio State University, Columbus, OH, USA G Peitz1, K Dvoracek2, J Sankaranarayanan3, M Balas4, K Olsen1 1University of Nebraska Medical Center, Omaha, NE, USA; 2Avera McKennan Hospital and University Health Center, Sioux Falls, SD, USA; 3University of Conneticut/Hartford School of Pharmacy, Storrs, CT, USA; 4The Ohio State University, Columbus, OH, USA y Critical Care 2014, 18(Suppl 1):P409 (doi: 10.1186/cc13599) Introduction We previously demonstrated a signifi cant increase in the number of ventilator-free days and reduced the rate of delirium in ICU patients treated with the Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility (ABCDE) bundle. This study investigated whether implementation of the ABCDE bundle was cost- eff ective at an academic medical center.f Conclusion PMX-DHP directly decreased the number and percentage of Tregs in peripheral blood circulating CD4+ T cells in patients with septic shock. PMX-DHP improved IFNγ production by natural killer (NK)/NKT cells in patients with septic shock. Therefore, PMX-DHP could improve sepsis-related immunosuppression. Methods A post-hoc cost-eff ectiveness study was done following the before–after ABCDE bundle implementation study. The bundle S148 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 An assessment of long-term sleep quality using actigraphy in survivors of critical illness The Actigraph results showed patients’ average total sleep time was 6.15 hours, with a sleep effi ciency of 78%. The mean time to fall asleep was 12 minutes. Patients had an average of 11 awakenings per night and were awake for an average of 7 minutes during the awakenings. There were no associations found between patients’ perceived sleep quality and total sleep, sleep effi ciency or sleep disruptions. Patients’ severity of illness, as measured by the APACHE II score, was statistically associated with lower total sleep time (β = –12.6, P = 0.019), reduced sleep effi ciency (β = –1.18, P = 0.042) and higher number of sleep disruptions (β = 0.64, P = 0.023). The number of days ventilated or ICU and hospital length of stay were not statistically associated with the Actigraph sleep parameters. P414 Haemodynamic eff ects of clonidine in an ovine model of severe sepsis with septic acute kidney injury P Calzavacca1, Y Lankadeva2, L Booth2, S Bailey2, M Bailey3, R Bellomo4, CN May2 1AO Melegnano, PO Uboldo, Cernusco sul Naviglio, Italy; 2Melbourne University, Parkville, Australia; 3Monash University, Clayton, Australia; 4Austin Health, Heidelberg, Australia Critical Care 2014, 18(Suppl 1):P414 (doi: 10.1186/cc13604) References 1. Bourne RS, et al.: Crit Care 2007, 11:226. 2. Kamdar et al.: Am J Crit Care 2012, 21:261-269. 2. Kamdar et al.: Am J Crit Care 2012, 21:261-269. Figure 1 (abstract P411). Figure 1 (abstract P411). g Conclusion Survivors of critical illness have high levels of sleep dysfunction as measured by actigraphy. Patients’ severity of illness while critically ill appears to increase the level of long-term sleep dysfunction experienced. There is discordance between objective and subjective measures of sleep quality, which has been shown previously [5]. Objective measures of sleep quality are needed on a larger number of patients to confi rm these fi ndings. Figure 1 (abstract P411). S149 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P412i nursing staff reported they were signifi cantly more confi dent in titrating opioids after implementation (3 (2; 3) and 3 (3; 4) respectively; P <0.01), despite a lack of signifi cant diff erence in their reported confi dence to assess pain. Compliance was good, with a median daily documentation rate of 66% with the standard being once per 8-hour shift. Median BPS score rank was signifi cantly higher on patient movement (2 (1; 2)) compared with at rest (1 (1; 2); P <0.001) (1 = no pain, 2 = mild pain). No statistically signifi cant diff erence was seen in the length of stay, duration of mechanical ventilation, VAP rate or median sedation exposure. Pre-implementation median opioid administration when looking at morphine (mg) equivalence was 455.8 (203.1; 1,174.8), and post implementation the median increased to 620.3 (218.1; 1,502); however, this did not reach statistical signifi cance (P = 0.235). There was no statistically signifi cant change in the prescribing of analgesic adjuncts. The sample size was underpowered to detect signifi cant diff erences. nursing staff reported they were signifi cantly more confi dent in titrating opioids after implementation (3 (2; 3) and 3 (3; 4) respectively; P <0.01), despite a lack of signifi cant diff erence in their reported confi dence to assess pain. Compliance was good, with a median daily documentation rate of 66% with the standard being once per 8-hour shift. Median BPS score rank was signifi cantly higher on patient movement (2 (1; 2)) compared with at rest (1 (1; 2); P <0.001) (1 = no pain, 2 = mild pain). Haemodynamic eff ects of clonidine in an ovine model of severe sepsis with septic acute kidney injury y y P Calzavacca1, Y Lankadeva2, L Booth2, S Bailey2, M Bailey3, R Bellomo4, CN May2 1AO Melegnano, PO Uboldo, Cernusco sul Naviglio, Italy; 2Melbourne University, Parkville, Australia; 3Monash University, Clayton, Australia; 4Austin Health, Heidelberg, Australia Critical Care 2014, 18(Suppl 1):P414 (doi: 10.1186/cc13604) Introduction In sepsis, sympathetic nerve activity is increased, which helps maintain arterial pressure in the face of nitric oxide-induced vasodilatation. Accordingly, we investigated the haemodynamic eff ects of the centrally acting α-adrenoceptor agonist clonidine in an ovine model of severe sepsis. Conclusion Using standard EEG in this small cohort of mechanically ventilated ICU patients, a deep sedation level was frequently observed. Our preliminary data suggest that simplifi ed EEG with the SedLine system is less accurate than the standard 19-channel EEG to assess the depth of sedation in the ICU setting. Methods A prospective interventional blinded crossover study in 12 Merino ewes with cardiac and renal fl ow probes implanted to continuously measure cardiac output and renal blood fl ow. Arterial pressure was continuously monitored and blood and urine samples were taken. After 24 hours of control, sepsis was induced by an intravenous bolus and continuous infusion of live Escherichia coli for 32 hours. After 24 hours of sepsis, animals were randomly and blindly allocated to vehicle infusion or clonidine (1 μg/ml/kg/minute) for 8 hours. The E. coli infusion was then discontinued, gentamycin 150 mg given i.m. and the animals were followed for 16 hours during recovery. The animals that survived were crossed over to the alternative treatment 2 weeks later. Implementation of the Behavioural Pain Scale in sedated mechanically ventilated patients in a UK ICU J Jennings, R Bourne Sheffi eld Teaching Hospital, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P413 (doi: 10.1186/cc13603) Implementation of the Behavioural Pain Scale in sedated mechanically ventilated patients in a UK ICU J Jennings, R Bourne Sheffi eld Teaching Hospital, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P413 (doi: 10.1186/cc13603) Results Complete data were collected on eight animals/group, three animals died/group. Hyperdynamic sepsis with hypotension and acute kidney injury of similar degree developed in the two groups. Haemodynamic and renal eff ects of clonidine are shown in Figure 1. Conclusion In ovine hyperdynamic sepsis, clonidine transiently increased urine output without aff ecting creatinine clearance. Results Complete data were collected on eight animals/group, three animals died/group. Hyperdynamic sepsis with hypotension and acute kidney injury of similar degree developed in the two groups. Haemodynamic and renal eff ects of clonidine are shown in Figure 1. Introduction Pain is a common problem in mechanically ventilated patients and assessing pain in the sedated patient is diffi cult, as patients are often unable to verbalise [1]. Behavioural pain assessment tools have been developed and validated for the assessment of pain in this patient group. A behavioural pain assessment tool was implemented as part of a wider ventilator-associated pneumonia (VAP) prevention programme within a large UK ICU. Conclusion In ovine hyperdynamic sepsis, clonidine transiently increased urine output without aff ecting creatinine clearance. Simplifi ed versus standard EEG to measure the depth of sedation in mechanically ventilated ICU patients Introduction The accurate measure of sedation depth among mechanically ventilated ICU patients remains challenging. The Patient State Index (PSI) is a quantitative measure calculated using an algorithm derived from a simplifi ed four-channel EEG. The aim of this study was to examine the value of the PSI to assess the level of sedation, and to verify its accuracy in comparison with the quantitative spectral analysis derived from a standard 19-channel EEG. Methods Using the SEDLine four-channel simplifi ed EEG system (Masimo Corporation, Irvine, CA, USA), which assessed depth of sedation through two frontal leads, we prospectively studied mechanically ventilated sedated ICU patients and examined whether the PSI was accurate to quantify the individual level of sedation. Pain stimuli were applied and changes in PSI were examined and compared with changes in electrical activity (% of delta power), measured by the frontal Fp2–Fp1 electrodes using a standard 19-channel EEG system (Viasys Neurocare, Madison, WI, USA). EEG recordings were performed simultaneously during 20 minutes, and the relationship between PSI and % of delta power was analyzed with the Pearson’s correlation coeffi cient. f Conclusion The BPS was successfully implemented into routine nursing practice and signifi cantly improved nurse confi dence in opioid infusion titration. Analgesic use was not signifi cantly diff erent between evaluation periods, but the results indicate that further education on anticipating pain and treating pain pre-emptively with timely administration of opioids is needed. References References 1. Puntillo K, et al.: Chest 2009; 135:1069-1074. 2. Payen JF, et al.: Crit Care Med 2001; 29:2258-2263. 2. Payen JF, et al.: Crit Care Med 2001; 29:2258-2263. fi Results Ten consecutive patients (mean age 59 ± 12 years) were included. EEG recordings were performed on average 6 ± 6 days from ICU admission. Sedation consisted of propofol (seven patients), midazolam (two patients) or both (one patient). The median PSI was 54 (range 10 to 97), indicating large individual variations in PSI values. In contrast, the median delta power was 86.3% (range 55.2 to 99.4), indicating a deep sedation level. Pain stimuli were associated with a signifi cant increase of PSI value from baseline 51 (range 16 to 96) to 68 (42 to 95) (P = 0.009). However, we found no correlation between the PSI and the % delta power, both at baseline (R = 0.32, P = 0.37) and after pain stimulation (R = 0.17, P = 0.63). The % delta power from frontal electrodes (Fp2–Fp1) was well correlated with that obtained from posterior electrodes (P4–Pz; R = 0.42, P < 0.0001), and remained unchanged after pain stimulation, indeed confi rming a deep sedation level in our patient cohort. References No statistically signifi cant diff erence was seen in the length of stay, duration of mechanical ventilation, VAP rate or median sedation exposure. Pre-implementation median opioid administration when looking at morphine (mg) equivalence was 455.8 (203.1; 1,174.8), and post implementation the median increased to 620.3 (218.1; 1,502); however, this did not reach statistical signifi cance (P = 0.235). There was no statistically signifi cant change in the prescribing of analgesic adjuncts. The sample size was underpowered to detect signifi cant diff erences. P412 Simplifi ed versus standard EEG to measure the depth of sedation in mechanically ventilated ICU patients T Suys, P Bouzat, A Rossetti, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2014, 18(Suppl 1):P412 (doi: 10.1186/cc13602) P415f Methods The Behavioural Pain Scale (BPS) [2] was selected and implemented into daily clinical practice supported by a tailored education programme and incorporation into the clinical information system (MetaVision). Questionnaires pre and post BPS implementation were used to assess nursing staff opinions on pain assessment and opioid infusion titration. Four months of data were compared pre and post implementation, examining aspects of patient sedation and analgesia exposure and ventilated patient outcome parameters. Off -label use of clonidine for sedation in Dutch ICUs J Van der Valk, M Zeeman, M Arbouw, H Van den Oever Deventer Hospital, Deventer, the Netherlands Critical Care 2014, 18(Suppl 1):P415 (doi: 10.1186/cc13605) Off -label use of clonidine for sedation in Dutch ICUs J Van der Valk, M Zeeman, M Arbouw, H Van den Oever Deventer Hospital, Deventer, the Netherlands Critical Care 2014, 18(Suppl 1):P415 (doi: 10.1186/cc13605) Off -label use of clonidine for sedation in Dutch ICUs J Van der Valk, M Zeeman, M Arbouw, H Van den Oever Deventer Hospital, Deventer, the Netherlands Critical Care 2014, 18(Suppl 1):P415 (doi: 10.1186/cc13605) Introduction Clonidine is an α2-agonist, licensed in most countries for treatment of hypertension. Other pharmacologic characteristics of α2-agonists are sedation, hypnosis, anxiolysis, sympatholysis, and analgesia [1]. Because of these central nervous eff ects, clonidine can Results In the pre-implementation and post-implementation question- naire (response rate of 38% and 37% respectively of nurses surveyed), S150 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P414). Haemodynamic and renal eff ects of clonidine in established sepsis. li h d i Figure 1 (abstract P414). Haemodynamic and renal eff ects of clonidine in established sepsis. varied 10-fold and loading doses 15-fold. Clinical studies are required to establish the safety and effi cacy of clonidine in the ICU setting. References varied 10-fold and loading doses 15-fold. Clinical studies are required to establish the safety and effi cacy of clonidine in the ICU setting. References be used off -label to augment sedation and delirium treatment. To our knowledge there have been no publications evaluating the effi cacy and safety of clonidine in ventilated critically ill adults. A survey in German ICUs showed that clonidine was used for sedation in 62% of units [2]. We undertook an enquiry to investigate the off -label use of clonidine in Dutch ICUs. 1. Blaudszun et al.: Anesthesiology 2012, 116:1312-1322. 2 Martin et al : Crit Care 2005 9:117-23 Methods An inventory was sent by email to 25 ICUs in the Netherlands, determined by the diffi culty to identify a contact person. Comparison of dexmedetomidine and propofol for sedation in patients with traumatic brain injury Results The response rate was 56%. The results are summarized in Table 1. Clonidine was used in all 14 responding ICUs; in 36% this use was reported as often. Licensed use (for hypertension) was 50%. Indications for off -label use were: substance withdrawal (50%), delirium (71%), and sedation (29%). The route of administration was intravenous in all cases. Nine ICUs reported the use of a loading dose, irrespective of indication, varying from 10 to 150 μg, median 150 μg. Ten ICUs reported the use of continuous infusion, varying from 10 to 100 μg/ hour, median 50 μg/hour.f Introduction Both propofol and dexmedetomidine decrease systemic blood pressure, heart rate, and cardiac output in a dose-dependent manner. The aim of this study was to compare their safety and effi cacy for intravenous sedation during mechanical ventilation. Introduction Both propofol and dexmedetomidine decrease systemic blood pressure, heart rate, and cardiac output in a dose-dependent manner. The aim of this study was to compare their safety and effi cacy for intravenous sedation during mechanical ventilation. Conclusion Off -label use of clonidine for sedation and treatment of withdrawal symptoms and delirium was common practice in Dutch ICUs. There was a wide range of dosing regimens: infusion schedules Methods Eighty-four patients with traumatic brain injury (Glasgow scale 7 to 8) entered the study, mean age 44 ± 13.37 years. All patients S151 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P415). Indication and doses of clonidine used in Dutch ICUs Table 1 (abstract P415). Indication and doses of clonidine used in Dutch ICUs IC levela Clonidine use Indication Loading dose (μg) Continuous infusion dose (μg/hour) 3 Often Hypertension sedation ns ns 3 Often Hypertension withdrawal; hypertension delirium; sedation 40 to 120 10 to 80 3 Often Substance withdrawal; sedation 150 50 3 Often Substance withdrawal 75 to 150 50 3 Often Substance withdrawal; hypertension; delirium 10 40 to 100 3 Sometimes Delirium ns 30 to 100 3 Sometimes Hypertension; delirium 150 ns 3 Sometimes Substance withdrawal; delirium ns 40 2 Sometimes Substance withdrawal; delirium ns 20 to 50 2 Sometimes Hypertension; sedation 150 40 2 Sometimes Substance withdrawal; hypertension; delirium 150 12 to 25 1 Sometimes Hypertension; delirium 150 ns 1 Sometimes Delirium 50 50 to 100 1 Sometimes Delirium ns ns ns, not specifi ed. Comparison of dexmedetomidine and propofol for sedation in patients with traumatic brain injury aLevel of ICU care, with level 3 being the most advanced. Loading dose (μg) Continuous infusion dose (μg/hour) dexmedetomidine, by evaluation of the systemic vascular system and cardiac function. dexmedetomidine, by evaluation of the systemic vascular system and cardiac function. underwent mechanical lung ventilation. Patients were divided into two groups depending on the type of intravenous sedation. In the fi rst group (n = 42), sedation was performed by intravenous propofol infusion at a dose of 4 to 12 mg/kg/hour. In patients of the second group (n = 42), sedation was carried out by intravenous infusion of dexmedetomidine at a dose of 0.2 to 1.4 mg/kg/hour. The level of sedation was assessed with bispectral index monitoring, targeting index 70. For comparison of methods we evaluated heart rate, blood pressure, SpO2 30, 90 and 180 minutes after the start of sedation. Both groups were matched for sex, age and comorbidity. Methods After animal preparation and under PiCCO monitoring (BL), endotoxemic shock was induced by an intravenous bolus of lipopolysaccharide (LPS, 055:B5) in ketamine-anesthetized rabbits. After fl uid resuscitation and norepinephrine infusion (SD0), animals were randomized to propofol (PROP, n = 8) or dexmedetomidine (DEX, n = 8) sedation, with two incremental doses (SD1 and SD2). The mean arterial pressure (MAP) and central vein pressure (CVP) were monitored. Pulse pressure variation (PPV) and stroke volume variation (SVV) were assessed to evaluate the preload dependency. Global end-diastolic volume, vascular resistances, mean systemic fi lling pressure, cardiac functional index and vascular resistances were assessed at each time point. Normality was assessed by the Kolmogorov–Smirnov test. Two- way analysis of variance for repeated measures was applied, and the Student–Newmann–Keuls post-hoc test when indicated. P <0.05 was considered signifi cant. Results Thirty minutes after the start of sedation in patients of the fi rst group, HR was 83 ± 11.31 beats/minute, blood pressure was 127 ± 12.87/64 ± 8.54 mmHg, SpO2 was 97 ± 3.01%. In patients of the second group 30 minutes after the beginning of sedation, HR was 87 ± 10.01 beats/minute, blood pressure was 131 ± 11.67/68 ± 8.19 mmHg, SpO2 was 97 ± 2.98%. After 90 minutes in the fi rst group of patients, we observed HR was 81 ± 6.27 beats/minute, blood pressure was 119 ± 11.46/59 ± 4.29 mmHg, SpO2 was 98 ± 2.35%. Comparison of dexmedetomidine and propofol for sedation in patients with traumatic brain injury In the second group, HR was 82 ± 7.31 beats/minute, blood pressure was 94 ± 13.62/55 ± 7.81 mmHg, SpO2 was 97 ± 2.76%. In the fi rst group 180 minutes after the start of sedation, HR was 86 ± 6.19 beats/minute, blood pressure was 105 ± 10.34/54 ± 4.28 mmHg, SpO2 was 98 ± 1.32%. In the second group, HR was 75 ± 6.27 beats/minute, blood pressure was 92 ± 12.54/51 ± 6.91 mmHg, SpO2 was 96 ± 2.91%. i Results At the increasing dose of propofol, both PPV and SVV signifi - cantly increased in SD1 versus SD0 (P <0.01) and SD2 versus SD0 (P <0.001), but only PPV in SD2 versus SD1 (P = 0.024). Dexmedetomidine infusion did not aff ect PPV and SVV. At SD1 and SD2, PPV and SVV were higher in PROP with respect to DEX (P <0.001). Moreover, propofol infusion increased the heart rate and had no eff ects on cardiac contractility and vascular resistances. On the contrary, in the DEX group we recorded a signifi cant decrease in heart contractility and increment of vascular resistances at SD2. Nonetheless, both drugs aff ected the MAP, CVP, mean systemic fi lling pressure, global end-diastolic volume and venous return. 2 Conclusion Using dexmedetomidine at a dose of 0.2 to 1.4 mg/kg/hour for intravenous sedation is safe in terms of hemodynamic stability and blood oxygenation for sedation during mechanical lung ventilation in traumatic brain injury patients. Conclusion In an endotoxemic shock model, after fl uid resuscitation and during norepinephrine infusion, propofol increased more PPV and SVV in comparison with dexmedetomidine. At high dosage the vascular resistances and heart contractility were infl uenced by dexmedetomidine, but not by propofol. Propofol: monitoring for complications Propofol: monitoring for complications F Yau, M Healy Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P418 (doi: 10.1186/cc13608) P418 T Yu1, C Pan1, S Liu1, F Guo1, F Longhini2, Y Yang1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P417 (doi: 10.1186/cc13607) T Yu1, C Pan1, S Liu1, F Guo1, F Longhini2, Y Yang1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P417 (doi: 10.1186/cc13607) P417f P417 Diff erent eff ects of propofol and dexmedetomidine on preload dependency in endotoxemic shock with norepinephrine infusion: a randomized case–control study T Yu1, C Pan1, S Liu1, F Guo1, F Longhini2, Y Yang1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P417 (doi: 10.1186/cc13607) P418 Infl uence of increased intracranial pressure on sevofl urane-fentanyl anesthesia in major abdominal surgery N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Critical Care 2014, 18(Suppl 1):P419 (doi: 10.1186/cc13609) Infl uence of increased intracranial pressure on sevofl urane-fentanyl anesthesia in major abdominal surgery N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Critical Care 2014, 18(Suppl 1):P419 (doi: 10.1186/cc13609) N Trembach, I Zabolotskikh Introduction Increased intracranial pressure (ICP) may adversely aff ect sevofl urane anesthesia and the recovery period due to a disturbed cerebral blood fl ow. Eff ect of sevofl urane on cerebral hemodynamics remains controversial and depends on the extent of the initial value of ICP [1]. This study was designed to evaluate the safety of sevofl urane- fentanyl anesthesia during major abdominal surgery in patients with increased ICP. Methods A total of 124 ASA 3 patients, undergoing major abdominal surgery (duration 5.6 (4.1 to 6.4) hours), were divided into two groups: with normal ICP (≤12 mmHg) (N group, 70 patients) and with ICP >12 mmHg (H group, 54 patients). Initial ICP was evaluated by venous ophthalmodynamometry [2]. ICP, mean arterial pressure (MAP) and cerebral perfusion pressure (CPP) were assessed every hour of anesthesia. Time of recovery of consciousness after anesthesia, complications and length of stay in the ICU and in the hospital were also evaluated. Conclusion Deep sedation resulted in greater physician satisfaction with procedural conditions as well as lower BIS scores but no signifi cant diff erence in patient satisfaction compared with conscious sedation. Patients with no recall of the procedure had lower BIS scores at the nadir and end of the procedure. BIS may be a novel tool to monitor procedural depth, allowing proceduralists to better monitor the narrow window between adequate sedation and dangerous oversedation. Results Initial ICP was 8 ± 3 mmHg in the N group and 15 ± 2 mmHg in the H group. During the anesthesia ICP increased in the H group with a total increase of 33% (from 15 ± 2 to 20 ± 3 mmHg (P <0.05)). In the N group ICP was stable without any signifi cant change. Decrease of MAP after induction of anesthesia was similar in the two groups and was stable during anesthesia. CPP was stable in the N group (above 70 mmHg during the anesthesia), but in the H group CPP decreased signifi cantly (from 82 mmHg to 63 mmHg (P <0.05)). Time of recovery of consciousness in the H group was higher (32 ± 6 minutes vs. 20 ± 4 minutes (P <0.05)). P419l Results Twenty-six procedures were monitored, 20 with CS and six endobronchial ultrasound procedures with DS. There was no diff erence with respect to age or gender. The mean doses of midazolam and fentanyl were 5 mg and 85 μg, respectively. BIS values were lower at all predefi ned points of the procedure for DS cases versus CS. Physicians were more satisfi ed with sedation and the lack of cough with DS, but there was no signifi cant diff erence in patient satisfaction between the two groups with regards to overall sedation, procedure-related symptoms or willingness to have repeat bronchoscopy. Patients with no recall had lower nadir BIS scores (46 vs. 71, P = 0.03) and scores at procedure end (76 vs. 94, P = 0.04) compared with those with any recall. There was no diff erence in the doses of midazolam or fentanyl in CS cases despite statistically signifi cant diff erences in patient recall and BIS scores. Junior fellows scored greater satisfaction with sedation, were less bothered by cough and more often felt able to perform the intended procedure compared with senior fellows. Quantifying sedation satisfaction during bronchoscopy using the Bispectral Index Quantifying sedation satisfaction during bronchoscopy using the Bispectral Index Q y g g py g Bispectral Index J Palminteri, J Dziodzio, D Seder, J Zuckerman, R Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P420 (doi: 10.1186/cc13610) J Palminteri, J Dziodzio, D Seder, J Zuckerman, R Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P420 (doi: 10.1186/cc13610) Introduction Sedation monitoring with the Bispectral Index (BIS) is uncommon during bronchoscopy but allows for a more objective measure of sedation; we hypothesized that higher BIS scores would correlate with lower patient satisfaction and that lower BIS scores (deep sedation) would result in better patient and physician satisfaction. ) p p y Methods Between October 2012 and August 2013, bronchoscopy using conscious (CS) or monitored anesthetic deep sedation (DS) was monitored with BIS. Medication administration and procedures were time-stamped. Sedation was administered blinded to BIS score. DS cases used propofol infusions, CS cases used bolus midazolam and fentanyl. Providers rated sedation satisfaction, cough, and the ability to perform the intended procedure using a 100 mm visual analog scale at the end of the procedure blind to BIS (0 unsatisfi ed to 100 satisfi ed). Patients were surveyed at 1 hour and 24 hours regarding overall sedation, symptoms, and procedure recall (unpleasant recall 1, no recall 4). Group diff erences were considered statistically signifi cant at P <0.05. Conclusion Awareness of complications from prolonged propofol sedation is increasing amongst our clinicians. This is refl ected in the increased frequency of lipid profi le and ECG monitoring compared with 2011. However, we feel that more regular and routine monitoring is essential to aid early detection of this potentially fatal complication. We recommend that all patients at risk of developing PRIS should have these parameters monitored on a daily basis as standard. Reference 1. Jacobi J, et al.: Clinical practice guidelines for the sustained use of sedative and analgesics in the critically ill adult. Crit Care Med 2002, 30:119-141. 1. Dahyot-Fizelier C, et al.: Ann Fr Anesth Reanim 2012, 31:e229-e234. 2. Firsching R, et al.: J Neurosurg 2011, 115:371-374. Infl uence of increased intracranial pressure on sevofl urane-fentanyl anesthesia in major abdominal surgery N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Critical Care 2014, 18(Suppl 1):P419 (doi: 10.1186/cc13609) The incidence of postoperative delirium was higher in the H group (22.2% vs. 12.8% in the N group (P <0.05)). There were no signifi cant diff erences between two groups in other complications. Total length of stay in the ICU and in the hospital was Propofol: monitoring for complications F Yau, M Healy Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P418 (doi: 10.1186/cc13608) Propofol: monitoring for complications F Yau, M Healy Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P418 (doi: 10.1186/cc13608) Introduction Septic shock is a status characterized by the simultaneous use of vasopressors and sedative drugs in critical care patients. Little is known about the possible interference of these drugs during shock status. We aimed to clarify how propofol could diff erently aff ect the preload dependency in an endotoxemic model in comparison with Introduction This re-audit aims to test our strategy for monitoring patients on propofol sedation for prolonged periods. Propofol infusion syndrome (PRIS), once established, is diffi cult to treat and currently there is limited guidance on how best to monitor for this potentially Introduction This re-audit aims to test our strategy for monitoring patients on propofol sedation for prolonged periods. Propofol infusion syndrome (PRIS), once established, is diffi cult to treat and currently there is limited guidance on how best to monitor for this potentially S152 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 life-threatening complication [1]. An audit done in 2011 highlighted that lipid profi le and electrocardiograms (ECGs) were rarely monitored. We recommended regular monitoring of these parameters when propofol sedation is used for over 3 days and that propofol-sparing agents are considered in these patients at risk of developing PRIS. higher in the H group (6 ± 2 days vs. 4 ± 2 days (P <0.05) and 15 ± 3 days vs. 11 ± 2 days in N group (P <0.05)).l Conclusion Sevofl urane-fentanyl anesthesia in patients with increased ICP was characterized by a delayed recovery with a higher incidence of postoperative delirium and higher length of stay in the ICU and in the hospital. The main cause of this is a decrease of CPP in patients with high ICP due to low craniocerebral compliance. References g p p g Methods In patients who required propofol sedation for 4 days or more, we prospectively monitored: frequency of performing lipid profi le and 12-lead ECG; and frequency of co-administration of a propofol-sparing agent. 1. Dahyot-Fizelier C, et al.: Ann Fr Anesth Reanim 2012, 31:e229-e234. 2. Firsching R, et al.: J Neurosurg 2011, 115:371-374. Results We collected data from 25 patients. The duration of propofol infusion was 4 to 15 days (mean 8.5 days). Mean propofol dose in the fi rst 4 days was 2.2 mg/kg/hour. Maximum daily propofol dose ranged from 2.1 to 3.8 mg/kg/hour. Lipid profi le was checked in 16/25 (64%) patients (20% in 2011) whilst on propofol sedation. However, these were checked following 3 days continuous infusion in only three patients. The triglyceride level was ≥2.2 mmol/l (very high) on fi rst testing in 75% of patients. All patients had a 12-lead ECG done on admission. Seventeen of 25 (68%) had a further ECG performed whilst on continuous propofol infusion (45% in 2011). Additional ECGs were done in 13/17 patients (zero in 2011). ECG changes that we feel were attributable to propofol occurred in two patients, one of whom developed severe PRIS. Fourteen of 25 (56%) patients (55% in 2011) were on concomitant sedative agents. This included the patient who developed PRIS. P420 P421 P423 P423 Delirium screening, prevention and treatment in the ICU: a systematic review of implementation strategies Z Trogrlic1, M Van der Jagt1, J Bakker1, MC Balas2, EW Ely3, PH Van den Voort4, E Ista5 1Erasmus MC University Medical Center Rotterdam, the Netherlands 2The Ohio State University, Columbus, OH, USA; 3Vanderbilt University Medical Center, Nashville, TN, USA; 4Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands; 5Erasmus MC University Medical Center Rotterdam – Sophia Children’s Hospital, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P423 (doi: 10.1186/cc13613) P423 Delirium screening, prevention and treatment in the ICU: a systematic review of implementation strategies Z Trogrlic1, M Van der Jagt1, J Bakker1, MC Balas2, EW Ely3, PH Van den Voort4, E Ista5 1Erasmus MC University Medical Center Rotterdam, the Netherlands 2The Ohio State University, Columbus, OH, USA; 3Vanderbilt University Medical Center, Nashville, TN, USA; 4Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands; 5Erasmus MC University Medical Center Rotterdam – Sophia Children’s Hospital, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P423 (doi: 10.1186/cc13613) g p Methods A total of 932 patients were admitted to our PICU from 1 October 2011 to 31 March 2013. Of these, 127 paediatric patients were supported on mechanical ventilation on the fi rst day in the PICU and received intravenous analgesics and sedatives. A retrospective review of a prospectively collected database. The statistical method was the Mann–Whitney U test, and P <0.05 was considered statistically signifi cant. Results Twenty-eight patients were scored with the WAT-1 during weaning from the drugs. Median age was 14 months (1 to 73 months) and the most common reasons for ventilation were airway trouble and pneumonia. Eight of 28 were diagnosed withdrawal syndrome, 20 were not. There were no signifi cant diff erences in age, body weight, cumulative morphine and benzodiazepine dose before weaning. In the withdrawal group, the lactate level, catecholamine index, Paediatric Index of Mortality 2 and heart rates were greater when they were admitted to hospital. Further, there are longer length of PICU stay and shorter ventilator-free days for patients with WAT-1 score >3. But all of them are alive at 28 days, so there is no diff erence in 28-day mortality. Conclusion Our study suggest that severely ill paediatric patients tended to suff er from withdrawal syndrome and then resulted in short ventilator-free days and longer PICU stay. But there is no diff erence in mortality. y Reference Reference Reference 1. Franck LS, et al.: Pain 2012, 153:142-148. Reference 1. Franck LS, et al.: Pain 2012, 153:142-148. P421 P421 Risk factor of withdrawal syndrome in the paediatric ICU C Tanaka1, N Shimizu2, O Staito2, M Motomura2, I Watanabe2 1Japanese Musashino Red Cross Hospital, Tokyo, Japan; 2Tokyo Metropolitan Children’s Centre, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P421 (doi: 10.1186/cc13611) Introduction Critically ill paediatric patients supported on mechanical ventilation frequently receive analgesia and sedation. Iatrogenic withdrawal syndrome occurs with the abrupt discontinuation or too rapid weaning of opioids and benzodiazepines. We induce the Introduction Critically ill paediatric patients supported on mechanical ventilation frequently receive analgesia and sedation. Iatrogenic withdrawal syndrome occurs with the abrupt discontinuation or too rapid weaning of opioids and benzodiazepines. We induce the S153 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 merits further investigation and the decision to insert the epidural catheter should be discussed with the patient considering both local experience and legal dispute in case of medical complications. Withdrawal Assessment Tool-1 (WAT-1) [1] to evaluate children during weaning from analgesics and sedatives. The patient is diagnosed with withdrawal syndrome when the score is 3 or >3. We compared the subjects who ever had a score over 3 and those with lower scores and assessed the risk factors and outcome of withdrawal syndrome between two groups. Withdrawal Assessment Tool-1 (WAT-1) [1] to evaluate children during weaning from analgesics and sedatives. The patient is diagnosed with withdrawal syndrome when the score is 3 or >3. We compared the subjects who ever had a score over 3 and those with lower scores and assessed the risk factors and outcome of withdrawal syndrome between two groups. P424f P424f P421 Introduction The occurrence of delirium heralds a circumstance of higher risk of death, longer stay, higher cost, and greater likelihood of long-term brain dysfunction, and yet the majority of ICU patients worldwide do not get routinely monitored for delirium, thus obstructing timely prevention and management strategies. Determination of eff ective implementation strategies regarding screening, treatment and prevention of delirium is critical to address needed modifi cations in the culture of patient care in the ICU. The aim of this study was to summarize the effi cacy of and the barriers to implementation of delirium management. yf y y Conclusion Our study suggest that severely ill paediatric patients tended to suff er from withdrawal syndrome and then resulted in short ventilator-free days and longer PICU stay. But there is no diff erence in mortality. g Methods We searched PubMed, Embase, PsychINFO, Cochrane and CINAHL for studies published between January 2000 and October 2012, and included them when implementation strategies and their effi cacy and/or potential barriers of implementation were described. Results In all studies (n = 34) multifaceted education strategies were used and combined (median: 5; IQR: 4 to 6). Positive results were reported for both process and clinical outcomes: adherence to delirium screening – improvement after implementation compared with the before measurement, by 15 to 57%; only measured after implementation, between 84 and 92%; delirium knowledge (on a 10-point scale; improved from 6.1 and 6.2 before to 8.2 and 7.4 respectively (P <0.001) after implementation); length of stay in the ICU (4.1 vs. 5.9 days; P = 0.21 and 6.3 to 5.35 days; P < 0.009) and hospital stay (12 days vs. 18 days; P = 0.036 and from 55 to 27 days; P <0.0001); and decreased mortality (29.4% vs. 22.9%; P = 0.009; and OR = 0.45; 95% CI = 0.22 to 0.92; P = 0.03). The major barriers found impairing implementation concerned clinicians’ attitude and healthcare professionals’ knowledge. P422 Epidural analgesia reduces perioperative myocardial infarction and all-cause mortality after cardiac surgery: but at least 25 epidural hematomas have already happened G Landoni, M Greco, I Francesca, D Taddeo, O Saleh, A Belletti, A Putzu, R Lembo, A Zangrillo Vita-Salute San Raff aele University, Milan, Italy Critical Care 2014, 18(Suppl 1):P422 (doi: 10.1186/cc13612) Introduction Epidural analgesia on top of general anesthesia in cardiac surgery might improve relevant clinical outcomes but the incidence of epidural hematoma is under-reported. Introduction Epidural analgesia on top of general anesthesia in cardiac surgery might improve relevant clinical outcomes but the incidence of epidural hematoma is under-reported. Methods An international web-based, online, viral, anonymous survey, a systematic review of the literature, and a meta-analysis of randomized and matched studies were performed.i p g Conclusion Implementation strategies used suggest a strong potential for multifaceted strategies to be able to aff ect both process and clinical outcomes. The majority of studies focused only on implementation of delirium screening. There is a knowledge defi cit regarding effi cacy of well-prepared implementation of integral delirium management considering screening, prevention, and treatment preceded by analysis of barriers. Further research on the extent and contents of implementation interventions aimed at integral delirium management such as those described in the recent ABCDE bundle and PAD guideline are necessary. Results Nine epidural hematomas were identifi ed in 198 published manuscripts. The risk of epidural hematoma (9:13,429) calculated on all published evidence might be therefore estimated to be 1:1,492 (95% confi dence interval (CI) = 1:2,857 to 1:833). Through an anonymous, web-based, viral, international survey, we identifi ed at least 16 further nonpublished epidural hematomas together with at least 72,400 epidural analgesia catheters positioned in cardiac surgery in the last 20 years. The risk of epidural hematoma (25:85,829) is therefore 1:3,436 (95% CI = 1:2,325 to 1:5,076) including both published and unpublished data. Out of the 66 randomized and case-matched studies, 57 trials reported the incidence of all-cause mortality at the longest available follow-up with a signifi cant reduction in the epidural group (59/3,137 (1.9%) in the epidural group vs. 108/3,246 (3.3%) in the control arm, RR 0.64 (95% CI 0.48 to 0.85), P = 0.002, NNT = 69). A nutritional protocol and personalized support reduce the cumulative caloric defi cit of cardiac surgery patients A nutritional protocol and personalized support reduce the cumulative caloric defi cit of cardiac surgery patients E De Waele, K De Bondt, J Czapla, J Nijs, D Nguyen, PM Honoré, H Spapen Universitair Ziekenhuis Brussel, Jette, Belgium Critical Care 2014, 18(Suppl 1):P427 (doi: 10.1186/cc13617) i g y p E De Waele, K De Bondt, J Czapla, J Nijs, D Nguyen, PM Honoré, H Spapen Universitair Ziekenhuis Brussel, Jette, Belgium Critical Care 2014, 18(Suppl 1):P427 (doi: 10.1186/cc13617) Introduction Providing adequate feeding in cardiac surgery patients is gaining importance [1]. The aims were to assess the preoperative nutritional status, to compare postoperative energy needs with eff ectively delivered amounts, and to evaluate the impact of a dietitian on optimizing postoperative energy balances. Methods Following ethical approval, the practice of measuring GRVs and the subsequent management of EN was retrospectively reviewed over a 3-month period, September to December 2012, and prospectively reviewed over a 3-month period, September to December 2013, following the change in practice. Recorded parameters included: length of ICU admission; admitting diagnosis; duration of EN; GRVs; prescribed calories; delivered calories via an enteral route; requirement for PN; duration of PN; and cost of total parenteral nutrition (TPN). Methods A prospective interventional study in adult patients after elective CABG and/or heart valve surgery. The patients’ nutritional risk was determined by the NRS 2002 and the Malnutrition Universal Screening Tool (MUST) [2]. A dedicated dietitian managed and assisted the nutritional approach. For each patient, global energy intake (intentional and non-intentional) and balance (diff erence between energy target and global caloric intake) were calculated daily during and after ICU stay until hospital discharge. The Harris–Benedict equation was used to calculate daily energy requirements. When energy delivery did not reach 60% of calculated needs, a protocol- driven nutritional intervention was initiated. Results The change of practice in terms of GRVs and management of EN led to an increased percentage of prescribed calories that was delivered. There was a reduced requirement for PN and reduced cost of TPN also associated with this change in practice. Conclusion Increasing the threshold of GRV prior to reducing or stopping EN can result in increased calories delivered to ICU patients and a reduced requirement for and cost of TPN. Results Two hundred patients were enrolled during a 10-month period, representing 2,690 study-days. Mean age was 67 ± 11 years. Reference 1. Estívariz CF, et al.: J Parenter Enteral Nutr 2008, 32:389-402. Increased threshold for gastric residual volumes and impact on nutrition in the ICU J Brohan, M Richardson, S O Riain Limerick Regional Hospital, Limerick, Ireland Critical Care 2014, 18(Suppl 1):P425 (doi: 10.1186/cc13615) Conclusion Although the initiation of early enteral feeding seems adequate for a good number of septic patients on D0, is still far off for a signifi cant percentage of those patients on D3 and is even worse on D7. The caloric goal achievements were better on D3 but very suboptimal on D7. There was no association, however, between nutritional status and compliance with the feeding protocols. It is therefore mandatory to follow daily the nutritional therapy of the septic patient and not rely only on the feeding protocols. Introduction Gastric residual volumes (GRVs) as measured at regular intervals are considered a marker of tolerance of enteral nutrition (EN). There is controversy surrounding this practice, however. The absolute value for the designated cutoff value for GRVs varies widely in the literature. No prospective randomized control trials have suggested that their use improves patient outcomes in the ICU. It has also not been shown, therefore, that GRVs are an accurate predictor of aspiration or pneumonia. However, the use of GRVs as a guide to the continuation of EN can result in a reduction in the percentage of goal calories received by patients. Prior to February 2013, in our ICU the threshold of GRVs prior to withholding of EN was 200 ml. This tolerated volume was increased to 500 ml in February 2013, in compliance with recent guidelines. By increasing the acceptable GRVs to this level, the aim was to increase the percentage of goal calories received by patients via the enteral route. P427 A nutritional protocol and personalized support reduce the cumulative caloric defi cit of cardiac surgery patients In total, 42.5% of the patients had a NRS 2002 score >3 and were considered to be nutritionally at risk. MUST identifi ed a high, medium, or low risk in respectively 1%, 4% and 95% of subjects. Mean energy requirement was 2,046 ± 347 kcal and mean daily intake 1,452 ± 335 kcal. Sixty-two percent of the caloric need was met during the entire hospital stay. Nutritional intervention was necessary in 52% of cases: oral feeding supplementation for 546 study-days, enteral feeding: 401 days, parenteral feeding: 367 days. This reduced the mean cumulative caloric defi cit to 9,085 kcal. Early enteral feeding in the septic critically ill patient: evaluation of our feeding protocol h d k l l b l k M Theodorakopoulou, I Dimopoulou, S Karambi, A Strilakou, A Diamantakis, S Orfanos, A Armaganidis University Hospital of Athens Greece ‘Attikon’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P426 (doi: 10.1186/cc13616) M Theodorakopoulou, I Dimopoulou, S Karambi, A Strilakou, A Diamantakis, S Orfanos, A Armaganidis University Hospital of Athens Greece ‘Attikon’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P426 (doi: 10.1186/cc13616) Introduction It has been established that early enteral nutrition in critically ill patients improves overall outcome and mortality. In our unit, feeding protocols were established based on the ESPEN recommendations and have been implemented for the last 2 years. The purpose of this study was to evaluate the compliance of our septic patients’ nutritional approach with our feeding protocols. Conclusion Close monitoring of caloric intake shouldered by interventions based on a predefi ned algorithm under supervision of a dedicated nutrition team restricts the caloric defi cit in cardiac surgery patients. References P424 Eff ect of enteral and/or parenteral glutamine supplementation on mortality and morbidity in the critically ill H Sungurtekin, I Ozturk, B Beder, S Serin Pamukkale University Medical Faculty, Denizli, Turkey Critical Care 2014, 18(Suppl 1):P424 (doi: 10.1186/cc13614) P424 Eff ect of enteral and/or parenteral glutamine supplementation on mortality and morbidity in the critically ill H Sungurtekin, I Ozturk, B Beder, S Serin Pamukkale University Medical Faculty, Denizli, Turkey Critical Care 2014, 18(Suppl 1):P424 (doi: 10.1186/cc13614) Conclusion Epidural analgesia on top of general anesthesia in cardiac surgery might reduce the incidence of all-cause mortality (NNT 69). The incidence of epidural hematoma in this setting is 1:3,436 (95% CI = 1:2,325 to 1:5,076) including both published and unpublished data. In fact, we identifi ed at least 25 epidural hematomas that occurred so far from the following countries: Belgium (n = 1), Brazil (n = 1), France (n = 1), Germany (n = 2), India (n = 2), Italy (n = 1), Japan (n = 2), Korea (n = 1), Malaysia (n = 1), Norway (n = 2), Russia (n = 3), Sweden (n = 1), the UK (n = 3), and the USA (n = 4). Even if from the public health point of view the benefi ts seem to encourage the use of epidural analgesia in cardiac surgery with a possible reduction in perioperative mortality, this topic Introduction In this study we aimed to compare the eff ectiveness of enteral, parenteral and combined enteral–parenteral glutamine supplementations in the nutrition of critical care patients. Methods This is a single-center, randomized controlled clinical trial. During the 5-day study period, all patients received standard enteral nutrition product and were divided into three groups, including parenteral glutamine (Group I), enteral glutamine (Group II) and enteral + parenteral glutamine (Group III) supplementations. Blood S154 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1. Estívariz CF, et al.: J Parenter Enteral Nutr 2008, 32:389-402. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 met the CCM criteria for sepsis upon admission to the ICU. APACHE II score, SOFA score, weight, BMI and nutritional status were calculated. Patients were initiated for enteral feeding based on the established feeding protocol within 48 hours of admission. The feeding status of the patient was recorded on the start day (D0), day 3 (D3) and day 7 (D7). Factors aff ecting the feeding process and its progression were also recorded biochemistry, rates of infections, length of stay in the ICU and duration of mechanical ventilation were evaluated. biochemistry, rates of infections, length of stay in the ICU and duration of mechanical ventilation were evaluated. Results Sixty patients were included in this study. There was no statistically signifi cant diff erence for biochemical values between the diff erent feeding groups. Frequency of infections ranged as Group II >Group III >Group I and mortality as Group II = Group III >Group I. Length of stay in the ICU and duration of mechanical ventilation were signifi cantly longer in Group II than the others. Results The patient mean age was 71.4 ± 12.2. LOS in the ICU was 9 to 21 days. Based on BMI, 18% of the patients were malnourished upon admission. APACHE II was 26 ± 7.8 and SOFA was 9.2 ± 4.6. The mortality rate was 42.5%. Enteral nutrition started early in 64 (77.1%) of the patients (D0), on day 3 (D3) 29 (45.31%) patients met their caloric goals and on day 7 (D7) only 18 (28.1%) patients achieved their caloric goals. Discontinuation of enteral feeding was mainly due to procedures, whereas late start and/or decreased hourly intake were due to GI complications, GI intolerance, excessive diarrhoea and hemodynamic instability. There was no association between compliance with the feeding protocol and the LOS, nutritional status, severity or disease progression. gi y g p Conclusion Although mortality was not signifi cantly diff erent between groups, parenteral glutamine administration causes less stay of ICU and mechanical ventilation. This needs a more powerful randomized controlled study [1]. P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) Introduction The renal system compensates respiratory disorders of acid–base equilibrium by modifying the urinary electrolyte composition [1]. Such a condition mainly involves changes in urinary ammonium excretion as a reaction to prolonged periods of acid–base disequilibrium, leaving the renal response to acute derangements unexplored. We aimed to determine the acute variations of urinary ammonium [NH4+]u, sodium [Na+]u, potassium [K+]u and chloride [Cl–]u concentrations following controlled minimal respiratory acid– base imbalances, and to further investigate whether urinary anion gap ([AG]u = [Na+]u + [K+]u – [Cl–]u) and sodium and chloride diff erence ([Na+]u – [Cl–]u) might unveil an early activation of the renal response. Conclusion The level of vitamin C was markedly decreased. Replacement of vitamin C should be considered for the homeless who visit the emergency department after alcohol ingestion. u u Methods Patients admitted to the ICU after major surgery were enrolled, during intubation and sedation. Patients with chronic renal failure were excluded. A urinary catheter was connected to the quasi- continuous urinary analyzer KING®, measuring [NH4+]u, [Na+]u, [K+]u and [Cl–]u over time. Based upon the arterial pH (pHa) at entrance, patients were randomly assigned to controlled hypoventilation (30% reduction of minute ventilation if pHa ≥7.40) or hyperventilation (30% increase of minute ventilation if pHa <7.40) for 2 hours. Samples for blood gas analysis were collected every 30 minutes. P429 Acid–base disorders according to the Stewart approach in septic patients J Szrama, P Smuszkiewicz, I Trojanowska University Hospital, Poznan, Poland Critical Care 2014, 18(Suppl 1):P429 (doi: 10.1186/cc13619) Introduction ICU patients often develop acid–base disorders. In clinical practice, there are several methods (for example, Henderson– Hasselbalch) used to interpret acid–base data, but most of them provide little information and fail to identify the cause of the problem. Stewart’s approach which is based on physicochemical principles has growing popularity among clinicians in critical care. This method includes three independent variables that determine plasma pH (strong ion diff erence (SID), PCO2, and total weak acid concentration – mainly albumins and phosphate). Introduction ICU patients often develop acid–base disorders. In clinical practice, there are several methods (for example, Henderson– Hasselbalch) used to interpret acid–base data, but most of them provide little information and fail to identify the cause of the problem. y y Results Thirty patients were enrolled; 20 were hypoventilated, 10 hyperventilated. References 1. Cresci G, et al.: Nutrition intervention in the critically ill cardiothoracic patient. Nutr Clin Pract 2012, 27:323-334. Methods A prospective study was done on a 24-bed mixed ICU over a period of 18 months. Eighty-three patients ≥18 years were included in the study. All patients were dependent on mechanical ventilation and 2. Lomivorotov VV, et al.: Prognostic value of nutritional screening tools for patients scheduled for cardiac surgery. Interact Cardiovasc Thorac Surg 2013, 16:162-168. patients scheduled for cardiac surgery. Interact Cardiovasc Thorac Surg 2013, 16:162-168. S155 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P428 ninety-three ABG results met the criteria of metabolic acidosis, 473 were categorized into the metabolic alkalosis group and 324 results were within the range value of BE according to the Henderson–Hasselbalch concept. In the metabolic acidosis group (BE <–2), 34.7% of the results had elevated lactate concentration, 100% revealed hypoalbuminemia, 96.9% had Cl/Na ratio >0.75 revealing SID acidosis, while 42.5% met the criteria of SIG acidosis. In the normal range BE group, 21.3% revealed lactate concentration >2 mmol/l, 100% had hypoalbuminemia, 98.4% had Cl/Na ratio >0.75 revealing SID acidosis and hyperchloremia, while 14.5% showed SIG acidosis. The analysis of the ABG with BE >2 group showed that 18.4% had elevated lactate concentration, 99.1% revealed hypoalbuminemia, 88.8% had Cl/Na ratio >0.75 (SID acidosis) and 4.6% showed SIG acidosis. ninety-three ABG results met the criteria of metabolic acidosis, 473 were categorized into the metabolic alkalosis group and 324 results were within the range value of BE according to the Henderson–Hasselbalch concept. In the metabolic acidosis group (BE <–2), 34.7% of the results had elevated lactate concentration, 100% revealed hypoalbuminemia, 96.9% had Cl/Na ratio >0.75 revealing SID acidosis, while 42.5% met the criteria of SIG acidosis. In the normal range BE group, 21.3% revealed lactate concentration >2 mmol/l, 100% had hypoalbuminemia, 98.4% had Cl/Na ratio >0.75 revealing SID acidosis and hyperchloremia, while 14.5% showed SIG acidosis. The analysis of the ABG with BE >2 group showed that 18.4% had elevated lactate concentration, 99.1% revealed hypoalbuminemia, 88.8% had Cl/Na ratio >0.75 (SID acidosis) and 4.6% showed SIG acidosis. Ramadoss J, et al.: Can J Physiol Pharmacol 2011, 89:227-231. P430 P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) Table 1 (abstract P428). Reference Lower than Higher than Mean (SD) range reference (%) reference (%) Vitamin B1 158.1 (80.2) 59 to 213 3.1 13.8 Vitamin B12 741.5 (432.2) 200 to 950 2.7 22.6 Vitamin B6 51.9 (53.8) 20 to 202 23.1 1.9 Vitamin C 15.57 (13.4) 26.1 to 84.6 82.7 0 Conclusion The level of vitamin C was markedly decreased. Replacement of vitamin C should be considered for the homeless who visit the emergency department after alcohol ingestion. References P428 Vitamin B and C levels of homeless patients who visit the emergency department with alcohol ingestion HJ Lee, JH Shin, E Kang, J Jung, DK Kim SMG-SNU Boramae Medical Center, Seoul, South Korea Critical Care 2014, 18(Suppl 1):P428 (doi: 10.1186/cc13618) Introduction Vitamins are essential micronutrients and depletions are reported for chronically ill patients. It is well known that the general nutrition status of the homeless is poor, especially for heavy alcoholics. But there were few data about the actual vitamin status of homeless patients. We want to evaluate the vitamin levels of homeless patients. Methods This study was conducted at a single urban teaching hospital emergency department. We performed a retrospective chart review of blood vitamin B1, B12, B6 and C levels of homeless patients. These vitamins are a common supplement in our center and sometimes blood levels are drawn if the patient is drunk, needs i.v. hydration and has cachexic features. Conclusion In critically ill patients with the BE values <–2, with BE in the range of normal value and BE >2 we observed complex acid–base disturbances with coexistence of hyperchloremic acidosis, hyperlactatemia, SIG acidosis and alkalosis caused by hypoalbuminemia. The Stewart approach is more eff ective in detecting acid–base disturbances and quantifying individual components of acid–base abnormalities and provides a detailed insight into their pathogenesis. Results During study periods, vitamin levels were checked for 156 patients. The number of male patients was 146 (94%) and the mean age was 50 ± 10.2. Vitamin C levels were 15.8 ± 1.3 mg/l. For 84 patients, levels of vitamin C were decreased. For vitamin B1 (152 ± 7.2 nmol/l), vitamin B12 (725.5 ± 35.4 pg/ml), and vitamin B6 (50.3 ± 5.5 ng/ml), there were three, two and 23 patients below the reference ranges respectively. See Table 1. P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) At 2 hours from ventilation change, pHa was respectively decreased from 7.44 ± 0.02 to 7.34 ± 0.02 and increased from 7.37 ± 0.03 to 7.44 ± 0.02 (P <0.001) in the two groups. Mean [NH4+]u rose by 2.6 ± 3.3 mEq/l in hypoventilated patients and fell by 2.5 ± 2.4 mEq/l in the hyperventilated (P <0.001). No diff erence in mean [K+]u was observed at any time, while [Na+]u and [Cl–]u progressively decreased in both groups (P <0.05). [Na+]u was reduced by 48 ± 43 mEq/l during hypoventilation and by 32 ± 41 mEq/l during hyperventilation (P = 0.14), while [Cl–]u decreased by 29 ± 69 and by 46 ± 66 mEq/l respectively (P = 0.93). Whereas [AG]u did not diff er between groups, [Na+]u – [Cl–]u variation of hyperventilated patients was greater than that of hypoventilated (17 ± 34 vs. –18 ± 54 mEq/l, P <0.05). Stewart’s approach which is based on physicochemical principles has growing popularity among clinicians in critical care. This method includes three independent variables that determine plasma pH (strong ion diff erence (SID), PCO2, and total weak acid concentration – mainly albumins and phosphate). Methods The prospective analysis of arterial blood gases (ABG) was performed according to the Henderson–Hasselbalch approach and the Stewart method. The results were categorized into three groups according to the Henderson–Hasselbalch concept and the BE values: BE <–2, metabolic acidosis; BE between –2 and 2, normal values; BE >2, metabolic alkalosis. The aim of the study was to compare the effi cacy of the traditional Henderson–Hasselbalch approach with acid–base disturbances with the Stewart concept in the population of critically ill septic patients. Conclusion During acute respiratory modifi cations, changes in urinary ammonium can be observed within the fi rst 2 hours even while maintaining arterial pH within a physiologic range. This response seems to be better associated with changes in the diff erence between urinary sodium and chloride rather than anion gap. Reference Results The analysis included 990 arterial blood gases taken from 43 consecutive septic patients admitted to the ICU. One hundred and Ramadoss J, et al.: Can J Physiol Pharmacol 2011, 89:227-231. P431 Admission hypomagnesemia as a mortality predictor in medical critically ill patients P431 Admission hypomagnesemia as a mortality predictor in medical critically ill patients y p A Permata Sari, C Pitoyo, D Aditianingsih, C Rumende Medical Faculty Indonesia University, Jakarta Pusat, Indonesia Critical Care 2014, 18(Suppl 1):P431 (doi: 10.1186/cc13621) y A Permata Sari, C Pitoyo, D Aditianingsih, C Rumende Medical Faculty Indonesia University, Jakarta Pusat, Indonesia Critical Care 2014, 18(Suppl 1):P431 (doi: 10.1186/cc13621) Introduction Magnesium is the second most abundant intracellular cation and serves as a cofactor in more than 300 enzymatic reactions. Hypomagnesemia is a common electrolyte imbalance in critically ill patients; yet it is frequently overlooked. Previous studies highlighted the relationship between hypomagnesemia and mortality in these patients [1,2]. This study was carried out on patients admitted to the critical care unit and seeks to fi nd admission hypomagnesemia’s role as a 28-day mortality predictor in medical critically ill patients. P432 Impact of reduced frequency of phosphate testing on detected phosphate levels and phosphate prescription in critical care D Hepburn, H Roberts, S Zouwail University Hospital of Wales, Cardiff , UK Critical Care 2014, 18(Suppl 1):P432 (doi: 10.1186/cc13622) Eff ect of albumin and total protein concentration on plasma sodium measurements in the ICU Results This study was conducted consecutively from April to July 2013. Most subjects were male (62%) with mean age 52.6 ± 15.93 years. The occurrence of sepsis (33.3%) and cardiac disturbances (24.7%) were the most common problems among patients. The mean MSOFA score for the hypomagnesemics was higher than the normal group (4.91 ± 4.19 vs. 3.77 ± 3.52). Subgroup analysis in the MSOFA 0 to 7 group had signifi cant P = 0.015 (chi-square) and crude RR = 2.2 (95% CI = 1.19 to 4.06). From survival analysis, the survival mean of hypomagnesemia patients was lower than the normal group (19.4 vs. 22.8 days), and so was the survival percentage (52.7% vs. 74.7%). Introduction Direct ion-selective electrode without dilution is the most eff ective method for determination of the concentration of the ionized fraction of sodium [1]. We tested the hypothesis that the diff erence between indirect and direct sodium assays would be related to the plasma albumin concentration or the total protein concentration. Methods A retrospective observational study was conducted in which plasma sodium concentrations, from 101 paired venous and arterial samples from patients admitted to the ICU, were respectively analyzed on the arterial blood gas (ABG) analyzers (direct ion-selective electrode) and the central laboratory auto-analyzers (indirect ion-selective electrode). A paired t test was performed comparing the central laboratory and ABG measurements. Correlation and regression analysis were performed between total protein concentration, albumin and the diff erences between the central laboratory and ABG assays for sodium. Conclusion There was a high prevalence of hypomagnesemia in medical critically ill patients and it was associated with a high mortality rate. Reference Reference 1. Burtis C, et al.: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Elsevier; 2006:983-1018. Reference 1. Burtis C, et al.: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Elsevier; 2006:983-1018. P434 p p p p p p Results A total of 4,253 tests were performed in phase 1, and 3,641 in phase 2 – a reduction of 612 (14.4%). The ICU workload was similar in both phases. There was no signifi cant diff erence in mean phosphate levels or detected incidence of abnormal phosphate levels in phase 1 versus phase 2. Mean level was 1.13 ± 0.40 mmol/l versus 1.14 ± 0.43 mmol/l (P = 0.42). Severe hypophosphatemia (<0.4 mmol/l) was relatively uncommon: n = 18 in phase 1 (0.42% of tests) versus 19 (0.52%), P = 0.42, in phase 2. Mild hypophosphatemia (0.4 to 0.7 mmol/l) was frequent, with 1,203 episodes (28.2% of tests) versus 1,088 (29.8%), P = 0.42. Hyperphosphatemia (>1.5 mmol/l) was also common in both phases with 608 detected episodes (14.3% of tests) versus 572 (15.7%), P = 0.49. Pharmacy data show phosphate replacement fell signifi cantly, from 687 prescriptions in phase 1 to 395 in phase 2, with drug cost- savings estimated at £1,430. Main causes of water–electrolyte disturbances in patients with acute brain injury: central diabetes insipidus and cerebral salt wasting syndrome L Tsentsiper, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint Petersburg, Russia Critical Care 2014, 18(Suppl 1):P434 (doi: 10.1186/cc13624) 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) S156 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P431 Conclusion Reducing testing from daily to three times weekly was not associated with a signifi cant change in mean phosphate levels nor with detection of abnormally high/low phosphate levels. Daily testing, however, is associated with higher rates of phosphate prescription. It is known that there is signifi cant diurnal variation in serum phosphate [2] and we speculate that mild hypophosphataemia self-corrects without intervention. Treating mild hypophosphatemia may therefore not be indicated. P431 Admission hypomagnesemia as a mortality predictor in medica critically ill patients A Permata Sari, C Pitoyo, D Aditianingsih, C Rumende Medical Faculty Indonesia University, Jakarta Pusat, Indonesia Critical Care 2014, 18(Suppl 1):P431 (doi: 10.1186/cc13621) References References 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. y y Results The central laboratory sodium measurement was, on average, 1.46 mmol/l more than the ICU assay, limits of agreement 1.18 to 1.74 mmol/l greater, P <0.001. Bland–Altman analysis of the central laboratory result minus the ICU sodium measurement had limits of agreement of 1.3 to –4.2 mmol/l. The correlation between the assay diff erences and total protein concentration and albumin were respectively r = 0.24 (P = 0.01) and r = 0.20 (P = 0.04). The diff erence in plasma sodium concentration between the assays increased as the plasma concentration albumin or total protein concentration decreased (respectively: r2 = 0.04 and r2 = 0.06).f Impact of reduced frequency of phosphate testing on detected phosphate levels and phosphate prescription in critical care D Hepburn, H Roberts, S Zouwail University Hospital of Wales, Cardiff , UK Critical Care 2014, 18(Suppl 1):P432 (doi: 10.1186/cc13622) D Hepburn, H Roberts, S Zouwailf University Hospital of Wales, Cardiff , UK Introduction Phosphate is essential for cell and bone function [1]. In critical illness, hypophosphatemia is common and practice is often to correct even mild derangement [2]. Its supplementation has signifi cant cost and risk including hypotension and hypocalcaemia. We investigated whether changing frequency of routine serum phosphate testing had eff ects on the detected incidence of abnormal plasma levels and prescription of phosphate. y Conclusion The diff erence between indirect and direct sodium assays was found to be statistically related to the plasma albumin concentration and the total protein concentration. Although the relationship was found to be weak, the total protein concentration should be monitored when measuring sodium by indirect ion-selective electrode. Methods This was a service improvement project using observational, anonymous data. We collected data on serum phosphate levels in a 33-bed ITU over two 6-month periods before and after introduction of a new testing regime (phases 1 and 2). In phase 1, phosphate levels were tested daily. In phase 2, phosphate levels were tested three times per week. Replacement was at clinical discretion. Pharmacy data on phosphate prescription were compared for both phases. Reference References References 1. Geerse et al.: Treatment of hypophosphatemia in the intensive care unit. Crit Care 2010, 14:R147. 1. Geerse et al.: Treatment of hypophosphatemia in the intensive care unit. Crit Care 2010, 14:R147. 2. Pocock et al.: Diurnal variations in serum biochemical and haematological measurements. J Clin Pathol 1989, 42:172-179. 2. Pocock et al.: Diurnal variations in serum biochemical and haematological measurements. J Clin Pathol 1989, 42:172-179. y y y Methods This is a cohort prospective study with prognostic research recruiting 150 critically ill patients in a major tertiary hospital. Blood samples were collected for estimation of serum total magnesium on admission, and then the patients were followed over 28 days. Cardiac surgery alters the sensitivity of the dynamic interaction between the pituitary and adrenal glands Methods Inclusion criteria: mechanical ventilation previewed at ICU admission >8 days and mortality predicted at ICU admission over 13% (SAPS II >32 points). After the clinical run-in period of 48 hours, samplings were taken to measure baseline blood melatonin and TAC, beginning from the third ICU night and day (midnight and 02:00 p.m.). At 8:00 p.m. of the third ICU day, treatment with 3 mg + 3 mg melatonin (Group M) or placebo (Group P) began: each patient received two tablets per day, at 8:00 p.m. and midnight, until ICU discharge. Further samplings were taken during the early (fourth night and day) and the late (eighth night and day) treatment phases. Melatonin was measured through an ELISA essay; TAC was measured with a specifi c kit. B Gibbison1, J Walker1, G Russell1, K Stevenson2, Y Kershaw1, G Asimakopoulos2, GD Angelini1, SL Lightman1 1University of Bristol, UK; 2University Hospitals Bristol NHS FT, Bristol, UK Critical Care 2014, 18(Suppl 1):P435 (doi: 10.1186/cc13625) Introduction Both ACTH and cortisol are secreted in a diurnal rhythm. Underlying this is an ultradian rhythm of discrete pulses [1] as a result of the feedforward:feedback interactions between cortisol and ACTH [2]. These pulses are critical for normal function; pulsatile and constant infusions yield diff erent transcriptional responses [3] and patients on optimal (nonpulsatile) glucocorticoid replacement have twice the age-related mortality of the general population [4]. We have now characterised the ultradian rhythm and pituitary–adrenal interaction of patients undergoing coronary artery bypass grafting (CABG). Results Sixty-four critically ill patients were enrolled. Endogenous melatonin was shown decreased in the run-in period and in the placebo group compared with healthy subjects. All patients reached satisfying pharmacological values with enteral administration: peak of blood melatonin value (pg/ml) was 2,514 (982 to 7,148) for the M group versus 20 (15 to 62) for the P group (P <0.001) during the fi rst treatment night, while maintaining signifi cant diff erences also during the daytime: 51 (23 to 180) M group versus 14 (11 to 24) P group (P = 0.001). The same trend was observed in the late treatment samples (eighth ICU day). Regarding TAC values (nmol Trolox equivalent/μl plasma), a signifi cant diff erence was highlighted during the night (107 (97 to 123) M group vs. 61 (42 to 89) P group, P <0.001), but not during the daytime (37 (30 to 69) M group vs. Cardiac surgery alters the sensitivity of the dynamic interaction between the pituitary and adrenal glands 28 (25 to 50) P group, P = 0.092). Correlation between melatonin and TAC: Spearman’s rho = 0.33 (P <0.001). Methods Twenty male patients presenting for elective CABG (on-pump and off -pump) were recruited. Blood samples were taken for 24 hours from placement of the fi rst venous access. Cortisol was sampled every 10 minutes, ACTH was sampled every hour and cortisol binding globulin (CBG) was sampled at baseline, at the end of operation and at the end of the 24-hour period. Results Cortisol and ACTH were pulsatile throughout the perioperative period and the cortisol–ACTH interaction persists (Figure 1). The sensitivity of this interaction (calculated by the ratio of cortisol to ACTH pulse amplitude) changed at about 8 hours post surgery such that the adrenal sensitivity to ACTH increased. Conclusion Enteral administration of melatonin was adequate in the early phase of critically illness, with pharmacokinetics similar to published data [2]. The administration of melatonin seems to increase the TAC, with a possible meaningful role in critically ill patients. References Figure 1 (abstract P435). Figure 1 (abstract P435). 1. Mistraletti G, et al.: Intensive Care Med 2011, 37:S181. 2. Mistraletti G, et al.: J Pineal Res 2010, 48:142-147. 1. Mistraletti G, et al.: Intensive Care Med 2011, 37:S181. 2. Mistraletti G, et al.: J Pineal Res 2010, 48:142-147. References 1. Veldhuis JD, et al.: Am J Physiol 1989, 257:E6-E14. 2. Walker JJ, et al.: Proc Biol Sci 2010, 277:1627-1633. g Results Central diabetes insipidus (CDI) developed in 106 patients. Against the background of substitution therapy, we did not observe the development of complications, hypovolemia, hypernatremia, or hyperosmolality. Out of 48 cases of CSWS, in 12 cases the symptoms interleaved with the symptoms of CDI. For 24 patients, CSWS was complicated by depressed consciousness, for three patients by convulsions against the background of hypovolemia, hyponatremia, or hypo-osmolality. No correlation between BPN level and absence of CSWS symptoms was found. 3. Conway-Campbell BL, et al.: J Neuroendocrinol 2010, 22:1093-1100. 4. Bergthorsdottir R, et al.: J Clin Endocrinol Metab 2006, 91:4849-4853. 3. Conway-Campbell BL, et al.: J Neuroendocrinol 2010, 22:1093-1100. h d l l d l b Melatonin blood values and total antioxidant capacity in critically ill patients p LA D’Amato, G Mistraletti, D Longhi, IR Piva, F Marrazzo, C Villa, M Tozzi, R Paroni, E Finati, G Iapichino Università degli Studi di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P436 (doi: 10.1186/cc13626) niversità degli Studi di Milano, Milan, Italy g y Critical Care 2014, 18(Suppl 1):P436 (doi: 10.1186/cc13626) Conclusion In patients with acute brain trauma we observed two syndromes causing water–electrolyte disturbance – CDI and CSWS. CSWS generally is more severe than CDI. We could not determine a correlation between BPN level and the absence of presence of the CSWS symptoms. Introduction Endogenous melatonin is decreased in critically ill patients; oral supplementation could help them to cope with sleep disruption and sepsis. Among patients who took part in a trial on melatonin and need for sedation [1], some were studied with blood sampling to describe their melatonin blood values and the relationship with total antioxidant capacity (TAC) in plasma. Cardiac surgery alters the sensitivity of the dynamic interaction between the pituitary and adrenal glands B Gibbison1, J Walker1, G Russell1, K Stevenson2, Y Kershaw1, G Asimakopoulos2, GD Angelini1, SL Lightman1 1University of Bristol, UK; 2University Hospitals Bristol NHS FT, Bristol, UK Critical Care 2014, 18(Suppl 1):P435 (doi: 10.1186/cc13625) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Russian Polenov’s Neurosurgical Institute from 2001 to 2012. Patients were between 16 and 55 years old. A total of 142 patients were operated for brain tumor, 72 of them of basal–supratentorial localization; eight severe brain trauma; 62 of the hemorrhagic type of stroke, one herpes encephalitis. We excluded from this study the patients with heart and renal failure receiving diuretics. We measured BP, HR, CVP, hourly and daily urine output, level of K and Na in plasma, brain natriuretic peptide (BNP) one to four times a day, and levels of K and Na in urine in single and daily servings. All patients were receiving dexamethasone at a dose between 8 and 32 mg/day as an anti-edema therapy, and thus levels of ACTH and cortisol were not investigated. Conclusion Both cortisol and ACTH remain pulsatile during and after cardiac surgery and the pituitary–adrenal interaction persists, although the sensitivity of the adrenal glands changes throughout the perioperative period. Our study shows that endogenous glucocorticoid levels reach very high oscillating levels following cardiac surgery, which not only invalidate the interpretation of point measures of adrenal function to diagnose adrenal insuffi ciency but also demonstrate that constant infusions of hydrocortisone are unphysiological. References Main causes of water–electrolyte disturbances in patients with acute brain injury: central diabetes insipidus and cerebral salt wasting syndrome L Tsentsiper, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint Petersburg, Russia Critical Care 2014, 18(Suppl 1):P434 (doi: 10.1186/cc13624) Introduction Water–electrolyte disturbances are one of the most common complications of acute brain injury of various origins, threatening the life of the patient and requiring timely correction. In this work we studied the structure of water–electrolyte complications in patients in the neurological intensive care with acute brain injury. Methods We analyzed 259 cases of water–electrolyte disturbances that developed in patients treated in the Department of Intensive Care of S157 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Evaluation of blood glucose control in ICU patients with Space GlucoseControl: a European study Evaluation of blood glucose control in ICU patients with Space GlucoseControl: a European study J Blaha, on behalf of the SGC NIS Study Group General University Hospital, Prague, Czech Republic Critical Care 2014, 18(Suppl 1):P438 (doi: 10.1186/cc13628) p p g p Methods Adult surgical patients with planned ICU admission of ≥24 hours at four medical centers were consented. Following admission to the ICU, the skin of an upper arm was cleaned and a 6 mm diameter site was prepared with controlled micro-abrasion using the Symphony CGM system. A transdermal CGM sensor containing glucose oxidase was applied. Following a 1-hour warm-up period, a calibration was performed. Blood samples were obtained from a radial artery catheter approximately every hour, centrifuged to plasma, and glucose was measured using a YSI 2300 STAT Plus Glucose Analyzer (reference BG). A maximum of 30 reference BG samples were collected for each patient. Samples were collected as frequently as every 15 minutes for trend analysis. CGM was prospectively calibrated every 4 hours. All treatment decisions were based on BG alone. Safety was assessed by visual inspection of the site using a dermatological scale following sensor removal. A study was defi ned as evaluable for CGM sessions >16 hours. Results Thirty-two subjects completed the study. Additional subjects were not considered evaluable due to early discharge from the ICU, failure or early removal of the radial artery catheter, or administration of intravenous acetaminophen. The study cohort was 19% female, 28% diabetes, 56% cardiac surgery, with a mean age of 65 ± 13 years. Overall mean absolute relative diff erence between CGM and reference BG was 12.5%. Continuous glucose error-grid analysis, which assesses point and trend accuracy, showed 98.2% of readings in the A zone (clinically accurate) and 1.2% in the B zone (benign errors). Glucose values ranged from 49 to 324 mg/dl. No device or study-related adverse events were reported. y J Blaha, on behalf of the SGC NIS Study Group y p General University Hospital, Prague, Czech Republic y p , g , p Critical Care 2014, 18(Suppl 1):P438 (doi: 10.1186/cc13628) Introduction Regardless of the ongoing debate on optimum target ranges, glycaemia control (GC) remains an important therapeutic goal in critically ill patients. Dozens of diff erent insulin protocols for ICUs have been developed with diff erent complexity, eff ectiveness, blood glucose (BG) variability and safety. P437 Conclusion SGC is a safe and very effi cient system to control BG in ICU patients. P437 Continuous prediction of glucose-level changes using an electronic nose in critically ill patients JH Leopold, RT Van Hooijdonk, LD Bos, T Winters, PJ Sterk, A Abu-Hanna, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P437 (doi: 10.1186/cc13627) Introduction Many if not most critically ill patients are treated with insulin during their stay in the ICU [1]. Intensive monitoring of the blood glucose level is a prerequisite for effi cient and safe insulin titrations Introduction Many if not most critically ill patients are treated with insulin during their stay in the ICU [1]. Intensive monitoring of the blood glucose level is a prerequisite for effi cient and safe insulin titrations Figure 1 (abstract P435). S158 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 management (enteral, parenteral or both) was carried out at the discretion of the each centre. management (enteral, parenteral or both) was carried out at the discretion of the each centre. in these patients [2]. Current continuous glucose measurement techniques rely on subcutaneous glucose measurements [3] or measurements in blood [4]. We hypothesized that changes in volatile organic compound (VOC) concentrations in exhaled breath refl ect changes in the blood glucose level. Changes in VOC concentrations can be analyzed continuously using a so-called electronic nose (eNose) [5]. Our aim was to investigate exhaled breath analysis to predict changes in glucose levels in intubated ICU patients. Results Seventeen centres from nine European countries included a total of 508 patients. During the study a total of 29,575 BG values were entered into the SGCs and the same number of recommendations were rendered. The mean time-in-target was 77.5 ± 20.9%. The mean proposed next measurement time was 2.0 ± 0.5 hours. Only four episodes of hypoglycaemia <2.2 mmol/l occurred (0.01% of measurements).fi g p Methods Exhaled breath was analyzed in 15 intubated ICU patients who were monitored with a subcutaneous CGM device. eNose results were compared with subcutaneous glucose measurements and linear regression models were built, including subject-specifi c models, and whole-sample models. The models were validated using temporal validation by training the model on the fi rst 75% of measurements and prospectively testing on the last 25% of measurements. Performance of the models was measured using an R2 value, Clarke error grids (CEG) and rate-error grid analysis (R–EGA). P439 P439 Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients J Joseph1, M Torjman2, J Reich3, A Furnary4, S Nasraway3, M McNamara-Cullinane5, D Olson5, D Walton5 1Thomas Jeff erson University Hospital, Philadelphia, PA, USA; 2Cooper University Hospital, Camden, NJ, USA; 3Tufts Medical Center, Boston, MA, USA; 4Providence Heart and Vascular Institute, Portland, OR, USA; 5Echo Therapeutics, Philadelphia, PA, USA Critical Care 2014, 18(Suppl 1):P439 (doi: 10.1186/cc13629) Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients J Joseph1, M Torjman2, J Reich3, A Furnary4, S Nasraway3, M McNamara-Cullinane5, D Olson5, D Walton5 1Thomas Jeff erson University Hospital, Philadelphia, PA, USA; 2Cooper University Hospital, Camden, NJ, USA; 3Tufts Medical Center, Boston, MA, USA; 4Providence Heart and Vascular Institute, Portland, OR, USA; 5Echo Therapeutics, Philadelphia, PA, USA Critical Care 2014, 18(Suppl 1):P439 (doi: 10.1186/cc13629) Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients J Joseph1, M Torjman2, J Reich3, A Furnary4, S Nasraway3, M McNamara-Cullinane5, D Olson5, D Walton5 1Thomas Jeff erson University Hospital, Philadelphia, PA, USA; 2Cooper University Hospital, Camden, NJ, USA; 3Tufts Medical Center, Boston, MA, USA; 4Providence Heart and Vascular Institute, Portland, OR, USA; 5Echo Therapeutics, Philadelphia, PA, USA Critical Care 2014, 18(Suppl 1):P439 (doi: 10.1186/cc13629) Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients Conclusion Exhaled breath prediction of glucose levels seems promising. However, performance of the current models is too low to be used in daily practice. References 1. Schultz MJ, et al.: Intensive Care Med 2010, 26:173-174. 2. Preiser J-C, et al.: Intensive Care Med 2009, 35:1738-1748. 3. Kosiborod M, et al.: Crit Care 2013, 17(Suppl 2):462. 4. Schierenbeck F, et al.: Diabetes Technol Ther 2013, 15:26-31. Introduction Glycemic control in the ICU has been shown to reduce morbidity, mortality and length of stay. However, current methods of blood glucose (BG) monitoring are invasive, intermittent and labor- intensive. Continuous glucose monitoring (CGM) has potential to improve safety/effi cacy of BG control. The performance of a non- invasive, transdermal CGM system (Symphony CGM; Echo Therapeutics, Philadelphia, PA, USA) was evaluated in post-surgical ICU patients. 5. Röck F, et al.: Chem Rev 2008, 108:705-725. References 1. Jacobi J, et al.: Crit Care Med 2012, 40:3251-3276. 2. Cordingley JJ, et al.: Intensive Care Med 2009, 35:123-128. 3. Blaha J, et al.: Diab Care 2009, 32:757-761. 4. Amrein K, et al.: Diabetes Technol Ther 2012, 14:690-695. g y Results Changes in VOC concentrations were associated with changes in subcutaneous glucose levels. R2 performance had a mean value of 0.67 (0.34 to 0.98) for subject-specifi c models, and a mean value of 0.70 (0.52 to 0.96) for the model for the whole sample. However, when externally validating the model, the predictive performance dropped to a mean R2 of 0.19 (0.00 to 0.70) for subject-specifi c models, and 0.04 for the model for the whole sample. Point accuracy in CEG was mostly good with >99% in zones A and B; trend accuracy, as visualized with R–EGA, was low. Time-course evaluation of blood glucose changes in response to insulin delivery in critically ill patients Very Very Variable low Low Control High high Number of patients 11,471 70,106 521,839 77,423 187,553 ICU mortality* (%) 55 31 13 18 26 ICU LOS* 7 6 4 5 6 P <0.001. y y p F Bass1, S Bird1, N Hammond1, J Myburgh2, S Finfer1 1Royal North Shore Hospital, St Leonards, NSW, Australia; 2The George Institute for Global Health, Sydney, Australia Critical Care 2014, 18(Suppl 1):P440 (doi: 10.1186/cc13630) Introduction Intravenous insulin by infusion is commonly used for blood glucose control in the ICU and blood glucose is almost exclusively monitored by intermittent sampling. The rate of change in blood glucose concentration [BG] when the insulin infusion rate is changed is not known, and as a result the optimum time to measure [BG] after changing the infusion rate is unclear. during the fi rst 24 hours from admission were recorded. BG control value was defi ned as BG (≥4.0 ≤9.9 mmol/l). Other BG levels were defi ned as: very low, ≤2.2 mmol/l (≤40 mg/dl); low, >2.2 ≤3.9 mmol/l (40 to 70 mg/dl); high, ≥10.0 <11.1 mmol/l (180 to 200 mg/dl); and very high, ≥11.1 mmol/l (200 mg/dl). g g Methods Following institutional ethics approval and patient consent, using a GluCath Continuous Glucose Monitoring system sensor deployed via a radial artery catheter we studied the change in [BG] in response to a 1 unit/hour increase in the insulin infusion rate during the fi rst 48 hours after cardiac surgery. [BG] was recorded every 10 seconds. Insulin was infused at a concentration of 1 unit/5 ml/hour via a volumetric pump. We recorded [BG] for 2 hours after changing the insulin infusion rate. Data aff ected by artifacts produced by blood draws and subsequent fl ushing of the arterial catheter were excluded and linear interpolation was used to estimate missing [BG] data.i Results There was an increased incidence of mortality in those patients with at least one BG measure below 3.9 mmol/l (70 mg/dl) compared with those without (Table 1). There was a link between BG levels and LoS for surviving patients with the longest hospital stays (critical care and total hospital) experienced by those with BG levels below 2.2 mmol/l (40 mg/dl). g Conclusion There is a strong association between BG levels during admission and mortality and LoS outcomes. Time-course evaluation of blood glucose changes in response to insulin delivery in critically ill patients Although it is not possible to make the link with causation from our dataset, we present results from the largest single dataset of critical care unit patients [1,2]. Results There were fi ve episodes where the insulin infusion increased by 1 unit/hour. [BG] decreased modestly for 50 minutes, after which there was marked interpatient variability in subsequent [BG] trend (Figure 1). The median (range) change in BG (mmol/l) at 30 minutes: –0.5 (–0.7/+0.1), 60 minutes: –0.1 (–0.7/+1.0), 90 minutes: –0.3 (–0.7/+0.6) and 120 minutes: –0.5(–1.2/+2.9).i Acknowledgement Funding from Edwards Lifesciences. References 1. Van den Berghe G, et al.: Intensive insulin therapy in critically ill patients. N Engl J Med 2001, 345:1359-1367. g 2. The NICE-SUGAR Study Investigators: Intensive versus conventional glucose control in critically ill patients. N Engl J Med 2009, 360:1283-1297. Conclusion Within the fi rst hour the change in [BG] was consistent but beyond this time it was highly variable. Further studies are needed to understand the dynamics of [BG] in response to changes in insulin infusion rates, but these data suggest [BG] should be checked hourly until stable. 2. The NICE-SUGAR Study Investigators: Intensive versus conventional glucose control in critically ill patients. N Engl J Med 2009, 360:1283-1297. P440 P440 Time-course evaluation of blood glucose changes in response to insulin delivery in critically ill patients F Bass1, S Bird1, N Hammond1, J Myburgh2, S Finfer1 1Royal North Shore Hospital, St Leonards, NSW, Australia; 2The George Institute for Global Health, Sydney, Australia Critical Care 2014, 18(Suppl 1):P440 (doi: 10.1186/cc13630) Evaluation of blood glucose control in ICU patients with Space GlucoseControl: a European study Although comparison of existing protocols is diffi cult due to signifi cant diff erences in processes and outcome measures, computerized clinical decision support systems generally achieved better GC with consistently lower hypoglycaemia rates than that achieved with paper-based protocols [1]. The enhanced Model Predictive Control (eMPC) algorithm, developed by the CLINICIP group, is the eff ective clinically proven protocol, which has been successfully tested at multiple institutions on medical and surgical patients with diff erent nutritional protocols [2,3]. The eMPC models the behaviour of glucose and insulin in ICU patients with a variable sample interval based on the accuracy of the BG prediction. It has been integrated in the B.Braun Space GlucoseControl (SGC) system, which allows direct data communication between pumps and Space Control with the incorporated eMPC algorithm. Although SGC is already clinically used in dozens of ICUs worldwide, there are few only published experiences with its use [4]. Methods The primary objective of this multicentre European non- interventional study was to evaluate the performance (effi ciency) of SGC under routine conditions in adult ICU patients requiring BG control. The primary endpoint was the percentage of time within the target range, and secondary outcome measures were the frequency of hypoglycaemic episodes and BG measurement intervals. Patients in this trial were assigned to the target range 4.4 to 8.3 mmol/l. Nutritional Conclusion The Symphony CGM system demonstrated clinically relevant accuracy and excellent safety in a variety of patients and ICU environments. Future studies are needed to determine whether Symphony CGM can be used to direct therapy and improve BG control in this patient population. S159 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P441). Critical care outcomes Table 1 (abstract P441). Critical care outcomes P440 First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICUi First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICU N Scawn1, N Coulson1, I Kemp1, P Candolfi 2, F Andrews1, V Mather1, C McGee1, J Goldstein2 1The Liverpool Heart and Chest Hospital, Liverpool, UK; 2Edwards Lifesciences, Nyon, Switzerland Critical Care 2014, 18(Suppl 1):P443 (doi: 10.1186/cc13633) Introduction The aim of this study was to determine the safety and effi cacy of treating patients using a novel intravenous continuous glucose monitoring (CGM) system (GlucoClear™; Edwards Lifesciences). The practical benefi t of controlling blood glucose in the critically ill remains contentious, largely because of the lack of tools to adequately measure and therefore manage levels in real time. A system that is able to provide instant, constantly calibrated, accurate values is a major bonus to ICU care and we report here the fi rst clinical use of a novel CGM system directly used to manage postoperative glycaemia. y y y Methods All consecutive consenting adult patients undergoing cardiac surgery involving cardiopulmonary bypass and requiring postoperative insulin (>98% of patients) were enrolled in the study with a target enrolment of 100. Blood glucose was measured via a dedicated peripheral intravenous catheter with values reported every 5 minutes. The primary outcome was the number of data points in the target glycaemic control range (4.4 to 8.0 mmol/l), using a dynamic insulin protocol. Secondary endpoints include mean glucose levels, time in range and number of hypoglycaemic and hyperglycaemic episodes.i Results For the fi rst 45 patients, mean age was 67.7 years (male 52.8%), 54% had undergone valve surgery with or without CABG and the majority (67%) had no history of diabetes. The CGM sensor was typically sighted in the forearm or hand (90.5%) and was resited on 7.5% occasions. Median monitoring time in the ICU was 28.4 hours. Mean glycaemic value was 6.8 mmol/l. From the possible 18,609 glucose values (one per 5 minutes), 16,808 values were recorded (90.3%). Overall, 13,389/16,808 values (79.6%) were within the target range. No hypoglycaemic episodes (≤2.2 mmol/l) were recorded at any point. There were 219/16,808 values (1.3%) in the hyperglycaemic range (≥10.0 mmol/l). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Krinsley JS: Crit Care Med 2008, 36:3008-3013. 3. Krinsley JS: Crit Care 2011, 15:R173. 4. Krinsley JS: Crit Care 2013, 17:R37. 5. Mulavisala KP, Gopal PB, Crane B: Crit Care 2012, 16: P175. Impact of corticosteroid administration in septic shock on glycemic variability L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, D Mirea3, I Grintescu1 1Clinical Emergency Hospital of Bucharest, Romania; 2National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P445 (doi: 10.1186/cc13635) P443 P443 First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICU N Scawn1, N Coulson1, I Kemp1, P Candolfi 2, F Andrews1, V Mather1, C McGee1, J Goldstein2 1The Liverpool Heart and Chest Hospital, Liverpool, UK; 2Edwards Lifesciences, Nyon, Switzerland Critical Care 2014, 18(Suppl 1):P443 (doi: 10.1186/cc13633) Glycaemia and critical care outcomes 2 2 1Care Hospital Nampally, Hyderabad, India; 2STAR Hospitals, Hyderabad, India Critical Care 2014, 18(Suppl 1):P442 (doi: 10.1186/cc13632) 1Care Hospital Nampally, Hyderabad, India; 2STAR Hospitals, Hyderabad, India Critical Care 2014, 18(Suppl 1):P442 (doi: 10.1186/cc13632) M Cecconi1, C Ryan2, D Dawson2, N Di Tomasso2, G McAnulty2, B Philips2, A Rhodes2 Introduction While there is ongoing discussion of the optimal range for glycemic control in hospital intensive care, recent publications from Mackenzie and colleagues [1] and Krinsley [2-4] suggest not only that mean BG should be considered, but also that glucose variability and complexity may be equally important. This has increased the need for continuous systems which can provide early warnings of hypoglycemia and eff ectively measure variability. GlySure Ltd has developed an intravascular glucose monitoring system to simplify the application of hospital protocols for tight glycemic control (TGC) at the point of care. Experience with the original research-based instrumentation [5] has now been incorporated into a combined pre-production monitor and Critical Care 2014, 18(Suppl 1):P441 (doi: 10.1186/cc13631) Introduction The aim of this study was to determine the impact of preadmission or fi rst 24-hour blood glucose (BG) measurements in UK ICUs on mortality. Introduction The aim of this study was to determine the impact of preadmission or fi rst 24-hour blood glucose (BG) measurements in UK ICUs on mortality. Methods The Intensive Care National Audit & Research Centre case-mix programme database on adult admissions to general, neuroscience and cardiothoracic critical care units was used for analysis. Within the database, the highest and lowest blood glucose (BG) values measured Figure 1 (abstract P440). Blood glucose change after infusion change and after calibration. S160 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 refi ned to maximise the benefi t of GGM. Overall, the evidence from the fi rst applied clinical use of this novel CGM showed that proper safe, tight glycaemic control can be achieved. Further investigations are required to demonstrate the reduction in morbidity and mortality using CGM, and we plan to embark on further studies to address this. autocalibration unit. We have now completed a 34-patient trial using this device and present the data collected from this study. autocalibration unit. We have now completed a 34-patient trial using this device and present the data collected from this study. Methods The study used GlySure sterile, single-use sensors and a 5-lumen 9.5-Fr CVC device, allowing the fl uorescence optical-based sensor to be placed into the patient’s right internal jugular vein. The screen data were blinded to the bedside staff . Data from the monitor were later compared with sample measurement from the Yellow Springs (YSI) glucose analyzer. The data accuracy was measured using the mean absolute relative diff erence (MARD), an error calculation tool. Results The device met the primary safety and eff ectiveness endpoints of the trial. The 456 sample values recorded by the monitor based on 8-hour calibrations were correlated with samples taken from the YSI and the MARD for the study was 9.40%. The analysis showed that 89.23% of the data fell within the A zone of the Clark error grid, with the rest falling within the B zone. g Conclusion The results demonstrate a good correlation with the accepted standard of blood glucose determination in ICU practice. Early detection of glycemic excursions can provide carers with the opportunity for an early intervention and thus achieve the elusive target of TGC around the chosen target range. e e e ces Mackenzie I, Whitehouse T, Nightingale P: Intensive Care Med 2011, 1. Mackenzie I, Whitehouse T, Nightingale P: Intensive Care Med 2011, 37:435-443. 2. Krinsley JS: Crit Care Med 2008, 36:3008-3013. 3. Krinsley JS: Crit Care 2011, 15:R173. 4. Krinsley JS: Crit Care 2013, 17:R37. 5. Mulavisala KP, Gopal PB, Crane B: Crit Care 2012, 16: P175. 1. Mackenzie I, Whitehouse T, Nightingale P: Intensive Care Med 2011, 37:435-443. 2. Krinsley JS: Crit Care Med 2008, 36:3008-3013. 3. Krinsley JS: Crit Care 2011, 15:R173. 4. Krinsley JS: Crit Care 2013, 17:R37. 5. Mulavisala KP, Gopal PB, Crane B: Crit Care 2012, 16: P175. 37:435-443. 2. Anti-infl ammatory and antioxidant eff ects of ranolazine on primary cultured astrocytes Introduction Because of its ability to block late INa [1], ranolazine is used as an antianginal agent for the treatment of chronic angina pectoris when angina is not adequately controlled by other agents [2]. Besides its cardiovascular eff ects, ranolazine improves diff erent neuronal functions, and thus its use has been proposed for the treatment of pain and epileptic disorders [3,4]. Since astrocytes are involved in neuronal infl ammatory processes, and autoimmune and neurodegenerative diseases [5], we have investigated the anti- infl ammatory and antioxidant eff ects of ranolazine in primary cultured astrocytes. y Results There were no diff erences between the three groups at the beginning of the study regarding demographic data and the clinical characteristics, including BG value. BG levels were strongly correlated with severity of septic shock estimated by APACHE II score (r = +0.241; P = 0.005) or Simplifi ed Acute Physiology Score II (r = 0.280; P = 0.001) – Pearson correlation. The risk of death is signifi cantly increased if SD of BG is more than 20 mg/dl (67.7% vs. 20.8%, P = 0.000). A total 94.4% of deceased patients in group A registered a SD of BG more than 20 mg/dl versus 89.5% in group B or 40% in group C (P = 0.006). In total, 53.5% of patients in group A needed insulin therapy versus 25.5% in group B or 27.3% in group C. The dose was between 30.28 ± 6.65 UI/day in group A, 37.85 ± 11.95 UI/day in group B, and 14.28 ± 5.76 UI/day in group C (P >0.05). Methods We incubated diff erentiated rat astrocytes in primary culture (10 days of culture) [5] for 24 hours with ranolazine (10–5, 10–6, 10–7 M). We measured the protein expression levels of PPARγ and Cu/Zn-SOD by western blot technique. Protective eff ect of ranolazine on cell viability was assayed using MTT conversion assay. Finally, to evaluate the eff ect of ranolazine on the IL-1β cytokine and TNFα mediators, we used the enzyme-linked immunosorbent assay technique. Conclusion BG variability is highly associated with mortality compared with BG mean daily value or insulin dose. SD levels above 20 mg/dl were associated with a signifi cantly higher mortality rate relative to those with SD levels below 20 mg/dl. N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK C iti l C 2014 18(S l 1) P448 (d i 10 1186/ 13638) Introduction Tuberculous meningitis (TBM) is the least common extrapulmonary manifestation of tuberculosis. Although the UK incidence of TBM is relatively low, it carries a high mortality and morbidity [1]. Neurological deterioration continues to be an important reason for ICU admission [2]. Little is known about the outcomes for TBM patients requiring ICU admission. Our aim is to evaluate patient demographics, TBM clinical data, and the necessity for organ support, and whether this can be associated with outcome. Methods Ninety-six patients were studied. The following parameters were calculated during the fi rst week after ICU admission. (1) Maximum value of SOFA score (SOFAmax). (2) Mean, standard deviation, maximum, minimum, and diff erence of BG levels (BGm, BGsd, BGmax, BGmin, BGd (BGmax – BGmin), respectively). BG levels were measured basically every 6 hours. (3) Correlation between SOFAmax and BG parameters using two-dimensional (correlation coeffi cient rt) and linear regression analysis (rl). Methods A retrospective study at a tertiary centre of patients with TBM admitted to our ICU between 2000 and 2012. Data were retrieved on demographics, microbiology, radiology and pharmacological fi ndings and type and level of organ support. APACHE II and SOFA scores were calculated. Patients were stratifi ed into two groups: CSF PCR+ve and CSF PCR–ve. l Results (1) Mortality of the patients with SOFAmax 0 to 3, 4 to 5, 6 to 7, 8 to 9, and 10 or more were 0%, 14%, 23%, 40%, and 89%, respectively. (2) rt and rl (rt/rl) between SOFAmax and BG parameters: BGsd (0.49/0.36), BGmax (0.47/0.32), BGm (0.45/0.23), BGd (0.44/0.32), and BGmin (0.25/0.06). (3) BG ranges that indicate SOFAmax less than 5 calculated from the two-dimensional correlation curves between SOFAmax and BGsd, BGmax, BGm, and BGd were 35 ± 25, 250 ± 112, 150 ± 33, and 156 ± 115 mg/dl, respectively. Results Eight patients (six males:two females) were evaluated. Total mean age was 43.9 ± 8.09 years. A reduction in GCS was the main reason for ICU admission. All patients received ≥3 anti-TBM drugs and steroids. Two CSF PCR+ve patients had primary drug resistance to isoniazid. There was also a longer mean delay in the time of onset of anti-TBM treatment in CSF PCR+ve patients (4 ± 2.88 days). Higher APACHE II and SOFA scores on admission (mean = 23, 8.5) were Conclusion BG parameters except BGmin were two-dimensionally related to the severity. P448 g j 1Shisei Hospital, Saitama, Japan; 2Keiyo Hospital, Tokyo, Japan gl References References 1. Belardinelli L, et al.: J Pharmacol Exp Ther 2013, 344:23-32. 2. Siddiqui MA, et al.: Drugs 2006, 66: 693-710. 3. Park YY, et al.: J Neurophysiol 2013, 109:1378-1390. 4. Kahlig KM, et al.: Br J Pharmacol 2010, 161:1414-1426. 5. Vallés SL, et al.: Brain Pathol 2004, 14:365-371. 2. Siddiqui MA, et al.: Drugs 2006, 66: 693-710. 2. Siddiqui MA, et al.: Drugs 2006, 66: 693-710. 3. Park YY, et al.: J Neurophysiol 2013, 109:1378-1390. P446 q g 3. Park YY, et al.: J Neurophysiol 2013, 109:1378-1390. Anti-infl ammatory and antioxidant eff ects of ranolazine on primary cultured astrocytes y y Results Compared with control cells, treatment with ranolazine induced an increment of anti-infl ammatory PPARγ and reduced the proinfl ammatory mediators IL-1β and TNFα in primary cultured astrocytes. Ranolazine (10–6 M) also increased the expression of antioxidant protein Cu/Zn-SOD and caused a signifi cant increase in cell viability.l Blood glucose target in acute phase suggested by the analysis of the relationship between blood glucose profi le and the severity of the diseases y 4. Kahlig KM, et al.: Br J Pharmacol 2010, 161:1414-1426. 5. Vallés SL, et al.: Brain Pathol 2004, 14:365-371. M Hoshino1, Y Haraguchi2, K Oda1, S Kajiwara1 M Hoshino1, Y Haraguchi2, K Oda1, S Kajiwara1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods In a prospective, controlled study, 134 patients with septic shock were randomized into three study groups: group A (n = 43), 200 mg/day hydrocortisone hemisuccinate in four daily doses; group B (n = 47), same dose of hydrocortisone hemisuccinate in continuous administration; group C (n = 44), no hydrocortisone hemisuccinate. Patients with diabetes mellitus were excluded. The duration of hydrocortisone treatment was a maximum 7 days. The target blood glucose (BG) level was below 180 mg/dl. BG values were analyzed by calculating mean daily values, standard deviation (SD) of BG values as an index of glycemic variability, and insulin doses. The local ethics committee approved the study.f P447 P447 Anti-infl ammatory and antioxidant eff ects of ranolazine on primary cultured astrocytes FB El Amrani, S Guerra, D Aguirre-Rueda, MD Mauricio, P Marchio, JM Vila, SL Vallés, F Fernández, M Aldasoro University of Valencia, Spain Critical Care 2014, 18(Suppl 1):P447 (doi: 10.1186/cc13637) First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICUi Impact of corticosteroid administration in septic shock on glycemic variability L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, D Mirea3, I Grintescu1 1Clinical Emergency Hospital of Bucharest, Romania; 2National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P445 (doi: 10.1186/cc13635) Conclusion This new CGM system enabled signifi cantly improved glycaemic control despite the challenges of working with an entirely new system of glucose control in a unit with over 200 nurses. Performance notably improved with experience and also highlighted that even previously validated dynamic algorithms will need to be Introduction The purpose of this study was to assess the relation between glycemic control and the severity of sepsis in a cohort of patients with vasopressor-dependent septic shock treated with corticosteroids [1,2]. S161 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References 1. Waeschle RM, Moerer O, Hilgers R, et al.: The impact of the severity of sepsis on the risk of hypoglycemia and glycemic variability. Crit Care 2008, 12:R129.f Conclusion Ranolazine decreases infl ammatory mediators IL-1β and TNFα, and increases anti-infl ammatory PPARγ as well as the antioxidant Cu/Zn-SOD in astrocytes in culture. These results suggest that ranolazine could be useful as a neuroprotective drug in pathologies inducing infl ammatory damage and oxidant processes. f 2. Loisa P, Parviainen I, Tenhunen J, et al.: Eff ect of mode of hydrocortisone administration on glycemic control in patients with septic shock. A prospective randomized trial. Crit Care 2007, 11:R21. Tuberculous meningitis: a 10-year case analysis of critical care admissions p p y p y p Critical Care 2014, 18(Suppl 1):P446 (doi: 10.1186/cc13636) y y Critical Care 2014, 18(Suppl 1):P446 (doi: 10.1186/cc13636) Introduction Blood glucose (BG) control in acute illness improves outcome. However, how to manage BG levels, or BG target, is not clearly elucidated. In this study, the BG target was suggested by the analysis of the relationship between BG profi le and the severity of the diseases. Methods Ninety-six patients were studied. The following parameters were calculated during the fi rst week after ICU admission. (1) Maximum value of SOFA score (SOFAmax). (2) Mean, standard deviation, maximum, minimum, and diff erence of BG levels (BGm, BGsd, BGmax, BGmin, BGd (BGmax – BGmin), respectively). BG levels were measured basically every 6 hours. (3) Correlation between SOFAmax and BG parameters using two-dimensional (correlation coeffi cient rt) and linear regression analysis (rl). Introduction Blood glucose (BG) control in acute illness improves outcome. However, how to manage BG levels, or BG target, is not clearly elucidated. In this study, the BG target was suggested by the analysis of the relationship between BG profi le and the severity of the diseases. N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 to compare known clinical predictors of MV and determine predictors of prolonged MV. associated with a positive CSF PCR result. Increased requirements for mechanical ventilation (100%), tracheostomy (50%), inotropes (75%), neurosurgical intervention (predominantly CSF drainage) (100%) and enteral feeding (50%) were all signifi cant for this group. Mortality and long-term neurological morbidity were substantially higher for PCR+ve patients (75% and 25%). In contrast, the majority of culture-negative patients survived (75%), and experienced good recoveries at follow-up. Overall mortality was 50%. associated with a positive CSF PCR result. Increased requirements for mechanical ventilation (100%), tracheostomy (50%), inotropes (75%), neurosurgical intervention (predominantly CSF drainage) (100%) and enteral feeding (50%) were all signifi cant for this group. Mortality and long-term neurological morbidity were substantially higher for PCR+ve patients (75% and 25%). In contrast, the majority of culture-negative patients survived (75%), and experienced good recoveries at follow-up. Overall mortality was 50%. Methods We conducted a retrospective chart review of consecutive CSI admitted between 1 January 2005 and 1 March 2009. We recorded data related to the injury, the duration of MV, respiratory complications, ICU and hospital length of stay and patients’ outcomes. A review of the literature identifi ed known predictors (ASIA level, ISS, level of injury, and so forth). Univariate and multivariate logistic regression were used to identify predictors of MV and prolonged MV. y Conclusion This is the second documented case analysis of TBM and ICU admission in adult patients [1,2]. Findings show that poor outcomes are associated with positive CSF PCR results. Factors linked to poor outcomes include delays in initiating anti-TBM treatment, neurosurgical interventions, and an increased requirement for multiorgan support. Earlier drug susceptibility testing would be preferable particularly for patients with positive CSF PCR cultures. Given the potential severity of TBM, a high index of clinical suspicion remains critical towards optimizing outcomes. y p p g Results Of the 208 patients, 82% were male and the mean age was 51 years. Hospital mortality was 8.7%. Main causes of injury were motor vehicle accidents (39.7%) and falls (43.2%). Injuries below C4 level represented 51.5% of the population. A complete loss of motor function (ASIA level A and B) was found in 34.9% of patients. The mean and median ISS score was 20.7. In total, 78 patients required MV (37.5%) and 30 patients required prolonged MV (14.4%). P452 P452 Evaluation of the ocular microcirculation in brain-dead patients: fi rst step towards a new method of multimodal neuromonitoring? T Tamosuitis1, A Pranskunas1, N Balciuniene1, V Pilvinis1, EC Boerma2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Medical Centre Leeuwarden, the Netherlands Critical Care 2014, 18(Suppl 1):P452 (doi: 10 1186/cc13642) g y Methods During surgery, samples of cerebrospinal fl uid and arterial blood were simultaneously withdrawn to evaluate lactate concentration. Samples were collected at fi ve fi xed times during and after surgery: T1 (beginning of the intervention), T2 (15 minutes after aortic cross-clamping), T3 (just before unclamping), T4 (end of surgery), and T5 (4 hours after the end of surgery).l 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Medical Centre Leeuwarden, the Netherlands ritical Care 2014, 18(Suppl 1):P452 (doi: 10.1186/cc13642) Introduction Multimodal neuromonitoring is a part of goal- directed therapy in severe neurosurgical pathology that leads to better understanding and therefore accurate and timely correction of disturbances of cerebral perfusion. The aim of our study was to evaluate and compare microcirculation in the conjunctiva of the eye and sublingual mucosa in brain-dead patients and healthy volunteers. No studies to our knowledge with this purpose were performed previously. We hypothesized that direct videomicroscopic evaluation of conjunctival microcirculation is linked to cerebral blood fl ow. Results Mean lactate levels in cerebrospinal fl uid rose consistently from the beginning of the intervention steadily until after surgery (T1 = 1.83 mmol/l, T2 = 2.10 mmol/l, T3 = 2.72 mmol/l, T4 = 3.70 mmol/l, T5 = 4.31 mmol/l). Seven patients developed spinal cord injury; two of them had delayed injury occurring 24 hours after the end of surgery; the remaining fi ve had early onset. In this group of fi ve patients, preoperative cerebrospinal fl uid lactate levels were signifi cantly (P = 0.04) higher than those of the other 40 patients preoperatively (2.12 ± 0.35 vs. 1.79 ± 0.29 mmol/l). jl Methods We evaluated microcirculation of the eye conjunctiva and sublingual mucosa of 10 brain-dead diagnosed patients and 10 healthy volunteers. Brain-death diagnoses were certifi ed by cerebral angiography. Direct in vivo observation of the microcirculation was obtained with sidestream dark-fi eld imaging. Assessment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI), proportion of perfused vessels (PPV)), and diff usion distance (perfused vessel density (PVD) and total vessel density (TVD)) was performed according to international criteria. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 After multivariate analysis, four predictors of MV were identifi ed: pneumonia (OR = 52.83); ISS score >22 (OR = 4.09); age (OR = 1.02); level C1 to C4 (2.34); and two predictors of prolonged MV: ASIA score A and B (OR = 5.57) and pneumonia (OR = 8.76). References G Landoni, M Pieri, V Testa, S Silvetti, M Zambon, G Borghi, M Azzolini, AL Di Prima, L Nobile, R Lembo, A Zangrillo Vita-Salute San Raff aele University, Milan, Italy Critical Care 2014, 18(Suppl 1):P449 (doi: 10.1186/cc13639) 1. Krueger H, et al.: Chronic Dis Inj Can 2013, 33:113-122. 2. Devivo MJ: Spinal Cord 2012, 50:365-372. 3. Como JJ, et al.: J Trauma Inj Infect Crit Care 2005, 59:912-916. 4. Durga P, et al.: Anesth Anal 2010, 110:134-140. 5. Honarmand A, et al.: Ulus Travma Acil Cerrahi Derg 2008, 14:110-117. 6. Branco BC, et al.: J Trauma 2011, 70:111-115. Introduction The aim was to evaluate the role of intrathecal lactate as an early predictor of spinal cord injury during thoracoabdominal aortic aneurysmectomy. Forty-four consecutive patients were scheduled to undergo thoracoabdominal aortic aneurysmectomy. Two patients had a type B dissecting aneurysm; all other 42 patients suff ered from degenerative aneurysm. P452 Conclusion The preoperative cerebrospinal lactate concentration is elevated in patients who will develop early-onset spinal cord injury after thoracoabdominal aortic aneurysmectomy. This may allow a better stratifi cation of these patients, suggesting a more aggressive strategy of spinal cord function preservation and possibly guaranteeing them a better outcome. References 1. Pehlivanoglu F, et al.: Scientifi cWorldJournal 2012, 2012:169028. 2. Verdon R, et al.: Clin Infect Dis 1996, 22:982-988. 1. Pehlivanoglu F, et al.: Scientifi cWorldJournal 2012, 2012:169028. 2. Verdon R, et al.: Clin Infect Dis 1996, 22:982-988. Conclusion In our study ISS, cervical level and age were associated with MV but not with the need for prolonged MV, whereas pneumonia was an independent risk factor for both. This is a potentially preventable risk factor where specifi c strategies can be applied to improve patients’ outcome. N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK C iti l C 2014 18(S l 1) P448 (d i 10 1186/ 13638) Therefore, a part of the severe patients seemed to have lower BG levels and lower BG variability. Targeting those BG parameters in early phase within the abovementioned levels was considered to link to better outcome. S162 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 External validation of an early warning alert for elevated intracranial pressure in the Avert-IT database Introduction After severe traumatic brain injury (TBI), episodes of elevated intracranial pressure (ICP) are associated with poor outcome. Previously we developed a model to predict increased ICP, 30 minutes in advance, using the dynamic characteristics of routinely monitored minute-by-minute ICP and mean arterial blood pressure (MAP) signals [1]. The model was developed using data from the Brain-IT database [2]. Here we present external validation results of this model, on a more recent cohort of adult TBI patients from the AVERT-IT project [3]. y Results The k-support system was managed in 102 emergency patients, 65 patients (64%) were classifi ed as neurological disease, 41 (40%) as stroke, 11 (11%) as head injury, two (2%) as epilepsy. The detail of stroke was ischemia in 35 (34%), hemorrhage in four (4%) and SAH in two (2%). Two ischemic stroke patients were treated with intravenous thrombolysis of alteplase using the k-support system and a ‘drip- and-ship’ paradigm. One patient using alteplase showed complete recanalization of the middle cerebral artery. The consultations resulted in hospitalization in 42%, transfer in 37% and return home in 20%. p p j Methods A retrospective analysis of physiological data collected at the minute resolution, from 43 adult patients from the AVERT-IT project. A total of 67 episodes of ICP above 30 mmHg lasting at least 10 minutes were identifi ed in this cohort. Four-hour time series of ICP and MAP anteceding each episode by 30 minutes were analyzed. Additional time series not preceding elevated ICP episodes were used for validation. Results Table 1 summarizes the main fi ndings. Performance of the model in the original study [1] is reproduced in the fi rst column. The model retains identical performance for all criteria in the cohort of more recent TBI patients of the AVERT-IT database. Methods A retrospective analysis of physiological data collected at the minute resolution, from 43 adult patients from the AVERT-IT project. A total of 67 episodes of ICP above 30 mmHg lasting at least 10 minutes were identifi ed in this cohort. Four-hour time series of ICP and MAP anteceding each episode by 30 minutes were analyzed. References 1. Guiza et al.: Crit Care Med 2013, 41:554. 2. [http://www.brainit.org] 3. [http://www.avert-it.org] 1. Guiza et al.: Crit Care Med 2013, 41:554. 2. [http://www.brainit.org] 3. [http://www.avert-it.org] External validation of an early warning alert for elevated intracranial pressure in the Avert-IT database Additional time series not preceding elevated ICP episodes were used for validation.i p Conclusion Before introduction of the k-support system, the standard thrombolytic therapy using alteplase for acute ischemic stroke could not operate in the Kaifu area due to the absence of stroke specialists and the long distance to a neighboring stroke center. The telemedicine system using a mobile device as the k-support system can be used anytime, anywhere and by anyone. This system can communicate with the doctors, between general physicians in depopulated areas and specialists in urban areas, and may become a useful tool for acute patient management in not only stroke but also other emergency diseases g Results Table 1 summarizes the main fi ndings. Performance of the model in the original study [1] is reproduced in the fi rst column. The model retains identical performance for all criteria in the cohort of more recent TBI patients of the AVERT-IT database. Table 1 (abstract P453). Database Brain-IT Avert-IT AUROC 0.85 0.83 aBrier SS (%) 34 30 Cal large 0.00 0.01 Cal slope 0.99 1.03 Accuracy (%) 77 77 References 1. Takao H, et al.: Stroke 2012, 43:236-239. 2. Tekle WG, et al.: Stroke 2012, 43:1971-1974. References 1. Takao H, et al.: Stroke 2012, 43:236-239. 2. Tekle WG, et al.: Stroke 2012, 43:1971-1974. P453 thrombolytic therapy using alteplase for acute ischemic stroke [1,2]. Methods A system was consisted of communicating patient data and imaging between the hospital system and participating staff members in and out of the hospital using mobile devices. The system can transfer clinical data and large volumes of CT and MRI, and expert opinion in real time. We developed the system (k-support) in the Kaifu area, which is a typical depopulated area in Tokushima Prefecture, Japan, between the general physicians in Tokushima Prefectural Kaifu Hospital and specialists in stroke and cardiovascular disease at Tokushima University Hospital from February 2013. External validation of an early warning alert for elevated intracranial pressure in the Avert-IT database M Beckers1, F Güiza1, B Depreitere1, I Piper2, R Donald3, G Van den Berghe1, G Meyfroidt1 1University Hospitals Leuven, Belgium; 2Southern General Hospital, Glasgow, UK; 3University of Glasgow, UK Critical Care 2014, 18(Suppl 1):P453 (doi: 10.1186/cc13643) References References 1. Takao H, et al.: Stroke 2012, 43:236-239. 2. Tekle WG, et al.: Stroke 2012, 43:1971-1974. Eff ects of cardiac output-guided hemodynamic management on fl uid administration after aneurysmal subarachnoid hemorrhage B Bergmans, M Egal, J Van Bommel, J Bakker, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P455 (doi: 10.1186/cc13645) Conclusion The obtained external validation results, on a previously unseen cohort of adult TBI patients, confi rm the robustness of the model to accurately predict future increased ICP events 30 minutes in advance. The general applicability of the model is probably due to its sparseness, as it only uses two routinely monitored signals as input, namely ICP and MAP. These results are a large step forward in our work toward an early warning system for elevated ICP that can be used worldwide. Introduction In patients with aneurysmal subarachnoid hemorrhage (SAH), hypervolemic therapy may result in fl uid overload that may be associated with adverse clinical outcomes [1,2]. We hypothesized that a goal-directed transpulmonary thermodilution (TPT) monitoring protocol aiming for normovolemia may result in decreased fl uid intake while sustaining adequate volume status in poor-grade SAH patients. Methods Following the introduction of the hemodynamic protocol in 2011, 26 consecutive patients with SAH were included until 2013. Using TPT (PiCCO; Pulsion), cardiac output (CO), global end- diastolic volume index (GEDVI) and extravascular lung water index (EVLWI) were determined. Fluid administration was targeted at fl uid unresponsiveness. Indications for start of the protocol were: hypotension (in spite of fl uids), pulmonary edema or cardiac stunning, daily fl uid balance ≤–1 l, cerebral ischemia (DCI) with progressive symptoms. Data were collected on fl uid intake and output up to 3 days before and 3 days after the start of TPT. We assessed the course of fl uid input and output and hemodynamic parameters before and after the start of the protocol with the generalized estimating equation. P454 New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas T Kageji1, H Oka1, F Obata2, K Tani3, H Bando2, R Tabata3, M Kohno3 1Tokushima University Hospital, Tokushima, Japan; 2Tokushima Prefectural Kaifu Hospital, Kaifu, Japan; 3The University of Tokushima Graduate School, Tokushima, Japan Critical Care 2014, 18(Suppl 1):P454 (doi: 10.1186/cc13644) P450 Predictors of ventilatory outcome in cervical spinal injuries HT Wang, DW Williamson, MA Albert Hopital Sacre-Coeur de Montreal, Canada Critical Care 2014, 18(Suppl 1):P450 (doi: 10.1186/cc13640) y g Results All brain-dead patients required vasopressor support to sustain perfusion of donor organs. The MFI of small vessels was signifi cantly lower in brain-dead patients in comparison with healthy controls in ocular conjunctiva (2.6 (2.4 to 2.8) vs. 3.0 (3.0 to 3.0), P = 0.03) and in sublingual mucosa (2.8 (2.6 to 2.9) vs. 3.0 (3.0 to 3.0), P = 0.04). TVD and PVD of small vessels were signifi cantly lower in brain-dead patients in comparison with healthy controls in ocular conjunctiva (10.2 (5.2 to 14.8) vs. 17.0 (15.4 to 27.2) mm/mm2, P = 0.023 and 5.2 (2.9 to 7.2) vs. 10.1 (8.5 to 16.7) l/mm, P = 0.023), but there was no diff erence in sublingual mucosa.i Introduction Spinal cord injuries aff ect 50 persons per million every year in North America [1], with over 50% occurring at the cervical level [2]. Cervical spinal cord injuries (CSI) are at particular risk for mechanical ventilation (MV), pulmonary complications and increased length of hospital stay. A few small cohort studies looked at predictors of MV [3-6], and to our knowledge there are no studies addressing factors associated with prolonged MV. The purpose of this study was Conclusion We were able to identify a signifi cant reduction of capillary density in the eye conjunctiva but not in sublingual mucosa when S163 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 medical quality between the urban and depopulated areas. To improve the problem, telemedicine using a mobile device between general physicians and stroke specialists became important with the increasing demand for rapid and correct diagnosis for treatment of acute stroke. We developed a system for rapidly exchanging diagnostic images and clinical information in depopulated areas to develop the standard comparing microcirculation of brain-dead diagnosed patients and healthy volunteers. However, the presence of conjunctival fl ow in case of general cerebral fl ow is completely absent, making it diffi cult to use conjunctival fl ow as a substitute for brain fl ow. P454 New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas T Kageji1, H Oka1, F Obata2, K Tani3, H Bando2, R Tabata3, M Kohno3 1Tokushima University Hospital, Tokushima, Japan; 2Tokushima Prefectural Kaifu Hospital, Kaifu, Japan; 3The University of Tokushima Graduate School, Tokushima, Japan p g g q Results Mean age was 55 ± 16, and median Glasgow Coma Scale on admission was 8 (IQR 6 to 13). TPT was started at a median of 1 day after ICU admission (IQR 0 to 4). DCI developed in 70% and the in-hospital death rate was 45%. Compared with days preceding the protocol (day –3 to –1), TPT (day 0 to 3) was associated with decreased fl uid intake (compared with day 3 as reference; day –1: +1.14 ± 0.31 l, P <0.001; day 0: +0.68 ± 0.29 l, P = 0.019; day 1: +0.73 ± 0.31 l, P = 0.02), increased Critical Care 2014, 18(Suppl 1):P454 (doi: 10.1186/cc13644) Introduction Stroke is the main cause of deterioration of activity of daily living in Japan. The social problem in Japan is the diff erence in S164 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 PbrO2 (Licox, GMS) ICP, CPP, MABP, CVP, local brain temperature, body core temperature, PCO2, and blood glucose among others. The cerebral monitoring probes were inserted via a Bolt (ICP, PbrO2, microdialysis) and an additional burr hole (CBF). All probes were positioned in the penumbra and location was verifi ed by brain CT. The PbrO2 arm of this study and its signifi cance is still underway and will be announced later. Thirteen of the patients were successfully discharged from the ICU while eight did not survive. PbrO2 (Licox, GMS) ICP, CPP, MABP, CVP, local brain temperature, body core temperature, PCO2, and blood glucose among others. The cerebral monitoring probes were inserted via a Bolt (ICP, PbrO2, microdialysis) and an additional burr hole (CBF). All probes were positioned in the penumbra and location was verifi ed by brain CT. The PbrO2 arm of this study and its signifi cance is still underway and will be announced later. Thirteen of the patients were successfully discharged from the ICU while eight did not survive. P454 New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas fl uid output (day –2: –0.91 ± 0.46 l, P <0.05 compared with day 3), and consequently a strong decrease in fl uid balance (day –3: +2.10 ± 0.56 l, P <0.001; day –2: +2.66 ± 1.14 l, P = 0.02; day –1: +1.41 ± 0.37 l, P <0.001). The decreased fl uid intake and fl uid balances did not result in decreased CO, GEDVI or EVLWI on days 1 to 3 compared with day 0 (mean PCCI 3.5 l/minute/m2, mean GEDVI 761 ml/m2, mean EVLWI 10.8 ml/kg). Conclusion Our data suggest that in poor-grade SAH patients goal- directed fl uid management with TPT is feasible to decrease intake and increase diuresis without adverse eff ects on cardiac output or preload parameters. Future research should assess the eff ect of such a protocol on clinical outcomes. g Results The fi nal data are currently under statistical evaluation, which will be completed at the time of presentation. However, there is indication of a link between brain glucose levels and CBF values, but it is not clear as to the CBF–PbrO2 correlation that is the second part of this study under evaluation. This may be due to the fl uctuation of brain glucose because of brain ischemia, hyperemia, hypermetabolism or hypometabolism. So far we are able to establish a correlation of CBF and lactate/pyruvate ratio only in persistently low CBF values. References 1. Mutoh T, et al.: Stroke 2009, 40:2368-2374. 2. Ibrahim GM, Macdonald RL: Neurocrit Care 2013, 19:140. 1. Mutoh T, et al.: Stroke 2009, 40:2368-2374. 1. Mutoh T, et al.: Stroke 2009, 40:2368-2374. 2. Ibrahim GM, Macdonald RL: Neurocrit Care 2013, 19:140. y y y Conclusion This will be a fi nal report of a study in human patients with severe subarachnoid hemorrhage and traumatic brain injury. The results indicate correlations of varying signifi cance between the pooled data still under statistical analysis. We hope that the outcome of our study will be able to answer questions regarding the pathophysiology of severe brain injury and guide us in the titration of therapy, as it is needed by each individual patient [1-4]. y References g y Critical Care 2014, 18(Suppl 1):P456 (doi: 10.1186/cc13646) References 1. Morgan Stuart R, et al.: Neurocrit Care 2010, 12:188-198. 2. Tisdall MM, et al.: Br J Anaesth 2007, 99:61-67. 3. Nordström CH: Crit Care 2008, 12:R9I. 4. Jaeger M, et al.: Acta Neurochir (Wien) 2005, 147:51-56. References 1. Morgan Stuart R, et al.: Neurocrit Care 2010, 12:188-198. 2. Tisdall MM, et al.: Br J Anaesth 2007, 99:61-67. 3. Nordström CH: Crit Care 2008, 12:R9I. 4. Jaeger M, et al.: Acta Neurochir (Wien) 2005, 147:51-56. Introduction The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily that has been implicated in multiple neuronal and infl ammatory stress processes. In the present study, we investigated changes in RAGE immunoreactivity and its protein levels in the gerbil hippocampus (CA1 to 3 regions) after 5 minutes of transient global cerebral ischemia. 4. Jaeger M, et al.: Acta Neurochir (Wien) 2005, 147:51-56. New look at the 20 mmHg ICP threshold New look at the 20 mmHg ICP threshold Methods The ischemic hippocampus was stained with cresyl violet (CV), NeuN (a neuron-specifi c soluble nuclear antigen) antibody and Fluro-Jade B (a marker for neuronal degeneration). g FG Guiza1, BD Depreitere1, IP Piper2, GV Van den Berghe1, GM Meyfroidt1 1University Hospitals Leuven, Belgium; 2Southern General Hospital, Glasgow, UK FG Guiza1, BD Depreitere1, IP Piper2, GV Van den Berghe1, GM Meyfroidt1 1University Hospitals Leuven, Belgium; 2Southern General Hospital, Glasgow, UK Results Five days after ischemia–reperfusion, delayed neuronal death occurred in the stratum pyramidale (SP) of the CA1 region. RAGE immunoreactivity was not detected in any regions of the CA1 to 3 regions of the sham group. RAGE immunoreactivity was detected only in the CA1 region from 3 days post ischemia, and the RAGE immunoreactivity was newly expressed in astrocytes, not in neurons. In addition, the level of RAGE protein was highest at 5 days post ischemia. In brief, both the RAGE immunoreactivity and protein level were distinctively increased in astrocytes in the ischemic CA1 region from 3 days after transient cerebral ischemia. Critical Care 2014, 18(Suppl 1):P458 (doi: 10.1186/cc13648) Introduction We present a method based on minute-by-minute ICP monitoring and outcome (GOS) to visualize in a clinically useful way the dynamic aspects of secondary injury. Introduction We present a method based on minute-by-minute ICP monitoring and outcome (GOS) to visualize in a clinically useful way the dynamic aspects of secondary injury. Methods A retrospective analysis of data from 165 adult patients from the Brain-IT database [1]. A color-coded contour plot was made for the association between good outcome and the number of secondary insults of ICP during the ICU stay, as defi ned by continuous values of insult duration and thresholds. Conclusion These results indicate that the increase of RAGE expression in astrocytes at post ischemia may be related to the ischemia-induced activation of astrocytes in the ischemic CA1 region. Figure 1 (abstract P458). Eff ect of transient cerebral ischemia on the expression of receptor for advanced glycation end products in the gerbil hippocampus proper p p JH Cho, CW Park, HY Lee, MH Won, JY Lee, DJ Chung Kangwon National University, Chuncheonsi, South Korea Model of intracranial hypertension of tumor etiology in laboratory ratsi Model of intracranial hypertension of tumor etiology in laboratory rats AN Kolesnikov, VI Cherniy, KA Kardash, TA Mustafi n, VN Stasyuk, IV Koktishev Donetsk National Medical University of Maxim Gorky, Donetsk, Ukraine Critical Care 2014, 18(Suppl 1):P459 (doi: 10.1186/cc13649) AN Kolesnikov, VI Cherniy, KA Kardash, TA Mustafi n, VN Stasyuk, IV Koktishev AN Kolesnikov, VI Cherniy, KA Kardash, TA Mustafi n, VN Stasyuk, IV Koktishev Donetsk National Medical University of Maxim Gorky, Donetsk, Ukraine Critical Care 2014, 18(Suppl 1):P459 (doi: 10.1186/cc13649) Introduction The objective of this research is the creation of an experimental model of intracranial hypertension (ICH) of the tumor etiology in white nonlinear rats. Figure 1 (abstract P460). Methods Work was executed in the neurophysiology laboratory on 12 white nonlinear rats, weight 280 to 320 g, preselected according to criteria of bioethical rules. For modeling ICH in animals, standard anesthesia was carried out. A longitudinal section was spent in the projections of saggital suture from the frontal to occipital bone. The off ensive surgical stage anesthesia showed loss of palpebral and lingual refl exes. Further, according to the stereotactic atlas coordinates, a burr hole was performed, through which sterile two-component anti- allergen and depyrogenized gel in the amount of 0.025 ml was applied into the fourth ventricle with an intracerebroventricular microinjector. The injector was at an angle of 12° in order to avoid injuring vessels, its distal end entering a depth 4.5 mm in the fourth ventricle, and was installed and documented in the burr hole for measuring intracranial pressure. Monitoring during operations included: respiration, heart rate, ECG, thermometry, and control of diuresis. Results In all animals the heart rate decreased to an average of 180 beats/minute during 90 minutes after the introduction of the intracerebroventricular two-component gel. Also there was a decrease in respiration rate by an average of <70 breaths/minute. Behavioral responses of animals were evaluated. The average intensity and the duration of such actions as shaking, friction, licking and carding decreased. Decline in grooming was possibly associated with breached central regulation in the brain of studied rats. Macroscopically in the preparation we observed a pronounced hyperemia of obtained materials, which may indirectly indicate the syndrome of ICH. Survival of the animals was: 3 days – 100%, 7 days – 100%, 14 days – 92%, 21 days – 83%. Correlation of thermal Doppler fl owmetry and microdialysis values in patients with severe subarachnoid hemorrhage and traumatic brain injury DC Papadopoulos1, P Papamichalis1, As Filippidis2, D Karangelis3, TH Bekiari4, KN Fountas5, G Vretzakis5, KN Paterakis5, A Komnos1 1General Hospital of Larisa, Greece; 2Boston Medical Center, Boston, MA, USA; 3Onassis Cardiac Surgey Center, Athens, Greece; 4Prefectural Authority of Thessaly, Larisa, Greece; 5University Hospital of Larissa, School of Medicine, University of Thessaly, Larisa, Greece C i i l C 2014 18(S l 1) P457 (d i 10 1186/ 13647) y y Critical Care 2014, 18(Suppl 1):P457 (doi: 10.1186/cc13647) Introduction The purpose of this study is to investigate the relationship between continuously monitored regional cerebral blood fl ow (CBF) and microdialysis values in severe subarachnoid hemorrhage and traumatic brain injury patients. Methods Advanced multimodal neuromonitoring including measure- ments of CBF (QFlow, Hemedex) and brain lactate, pyruvate, lactate/ pyruvate ratio, glycerol and glucose values using microdialysis (CMA600, microdialysis) were performed in 21 patients with severe subarachnoid hemorrhage (n = 17) and traumatic brain injury (n = 4). Thirteen of the patients were successfully discharged from the ICU while eight did not survive. Additional recorded parameters include Figure 1 (abstract P458). S165 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Figure 1 visualizes in blue the region associated with good outcome for continuous values of thresholds and durations of secondary insults. The thick black line indicates the transition towards the negative association region in red. A best-fi t exponential curve is superimposed. This clearly shows that insults below 20 mmHg of ICP can be detrimental if sustained for long periods. Alternatively, they can be tolerated for higher thresholds but only for shorter periods. Figure 1 (abstract P460). Conclusion Secondary injury is dynamic such that not only thresholds but also insult duration are relevant. The proposed visualization goes beyond the static 20 mmHg ICP threshold introduced in [2] and provides a more accurate representation that could help the clinician in identifying when the patient’s outcome could be making a turn for the worse. Model of intracranial hypertension of tumor etiology in laboratory ratsi y Conclusion The main result of this work can be considered creation of a successful and workable model of ICH. The developed model is the testing ground to assess especially anesthesia protection in patients with the syndrome of ICH including tumor etiology and has no analogs. lactate, serum sodium and C-reactive protein (CRP) between arterial and jugular venous bulb blood. j g Methods An observational study. Between 1 January and 31 October 2013 we included neurocritical patients (NCP) with multimodal neuromonitoring (MMN). Daily samples of arterial blood and venous jugular bulb blood were obtained for measuring pCO2, lactate, serum sodium and CRP.i j g Methods An observational study. Between 1 January and 31 October 2013 we included neurocritical patients (NCP) with multimodal neuromonitoring (MMN). Daily samples of arterial blood and venous jugular bulb blood were obtained for measuring pCO2, lactate, serum sodium and CRP.i References References 1. [http://www.brainit.org] 2. Marmarou et al.: J Neurosurg 1991, 75:59-66. 1. [http://www.brainit.org] 2. Marmarou et al.: J Neurosurg 1991, 75:59-66. Effi cacy of terutroban in preventing delayed cerebral ischemia after subarachnoid hemorrhage: a functional isotope imaging study on a rat model D Lagier1, B Guillet2, L Velly1, N Bruder1, M Alessi2 D Lagier , B Guillet , L Velly , N Bruder , M Alessi 1CHU Timone, Marseille, France; 2Aix Marseille University, Marseille, France Critical Care 2014, 18(Suppl 1):P461 (doi: 10.1186/cc13651) 1CHU Timone, Marseille, France; 2Aix Marseille Univer g Methods Consecutive patients admitted to our ICU for aSAH were enrolled in the study. CBF-Vm and the pulsatility index (PI) were obtained by TCCS in both mean cerebral arteries (MCAs) at two scheduled time points, <3 days (T1) and 7 to 10 days (T10), and at least once every other day after bleeding. The highest Vm between left and right and the corresponding PI were chosen for analysis. All patients underwent brain MRI plus TOF-MRI angiography at T1 and T10 to assess radiologic VSP. Symptomatic VSP was defi ned as a new neurologic defi cit associated with radiological VSP. The occurrence of DCI was evaluated on DWI sequences. The C statistic (ROC curves) and nonparametric t test were used for analysis. Introduction After a subarachnoid hemorrhage (SAH), delayed cerebral ischemia (DCI) remains the principal cause of morbid mortality. F2-isoprotanes are recognized as biomarkers of DCI. These lipid metabolites, by fi xing the thromboxane and prostaglandin (TP) receptor, induce vasoconstriction and platelet aggregation. Our objective was to evaluate the effi cacy of terutroban (TER), a TP receptor- specifi c antagonist, in preventing DCI after SAH. i Methods Twenty rats were assigned to one of three groups: a double 250 μl intracisternal injection (ICI) was realized with saline in the CONTROLS group (n = 6) or with autologous arterial blood in the SAH (n = 8) and SAH+TER (n = 6) groups. Treated animals received an oral administration of 30 mg/kg/day TER during 5 days following blood injection. Rats were evaluated using a functional isotope imaging technique (high-resolution microSPECT). Brain capture of three 99m technetium radiolabeled tracers was evaluated: HMPAO at day (D) 0, 2 and 5 for cerebral perfusion quantifi cation, DTPA at D3 for blood–brain barrier (BBB) integrity study and annexin V-128 at D4 for apoptotic activity study. Radioactivity was measured in a predefi ned region of interest: cerebrum, cerebellum and brainstem. Statistical analysis: one- way ANOVA followed by Student’s t test. Results Forty-three consecutive patients were recruited. Thirty-seven patients had simultaneous TCCS measures and MRI (mean age 58 years, 28% WFNS 4 to 5). Arterial–jugular bulb diff erences in pCO2, lactate, serum sodium and C-reactive protein in neurocritical patients Arterial–jugular bulb diff erences in pCO2, lactate, serum sodium and C-reactive protein in neurocritical patients M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P460 (doi: 10.1186/cc13650) Results There were 45 NCP, six (13%) with MMN (fi ve men). Mean age was 37 ± 11 years (35 to 61). Diagnostics: two TBI, two SAH, one stroke, one lupus encephalitis. APACHE II was 27 ± 6.5 (25 to 39). Glasgow Coma Scale at admission was 14 ± 4 (4 to 14). pCO2 (mmHg): arterial 41 ± 6.3 versus jugular 45 ± 7.4 (r = 0.7, P = 0.007). Lactate (mg/dl): arterial 11 ± 5.6 versus jugular 13.5 ± 3.9 (r = 0.7, P = 0.9). Sodium (mEq/dl): Results There were 45 NCP, six (13%) with MMN (fi ve men). Mean age was 37 ± 11 years (35 to 61). Diagnostics: two TBI, two SAH, one stroke, one lupus encephalitis. APACHE II was 27 ± 6.5 (25 to 39). Glasgow Coma Scale at admission was 14 ± 4 (4 to 14). pCO2 (mmHg): arterial 41 ± 6.3 versus jugular 45 ± 7.4 (r = 0.7, P = 0.007). Lactate (mg/dl): arterial 11 ± 5.6 versus jugular 13.5 ± 3.9 (r = 0.7, P = 0.9). Sodium (mEq/dl): g Critical Care 2014, 18(Suppl 1):P460 (doi: 10.1186/cc13650) Introduction Several reports indicate the potential usefulness of monitoring brain metabolic parameters and their correlation with the system [1-4]. We want to establish diff erences and correlation in pCO2, S166 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 arterial 141 ± 4.5 versus 141 ± 4.4 (r = 0.8, P = 0.15). CRP (mg/dl): arterial 8 ± 7.4 versus 17 ± 11.6 (r = 0.9, P <0.001). The correlation and trend curves are shown in Figure 1. arterial 141 ± 4.5 versus 141 ± 4.4 (r = 0.8, P = 0.15). CRP (mg/dl): arterial 8 ± 7.4 versus 17 ± 11.6 (r = 0.9, P <0.001). The correlation and trend curves are shown in Figure 1. Conclusion We made the fi rst microSPECT scan description of a DCI rat model. After induction of SAH, TER improves cerebral perfusion, prevents BBB disruption in the brainstem and decreases apoptotic phenomenon in the cerebrum. g Conclusion A suitable correlation is observed for the arterial–jugular bulb in diff erent variables. Reference Figure 1 (abstract P461). HMPAO uptake at day 2 and day 5. P <0.01 versus day 0; #P <0.01 versus day 2; ‡P <0.01. 1. Carrera E, et al.: Transcranial Doppler for predicting delayed cerebral ischemia after subarachnoid hemorrage. Neurosurgery 2009, 65:316-324. use u ess. References 1. Futier et al.: Crit Care 2010, 14:R19. 1. Futier et al.: Crit Care 2010, 14:R19. F Di Corte, S Piff eri, L Capuano, S Magnoni, V Conte, F Ortolano, N Stocchetti Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P462 (doi: 10.1186/cc13652) 2. Chieregato et al.: Acta Neurochir 2005, Suppl 95. 3. Stochetti et al.: Anesthesiology 2005, 103:957. 4. Chieregato et al.: J Neurosurg Anesthesiol 2007, 19:222. Introduction Cerebral vasospasm (VSP) and delayed cerebral ischemia (DCI) play a central role in worsening the neurological outcome in patients with aneurismal subarachnoid hemorrhage (aSAH) [1]. Transcranial color-coded duplex sonography (TCCS) is a non-invasive tool to detect VSP and predict DCI, but its clinical utility is limited by low accuracy. Our aim was to perform a sensitivity/specifi city analysis of TCCS in predicting clinical VSP and DCI. Arterial–jugular bulb diff erences in pCO2, lactate, serum sodium and C-reactive protein in neurocritical patients There is a signifi cant diff erence in CRP and pCO2 values being persistently higher in the jugular, particularly for CRP. Studies are required to defi ne its interpretation and potential usefulness. f P462 Accuracy of transcranial color-coded duplex sonography in predicting clinical vasospasm and delayed cerebral ischemia in patients with subarachnoid hemorrhage F Di Corte, S Piff eri, L Capuano, S Magnoni, V Conte, F Ortolano, N Stocchetti Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P462 (doi: 10.1186/cc13652) Effi cacy of terutroban in preventing delayed cerebral ischemia after subarachnoid hemorrhage: a functional isotope imaging study on a rat model Eleven patients (30%) developed symptomatic VSP and 26 (70%) did not (NVSP). Vm increased signifi cantly from T1 to T10 in both groups (VS: P = 0.03, NVSP P = 0.01). The accuracy of TCCS at T10 (Vm) for predicting vasospasm was described by an AUC of 0.86 (CI = 0.74 to 0.98) and a cutoff value >115 cm/seconds (90% sensitivity, 73% specifi city). Interestingly, a cutoff value >168 cm/second corresponded to the best specifi city (92% specifi city), but low sensitivity (45%). The accuracy of PI for predicting VSP was lower (AUC 0.75). The accuracy of TCCS for predicting DCI was also poor (AUC 0.57). Considering larger DCI only (volume >2 ml), the accuracy was slightly increased (AUC 0.75). Conclusion TCCS is useful to predict clinical VSP with good accuracy. The Vm cutoff value >115 cm/second might be considered as a warning threshold, while higher values (>168 cm/second) identify high-risk patients. The accuracy of TCCS in predicting DCI is low, which indicates that mechanisms other than VSP are likely to play a role. Reference y y Results Brain HMPAO perfusion microSPECT (Figure 1) reveals a transient hypoperfusion after ICI (D2) and a lasting hypoperfusion in the SAH group. TER curtailed the SAH-induced decrease of the HMPAO uptake. TER also signifi cantly counteracted the SAH-induced increase of DTPA in the brainstem (SAH 2.6 ± 0.7 vs. SAH+TER 1.3 ± 0.1 ppm/ mm3; P <0.05) and increase of annexin V-128 in the cerebrum (SAH 1.2 ± 0.1 vs. SAH+TER 1 ± 0.06 ppm/mm3; P <0.05). Brain death determination in Europe: one condition with too many nuances IA Crippa, A Bronco, A Vargiolu, G Citerio San Gerardo Hospital, Monza, Italy Critical Care 2014, 18(Suppl 1):P464 (doi: 10.1186/cc13654) y y gi Results Of a total 1,221 patients admitted to our ICU, 37 patients (18 men, 19 women, age 65 ± 20) were clinically diagnosed with brain death. Primary diagnoses were 18 post-cardiac arrest syndromes, 14 cerebrovascular diseases, and four traumatic subarachnoid hemorrhages. GCS on admission was 4.5 ± 2.7 and days to cardiac arrest after diagnosed brain death was 8.6 ± 13.2. Urine volume for the last 24 hours was 997 ± 1,712 and serum potassium level was 4.8 ± 1.7. PaO2 and PaCO2 were 131.9 ± 80.6 and 48.9 ± 13.8, respectively. The fi nal causes of cardiac arrest were 20 cardiac failures, 11 renal failures and six respiratory failures, although with mechanical ventilator support. The fi nal cause of cardiac arrest due to respiratory failure had signifi cantly longer stay in the ICU and days after diagnosis of brain death than cardiac failure or renal failure (27.2 ± 22.6 days vs. 9.2 ± 10.0 and 8.1 ± 4.6, P <0.05; 23.2 ± 24.0 vs. 6.0 ± 9.0 and 5.4 ± 5.2, P <0.05, respectively).ii Introduction A patient is declared brain dead (BD) when physicians determine permanent loss of brain functions. Unfortunately, criteria for defi ning BD vary across diff erent countries [1]. We therefore decide to survey BD diagnostic modalities in Europe in order to describe diff erences. Methods A multiple-choice questionnaire was developed on an online platform [2]. Direct link was sent to national representatives of the European Society of Intensive Care Medicine and NeuroIntensive Care section’s members. Thirty-three countries were contacted. Answers were reviewed. In cases of discrepancies or missing data, participants were contacted for further clarifi cation. Descriptive statistics have been applied. Results Twenty-eight participants returned the questionnaire (85%). Every country has either specifi c law (93%) or guidelines issued by the scientifi c society (89%). Clinical examination, essential to the diagnosis, is the only requirement in 50% of countries. Coma, apnea, absence of corneal and cough refl exes are always necessary. Blood pressure and electrolytes are checked in 64% as mandatory prerequisites. The apnea test is legally defi ned in 86% of countries. Eighty-two percent of countries require achievement of a target paCO2 level while the Netherlands’ law states target apnea duration. Acute and long-term outcomes of ICU-acquired weakness: a cohort study and propensity matched analysis Acute and long-term outcomes of ICU-acquired weakness: a cohor study and propensity matched analysis G Hermans1, H Van Mechelen2, B Clerckx2, T Vanhullenbusch2, D Mesotten2, A Wilmer1, MP Casaer2, P Meersseman1, Y Debaveye2, PJ Wouters2, R Gosselink2, G Van den Berghe2 1UZ Leuven, Belgium; 2KU Leuven, Belgium Critical Care 2014, 18(Suppl 1):P466 (doi: 10.1186/cc13656) y y y G Hermans1, H Van Mechelen2, B Clerckx2, T Vanhullenbusch2, D Mesotten2, A Wilmer1, MP Casaer2, P Meersseman1, Y Debaveye2, PJ Wouters2, R Gosselink2, G Van den Berghe2 1UZ Leuven, Belgium; 2KU Leuven, Belgium Critical Care 2014, 18(Suppl 1):P466 (doi: 10.1186/cc13656) G Hermans1, H Van Mechelen2, B Clerckx2, T Vanhullenbusch2, D Mesotten2, A Wilmer1, MP Casaer2, P Meersseman1, Y Debaveye2, PJ Wouters2, R Gosselink2, G Van den Berghe2 1UZ Leuven, Belgium; 2KU Leuven, Belgium Critical Care 2014, 18(Suppl 1):P466 (doi: 10.1186/cc13656) Introduction ICU-acquired weakness is a frequent complication of critical illness. It is unclear whether it is a marker or mediator of poor outcomes. We aimed to determine acute and long-term outcomes and costs of ICU-acquired weakness among long-stay (≥8 days) ICU patients and to assess the impact of recovery of weakness at ICU discharge. Methods Data were prospectively collected during an RCT (Clinical trials.gov: NCT00512122) [1,2]. Impact of weakness (MRC sum <48) on outcomes and costs was analyzed with 1:1 propensity score-matching for baseline characteristics, illness severity and risk factor exposure prior to assessment. Among weak patients, impact of persisting weakness at ICU discharge on risk of death after 1 year was examined with multivariable Cox proportional-hazard analysis. l Conclusion There are still areas of uncertainty and disparities in brain death diagnosis in European countries. This predisposes to misdiagnosis and confusion both for clinicians and families. Measures to promote uniformity of brain death procedures and clinical practice are therefore desirable. References 1. Wijdicks EF: Brain death worldwide: accepted fact but no global consensus in diagnostic criteria. Neurology 2002, 58:20-25. 2. [www.surveyplanet.com] p p y Results A total 227 of the 405 (56%) long-stay assessable ICU patients were weak; 122 weak patients were matched to 122 not-weak patients. As compared with matched not-weak patients, weak patients had a lower likelihood at any time for live weaning from mechanical ventilation (HR: 0.709 (0.548 to 0.918), P = 0.009), live ICU (HR: 0.738 (0.570 to 0.955), P = 0.021) and hospital discharge (HR: 0.682 (0.521 to 0.893), P = 0.005). In-hospital costs/patient (+30.5%, €+5,443/patient, P = 0.04) and 1-year mortality (30.6% vs. 17.2%, P = 0.015) were also higher. The 105/227 (46%) weak patients not matchable to not-weak patients had even worse prognosis and higher costs. At any time within the fi rst year following ICU admission, compared with patients who recovered from weakness and adjusted for potential confounders, those with persistent weakness and MRC sum between 36 and 47 at 1. Wijdicks EF: Brain death worldwide: accepted fact but no global consensus in diagnostic criteria. Neurology 2002, 58:20-25. 2 [ l t ] Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 of the legal issues involved. Therapy is continued except in the case of donation of organs for transplantation from brain-dead patients until their cardiac arrest. Consequently, we may determine what the fi nal cause of death is in brain-dead patients. Our hypothesis is that brain- dead patients ultimately die of cardiac failure. of the legal issues involved. Therapy is continued except in the case of donation of organs for transplantation from brain-dead patients until their cardiac arrest. Consequently, we may determine what the fi nal cause of death is in brain-dead patients. Our hypothesis is that brain- dead patients ultimately die of cardiac failure. Results Wernicke’s area activation was observed in nine patients (eight patients with TBI, one with hypoxia). During the subsequent examination, which lasted from 3 to 12 months, seven patients with activated Wernicke’s area showed further consciousness expansion up to the minimal consciousness state (further consciousness expansion was seen in two patients). Two other patients with activated Wernicke’s area did not show any signs of consciousness. Two patients revealed signifi cant activity in the Broca’s area. p y Methods From January 2011 to December 2012, brain-dead adult patients in our ICU were investigated. The declaration of brain death is determined by clinical neurological examinations, electroencephalography, and the auditory brain-stem response test. Age, sex, primary diagnosis, Glasgow Coma Scale (GCS) on admission, the number of days from the diagnosis of brain death until cardiac arrest (days), the fi nal cause of cardiac arrest, urine volume for the last 24 hours (ml), serum potassium levels (mEq/l), PaO2 (mmHg) and PaCO2 (mmHg) on the last day were evaluated. Values are expressed as mean ± SD. Data were analyzed by Kruskal–Wallis test and Mann– Whitney U test. P <0.05 was considered statistically signifi cant. gi y Conclusion According to the fi rst results of the study one can conclude that behind the outwardly similar clinical symptoms in patients in VS lies a diverse (due to the organization of brain functions) group of patients. fMRI enables one to reveal the fi rst signs of cognitive activity; that is, reveals the linguistic value of speech addressed to the patient which cannot be detected during routine neurological examination. Brain death determination in Europe: one condition with too many nuances IA Crippa, A Bronco, A Vargiolu, G Citerio San Gerardo Hospital, Monza, Italy Critical Care 2014, 18(Suppl 1):P464 (doi: 10.1186/cc13654) Number of physicians (median 2, range 1 to 4), number of clinical examinations (median 2, 1 to 3), and minimum observation time (median 3 hours, 0 to 12) are variable requisites in diff erent countries. In 50% of nations, additional tests are required. Hypothermia (4%), anoxic injury (7%), inability to complete clinical examination (61%), toxic drug levels (57%), and inconclusive apnea test (54%) are legal indications to perform additional tests. Cerebral blood fl ow investigation is mandatory in 18% of countries, while it is either optional or used only in selected cases in 82%. Conventional angiography is still the preferred method (50%), followed by transcranial Doppler (43%), angioCT (39%), CT perfusion and angioMR (11%). EEG is always (21%) or optionally (14%) recorded. Russia and Croatia evaluate both EEG and cerebral blood fl ow (7%). Conclusion There were signifi cantly longer days until fi nal cardiac arrest that was caused by respiratory failure than by cardiac failure or renal failure. Many diff erent observations in each case were shown, such as urine volume or potassium level on the last day. Not only cardiac failure but also other failures might lead to fi nal cardiac arrest. P466 Functional MRI examination results in patients with vegetative state: revealing of the Wernicke’s area EA Kondratyeva1, SV Diment2, SA Kondratyev EA Kondratyeva1, SV Diment2, SA Kondratyev1 1Russian Polenovs Neurosurgical Institute, St Petersburg, Russia; 2International Institute of Biological Systems, St Petersburg, Russia Critical Care 2014, 18(Suppl 1):P463 (doi: 10.1186/cc13653) Introduction The aim was to evaluate the prognostic importance of the MRI examination using Block Design fMRI in patients in a vegetative state (VS). Introduction The aim was to evaluate the prognostic importance of the MRI examination using Block Design fMRI in patients in a vegetative state (VS). Methods Twenty-two patients corresponding to the international standards of VS underwent high-resolution MRI (T2 and T1, isomatrix with slice thickness 1.00 mm, T2 FLAIR FS, DWI, SWI) examination and Block Design fMRI (paradigm of passive speech). The patients were aged from 4 to 42 years. Causes of VS: TBI in 19 patients, hypoxia in three patients. By the time of the examination the patients were in VS from 1 to 4 months. Figure 1 (abstract P461). HMPAO uptake at day 2 and day 5. *P <0.01 versus day 0; #P <0.01 versus day 2; ‡P <0.01. S167 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P467 Early electrophysiological diagnosis of ICU-acquired weakness L Wieske, C Verhamme, E Witteveen, A Bouwes, MJ Schultz, IN Van Schaik, J Horn Academic Medical Center, University of Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P467 (doi: 10.1186/cc13657) Introduction Cerebral near-infrared spectroscopy (NIRS) represents an exciting prospect for the non-invasive monitoring of cerebral tissue oxygenation in traumatic brain injury (TBI). Earlier attempts at clinical application of cerebral NIRS demonstrated that further work was needed [1]. The basic layout of the probe portion of these devices consists of a light source and a light detector, arranged at calculated distances and confi gurations in order to observe target tissue. We aim to determine which commercially available NIRS probe represents the most sensitive layout of sources/detectors for the greatest sensitivity in observing tissue oxygenation at the optimal depth for TBI. Early electrophysiological diagnosis of ICU-acquired weakness L Wieske, C Verhamme, E Witteveen, A Bouwes, MJ Schultz, IN Van Schaik, J Horn Academic Medical Center, University of Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P467 (doi: 10.1186/cc13657) Introduction An early diagnosis of ICU-acquired weakness (ICU-AW) is diffi cult because disorders of consciousness preclude strength assessment [1]. Electrophysiological (EMG) studies may be an alternative approach [2]. In this study we investigated feasibility and diagnostic accuracy of EMG studies to diagnose ICU-AW in unconscious patients. y Methods The optimal depth for grey matter target tissue (grey–white matter junction) was ascertained by reviewing a series of brain CT scans of patients who had sustained a TBI. Set (average) measurements were derived from these identifying the target depth of the grey matter strip from the point of probe placement. Currently there are fi ve commercially available cerebral NIRS systems. Table 1 presents the variety of confi gurations off ered by each device. Source detector layouts were modelled and simulated using the NIRFAST® computational light modelling software (developed at the University of Birmingham) [2]. The novel approach of this modelling system makes no extrapolated assumptions based on subtraction. p Methods Newly admitted unconscious ICU patients (RASS <–3), ventilated for ≥2 days, were included in this single-center prospective cohort study. EMG testing included ulnar (motor/sensory), peroneal (motor) and sural (sensory) studies. Myography was performed when coagulation was normal (dorsal interossei I/II, deltoid and tibial muscles). Reliability of results was checked by an experienced neurophysiologist, blinded for strength. Motor/sensory studies were abnormal if amplitudes were below the 2.5th percentile reference values [3]. P468 P468 Choosing a cerebral near-infrared spectroscopy system for use in traumatic brain injury: deriving the ideal source detector layout D Davies1, M Clancy2, Z Su1, H Denghani2, A Belli1 1National Institute for Health Research Surgical Reconstruction and Microbiology, Edgbaston, Birmingham, UK; 2University of Birmingham – School of Computational Science, Birmingham, UK Critical Care 2014, 18(Suppl 1):P468 (doi: 10.1186/cc13658) Acknowledgement GH and HVM contributed equally to this study. R f Acknowledgement GH and HVM contributed equally to this study. References 1. Casaer M, et al.: N Engl J Med 2011, 365:506-517. 1. Casaer M, et al.: N Engl J Med 2011, 365:506-517. 2. Hermans G, et al: Lancet Respir Med 2013, 1:621-629. 1. Casaer M, et al.: N Engl J Med 2011, 365:506-517. 2. Hermans G, et al: Lancet Respir Med 2013, 1:621-629. 2. Hermans G, et al: Lancet Respir Med 2013, 1:621-629. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ICU discharge had a higher likelihood of death (HR: 1.937, 95% CI: 1.048 to 3.581, P = 0.035). This likelihood of late death was even higher for those patients with a more severe degree of persistent weakness (MRC sum <36) (HR: 1.815, 95% CI: 3.693 to 7.514, P <0.001). References References 1. Crit Care Med 2009, 37:S299-S308. 2. Crit Care Med 2009, 37:2632-2637. 3. J Neurol 2004, 251:1491-1497. Conclusion Patients with ICU-acquired weakness had worse acute morbidity outcomes, consumed more resources and revealed higher mortality after 1 year than patients without weakness. Persistence of weakness at ICU discharge further increased late mortality. 65 What do brain-dead patients ultimately die of? What do brain-dead patients ultimately die of? A Kawasaki, M Hotta, K Mochiduki, N Saito, M Namiki, M Takeda, T Harada, A Yaguchi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P465 (doi: 10.1186/cc13655) Introduction After a patient is declared brain dead, the cessation or withdrawal of therapy in Japan is quite a diffi cult operation because Introduction After a patient is declared brain dead, the cessation or withdrawal of therapy in Japan is quite a diffi cult operation because S168 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P467 P469 Single-subject assessment of the distribution of white matter abnormalities measured by diff usion tensor imaging in patients with severe traumatic brain injury S Magnoni1, M Macrì2, F Stretti2, C Mac Donald3, R Biffi 2, E Zanier4, A Snyder3, J Shimony3, N Stocchetti1, D Brody3 1Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Milan University, Milan, Italy; 3Washington University, St Louis, MO, USA; 4IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy Critical Care 2014, 18(Suppl 1):P469 (doi: 10.1186/cc13659) P469 Single-subject assessment of the distribution of white matter abnormalities measured by diff usion tensor imaging in patients with severe traumatic brain injury Introduction Traumatic axonal injury (TAI) is a major contributor to adverse outcomes following traumatic brain injury (TBI). Diff usion tensor imaging (DTI) is a magnetic resonance imaging technique, which provides a robust measure of white matter integrity in patients with TBI. Certain brain regions have been identifi ed as particularly susceptible to TAI by means of DTI. Few studies, however, have focused on describing the distribution of DTI abnormalities in individual TBI patients. The aim of this project was to conduct an exploratory analysis of the extent and distribution of axonal injury in TBI patients at a single- subject level. Methods Patients admitted to the ICU for severe TBI underwent brain MRI and DTI (32 directions, b = 1,000, voxel size 2 × 2 × 2 mm3) between 2 weeks and 3 years after injury. For each individual patient, we enrolled fi ve age-and-sex-matched healthy volunteers who underwent the same DTI protocol and were used as controls (31 total). We used a region of interest (ROI) automated analysis [1] to quantify white matter integrity. The fractional anisotropy (FA) maps were segmented using a white matter parcellation map covering the entire brain. Our primary outcome was the normalized diff erence in FA between each patient and the controls. Abnormalities were defi ned as values that were more than 2 SD below the mean of the values for the matched controls for each ROI. Figure 2 (abstract P468). CT measurements and sensitivities at depth. Results Twelve TBI patients with a median age of 30 years (range 20 to 38) and a median GCS of 5 (range 5 to 7) were included. The majority of them had diff use axonal injury according to CT and conventional MRI. Reference [www.mristudio.org] [www.mristudio.org] 1. Wolf M, et al.: J Biomed Opt 2007, 12:062104. P470 Conclusion Based on the computational modelling of our work, the FORE-SIGHT NIRS device by CAS Medical source detector layout provides the greatest sensitivity at depth for the purposes of cortical monitoring in trauma. Variations in the layout have a signifi cant impact on the quality of signal detected. Long-term outcome after severe traumatic brain injury M Ciapetti, M Migliaccio, A Cecchi, M Bonizzoli, A Peris Intensive Care Unit, Florence, Italy Critical Care 2014, 18(Suppl 1):P470 (doi: 10.1186/cc13660) P467 Analysis of individual ROIs revealed that all subjects had numerous abnormal ROIs, varying from 14 to 64 (median 21) out of 78 regions assessed. The most frequently altered regions were the frontal lobe, orbital–frontal regions, corpus callosum, internal capsules, superior and inferior longitudinal fasciculi, cingulum, cerebellar structures and corona radiata. Thalamus, hippocampus and occipital lobes were less frequently aff ected. q yf Conclusion The extent and distribution of TAI varies on a patient basis. These fi ndings highlight the need for standardized methods to precisely assess TAI in single subjects. Such single-subject methods will probably improve prognostic accuracy and may be useful in clinical trials. Results We reviewed 32 trauma series CT brain images and the average depth to grey matter was derived as 21.37 mm (range 16.59 to 31.03 mm, SD 2.33) from the surface (Figure 1). The spectrum of sensitivity of the fi ve probes was modelled (Figure 2). As is apparent here, the FORE-SIGHT (CAS Medical, CT, USA) probe currently off ers the greatest sensitivity at our derived target depth. 2. Dehghani H, et al.: Commun Numer Methods Eng 2008, 25:711-732. P467 Myography was abnormal if spontaneous abnormal activity was found in ≥1 muscles. Upon awakening, strength was assessed (ICU- AW: average MRC <4 [1]), blinded for EMG. Feasibility was determined as the percentage of measurements that could be performed and were reliable. Diagnostic accuracy was analyzed using sensitivity and specifi city. Figure 1 (abstract P468). Light scatter predictions of available systems/ probes illustrated. i Results We included 35 patients (ICU-AW: 17). EMG testing was done on day 4 (IQR: 3 to 6). Feasibility was 94%, 89%, 77%, 34% and 31% for ulnar motor, peroneal motor, ulnar sensory, sural sensory and myography studies, respectively. Figure 1 displays amplitude values. Sensitivity/specifi city was 100%/0%, 100%/31%, 31%/100%, 50%/25% and 67%/38%, respectively. Conclusion Feasibility of ulnar and peroneal studies was acceptable; feasibility of sural and myography studies was low. Diagnostic accuracy was low for all studies. This may be improved with new reference values. Figure 1 (abstract P467). Dotted lines represent 2.5th percentile reference values [3]. Figure 1 (abstract P468). Light scatter predictions of available systems/ probes illustrated. Figure 1 (abstract P468). Light scatter predictions of available systems/ probes illustrated. Figure 1 (abstract P467). Dotted lines represent 2.5th percentile reference values [3]. S169 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P468). Available source detector layouts Probe Wavelengths (nm) Sources Detectors Spacing (mm) Peak depth sensitivity Optiplex TX (ISS, IL, USA) 690, 830 4 1 30, 35, 40, 45 8 INVOS (Covidien, MA, USA) 730, 810 1 2 30, 40 8 EQUANOX (Nonin, MN, USA) 730, 810, 880 2 2 20, 40 7 FORE-SIGHT (CAS Medical, CT, USA) 690, 780, 805, 850 1 2 20, 50 10 NIRO-200NX (Hamamatasu, Japan) 735, 810, 850 1 2 19.2, 20 9 Table 1 (abstract P468). Available source detector layouts Figure 2 (abstract P468). CT measurements and sensitivities at depth. Vitamin D level could aff ect the recovery rate in traumatic brain injury: a retrospective study Introduction Severe traumatic brain injury (TBI) is a major cause of death in people between 19 and 45 years old. Gastrointestinal dysfunction is the most common complication due to mucosal ischemia, motility disorders, and disruption of the gut barrier, with severe consequences: malnutrition, weight loss, and high risk of infections [1]. Therefore, maintaining the intestinal barrier function is a systematic engineering project. Selected new probiotics due to the capacity to bind and neutralize toxins, and to interfere with pathogen adherence, by immunomodulatory properties, mopping up the infection, could improve recovery of critically ill patients [2]. Our aim was to assess the eff ects of a new probiotic in an early enteral regimen on clinical outcome of severe TBI patients, in terms of VAP incidence, tolerance to enteral nutrition, duration of mechanical ventilation, and mortality rate. FI Socci, S Di Valvasone, A Cecchi, M Bonizzoli, A Terreni, A Peris AOU Careggi, Florence, Italy FI Socci, S Di Valvasone, A Cecchi, M Bonizzoli, A Terreni, A Peris AOU Careggi, Florence, Italy FI Socci, S Di Valvasone, A Cecchi, M Bonizzoli, A Terreni, A Peris AOU Careggi, Florence, Italy gg y Critical Care 2014, 18(Suppl 1):P471 (doi: 10.1186/cc13661) Critical Care 2014, 18(Suppl 1):P471 (doi: 10.1186/cc13661) Introduction Recent studies have shown that 1,25-dihydroxyvitamin D3 (vitamin D) defi ciency may aff ect negatively the clinical course of traumatic brain injury (TBI) [1]. This problem becomes important with respect to the older patient considering a 50% prevalence of vitamin D defi ciency [2]. Data from the Third National Health and Nutrition Examination Survey [3] document more than 60% of Caucasians aff ected by D defi ciency [4] so that all patients with TBI of any age are theoretically at risk of unfavorable outcome [2]. The objective of this preliminary study was to determine whether low levels of vitamin D at admission to the ICU (<24 hours) could negatively aff ect neurological recovery of patients with TBI. y Methods A prospective randomized 1-year study of 64 patients 19 to 78 years old allocated to receive for 10 days either an early enteral diet plus a new probiotic (Bioent; Lactobacillus bulgaricus 10 trillion CFU/cp + activated charcoal) every 6 hours (Group A) or the same formula without probiotics (Group B). The diets were isocaloric and isonitrogenous, and there were no diff erences between groups in gender, age, and nutritional status. Vitamin D level could aff ect the recovery rate in traumatic brain injury: a retrospective study We assessed the VAP incidence, duration of mechanical ventilation, tolerance to enteral nutrition, length of ICU stay, duration of diarrhea episodes, and mortality rate. The ANOVA test and t test were carried out; P <0.05 was considered signifi cant. y p Methods We retrospectively analyzed the data of 46 patients aff ected by TBI (65% severe, 9.5% moderate, 28.5% moderate) both isolated or associated with other extracranial lesions. The sampling of vitamin D was carried out within 24 hours from ICU admission. We had registered GCS at the moment of presentation (GCS in) and at discharge (GCS out) and their diff erence (GCS diff ) compared with levels of vitamin D. Patients that died in the ICU were assigned a GCS out = 0. See Table 1. Results Our data, according to other studies [5], confi rm the presence of a defi ciency of vitamin D (Table 1); however, they do not demonstrate a statistical signifi cance correlation at the univariate regression (R = 0.04; P = 0.786) between vitamin D level and outcome from the ICU. There was no correlation stratifying patients for age, for TBI class, for Injury Severity Score and for BMI. Methods We retrospectively analyzed the data of 46 patients aff ected by TBI (65% severe, 9.5% moderate, 28.5% moderate) both isolated or associated with other extracranial lesions. The sampling of vitamin D was carried out within 24 hours from ICU admission. We had registered GCS at the moment of presentation (GCS in) and at discharge (GCS out) and their diff erence (GCS diff ) compared with levels of vitamin D. Patients that died in the ICU were assigned a GCS out = 0. See Table 1.i Results Our data, according to other studies [5], confi rm the presence of a defi ciency of vitamin D (Table 1); however, they do not demonstrate a statistical signifi cance correlation at the univariate regression (R = 0.04; P = 0.786) between vitamin D level and outcome from the ICU. There was no correlation stratifying patients for age, for TBI class, for Injury Severity Score and for BMI. i Results The infection rate was higher in group B, the duration of mechanical ventilation was shorter in group A, and the patients in group A received 91.7% of total caloric needs by an enteral route versus 74.68% in group B. 1. Chatterjee M, et al.: Vitamin D and genomic stability Mutat Res 2001; 475:69-87. P471 , , Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2014 18(Suppl 1):P472 (doi: 10 1186/cc13662) Emergency Hospital Floreasca, Bucharest, Romania Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P472 (doi: 10.1186/cc13662) g y p Critical Care 2014, 18(Suppl 1):P472 (doi: 10.1186/cc13662) Could selected probiotics have benefi cial eff ects on clinical outcome of severe traumatic brain injury patients? D Pavelescu, L Mirea, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P472 (doi: 10.1186/cc13662) D Pavelescu, L Mirea, I Grintescu Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Plan and Operation of the Third National Health and Nutrition Examination Survey, 1988–94. DHHS publication No. (PHS)94-1308 – Vital and Health Statistics, series 1, no. 32. Hyattsville, MD: US Department of Health and Human Services, Public Health Service, CDC; 1994. Conclusion According to the other studies, our data confi rm that the severe traumatic brain injury is associated with a high mortality at 1 year. One-half of the survivors have a diff erent level of disability. References 4. Khazai N, et al.: Calcium and vitamin D: skeletal and extraskeletal health. Curr Rheumatol Rep 2008, 10:110-117. 4. Khazai N, et al.: Calcium and vitamin D: skeletal and extraskeletal health. Curr Rheumatol Rep 2008, 10:110-117. 5. Cecchi A, et al.: Vitamin D defi ciency in septic patients at ICU admission is not a mortality predictor. Minerva Anest 2011, 77:1184-1189. 1. Nichol AD, et al.: Measuring functional and quality of life outcomes following major head injury: common scales and checklists. Injury 2011, 42:281-287. 2. Roozenbeek B, et al.: Prediction of outcome after moderate and severe traumatic brain injury: external validation of the(IMPACT) and (CRASH) prognostic models. Crit Care Med 2012, 40:1609-1617. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (P471) profound impact of TBI is not only felt by the individuals who suff er the injury but also their caregivers and society as a whole. In this study we observed the long-term outcome of patients with severe traumatic brain injury admitted to our intensive care. TBI Descriptive statistics Isolated Associated P value Dead 8.70% Percentage 22 78 Male 87% GCS in 4 ± 3 8 ± 5 0.043 Age 50 ± 21 GCS out 10 ± 4 10 ± 5 0.933 BMI 26.36 ± 4.14 GCS diff 6 ± 4 3 ± 6 0.151 ISS 29 ± 15 Vitamin D (ng/ml) 17 ± 8.5 17 ± 9.8 0.597 Vitamin D 73.90% Length of 12 ± 7 8 ± 9 0.261 defi ciency stay ICU (days) (<30 ng/ml) j y Methods This study includes all patients (n = 160) with severe head trauma (GCS <9) admitted to the ICU of the emergency department of a tertiary referral center (Careggi Teaching Hospital, Florence, Italy) from 2009 to 2012. All patients will undergo a clinical assessment after 1 year, which is routine post-intensive follow-up. As an objective index of ability to function after injury, the Glasgow Outcome Scale (GOS) will be used. The neurological evaluation to determine the outcome of patients by GOS is performed in two diff erent modes. All eligible patients 1 year after discharge from the ICU are contacted by telephone by a nurse of the intensive care staff and invited to make a visit to the surgery follow-up, where a intensivist evaluates the patient and determines the GOS. For the patients who were still hospitalized at 6 months in rehabilitation departments, GOS assessment is performed by a doctor of the structure and notifi ed by telephone. 2. Milos C, et al.: Combination treatment with progesterone and vitamin D hormone may be more eff ective than monotherapy for nervous system injury and disease. Neuroendocrinology 2009, 30:158-172. yi y Results The ICU and hospital mortality was respectively 33.7% (n = 54) and 36.9% (n = 59). The mortality at 1 year was 44.4% (n = 71). The results of the neurological follow-up at 1 year were: GOS 2: 5.6% (n = 9), GOS 3: 10% (n = 16), GOS 4: 13.1% (n = 21), GOS 5: 26.9% (n = 43).i 3. References Introduction Traumatic brain injury is a major public health issue, which results in signifi cant mortality and long-term disability [1,2]. The 2. Dehghani H, et al.: Commun Numer Methods Eng 2008, 25:711-732. S170 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P474 Eff ect of blood alcohol level on outcome of patients with traumatic brain injury V Borovnik Lesjak, M Strnad, V Vujanović Popović, T Pelcl ZD dr. Adolfa Drolca Maribor, Slovenia Critical Care 2014, 18(Suppl 1):P473 (doi: 10.1186/cc13663) P474 Long-term outcome prediction using IMPACT and APACHE II in patients with traumatic brain injury treated in the ICU R Raj, R Kivisaari, J Siironen, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2014, 18(Suppl 1):P474 (doi: 10.1186/cc13664) Introduction We investigated whether blood alcohol levels (BAL) had any impact on presentation and outcome in patients with traumatic brain injury (TBI). Introduction The International Mission for Prognosis and Analysis of Clinical Trials (IMPACT) is currently the most robust prognostic model in patients with traumatic brain injury (TBI). No studies have compared this TBI-specifi c prediction model with general ICU models, such as the Acute Physiology and Chronic Health Evaluation II (APACHE II) [1,2]. This study investigates the performance and the correlation of the IMPACT and APACHE II models for long-term outcome prediction in patients with TBI. y Methods Forty-six patients with TBI requiring intubation between January 2000 and December 2012 were included. Patients were grouped into BAL-positive (>0.5‰; n = 24) and BAL-negative (<0.5‰; n = 22). Physiological parameters and outcome (survival to hospital discharge (STHD)) and neurological outcomes (Glasgow Outcome Scale (GOS), Cerebral Performance Category (CPC) and Glasgow Coma Scale (GCS)) were analyzed. Diff erences between groups were analyzed using Student’s t test and results presented as mean ± SD. Methods The study population consisted of TBI patients admitted to a designated ICU in southern Finland during a 4-year period (2009 to 2012). The IMPACT and APACHE II performances were assessed by calculating discrimination (by area under the curve), calibration (by GiViTI calibration test and Hosmer–Lemeshow C statistic) and precision (by Brier score). Correlation between the IMPACT and APACHE II was tested by Spearman’s rho. Primary outcome was 6-month mortality. Results Of the total 897 included patients, overall 6-month mortality was 22%. The IMPACT and APACHE II showed similar AUCs (0.81, 95% CI = 0.77 to 0.84; 0.80, 95% = CI 0.76 to 0.83) and Brier scores (0.134, 0.135). Calibration tests revealed signifi cant (P <0.05) deviations between observed and predicted risk for both models. Vitamin D level could aff ect the recovery rate in traumatic brain injury: a retrospective study There is a signifi cant diff erence in the number of diarrhea episodes, and the ICU length of stay was signifi cantly lower in group A; there was no signifi cant diff erence in the mortality rate between groups. See Figure 1. y Conclusion Vitamin D defi ciency is really prevalent in our TBI cases but does not seem to aff ect neurological recovery at ICU discharge; however, these preliminary results should be exposed to several criticisms and need to be confi rmed with prospective studies. R f Conclusion Early administration of new probiotics to severe TBI patients could have benefi cial eff ects in terms of reduction of GI dysfunction, VAP incidence and length of ICU stay. S171 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P472). Figure 1 (abstract P472). Figure 1 (abstract P472). Conclusion BAL did not have a signifi cant eff ect on presenting physiological parameters such as systolic BP and RR. It did, however, aff ect the presenting GCS score, which was signifi cantly lower in the BAL-positive group. BAL did not seem to have an eff ect on functional outcome or mortality measured by STHD. No favorable neuroprotective or deleterious eff ect was observed, demonstrated by no signifi cant diff erences in CPC, GOS or GCS scores at discharge. References 1. Bansal V, et al.: Traumatic brain injury and intestinal dysfunction. J Neurotrauma 2009, 26:1353-1359. Functional status after 3 years in ICU patients with traumatic brain injury Figure 1 (abstract P474). Area under the curve for the new models. Introduction The aim was to study the functional status at 1 year and after 3 years in ICU patients with traumatic brain injury (TBI). Introduction The aim was to study the functional status at 1 year and after 3 years in ICU patients with traumatic brain injury (TBI). Methods A prospective cohort study of patients with TBI admitted to Carlos Haya Hospital, Malaga, between 2004 and 2008. l d d Introduction The aim was to study the functional status at 1 year and after 3 years in ICU patients with traumatic brain injury (TBI). Methods A prospective cohort study of patients with TBI admitted to Carlos Haya Hospital, Malaga, between 2004 and 2008. after 3 years in ICU patients with traumatic brain injury (TBI). Methods A prospective cohort study of patients with TBI admitted to Carlos Haya Hospital, Malaga, between 2004 and 2008. was noted (Spearman’s rho = 0.566, P <0.001). The IMPACT and the APACHE II were found to identify slightly diff erent groups of patients that eventually do not survive (Figure 1). Ca os aya osp ta , a aga, bet ee 00 a d 008. Results We studied 531 patients, age 40.35 ± 19.75 years, APACHE II 17.94 ± 6.97, GCS admission 7.53 ± 3.83 points. Cranial tomography at admission was: diff use injury type I (10.4%), diff use injury type II (28.1%), diff use injury type III (24.5%), diff use injury type IV (8.3%), evacuated mass lesion (22.6%), non-evacuated mass lesion (6.2%). Hospital mortality was 28.6%. One-year mortality was 171 (32.2%) (missing: 6.6%). After 3 years, mortality was 181(34.1%) (16.2% missing). We followed 496 patients for 1 year after admission (35 were missing) and assessed their functional status using the GOS. Thus, 22.7% were normal and 20% presented some limitations but were self-suffi cient, representing 55.6% of those who survived hospital admission. A total of 57.3% presented bad evolution (died: 34.5%, vegetative: 3%, disabled not self-suffi cient: 19.8%). After 3 years we followed 445 patients and 86 (16.2%) were missing. Thus, 132 patients (29.7% of 445 patients) were normal and 15.3% presented some limitations but were self-suffi cient, representing 75.76% of those who survived hospital admission. Fifty-six percent of 445 patients presented bad evolution (died: 40.7%, vegetative: 2.2%, disabled not self-suffi cient: 12.1%). Functional status after 3 years in ICU patients with traumatic brain injury Of 445 patients studied after 3 years of admission 171 died, and of the 274 who survived 198 (72.2%) were in a similar functional situation after 1 year, 11 (4.01%) had worsened functional situation, of which 10 had died, and 65 (27.72%) had improved situation (P <0.001). y g Conclusion The IMPACT and the APACHE II models showed equal performance for 6-month mortality prediction. A moderately strong, positive correlation, with some major discrepancies between the models, was found. Thus, features of both the IMPACT and APACHE II models are valuable for optimal outcome prediction in patients with TBI treated in the ICU. Validating and comparing the CAM-ICU and the ICDSC in mild and moderate traumatic brain injury patients: a multicenter prospective study E Bebawi1, L Deslauriers1, AA Tessier1, AJ Frenette1, MM Perreault2, MS Delisle2, JC Bertrand1, M Desjardins2, F Bernard1, P Rico1, K Khwaja2, L Burry3, DR Williamson1 Conclusion This study shows that approximately 75% of the patients who survived after 3 years from admission to the ICU with traumatic brain injury presented good recovery with functional situation normal or with some limitations but self-suffi cient. Between 1 and 3 years after admission, approximately 25% of patients improved their functional situation. Introduction The Confusion Assessment Method for the ICU (CAM- ICU) and the Intensive Care Delirium Screening Checklist (ICDSC) are recommended for routine delirium screening. However, literature about delirium assessment in traumatic brain injury (TBI) remains scarce. The aim of our study was to evaluate the validity and reliability of the CAM-ICU and the ICDSC for delirium assessment in patients with mild to moderate TBI. P475 Validating and comparing the CAM-ICU and the ICDSC in mild and moderate traumatic brain injury patients: a multicenter prospective study E Bebawi1, L Deslauriers1, AA Tessier1, AJ Frenette1, MM Perreault2, MS Delisle2, JC Bertrand1, M Desjardins2, F Bernard1, P Rico1, K Khwaja2, L Burry3, DR Williamson1 1Hôpital du Sacré-Coeur de Montréal, Canada; 2McGill University Health Center, Montreal, Canada; 3Mount Sinai Hospital, Toronto, Canada Critical Care 2014, 18(Suppl 1):P475 (doi: 10.1186/cc13665) Validating and comparing the CAM-ICU and the ICDSC in mild and moderate traumatic brain injury patients: a multicenter prospective study References 1. Lingsma et al.: Lancet Neurol 2010, 9:543-554. 1. Lingsma et al.: Lancet Neurol 2010, 9:543-554. 2. Maas et al.: Lancet Neurol 2013, 12:1200-1210. P474 The overall kappa for inter-rater reliability for the CAM-ICU and ICDSC was 0.64 and 0.68, respectively. In subgroup analyses, the CAM- ICU and ICDSC showed increased sensitivity and decreased specifi city in moderate TBI as well as deeper levels of sedation (RASS –2 to –3). p Conclusion Criterion validity and inter-rater reliability of the CAM-ICU and ICDSC were good. Severity of TBI and depth of sedation infl uence delirium assessments. Clinicians should be aware of those limitations before using these clinical tools in this population. P474 A moderately strong, positive correlation between the IMPACT and the APACHE II g Results There were no signifi cant diff erences in gender and age distributions between the BAL-negative and BAL-positive groups (73% vs. 88% male; P = 0.218; 53 ± 21 vs. 43 ± 17 years; P = 0.098). There were also no diff erences in initial systolic blood pressure (BP; 144 ± 30 vs. 132 ± 37 mmHg; P = 0.241) and respiratory rate (RR; 13 ± 6 vs. 12 ± 7/ minute; P = 0.891) but BAL did aff ect the initial GCS score (7 ± 3 vs. 5 ± 2; P = 0.022). There was no eff ect on CPC (3.5 ± 1.9 vs. 2.8 ± 1.8; P = 0.185), GOS (2.4 ± 1.8 vs. 3.0 ± 1.7; P = 0.270), and GCS at discharge from the hospital (14 ± 2 vs. 14 ± 2; P = 0.801). There were also no diff erences in length of hospital stay (LOHS; 15 ± 23 vs. 23 ± 34; P = 0.372) and STHD (1.6 ± 0.5 vs. 1.3 ± 0.5; P = 0.083). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S172 Figure 1 (abstract P474). Area under the curve for the new models. Results During an 8-month period, 61 patients (mean age 56.4 ± 18.5 years, mean APACHE II score 11.4 ± 6.5, mean GCS 13 ± 2) were enrolled. The overall sensitivity and specifi city for CAM-ICU (62% and 74%, respectively) and ICDSC (64% and 79%, respectively) were similar. The overall kappa for inter-rater reliability for the CAM-ICU and ICDSC was 0.64 and 0.68, respectively. In subgroup analyses, the CAM- ICU and ICDSC showed increased sensitivity and decreased specifi city in moderate TBI as well as deeper levels of sedation (RASS –2 to –3). Conclusion Criterion validity and inter-rater reliability of the CAM-ICU and ICDSC were good. Severity of TBI and depth of sedation infl uence delirium assessments. Clinicians should be aware of those limitations before using these clinical tools in this population. Results During an 8-month period, 61 patients (mean age 56.4 ± 18.5 years, mean APACHE II score 11.4 ± 6.5, mean GCS 13 ± 2) were enrolled. The overall sensitivity and specifi city for CAM-ICU (62% and 74%, respectively) and ICDSC (64% and 79%, respectively) were similar. P476 P476 Functional status after 3 years in ICU patients with traumatic brain injury M Delange van der Kroft1, M Arias-Verdú2, E Banderas-Bravo2, E Curiel-Balsera2, A Muñoz-Lopez2, J Fernandez-Ortega2, R Rivera-Fernandez2, M Prieto-Palomino2 1County Hospital of Axarquia, Velez Malaga, Spain; 2Carlos Haya University Hospital, Malaga, Spain Critical Care 2014, 18(Suppl 1):P476 (doi: 10.1186/cc13666) Outcome measures in randomized controlled trials of patients with severe traumatic brain injury: a systematic review Outcome measures in randomized controlled trials of patients with severe traumatic brain injury: a systematic review G Lalonde1, M Shemilt1, F Lauzier1, P Desjardins1, A Boutin1, L Moore1, D Fergusson2, R Zarychanski3, A Turgeon1 1Laval University, Quebec, Canada; 2OHRI, Ottawa, Canada; 3University of Manitoba, Winnipeg, Canada Critical Care 2014, 18(Suppl 1):P478 (doi: 10.1186/cc13668) g Results A total of 1,625 patients were included. Overall 6-month mortality was 33%. The APACHE II and SAPS II-based models showed good discrimination (area under the curve (AUC) 0.79, 95% confi dence interval (CI) = 0.75 to 0.82; and 0.80, 95% CI = 0.77 to 0.83), calibration (P >0.05) and precision. The SOFA-based model showed poor discrimination (AUC 0.68, 95% CI = 0.64 to 0.72) and precision but good calibration (P >0.05). The adjusted SOFA model displayed better discrimination (AUC 0.79, 95% CI = 0.76 to 0.82). The reference model showed comparable performance with all scoring system-based models regarding discrimination (AUC 0.77, 95% CI = 0.74 to 0.80), precision and calibration. See Figures 1 and 2. severe traumatic brain injury: a systematic review G Lalonde1, M Shemilt1, F Lauzier1, P Desjardins1, A Boutin1, L Moore1, D Fergusson2, R Zarychanski3, A Turgeon1 1Laval University, Quebec, Canada; 2OHRI, Ottawa, Canada; 3University of Manitoba, Winnipeg, Canada Critical Care 2014, 18(Suppl 1):P478 (doi: 10.1186/cc13668) Introduction Traumatic brain injury (TBI) is a major cause of death and long-term disability worldwide, and understanding its eff ect on health is critical. Although mortality has been a gold standard for years, functional scales and quality of life have been used as main outcome measures over the last decades due to their usefulness and aid in decision-making for medical teams, patients, and families. Despite this preference, no consensus exists for the most optimal outcome measure. We systematically reviewed outcome measures used in randomized controlled trials (RCTs) in patients with severe TBI admitted to an acute care hospital. Conclusion A simple prognostic model, based only on age and GCS, displayed a fairly good prognostic performance in predicting 6-month mortality of ICU-treated patients with moderate-to-severe TBI. The use of more complex scoring systems added little to the prognostic performance. p References p Methods We searched MEDLINE, EMBASE, Cochrane Central, BIOSIS and references of eligible trials. RCTs published over the last 7 years (2006 to October 2013) in 18 selected journals (based on impact factor) were considered for inclusion. RCTs performed in adults with severe TBI (GCS ≤8) were eligible. The primary endpoint was the outcome measures used in RCTs. The secondary outcomes were the timing of assessment and the methodological quality of trials using the Cochrane risk of bias assessment tool. Two independent reviewers selected trials and collected data using a standardized form. 1. Rothen et al.: Curr Opin Crit Care 2008, 14:513-519. 1. Rothen et al.: Curr Opin Crit Care 2008, 14:513-519. 2. Woodhouse et al.: Curr Opin Crit Care 2009, 15:450-455. 3. Ridley et al.: Anaesthesia 1998, 53:1185-1194. 2. Woodhouse et al.: Curr Opin Crit Care 2009, 15:450-455. 3. Ridley et al.: Anaesthesia 1998, 53:1185-1194. 2. Woodhouse et al.: Curr Opin Crit Care 2009, 15:450-45 References Methods A Finnish multicenter ICU database was screened for TBI patients admitted in 2003 to 2012. Logistic regression was used for customization of the APACHE II, SAPS II and SOFA for 6-month mortality prediction. An adjusted SOFA model, including age, and a reference model, including only age and Glasgow Coma Scale, were built for comparison. A randomized split-sample technique was used for internal validation and prognostic performance was determined by assessing discrimination, calibration and precision. 1. Ministry of Health 2012 Health Facts Singapore [http://www.moh.gov.sg/ content/moh_web/home/statistics/Health_Facts_Singapore/Principal_ Causes_of_Death.html] 2. Chua KS, et al.: A brief review of traumatic brain injury rehabilitation. Ann Acad Med Singapore 2007, 36:31-42. 2. Chua KS, et al.: A brief review of traumatic brain injury rehabilitation. Ann Acad Med Singapore 2007, 36:31-42. 2. Chua KS, et al.: A brief review of traumatic brain injury rehabilitation. Ann Acad Med Singapore 2007, 36:31-42. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Outcome measures used to evaluate the eff ect of intervention in RCTs performed in patients with severe TBI in acute care are heterogeneous. A signifi cant proportion of trials did not consider evaluating the functional status or other clinically meaningful outcomes, and very few trials assessed outcomes beyond 6 months. Thus, a signifi cant proportion of RCTs in patients with severe TBI are based on outcome measures not clinically useful to guide or change practice in this population. demographic profi les of severe TBI in the local context, with implications in the management of severe TBI, particularly the utilisation of critical care resources. Results A total of 780 patients were admitted with TBI during the study period, of which 365 patients (46.8%) sustained severe TBI. The majority (75.3%) of the severe TBI patients were male. There was a bimodal preponderance of severe TBI cases in young adults (age 21 to 40) and older people (age >61). Motor vehicle accidents (48.8%) and falls from <2 m (35.1%) were the main mechanisms of injury. Invasive monitoring was frequently employed in these patients with severe TBI: arterial blood pressure monitoring in 298 patients (81.6%), central venous pressure monitoring in 219 patients (60.0%), and intracranial pressure monitoring in 173 patients (47.4%). The incidence of use of tiered therapy such as sedation, mild hyperventilation, osmotherapy with mannitol, cerebrospinal fl uid drainage, barbiturate coma and decompressive craniectomy to control ICP converged with international practices. P479 P479 Predicting 6-month mortality of patients with traumatic brain injury: usefulness of common severity scores MB Skrifvars1, R Raj1, S Bendel2, T Selander2, R Kivisaari1, J Siironen1, M Reinikainen3 1Helsinki University Central Hospital, Helsinki, Finland; 2Kuopio University Hospital, Kuopio, Finland; 3North Karelia Central Hospital, Joensuu, Finland Critical Care 2014, 18(Suppl 1):P479 (doi: 10.1186/cc13669) Conclusion Young adults and older people involved mainly in motor vehicle accidents and falls respectively were among the high-risk groups for severe TBI. Management of these patients goes beyond the ICU and involves, but is not limited to, social support, emotional motivation and community reintegration of these patients [2]. TBI among the high-risk groups is largely preventable. Public awareness and prevention programmes will go some way in reducing the incidence of TBI amongst the high-risk groups. Introduction Severity of illness scoring systems is paramount for the evaluation of quality of care of critically ill and trauma patients [1-3]. The purpose of the present study was to evaluate the usefulness of the Acute Physiology and Chronic Health Evaluation II (APACHE II), Simplifi ed Acute Physiology Score II (SAPS II) and Sequential Organ Failure Assessment (SOFA) scores in predicting long-term outcome of patients with moderate-to-severe traumatic brain injury (TBI). P477 P477 Demographic profi les and extent of critical care resources utilisation in patients with severe traumatic brain injury: a Tan Tock Seng Hospital National Neuroscience Institute study J Yang1, JZ Wee2, CT Chong1 1Tan Tock Seng Hospital, Singapore; 2Ministry of Health, Singapore Critical Care 2014, 18(Suppl 1):P477 (doi: 10.1186/cc13667) Methods A prospective observational study of mild to moderate TBI adult patients in two critical care trauma centers. Patients underwent delirium assessment on days 3, 5 and 7 with the CAM-ICU and the ICDSC. Psychiatrists or neurointensivists evaluated delirium using the DSM-IV criteria for delirium. Assessments results were blinded and performed independently. Criterion validity of the CAM-ICU and the ICDSC were calculated from 2 × 2 frequency tables using standard defi nitions of sensitivity, specifi city, positive and negative predictive value, and overall accuracy. Because delirium was assessed repeatedly, estimates of 95% CIs for binary repeated data using generalized estimating equation in conjunction with the Huber–White estimator were performed. Inter-rater reliability for the CAM-ICU and ICDSC was assessed with the kappa coeffi cient. Introduction Trauma is the fi fth principal cause of death in Singapore, with traumatic brain injury (TBI) being the leading specifi c subordinate cause [1]. Introduction Trauma is the fi fth principal cause of death in Singapore, with traumatic brain injury (TBI) being the leading specifi c subordinate cause [1]. Methods This 8-year retrospective review of patients with severe TBI admitted to the neuroICU (NICU) of the National Neuroscience Institute, Tan Tock Seng Hospital between 2004 and 2011 reports the S173 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P480 Work activities after 3-year follow-up in ICU patients with traumatic brain injury j y M Arias-Verdu1, M Delange van der Kroft2, E Curiel-Balsera1, A Muñoz-Lopez1, J Fernandez-Ortega1, R Rivera-Fernandez1, M Prieto-Palomino1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain Critical Care 2014, 18(Suppl 1):P480 (doi: 10.1186/cc13670) j y M Arias-Verdu1, M Delange van der Kroft2, E Curiel-Balsera1, A Muñoz-Lopez1, J Fernandez-Ortega1, R Rivera-Fernandez1, M Prieto-Palomino1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain Critical Care 2014, 18(Suppl 1):P480 (doi: 10.1186/cc13670) Results From 5,602 citations retrieved after removal of duplicates, 36 RCTs met eligibility criteria. The outcome measures most frequently used were neurophysiologic indices (n = 18, 50%), the Glasgow Outcome Scale (n = 17, 47%), mainly at 6 months, nonspecifi c complications (n = 15, 42%), mortality (n = 12, 33%), and biomarkers (n = 10, 28%). Nine trials reported only physiologic indices and did not present any clinical or functional outcome measures. The methodological quality of included RCTs was heterogeneous. We observed a low risk of bias for sequence generation (n = 29, 80%), allocation concealment (n = 20, 55%), complete data reporting (n = 30, 83%), selective reporting (n = 36, 100%), and sample size (n = 21, 58%) but a high risk of bias for blinding (n = 20, 55%). Introduction The aim was to study work activities after 3 years in patients admitted to the ICU with traumatic brain injury (TBI). Introduction The aim was to study work activities after 3 years in patients admitted to the ICU with traumatic brain injury (TBI). Methods A prospective cohort study in an ICU of a reference hospital. We studied ICU-admitted patients consecutively with TBI between 2004 and 2008. We used validated scales including the Glasgow Outcome Scale (GOS) and the Project for the Epidemiological Analysis of Critical Care Patients Quality of Life questionnaire. Methods A prospective cohort study in an ICU of a reference hospital. We studied ICU-admitted patients consecutively with TBI between 2004 and 2008. We used validated scales including the Glasgow Outcome Scale (GOS) and the Project for the Epidemiological Analysis of Critical Care Patients Quality of Life questionnaire. S174 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 2 (abstract P479). Discrimination. Results We studied 531 patients, age 40.35 ± 19.75 years, APACHE I Figure 1 (abstract P479). Calibration. Figure 2 (abstract P479). Discrimination. (16.2% missing). P480 Work activities after 3-year follow-up in ICU patients with traumatic brain injury Regarding work activities, after 1 year, 28.5% of 326 patients evaluated have no diffi culties with work, 4.6% have diffi culties but work as before, 10.1% work only part-time or have changed to a job requiring minimum eff ort and 56.7% of patients do not work. After 3 years, 41.2% of 238 patients evaluated have no diffi culties with work, 4.6% have diffi culties but work as before, 12.6% work only part-time or have changed to a job requiring minimum eff ort and 41.6% of patients do not work. Evolution between 1 and 3 years by the McNemar test was statistically signifi cant (P <0.001). A total of 173 patients were in similar situation, only one had deteriorated and in 62 (26.05%) patients the evaluation of work activity had improved. y Conclusion After 1 year of admission from the ICU with TBI, more than 50% of patients have diffi culties with work. After 3 years the number of patients who work has increased although approximately 40% of the surviving patients do not work. Enteral administration of antiepileptic agents could have effi cacy for prevention of post-traumatic seizures in severe traumatic brain injury j y K Mocjiduki, H Suzuki, A Kawasaki, M Hotta, M Namiki, T Harada, M Takeda, A Yaguchi K Mocjiduki, H Suzuki, A Kawasaki, M Hotta, M Namiki, T Harada, M Takeda, A Yaguchi Introduction Antiseizure prophylaxis is recommended for preventing only early post-traumatic seizures (PTS) in the guidelines for the management of severe traumatic brain injury (TBI) by the Brain Trauma Foundation. Phenytoin is recommended to reduce the incidence of early PTS prophylaxis. Early enteral nutrition has recently shown theoretical advantages for prevention of bacterial translocation to maintain normal turnover of gut mucosa and is commonly used for TBI patients. Our hypothesis is that the enteral administration of antiepileptic agents is also useful for early PTS. Figure 1 (abstract P479). Calibration. Figure 1 (abstract P479). Calibration. Results We studied 531 patients, age 40.35 ± 19.75 years, APACHE II 17.94 ± 6.97 points, Glasgow Coma Scale at admission 7.53 ± 3.83 points. Cranial tomography at admission was: diff use injury type I (10.4%), diff use injury type II (28.1%), diff use injury type III (24.5%), diff use injury type IV (8.3%), evacuated mass lesion (22.6%), non-evacuated mass lesion (6.2%). Hospital mortality was 28.6%, 171 (32.2%) patients died after 1 year (6.6% missing) and 181 (34.1%) died after 3 years p p g y Methods This retrospective observational study included all adult patients admitted to our tertiary academic medico-surgical ICU due to TBI from September 2011 to August 2012. Patients who have epilepsy as a past history were excluded. Clinical data were collected from electrical medical archives. The baseline characteristics collected were age, gender, diagnosis, antiepileptic agents, timing of start and adverse eff ects of those agents, and methods of administration. S175 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Of 65 patients with TBI, 25 patients (18 men, seven women; mean age 56.7 ± 20.1) who were administered antiepileptic agents for PTS prophylaxis were studied. Fifteen cerebral contusions, 10 acute subdural hematomas, nine traumatic subarachnoid hemorrhages, two cerebral infarctions, two pneumocephalus and one traumatic intracerebral hemorrhage were shown in 25 patients. All patients were alive 28 days after the injury. Fourteen patients (56%) were intravenously administered (13 phenytoin and one phenobarbital), while 11 patients (44%) were administered with enteral feeding (four valproates, four carbamazepine and three zonisamides) as PTS prophylaxis. Simulation-based education for cardiopulmonary resuscitation and airway management protocols: a brief report of a systematic review and meta-analysis Simulation-based education for cardiopulmonary resuscitation an airway management protocols: a brief report of a systematic revie and meta-analysis A Cortegiani, V Russotto, A Naro, G Sanzo, G Grutta, SM Raineri, A Giarratano University of Palermo, Italy Critical Care 2014, 18(Suppl 1):P482 (doi: 10.1186/cc13672) y A Cortegiani, V Russotto, A Naro, G Sanzo, G Grutta, SM Raineri, A Giarratano University of Palermo, Italy Critical Care 2014, 18(Suppl 1):P482 (doi: 10.1186/cc13672) y A Cortegiani, V Russotto, A Naro, G Sanzo, G Grutta, SM Raineri, A Giarratano University of Palermo, Italy Critical Care 2014, 18(Suppl 1):P482 (doi: 10.1186/cc13672) f Methods All ambulances of the Hiroshima City Fire Department are equipped with a specially designed transmission device (RVT-SD200; Sony) that transmits high-resolution visual images and patient vital data using video cameras and a bio-monitor. We analyzed video data of OHCA patients transported by ambulance from November 2012 through December 2012, and evaluated the performance of CPR during transportation in accordance with the 2010 guidelines. The hands-off time was calculated as the time without chest compressions divided by the total CPR time. Introduction We aimed to summarize the effi cacy of simulation-based education in cardiopulmonary resuscitation and airway management [1]. Methods We searched the MEDLINE, Scopus and EMBASE databases for all peer-reviewed articles enrolling physicians/medical students in a simulation of either cardiopulmonary resuscitation or airway management protocols compared with no intervention or traditional teaching methods. We categorized the outcomes of the studies into four groups: task success, process skill, time skill, knowledge. Task success was defi ned as evaluation of successful completion of the task, process skill as evaluation of the procedure, time skill as the time required to complete the task, and knowledge as the objective assessment of conceptual understanding. When studies investigated more than one outcome, we considered the primary outcome, the overall measure or the most clinically relevant outcome. Results Thirty-two resuscitation episodes during transportation by ambulance were analyzed. Median CPR time per episode was 846 seconds (range 126 to 1,833 seconds). In total, the fraction of time without chest compression was 19.5 ± 7.6% (mean ± SD). Enteral administration of antiepileptic agents could have effi cacy for prevention of post-traumatic seizures in severe traumatic brain injury The average start day of PTS prophylaxis was 2.6 days after the injury by intravenous administration, and 2.2 days by enteral administration, respectively. Two patients with phenytoin showed hepatic dysfunction as an adverse eff ect and no patient showed early PTS both by intravenous and by enteral administrations. favor of simulation. Pooled eff ect size for process skill was 0.48 (0.11 to 0.84), for time skill was 0.29 (0.13 to 0.73) and for knowledge was 0.41 (0 to 0.84). Conclusion Simulation is an eff ective educational method to improve performance of physicians/medical students in the application of protocols for cardiopulmonary resuscitation and airway management. Reference 1. McGaghie WC, et al.: A critical review of simulation-based medical education research: 2003–2009. Med Educ 2010, 44:50-63. P483 Video analysis of cardiopulmonary resuscitation performance of ambulance crews during transportation g p H Giga1, T Otani1, T Sadamori1, K Une1, Y Kida1, K Ota1, J Itai1, S Yamaga1, S Kusunoki2, S Ohshimo1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 1Hiroshima University, Hiroshima, Japan; 2Critical Care Medical Center, Hiroshima Prefectural Hospital, Hiroshima, Japan Critical Care 2014, 18(Suppl 1):P483 (doi: 10.1186/cc13673) y y Conclusion The present study has some limitations because it is a single-center retrospective analysis. However, enteral administration of antiepileptic agents could be useful for PTS prophylaxis. Considering cost, adverse eff ects and serum monitoring, there is a possibility of enteral administration of antiepileptic agents as an alternative to intravenous phenytoin. Introduction High quality of chest compressions during cardio- pulmonary resuscitation (CPR) is a critical determinant of outcome from out-of-hospital cardiac arrest (OHCA). Unfortunately, however, victims often do not receive adequate chest compression for various reasons, particularly during transportation. Recent studies have demonstrated the interruption time of chest compression using transthoracic impedance analysis, but more information is needed to evaluate the performance of CPR provided by ambulance crews and reveal reasons for hands off chest during CPR. Simulation-based education for cardiopulmonary resuscitation and airway management protocols: a brief report of a systematic review and meta-analysis Reasons for interruption and its fraction of time in total hands-off time were as follows: 36% accounted for rhythm analysis/pulse check, 31% for ventilation, 11% for setting up automated chest compression devices, 8% for tracheal intubation/placement of supraglottic airway devices, 4% for intravenous line placement/administration of adrenaline, 3% for rescuer change, and 7% for adjustment of patient position/correction of rescuer posture and others. y Results From 8,528 articles, we selected 24 studies (13 randomized controlled studies, eight pre–post studies, three case–control studies) involving 1,149 participants. Compared with no intervention or traditional teaching methods, simulation was associated with a signifi cant improvement from mild to moderate of all outcomes (Figure 1). Log of odds ratio for task success was 2.03 (0.46 to 3.59) in Conclusion The fraction of time without chest compression observed in this study was comparative with those found in other studies in spite of the diffi cult situations, such as during transportation. Most frequent reasons for hands-off time were rhythm analysis and ventilation even though the ambulance crews strictly adhered to the guidelines. Figure 1 (abstract P482). S176 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P484 with 100% survival and in-hospital ICU transfer. The cardiac arrest (CA) group depended on cause of CA: cardiac arrhythmia (n = 12), survival 50%; surgical etiology (nontraumatic), survival 50%, mean age 70; myocardial infarction (n = 12), survival 83%, mean age 61; sepsis (n = 6), survival 67%, mean age 48; drugs overdose (n = 2), survival 100%; traumatic cardiac arrest (n = 15), 89% mortality, mean age 41; and CA with unknown etiology, survival 38%, mean age 68. P484 Implementation of the PulsePoint smartphone application for crowd-sourcing bystander resuscitation SC Brooks1, H Worthington1, T Gonedalles2, B Bobrow3, LJ Morrison4 1Queen’s University, Kingston, Canada; 2St Michael’s, Toronto, Canada; 3University of Arizona Medical College, Phoenix, AZ, USA; 4University of Toronto, Canada Critical Care 2014, 18(Suppl 1):P484 (doi: 10.1186/cc13674) Conclusion Emergency room cardiac arrest arrivals are not correlated with ER-CPA total activity. Good survival rates are probably related to a special quick response protocol and trained teams. Outcomes research to detect potential improvement in nurse care is needed, and should be evidence guided. Worst results in survival rates must be detected as the main topics for nursing care development. What is the role of head computed tomography in post-resuscitation care? What is the role of head computed tomography in post-resuscitation care? Y Valzani1, A Marudi2, S Baroni2, G De Grandis2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P486 (doi: 10.1186/cc13676) p Y Valzani1, A Marudi2, S Baroni2, G De Grandis2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P486 (doi: 10.1186/cc13676) Y Valzani1, A Marudi2, S Baroni2, G De Grandis2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P486 (doi: 10.1186/cc13676) Methods We conducted a structured survey delivered via Survey Monkey optimized for mobile devices. All alerted PulsePoint users between 28 June 2012 and 1 November 2013 were sent an invitation to participate in the survey. Survey responses associated with test activations and emergency medical services (EMS) professionals on active duty were excluded from the analysis. Introduction Guidelines recommend to perform head computed tomography (CT) in nontraumatic cardiac arrest only if the patient remains unconscious after return of spontaneous circulation [1]. Nevertheless, in cardiac arrest due to neurological causes, head CT would dramatically change the therapeutic strategy in these patients [2]. The aim of this study is to present the experience of our ICU in nontraumatic out-of-hospital cardiac arrest (OHCA) patients and to demonstrate that neurological abnormalities can be the primary cause of cardiac arrest. y y Results Of 4,827 survey requests sent, 995 responded and completed our survey (response rate 21%). Respondents identifi ed themselves as fi refi ghters (30%), paramedics (19%), EMTs (16%), nurses (4.4%), MDs (0.83%) and other professions (50%). Of those who received a PulsePoint alert, 23% (157/690) responded by making their way towards the location of the emergency. Of those who responded, 70% (110/157) arrived on scene. Most of those who did not arrive said they saw professional rescuers already on scene and turned back (52%, 24/47). Of those who arrived on scene, only 34% (37/110) found a person unconscious and not breathing normally. The majority found a person on scene who was not suff ering cardiac arrest. Of those who arrived on scene prior to EMS and also found a cardiac arrest victim on scene who required resuscitation, 80% (8/10) performed bystander CPR. To see or not to see: does video CPR perform better than telephone CPR? To see or not to see: does video CPR perform better than telephone CPR? A Ghuysen1, A Delfosse1, S Stipulante2, A Donneau3, V D’Orio1 1CHU – Ulg Liège, Belgium; 2Federal Public Health Services, Liege, Belgium; 3Liège University, Liege, Belgium Critical Care 2014, 18(Suppl 1):P487 (doi: 10.1186/cc13677) Introduction An organized team response and trained nursing staff in the emergency room critical patient area (ER-CPA) are the main factors to determine cardiopulmonary resuscitation (CPR) success rates. The objective was to evaluate outcomes in the emergency room after advanced life support (ALS) by reanimation teams, to improve nursing care, quality of resuscitation and survival. Introduction The ALERT algorithm, a simple and eff ective com- pression-only telephone CPR (t-CPR) protocol, has been previously demonstrated to help bystanders initiate CPR. According to their worldwide availability, mobile phone communications may play an increasing role in emergency calls. Preliminary studies suggest that they might improve dispatcher’s understanding of the rescuer’s situation. However, there is currently no validated protocol for videoconference-assisted CPR (v-CPR). We initiated the present study to validate an original protocol of v-CPR based on the ALERT algorithm and to evaluate the potential benefi t of this assistance in comparison with classical t-CPR. Introduction The ALERT algorithm, a simple and eff ective com- pression-only telephone CPR (t-CPR) protocol, has been previously demonstrated to help bystanders initiate CPR. According to their worldwide availability, mobile phone communications may play an increasing role in emergency calls. Preliminary studies suggest that they might improve dispatcher’s understanding of the rescuer’s situation. However, there is currently no validated protocol for videoconference-assisted CPR (v-CPR). We initiated the present study to validate an original protocol of v-CPR based on the ALERT algorithm and to evaluate the potential benefi t of this assistance in comparison with classical t-CPR. q y Methods We included all adult patients receiving ALS in the ER-CPA during 1 year. Cases not allowed CPR are excluded. A retrospective design, multivariate with ALS performed, gender, age, cause of arrest, precedence, outcome and hospital derivation, and 100% of cases studied. Results A total of 149 patients were attended in the ER-CPA with ALS maneuvers during the studied period (9.23% of a total 1,613). Thirty-four of them were nonheart-beating donors. The protocol was performed with 23 eff ective donors and 31 organs were obtained for transplant. Simulation-based education for cardiopulmonary resuscitation and airway management protocols: a brief report of a systematic review and meta-analysis Introduction Only a minority of patients suff ering out-of-hospital cardiac arrest receive any bystander cardiopulmonary resuscitation (CPR). Bystander CPR is associated with improved odds for survival. The PulsePoint smartphone application alerts users in the vicinity of a cardiac arrest to facilitate immediate citizen bystander resuscitation. Addressing implementation barriers may provide an opportunity to increase eff ectiveness of the application. PulsePoint is currently active in over 400 communities in the United States. Our objective was to identify modifi able barriers to optimal implementation of the PulsePoint smartphone application. P486 P485 Emergency room advanced life support after cardiac arrest: outcomes and survival, nursing care and team response A Villamor, A Tugas, Y Masguret, M Bayon, P Ceregido Barcelona Clinic Hospital, Barcelona, Spain Critical Care 2014, 18(Suppl 1):P485 (doi: 10.1186/cc13675) What is the role of head computed tomography in post-resuscitation care? Methods We collected data for all patients with nontraumatic OHCA admitted to the ICU from 1 January 2012 to 31 December 2012. We recorded mean age, male-to-female ratio, cause of cardiac arrest and mortality in the ICU. Results Thirty-four survive cardiac arrest patients were admitted to the ICU. Mean age was 66 years. The male-to-female ratio was 2:1. Sixteen patients died in the ICU. Head CT scans obtained in the immediate post- arrest period found signifi cant abnormalities in four patients (three subarachnoid hemorrhage and one ischemic ictus) with immediate modifi cation of treatment. Conclusion We observed a very high proportion of bystander CPR (80%) for victims of out-of-hospital cardiac arrest when PulsePoint users arrived before EMS. This suggests that optimized PulsePoint implementation may increase community bystander CPR rates. Alert specifi city for cardiac arrest may be too low (the cry wolf scenario) with current default activation criteria. Also, the current activation radius (0.5 mile) may be too large because EMS frequently arrive before the PulsePoint responder. i Conclusion Head CT abnormalities can be found in cardiac arrest patients. Further investigations are necessary to evaluate the impact of cardiac arrest from neurological causes and to establish the role of neuroimaging in these patients. f References References 1. Cocchi et al.: Intern Emerg Med 2010, 5:533-538. 2. Ball et al.: Can J Anesth 2005, 52:892-893. 1. Cocchi et al.: Intern Emerg Med 2010, 5:533-538. 1. Cocchi et al.: Intern Emerg Med 2010, 5:533-538. 2. Ball et al.: Can J Anesth 2005, 52:892-893. Initial anticoagulation strategy for extracorporeal cardiopulmonary resuscitation patients p Critical Care 2014, 18(Suppl 1):P489 (doi: 10.1186/cc136 Introduction Out-of-hospital cardiac arrest (OOHCA) causes 60,000 UK and 300,000 US deaths each year. Survival to hospital discharge in the developed world has historically been 7 to 10% with obvious cognitive impairment in 10% of survivors. Primary percutaneous coronary intervention (PPCI) and targeted temperature management (TTM) (or at least hyperthermia avoidance) have been shown to improve survival in comatose patients post OOHCA. There is no reliable method to predict poor outcome on presentation. We aimed to identify factors associated with poor outcome in our single-centre regional referral OOHCA population. Y Iwashita, M Matsuduki, M Yukimitsu, K Yokoyama, T Nakata, A Yamamoto, K Ishikura, H Imai Mie University Hospital, Tsu, Mie, Japan Critical Care 2014, 18(Suppl 1):P488 (doi: 10.1186/cc13678) Introduction Extracorporeal cardiopulmonary resuscitation (ECPR) is increasingly being used in emergency and critical care medicine in Japan. Although a major complication of this procedure is bleeding, the optimal heparin dose and activated coagulation time (ACT) remain unknown. p p Methods We performed a pragmatic single-centre retrospective review over 18 months commencing 1 January 2011 of all patients admitted to our regional OOHCA centre ICU following successful resuscitation from OOHCA. In keeping with guidelines, all patients were assessed for suitability for PPCI and TTM. A good outcome was defi ned by a Pittsburgh Cognitive Performance Category (CPC) score of 1 to 2 (independence, mild impairment) on hospital discharge. A poor outcome was defi ned as death or CPC 3 to 4 (moderate to severe impairment, coma) on hospital discharge. CPC scoring was determined Methods We retrospectively evaluated the initial heparin doses, ACT values, and complications of patients who received ECPR between February 2011 and November 2013 at the Emergency and Critical Care Center, Mie University Hospital, Japan. Results ECPR was performed in 45 patients, and the ACT was evaluated in 32 patients. All patients were administered 3,000 U unfractionated heparin at the time of priming the circuit. Patients for whom cannulation took a longer time received an additional 2,000 to 3,000 U unfractionated heparin. The average heparin dose administered Table 1 (abstract P489). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 was 53.6 U/kg body weight. The average ACT was 231.3 seconds. In 17 of the 32 patients, the ACT exceeded 200 seconds. Three patients experienced fatal bleeding in the chest wall, which could not be stabilized by conservative treatment. One patient developed a cerebral infarction. There were no signifi cant diff erences between the patients with fatal bleeding and those without fatal bleeding with regard to the heparin dose, ACT, and duration of CPR. every 2 minutes. A total of 120 students without prior CPR training were recruited from upper secondary school, during regular class hours, and randomly assigned to the t-CPR group (n = 60) versus the v-CPR group (n = 60). The Resusci®Anne SkillReporter™ manikin was used to evaluate CPR performance. Data were transferred from the manikin into a computerized database using the Laerdal SkillReporting System V.2.2.1 software. Further analysis was based on audio-recordings and video-recordings. Primary outcome measures were the results of the Cardiff evaluation test; the secondary measures were global scoring of a complete 7-minute period of CPR.i Conclusion According to the Extracorporeal Life Support Organization guidelines, the target ACT should be around 1.5 times the normal ACT. However, it is diffi cult to obtain the normal ACT in emergency situations. Many of our patients’ ACTs exceeded 200 seconds, and three patients experienced fatal bleeding that was possibly due to chest compression. Post-cardiac arrest patients often experience coagulopathy due to either cardiac arrest or hypothermia therapy. Therefore, an anticoagulation protocol other than the pulmonary extracorporeal membrane oxygenation protocol is required for ECPR patients. We evaluated our anticoagulation protocol for ECPR and observed that our patients’ ACTs frequently exceeded the target value and some experienced fatal bleeding. The anticoagulation protocol for post-CPR patients may need to be reconsidered. Results The mean chest compression rate increased signifi cantly in the v-CPR group as compared with t-CPR (110 ± 16 vs. 86 ± 28; P <0.0001), while depth remained constant (48 ± 13 mm vs. 47 ± 16 mm, P = NS). Hand positioning was correct in 91.7% of cases with v-CPR, but only in 68% with t-CPR (P = 0.001). The hands-off period was almost nonexistent in the v-CPR group (0 vs. 7 seconds; P = 0.0016), but the median no-fl ow time was signifi cantly greater in the v-CPR group (146 vs. 122 seconds, P < 0.0001). P488 l Hammersmith Hospital, London, UK Critical Care 2014, 18(Suppl 1):P489 (doi: 10.1186/cc13679) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 As a consequence, global evaluation of CPR performance revealed a signifi cant improvement in v-CPR group score as compared with the t-CPR group (6 vs. 5, P < 0.001). Conclusion Video-assisted CPR using this original algorithm allows bystanders to reach compression rates and depths close to international guidelines and to reduce hands-off events during CPR. To see or not to see: does video CPR perform better than telephone CPR? Respiratory arrest (n = 31) was the best outcome group, Methods We developed a strictly worded algorithm for v-CPR, adapted from the ALERT t-CPR protocol, with additional re-evaluation loops, S177 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Post Arrest Consult Team: a knowledge translation strategy for post-cardiac arrest care Results Of the 51 patients, 21 were S. Mean age was 70 years (± 15) in the S, of which 10 (48%) were male, and in the nonsurvivors (NS) mean age was 70 years (± 17) (P = 0.916) with 23 (77%) male patients (P = 0.042). The initial rhythm was asystole in 11 S and in 20 NS (P =0.386), pulseless electrical activity in two S and NS (P = 1), and ventricular fi brillation in eight S and six NS (P = 0.207). The arrest was witnessed in 15 NS and 16 S (P = 0.083). Lay rescuer BLS was performed in 17 NS and nine S (P = 0.4). A signifi cant diff erence in time of EC and start of ALS was observed between S (12 minutes; 8 to 15) and NS (14 minutes; 12 to 17) (P = 0.03). Mean initial rSO2 was 34% (± 23) and 24% (± 12.5) in S and NS (P = 0.077). We observed a negative correlation between mean initial rSO2 and time between EC and ALS (correlation coeffi cient –0.243; P = 0.041). Critical Care 2014, 18(Suppl 1):P492 (doi: 10.1186/cc13682) Critical Care 2014, 18(Suppl 1):P492 (doi: 10.1186/cc13682 Introduction Lack of standardized care contributes to low survival in admitted out-of-hospital cardiac arrest (OHCA) patients. The objective of our study was to implement a Post Arrest Consult Team (PACT) and improve the quality of care for admitted OHCA patients. Introduction Lack of standardized care contributes to low survival in admitted out-of-hospital cardiac arrest (OHCA) patients. The objective of our study was to implement a Post Arrest Consult Team (PACT) and improve the quality of care for admitted OHCA patients. p q y p Methods We conducted a prospective cohort study with concurrent controls from February 2011 to February 2013 in a network of 29 Toronto-area hospitals. The PACT was implemented in two hospitals and functioned as a consult service with a nurse and physician on- call 24/7. Patients from other network hospitals acted as concurrent controls. The PACT focused on four key processes of care: targeted temperature management (TTM); coronary angiography; avoidance of premature withdrawal of life-sustaining therapy (WLST <72 hours) on the basis of neuroprognostication; and electrophysiology assessment. We included nontraumatic OHCA patients who were >18 years old, survived at least 6 hours, and were comatose. We excluded patients with do-not-resuscitate orders, intracranial or other severe bleeding. P490 Results There were 247 admissions to the ICU following OHCA from 2010 to 2012. Seven patients were excluded for missing/incomplete data. In total, 102 patients (42.5%) survived to discharge. PAR ranged from –2 to 18. Zero was the most frequent score. Only one of 15 admissions with PAR >5 (PAR 13) survived. A total of 101 patients with PAR ≤5 survived (45%). Acute myocardial infarction was identifi ed as the precipitating event in 111 patients (46%). Mean initial cerebral saturation and time to start advanced life support in out-of-hospital cardiac arrest: are they correlated? C Genbrugge, C De Deyne, I Meex, F Jans, W Boer, J Dens ZOL, Genk, Belgium Critical Care 2014, 18(Suppl 1):P490 (doi: 10.1186/cc13680) Introduction During advanced life support (ALS) it is still impossible to predict return of spontaneous circulation (ROSC) or outcome. Cerebral saturation (rSO2) measurements with near-infrared spectroscopy can be used in cardiac arrest circumstances and could play a role in predicting ROSC or neurologic outcome [1]. It is known that an initial rhythm of asystole and a long no/low-fl ow time has a worse outcome. We measured rSO2 during ALS in out-of hospital cardiac arrest (OHCA) patients and compared the mean rSO2 during the fi rst minute with the time between the emergency call (EC) and start of ALS. Conclusion Only one of 240 OHCA patients admitted to the ICU over a 3-year period with PAR >5 survived until discharge. PAR scoring has been shown to be useful in helping decide the appropriateness of ICU admission for combined IHCA/OHCA. Our data support its use in isolated OHCA. PAR ≤5 appears to be poorly predictive of survival. However, PAR >5, combined with clinical evaluation, could help identify OHCA nonsurvivors and avoid ICU admissions that do not benefi t the patient. References 1. Bernard SA: Emerg Med 1998, 10:25-29. y Methods With IRB approval, rSO2 was prospectively measured between December 2011 and November 2013 in 51 OHCA cases with presumed cardiac cause during ALS. One sensor of the EQUANOX Advance was applied to the right side of the patient’s forehead when the medical emergency team arrived. The measurement was continued until death of the patient or arrival at the ICU. Survivors (S) are defi ned as sustained ROSC longer than 20 minutes. CPR data were collected using the Utstein CPR data registration. Post Arrest Consult Team: a knowledge translation strategy for post-cardiac arrest care We used generalized linear mixed models to assess whether PACT implementation was associated with higher odds of achieving each of the four targeted processes of care while adjusting for secular trends unrelated to the intervention. Conclusion A tendency towards higher mean initial rSO2 in S compared with NS was observed together with a negative correlation between mean initial rSO2 and time between the EC and start of ALS. Also a signifi cant diff erence in time between the EC and start of ALS between S and NS is observed. Larger studies are needed to confi rm the possible function of rSO2 as a surrogate for time between the EC and start of ALS. Reference 1. Ito N et al.: Resuscitation 2012, 83:46-50. P490 The Mann–Whitney test and Pearson chi- square were utilized for comparison of S and nonsurvivors (NS) data. The Pearson test was used to examine correlation. 2. Ebell MH: J Fam Pract 1992, 34:551-558. 3. O’Keefe S, et al.: Resuscitation 1994, 28:21-25. 4. Porter R, et al.: Crit Care 2011, 15:P299. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 has been fully validated for predicting survival following OHCA. The Prognosis After Resuscitation (PAR) score was developed from meta- analysis in 1992. A score >5 (from seven variables) predicts nonsurvival following in-hospital cardiac arrest (IHCA) [2,3]. Porter and colleagues demonstrated that PAR >5 is a useful predictor of nonsurvival for combined IHCA/OHCA ICU admissions [4]. We aim to evaluate PAR application to adult ICU admissions at University Hospitals Bristol (UHB), UK following OHCA. from physiotherapy and occupational therapy assessments in the clinical notes. Continuous data were analysed using a two-tailed t test or Mann–Whitney U test, categorical data with a chi-squared test. has been fully validated for predicting survival following OHCA. The Prognosis After Resuscitation (PAR) score was developed from meta- analysis in 1992. A score >5 (from seven variables) predicts nonsurvival following in-hospital cardiac arrest (IHCA) [2,3]. Porter and colleagues demonstrated that PAR >5 is a useful predictor of nonsurvival for combined IHCA/OHCA ICU admissions [4]. We aim to evaluate PAR application to adult ICU admissions at University Hospitals Bristol (UHB), UK following OHCA. from physiotherapy and occupational therapy assessments in the clinical notes. Continuous data were analysed using a two-tailed t test or Mann–Whitney U test, categorical data with a chi-squared test. Results Sixty-nine patients were admitted to our ICU following successful resuscitation from OOHCA in the 18-month period. Presenting features and outcomes are shown in Table 1. Conclusion Good outcome was associated with a shockable rhythm on presentation, cardiac cause, arrest occurring in daylight hours (08:00 to 20:00) and shorter time between collapse and ROSC. Poor outcome was associated with placement of an advanced airway device, lower pH, higher base defi cit and higher lactate. g Methods The Innovian (Dräger) electronic clinical information database was searched retrospectively for all OHCA ICU admissions from 2010 to 2012. Data were extracted using an electronic pro forma in Microsoft Excel. Missing/incomplete data were excluded. Survival was defi ned as survival to discharge from the acute hospital (UHB). Initial anticoagulation strategy for extracorporeal cardiopulmonary resuscitation patients Presenting features and outcome at hospital discharge of ICU OOHCA patients Good outcome (CPC 1 to 2) Poor outcome (dead or CPC 3 to 4) Total P value Age 63.3 ± 16.8 63.6 ± 14.4 63.5 ± 14.7 NS Male sex 21/26 (81%) 29/43 (67%) 50/69 (72%) NS Arrest occurring 08:00 to 20:00 14/19 (74%) 16/36 (44%) 30/55 (55%) <0.05 VF/VT as initial rhythm 25/26 (96%) 25/43 (58%) 50/69 (72%) <0.001 Bystander CPR 12/26 (46%) 24/43 (56%) 36/69 (52%) NS Time from collapse to ROSC (minutes) 18.3 ± 11 32.3 ± 26 27.1 ± 23 <0.05 Advanced airway management 14/24 (58%) 38/42 (90%) 52/66 (79%) <0.005 Cardiac cause 24/26 (92%) 30/43 (70%) 54/69 (78%) <0.05 Angiography 19/26 (73%) 23/43 (53%) 42/69 (61%) NS Therapeutic hypothermia 24/26 (92%) 41/43 (95%) 64/69 (93%) NS Presenting pH 7.21 ± 0.11 7.08 ± 0.18 7.13 ± 0.17 <0.01 Presenting base defi cit (mmol/l) –7.9 ± 6.2 –13.8 ± 5.9 –11.5 ± 6.7 <0.001 Presenting lactate (mmol/l) 4.3 ± 3.0 8.3 ± 4.5 6.8 ± 4.4 <0.0005 Table 1 (abstract P489). Presenting features and outcome at hospital discharge of ICU OOHCA patients Table 1 (abstract P489). Presenting features and outcome at hospital discharge of ICU OOHCA patients Good outcome (CPC 1 to 2) Poor outcome (dead or CPC S178 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P492 P492 Post Arrest Consult Team: a knowledge translation strategy for post-cardiac arrest care SC Brooks1, D Scales2, K Dainty2, S Gray2, R Pinto3, E Racz2, M Gaudio2, A Amaral2, A Baker2, M Chapman2, E Crystal2, P Dorian2, N Fam2, R Fowler2, J Friedrich2, M Madan2, G Rubenfeld2, O Smith4, LJ Morrison2 1Queen’s University, Kingston, Canada; 2University of Toronto, Canada; 3Sunnybrook Health Sciences Centre, Toronto, Canada; 4St Michael’s, Toronto, Canada Critical Care 2014 18(Suppl 1):P492 (doi: 10 1186/cc13682) P493 One-year assessment of in-hospital cardiac arrest M Ahmed, A Kochhar, O Rose Univeristy Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P493 (doi: 10.1186/cc13683) One-year assessment of in-hospital cardiac arrest M Ahmed, A Kochhar, O Rose Univeristy Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P493 (doi: 10.1186/cc13683) survivors of cardiac arrest (CA). Despite this, in our country there are still few centers that apply hypothermia post CA regularly. We describe a Chilean experience with endovascular hypothermia post CA, in three ICUs of the same university clinical center [1-3]. Introduction This retrospective audit evaluated adult patients who suff ered in-hospital cardiac arrest (IHCA) against the recent National Confi dential Enquiry into Patient Outcome and Death (NCEPOD) report [1]. It looked specifi cally at the recognition of the acutely unwell, the interventions made, the decisions taken from admission through to the post-arrest period and the outcomes following cardiopulmonary resuscitation (CPR). The audit aims to guide future improvements in preventing cardiac arrest and enhancing end-of-life care decision-making.f Methods A descriptive cohort study. All surviving comatose patients after CA were included, and underwent endovascular hypothermia management according to protocol. CoolGard™ internal cooling equipment was used. Variables: delay between CA and hypothermia (34°C), time in hypothermia, complications, ventilatory and hemo- dynamic management. Main outcome measures: mortality and neuro- logical follow-up to 6 months with Glasgow Outcome Score Extended (GOSE). Results Twenty-seven patients were managed in the ICU post CA (18 outpatients). Twenty-four were men (89%), mean age 33.5 ± 19 years (16 to 76). Delay was considered (time between CA and achieving target temperature of 34°C): median 10.5 ± 3.3 hours (3 to 18 hours). Hypothermic maintenance (from when it reaches 34°C until it returns to 36°C): 24 ± 18 hours (24 to 48 hours). Complications of hypothermia: fi ve hypokalemia (18.5%), three ventricular arrhythmias (11%), one vein thrombosis (3.7%). There were no deaths during hypothermia. Hospital mortality was two cases (7.4%). At 6 months it was three (11%). Neurological outcome at discharge and 6 months are shown in Figure 1. Good outcome (GOSE 5 to 8) occurred in 11 patients (40%) at discharge. Good outcome at 6 months occurred in 20 patients (74%). Methods Medical notes of adult patients suff ering IHCA, over a 1-year period, were identifi ed and data were collected using a validated NCEPOD audit tool. These data included patient demographics, initial clerking and consultant review, patient care during the 48 hours prior to cardiac arrest, the resuscitation status of the patient, the resuscitation attempt, the post-cardiac arrest care and survival to discharge rates. Knowledge and use of therapeutic hypothermia in cardiac arrest victims among healthcare staff in Greece Conclusion The results from this audit highlight persistent defi ciencies in the care pathway for the acutely unwell patient. Improvement will be focused on earlier consultant review and better recognition of warning signs with appropriate action taken. Furthermore, earlier routine and senior clinician-led discussions on appropriate end-of-life care are vital. References Knowledge and use of therapeutic hypothermia in cardiac arrest victims among healthcare staff in Greece P Manthou1, T Katsoulas1, A Korobeli1, L Kiriakou2, G Fildissis1 1Department of Nursing University, Athens, Greece; 2Gonk Agioi Anargiroi Hospital, Kifi ssia, Greece Critical Care 2014, 18(Suppl 1):P495 (doi: 10.1186/cc13685) P Manthou1, T Katsoulas1, A Korobeli1, L Kiriakou2, G Fildissis1 1Department of Nursing University, Athens, Greece; 2Gonk Agioi Anargiroi Hospital, Kifi ssia, Greece Critical Care 2014, 18(Suppl 1):P495 (doi: 10.1186/cc13685) i Critical Care 2014, 18(Suppl 1):P495 (doi: 10.1186/cc13685) 1. Findlay G, et al.: Time to Intervene? A Review of Patients who Underwent Cardiopulmonary Resuscitation as a Result of an In-hospital Cardiorespiratory Arrest. London: NCEPOD; 2012. [http://www.ncepod.org.uk/ 2012report1/ downloads/CAP_summary.pdf] Introduction Therapeutic hypothermia (TH) improves the neurologic outcome of patients who survive after cardiac arrest but suff er from severe secondary neurological damage [1]. In 2010, the use of TH after cardiac arrest was included in the ERC guidelines. The purpose of this study was to investigate the knowledge of the medical and nursing staff on the implementation of TH in patients after cardiac arrest [2]. 2. Meaney PA, et al.: Crit Care Med 2010, 38:101-108. 2. Meaney PA, et al.: Crit Care Med 2010, 38:101-108. f p p [ ] Methods Data were collected by an anonymous questionnaire designed for the purpose of research, addressed to medical and nursing staff of Greek hospitals. The questionnaire consisted of questions about knowledge and behavior related to TH induction, target temperature and duration of cooling. Information about the potential barriers to implementation of TH was also collected. Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score H Davies, A Loosely, S Dolling, R Eve University Hospitals Bristol NHS Foundation Trust, Bristol, UK Critical Care 2014, 18(Suppl 1):P491 (doi: 10.1186/cc13681) Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score H Davies, A Loosely, S Dolling, R Eve University Hospitals Bristol NHS Foundation Trust, Bristol, UK Critical Care 2014, 18(Suppl 1):P491 (doi: 10.1186/cc13681) Results The primary analysis included 162 patients from two intervention hospitals and 892 from 27 control hospitals. Thirty- two percent of the patients were female and the mean age was 65.3 ± 16.5 years. Almost one-half (46%) of patients had a shockable initial cardiac arrest rhythm, 41% had bystander CPR, and 5% had an AED applied. PACT did not improve use of TTM (ratio of ORs = 1.03, 95% CI = 0.89 to 1.20), angiography for patients without ST-elevation H Davies, A Loosely, S Dolling, R Eve H Davies, A Loosely, S Dolling, R Eve University Hospitals Bristol NHS Foundation Trust, Bristol, UK Critical Care 2014, 18(Suppl 1):P491 (doi: 10.1186/cc13681) Introduction Out-of-hospital cardiac arrest (OHCA) has a poor prognosis even after successful resuscitation [1]. No scoring system S179 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 myocardial infarction (ratio of ORs = 1.10, 95% CI = 0.87 to 1.40), or electrophysiology assessment (ratio of ORs = 1.06, 95% CI = 0.81 to 1.38) as compared with concurrent control hospitals. Patients in the intervention group were less likely to have life support withdrawn within 72 hours on the basis of neuroprognosis compared with patients in the concurrent control group (ratio of ORs = 0.62, 95% CI = 0.39 to 0.98). Conclusion PACT was associated with reduced WLST <72 on the basis of neuroprognostication but did not improve other important post-cardiac arrest processes of care. Further work is underway to identify factors that infl uenced implementation. This will guide future consideration of the PACT model in other settings. myocardial infarction (ratio of ORs = 1.10, 95% CI = 0.87 to 1.40), or electrophysiology assessment (ratio of ORs = 1.06, 95% CI = 0.81 to 1.38) as compared with concurrent control hospitals. Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score Patients in the intervention group were less likely to have life support withdrawn within 72 hours on the basis of neuroprognosis compared with patients in the concurrent control group (ratio of ORs = 0.62, 95% CI = 0.39 to 0.98). Conclusion PACT was associated with reduced WLST <72 on the basis of neuroprognostication but did not improve other important post-cardiac arrest processes of care. Further work is underway to identify factors that infl uenced implementation. This will guide future consideration of the PACT model in other settings. Figure 1 (abstract P494). P493 One-year assessment of in-hospital cardiac arrest M Ahmed, A Kochhar, O Rose Univeristy Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P493 (doi: 10.1186/cc13683) p p g Results Medical notes were available for assessment for 69 out of the 82 patients that were identifi ed as having IHCA between 1 October 2011 and 30 September 2012. The frequency of IHCA showed no correlation to day of the week or month. Initial clerking was incomplete in history- taking (16% vs. 14% in NCEPOD) and in examination (46% vs. 24% in NCEPOD). The majority of patients were appropriately escalated in a timely fashion (94% vs. 82% in NCEPOD), but fi rst consultant review was delayed beyond 12 hours in 49% of cases (48% in NCEPOD). A total 81% of patients suff ered cardiac arrest 24 hours after admission (68% in NCEPOD). Warning signs for cardiac arrest were considered present in 59% of cases (75% in NCEPOD), with a signifi cant proportion going unrecognised (27%) despite multiple medical reviews. Out-of- hours CPR attempts (68% vs. 59% in NCEPOD) seemed be associated with poorer survival. The survival to discharge rate after in-hospital cardiac arrest was 10.1%. This compares with 14.6% in the NCEPOD data and 20% in larger studies [2]. Ninety per cent of patients had no documentation of resuscitation status (78% in NCEPOD). Conclusion Our series shows a low mortality and a very good neurological outcome. There was no mortality or severe complications associated with endovascular hypothermia. It is a safe and feasible technique implemented in Latin American critical care units. Even the delay in achieving the objective of hypothermia is very long. R f 1. Kory et al.: Resuscitation 2011, 82:15-20. 1. Kory et al.: Resuscitation 2011, 82:15-20. 2. Seder et al.: Crit Care Med 2009, 37:S211-S222. 3. Holzer et al.: N Engl J Med 2010, 363:1256-1264. 1. Kory et al.: Resuscitation 2011, 82:15-20. 2. Seder et al.: Crit Care Med 2009, 37:S211-S222. 2. Seder et al.: Crit Care Med 2009, 37:S211-S222. 3. Holzer et al.: N Engl J Med 2010, 363:1256-1264. 3. Holzer et al.: N Engl J Med 2010, 363:1256-1264. P494 d Endovascular hypothermia after cardiac arrest in a Chilean ICU M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P494 (doi: 10.1186/cc13684) Endovascular hypothermia after cardiac arrest in a Chilean ICU M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P494 (doi: 10.1186/cc13684) Introduction For more than a decade, mild hypothermia has been a standard for enhancing the neurological prognosis in comatose Results We obtained 344 questionnaires from 16 hospitals. The population of the study was registered nurses (RN) (63.8%) and doctors S180 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 24 hours of targeting body temperature at 33°C or 36°C. These results suggest that hypothermia does not alter host immune response. Reference 24 hours of targeting body temperature at 33°C or 36°C. These results suggest that hypothermia does not alter host immune response. Reference 1. Nielsen N, et al.: Targeted temperature management at 33°C versus 36°C after cardiac arrest. N Engl J Med 2013, 369:2197-2206. 24 hours of targeting body temperature at 33°C or 36°C. These results suggest that hypothermia does not alter host immune response. Reference (36.2%). The majority of health staff (81.5%) had never implemented TH. A total 45.8% of respondents stated that the main reasons for not using TH were the lack of information and training about the method, the lack of nursing staff , the lack of available cooling methods and the required time. The most common methods of application were cold packs and intravenous fl uids. Only 30.2% of the doctors and 5.5% of the nurses (P <0.001) actually had the knowledge to implement TH, and this was demonstrated by correct answers. Of the respondents who answered that they did know the method, only 23.9% answered correctly; about the target temperature, the maintenance and rewarming phase. A total 59.1% of doctors, despite having attended the Advanced Cardiac Life Support seminar, were not able to answer correctly the knowledge questions. Continuous education of health professionals and the existence of a protocol were proposed by 65% of participants as the best way of increasing knowledge and adherence with ERC guidelines about TH. 1. Nielsen N, et al.: Targeted temperature management at 33°C versus 36°C after cardiac arrest. N Engl J Med 2013, 369:2197-2206. Endovascular hypothermia after cardiac arrest in a Chilean ICU M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P494 (doi: 10.1186/cc13684) P497 P497 Derived electromyography is an accurate measure of shivering burden during targeted temperature management T May, DS Seder, GF Fraser, BM McCrum, CF Hoover, RR Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P497 (doi: 10.1186/cc13687) Introduction Shivering complicates targeted temperature manage- ment (TTM) by increasing metabolism, oxygen consumption, rest ing energy expenditure, and carbon dioxide production and is associated with lower brain tissue oxygen levels; all of these may limit the eff ectiveness of TTM. However, the recognition and measurement of shivering are subjective and ill-defi ned. The Bedside Shivering Assessment Scale (BSAS) is the only validated tool to describe the intensity of shivering. We hypothesized that the derived electromyography (dEMG) value measured by the bispectral index monitor (BIS) would agree with energy expenditure due to shivering, compared with the BSAS. Introduction Shivering complicates targeted temperature manage- ment (TTM) by increasing metabolism, oxygen consumption, rest ing energy expenditure, and carbon dioxide production and is associated with lower brain tissue oxygen levels; all of these may limit the eff ectiveness of TTM. However, the recognition and measurement of shivering are subjective and ill-defi ned. The Bedside Shivering Assessment Scale (BSAS) is the only validated tool to describe the intensity of shivering. We hypothesized that the derived electromyography (dEMG) value measured by the bispectral index monitor (BIS) would agree with energy expenditure due to shivering, compared with the BSAS. Conclusion Therapeutic hypothermia is rarely used in Greek hospitals. The level of knowledge is mainly related to the lack of education and the lack of information about new techniques. Programs for continuing education are necessary for the use of new therapeutic techniques in the fi eld of health. i References 1. Erb JL, Hravnak M, Rittenberger JC: Therapeutic hypothermia after cardiac arrest. Am J Nurs 2012, 112:38-44. 1. Erb JL, Hravnak M, Rittenberger JC: Therapeutic hypothermia after cardiac arrest. Am J Nurs 2012, 112:38-44. 2. Metzger JC, Eastman AL, Pepe PE: Year in review 2010: critical care – cardiac arrest and cardiopulmonary resuscitation. Crit Care 2011, 15:239. 2. Metzger JC, Eastman AL, Pepe PE: Year in review 2010: critical care – cardiac arrest and cardiopulmonary resuscitation. Crit Care 2011, 15:239. 2. Metzger JC, Eastman AL, Pepe PE: Year in review 2010: critical care – cardiac arrest and cardiopulmonary resuscitation. Crit Care 2011, 15:239. Methods We measured continuous indirect calorimetry during a 2 to 5 hour time span during targeted temperature management in 12 patients being treated for hypoxic ischemic encephalopathy after cardiac arrest. Patients were excluded if seizing, requiring >FiO2 0.5, exhibiting early spasticity, or not shivering. The BSAS was measured every 15 minutes by a blinded observer and shivering was treated for a BSAS ≥1, as per institutional protocol. The association of dEMG and BSAS as a predictor of resting energy exposure (REE) was measured using linear regression and Pearson’s correlation. P498 hi P498 P496 Induced hypothermia of 33°C does not aff ect host response compared with maintaining 36°Cf Induced hypothermia of 33°C does not aff ect host response compared with maintaining 36°Cf C Beurskens, J Horn, E Van Leeuwen, N Juff ermans AMC, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P496 (doi: 10.1186/cc13686) g g Results The study population included 12 patients. Average age was 54 years, nine patients were male, eight patients had a CPC of 1 to 3 on hospital discharge. There were a total of 182 measurements of BSAS, dEMG, and REE. There is improved correlation between REE and dEMG compared with REE and BSAS (0.24 (CI = 0.10 to 0.37) vs. 0.10 (CI = –0.04 to 0.24); P <0.001 vs. 0.14). Each increase in dEMG resulted in an increase of 14 kcal of energy expenditure (P = 0.003). Introduction Induced hypothermia is applied in the ICU and in the operating theater to reduce ischemia–reperfusion injury. It is thought that induced hypothermia may hamper the immune response and therefore carry the risk of acquiring or aggravating an infection. We investigated the eff ect of hypothermia on host response by comparing survivors of cardiac arrest in which body temperature was kept at either 33°C or 36°C. Conclusion Continuous dEMG power measured by Covidien BIS monitors is a more accurate and less subjective measure of shivering burden compared with the intermittent BSAS. Introducing dEMG into clinical practice may improve recognition of shivering, allow quantifi cation of the metabolic cost of shivering, and serve as a more reliable research tool for diagnostic and treatment strategies of shivering. Methods As a substudy to the Target Temperature Management trial [1] in which cardiac arrest patients admitted to the ICU were randomized to maintaining either 33°C (n = 11) or 36°C (n = 9) for 24 hours, blood was drawn at the start and end of the target temperature phase as well as after reaching normotemperature. Host response was measured via monocyte human leukocyte antigen-DR (HLA-DR) expression and via whole blood stimulation with TLR ligands lipopolysaccharide (LPS) and lipoteicoic acid (LTA) for 24 hours. Plasma levels of interleukin (IL)-1β, IL-1RA, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNFα), macrophage infl ammatory proteins (MIP)-1, monocyte chemotactic protein (MCP)- 1 and soluble CD40 ligand levels were determined with ELISA or Luminex. Statistics were by unpaired Mann–Whitney U tests. P501 Serum phosphate concentration during the rewarming period after deep hypothermic circulatory arrest H Hayami1, O Yamaguchi2, S Ishida3, Y Koide4, T Gotoh2 1Yokohama Municipal Citizen’s Hospital, Yokohama, Japan; 2Yokohama City University Hospital, Yokohama, Japan; 3Yokohama City University Medical Center, Yokohama, Japan; 4Hayama Heart Center, Hayama, Japan Critical Care 2014, 18(Suppl 1):P501 (doi: 10.1186/cc13691) p Results Forty-three patients were admitted in Group 1, 28 in Group 2. Of these, 42% in both groups were following out-of-hospital (OOH) VF arrests. Cooling was attempted in 88% and 82% of OOH VF patients respectively. For patients with either in-hospital or non-VF/ VT cardiac arrests, the numbers cooled were 16% and 12.5%. Cooling initiation within 1 hour increased from 27 to 50%. Achievement of a target temperature of 32 to 34°C within 4 hours of ROSC was 55% and 50% respectively. Target maintenance for 12 to 24 hours after ROSC increased 79% to 100%. Avoidance of hypothermia <31°C for 48 hours after ROSC improved 95% to 100%. Slow rewarming at 0.25 to 0.5°C/ hour to 37°C was achieved in 76% and 90%. Avoidance of temperature >37.2°C for 48 hours after ROSC increased 84 to 100%. Of the patients cooled, survival with good neurological outcome was achieved in 52% in Group 1 and 88% in Group 2. Factors involved in prolonged eff ect of neuromuscular blockade in therapeutic hypothermia Factors involved in prolonged eff ect of neuromuscular blockade in therapeutic hypothermia p yp H Tanaka, T Mochizuki, S Ode, S Ishimatsu St Lukes International Hospital, Akashi-Chou Chuo-Ku, Japan Critical Care 2014, 18(Suppl 1):P500 (doi: 10.1186/cc13690) p yp H Tanaka, T Mochizuki, S Ode, S Ishimatsu St Lukes International Hospital, Akashi-Chou Chuo-Ku, Japan Critical Care 2014, 18(Suppl 1):P500 (doi: 10.1186/cc13690) Introduction Therapeutic hypothermia (TH) following out-of-hospital cardiac arrest (OHCA) improves neurological outcomes in such patients. During TH, neuromuscular blockade is used to control shivering in the patient. In our hospital, we use vecuronium as a neuromuscular blocker. However, occasionally, prolonged eff ects of vecuronium delay accurate evaluation of patients’ neurological function or extubation. Unfortunately, the factors involved in the prolonged eff ect of vecuronium in TH remain unclear. Conclusion The energy burden of shivering is underestimated by standard nutritional formulas in patients undergoing TTM after cardiac arrest. Subclinical shivering is associated with increased energy expenditure. Clinical recognition occurs long after the increase in metabolic activity, and persists for a signifi cant period of time after treatment. These fi ndings should infl uence how shivering is monitored and treated during TTM. Methods We conducted a retrospective cohort study of patients managed with TH following OHCA at our institution from April 2010 to September 2013. We defi ned full-muscle reaction to train-of-four stimulation (TOF) as the end of eff ects of vecuronium. In this study, the time from the end of vecuronium administration to full-muscle reaction to TOF was evaluated as the outcome. We calculated the adjusted hazard ratio (HR) for the outcome using Cox regression analysis after adjustment for age, gender, albumin levels, estimated glomerular fi ltration rate, temperature at the end of vecuronium administration, and total amount of vecuronium per kilogram of body weight. Temperature management following cardiac arrest: introducing a protocol improves compliance with targets P Creber, G Talling, M Oram Cheltenham General Hospital, Cheltenham, UK Critical Care 2014, 18(Suppl 1):P499 (doi: 10.1186/cc13689) Results In total, 52 patients were evaluated in this study (86.5% male; average age, 55.2 years). The median time from the end of vecuronium administration to full-muscle reaction to TOF was 660 minutes (maximum: 10,363 minutes; minimum: 72 minutes). Blood albumin levels on admission decreased the time (adjusted HR: 2.31, 95% CI: 1.073 to 5.004; P <0.05). However, other factors had no signifi cant diff erence on the time. The amount of vecuronium per kilogram of weight had no signifi cant diff erences (adjusted HR: 1.073, 95% CI: 0.869 to 1.325; P = 0.511). Introduction We audited the achievement of therapeutic hypothermia (TH) before and after the introduction of a cooling protocol. Instituting TH is recommended following the return of spontaneous circulation (ROSC) for many patients who survive a cardiac arrest [1,2]. The key intervention may be the avoidance of hyperthermia rather than cooling [3]. Conclusion The time from the end of vecuronium administration to full-muscle reaction to TOF decreased in relation to blood albumin levels on admission. Other factors had no signifi cant diff erence on the outcome time. Taken together, the outcome time may be determined by albumin levels and not by the administered amount of vecuronium per kilogram of body weight. Based on this result, we suggest that blood albumin levels on admission should be taken into consideration for appropriate use of vecuronium in TH. g Methods We conducted a chart review of all patients admitted to the Department of Critical Care (DCC) at our hospital following cardiac arrest over 2 years in 2010 to 2012 (Group 1). We recorded compliance with key recommendations produced by the Royal College of Anaesthetists [4] although we defi ned post-ROSC hyperthermia as >37.2°C rather than >38°C. A TH protocol was designed and personnel in the emergency department and DCC educated as to its use. Recommended practice was the infusion of cold i.v. normal saline (1 to 2 l) followed by the use of an intravascular cooling device (Alsius CoolGard™). Data collection was then undertaken after introduction of the protocol for all patients admitted to the DCC following cardiac arrest in November 2012 to 2013 (Group 2). Shivering during targeted temperature management after cardiac arrest: a physiologic description Shivering during targeted temperature management after cardiac arrest: a physiologic description T May, DS Seder, GF Fraser, CF Hoover, RR Riker, BM McCrum Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P498 (doi: 10.1186/cc13688) T May, DS Seder, GF Fraser, CF Hoover, RR Riker, BM McCrum Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P498 (doi: 10.1186/cc13688) y p y Results HLA-DR expression was decreased compared with healthy controls, without diff erences between 33°C and 36°C. Following whole blood stimulation with LPS, TNFα and IL-6 production was lower after cardiac arrest compared with healthy controls. The 33°C and 36°C groups diff ered at baseline in TNFα levels after LPS whole blood stimulation. After 24 hours of temperature management, there was no diff erence in both TNFα and IL-6 production between the groups following TLR ligand stimulation. After cessation of temperature management only TNFα levels increased after LTA stimulation. Plasma levels of IL-1RA, IL-8 and IL-10 were decreased after cardiac arrest compared with healthy controls. In plasma levels of IL-1β, MIP-1 and soluble CD40 ligand there were no diff erences between the 33°C and 36°C groups at all time points. Apart from baseline diff erences, there were no diff erences between 33°C and 36°C in plasma levels of IL-1RA, IL-8, IL-10 and MCP-1.l Introduction Targeted temperature management (TTM) is used to treat hypoxic ischemic encephalopathy (HIE), elevated intracranial pressure, status epilepticus, and other brain injuries. Shivering complicates TTM, and the associated energy burden and its response to treatment are poorly understood. We describe the pattern of shivering and response to neuromuscular blockade in a series of patients undergoing TTM after cardiac arrest using continuous indirect calorimetry. Methods With IRB approval, we studied 16 patients undergoing TTM for HIE with continuous calorimetry during their treatment. The calorimeter measures inspired and expired oxygen and carbon dioxide, which are used in the Weir equation to calculate resting energy expenditure (REE). We excluded patients known to be actively seizing, requiring FiO2 >0.5, with early spasticity, or who did not shiver. All patients received counterwarming, moderate analgosedation, and bolused vecuronium in response to visible shivering, measured hourly by nurses using the Bedside Shivering Assessment Scale. Conclusion Cardiac arrest induces a decrease in proinfl ammatory response. Shivering during targeted temperature management after cardiac arrest: a physiologic description There were no diff erences in immune host response after S181 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Sixteen patients of average age 57 included 12 men. Twelve patients had CPC of 1 to 3 on hospital discharge. Sixteen shivering events were monitored with calorimetry among the 12 patients. The average rate of energy expenditure (without shivering) leading up to and following paralytic treatment was 1,425 kcal/hour (± 489 kcal/ hour) and 1,386 kcal/hour (± 235 kcal/hour). This was signifi cantly greater than the predicted basal energy expenditure (1,089 ± 222 kcal; P = 0.007 and P <0.001). Time from a change in baseline energy expenditure to recognition and treatment of clinical shivering was 57 (± 64) minutes, and from treatment with neuromuscular blockade to baseline energy expenditure was 30 (± 20) minutes. This accounts for a total diff erence of 15,223 kcal (± 10,997 kcal) before treatment and 7,113 kcal (± 3,706 kcal) after treatment for each shivering episode compared with baseline (P = 0.01 and P = 0.003). References References References 1. N Engl J Med 2002, 346:549-555. 2. Crit Care Med 2011, 39:1113-1125. 1. N Engl J Med 2002, 346:549-555. 1. N Engl J Med 2002, 346:549-555. Infl uence of baseline magnesium concentrations on shivering in therapeutic temperature modulation Infl uence of baseline magnesium concentrations on shivering in therapeutic temperature modulation KL Johnson HCMC, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P502 (doi: 10.1186/cc13692) g Results Twenty patients receiving TTM for TBI were evaluated (March to October 2013). One-half of the patients maintained targeted BSAS scores <1 for the full duration of TTM (n = 10 of 20). Serum Mg levels at the initiation of TTM were observed to negatively correlate with the level of shivering, as indicated by the BSAS scoring system (P = 0.02). See Figure 1. Introduction Therapeutic temperature modulation (TTM) is widely used in the care setting to improve outcomes of patients with traumatic brain injury (TBI). Through fever prevention, both oxygen utilization and caloric expenditure are reduced, so metabolic effi ciency can be maximized [1]. However, patient cooling is not without consequences and shivering is experienced by more than 70% of patients achieving TTM. Because shivering triggers an increase in metabolic demand, causing additional oxygen consumption and the promotion of catabolism, its prevention is ideal [2]. We set out to review the data surrounding the anti-shivering component of a normothermia protocol in the surgical ICU (SICU) of one Minnesota hospital. g Conclusion The literature suggests the positive impact of TTM on patient outcomes can be maximized with shivering prevention [2]. Current SICU practices provide a similar Mg loading dose for all patients, regardless of baseline Mg levels. In our observed patients, achieving a baseline serum Mg level >2 was associated with lower shivering scores throughout the TTM course. This supports the hypothesis that serum Mg concentrations prior to TTM are important predictors of shivering reduction, and suggests that loading doses of Mg should be tailored to the individual patient to achieve such levels. R f Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P502). Occurrence of SND was defi ned as neurological disarrangement such as irritability, confusion, or delirium within 48 hours after operation. Occurrence of SND was defi ned as neurological disarrangement such as irritability, confusion, or delirium within 48 hours after operation. as irritability, confusion, or delirium within 48 hours after operation. Results Durations of CPB, aortic clamp, and circulatory arrest were 194 ± 51, 136 ± 53, and 70 ± 17 minutes respectively. Nine patients underwent ACP (89 ± 26 minutes) and 11 patients underwent RCP (56 ± 25 minutes). Concentration of ALac, ScvO2, and P changed at 2, 4, 6, 10 hours after CPB as follows: ALac 4.0 ± 1.9, 5.1 ± 2.6, 5.3 ± 3.3, 4.0 ± 2.8 mmol/l, ScvO2 71 ± 6.7, 67 ± 10, 67 ± 9.7, 65 ± 11%, and P 2.9 ± 0.9, 2.4 ± 0.7, 2.2 ± 1.0, 1.8 ± 0.8 mg/dl, respectively. Serum P recovered to 2.6 ± 1.4 at 18 hours after CPB. The incidence of hypophosphatemia (<2.6 mg/dl) and SND in our series were 18/20 (90%) and 14/20(70%) respectively. There was a correlation between minimum P and time to confi rm M6 on the GCS (P = 0.508, Fisher r-to-z transformation), but no signifi cant correlation between SND. The mortality rate during the fi rst 28 days was 5% (1/20). y Conclusion Serum phosphate decreased dramatically during the rewarming period after deep hypothermic circulatory arrest. There was a moderate correlation between minimum phosphate concentration and time to confi rm M6 on the GCS. Monitoring phosphate concentration might predict neurological recovery after deep hypothermic circulatory arrest. Figure 1 (abstract P502). attention paid to the anti-shivering portion of the protocol. Serum magnesium (Mg) levels were assessed prior to initiation of TTM and Bedside Shivering Assessment Scale (BSAS) scores were collected. Serum phosphate concentration during the rewarming period after deep hypothermic circulatory arrest H Hayami1, O Yamaguchi2, S Ishida3, Y Koide4, T Gotoh2 1Yokohama Municipal Citizen’s Hospital, Yokohama, Japan; 2Yokohama City University Hospital, Yokohama, Japan; 3Yokohama City University Medical Center, Yokohama, Japan; 4Hayama Heart Center, Hayama, Japan Critical Care 2014, 18(Suppl 1):P501 (doi: 10.1186/cc13691) H Hayami1, O Yamaguchi2, S Ishida3, Y Koide4, T Gotoh2 1Yokohama Municipal Citizen’s Hospital, Yokohama, Japan; 2Yokohama City University Hospital, Yokohama, Japan; 3Yokohama City University Medical Center, Yokohama, Japan; 4Hayama Heart Center, Hayama, Japan Critical Care 2014, 18(Suppl 1):P501 (doi: 10.1186/cc13691) Introduction Short-term neuropsychologic dysfunction (SND) is often observed during the postoperative period of open total arch repair (TAR) under deep hypothermic circulatory arrest (DHCA). We supposed that there might be a large consumption of high-energy phosphates during the rewarming period, and this might be associated with awakening and SND. Due to diffi culty in measuring such high-energy phosphates at the bedside, we measured the serum phosphate concentration (P) as a substitute, and assessed its infl uence on awakening and postoperative SND. Conclusion The institution of a temperature management protocol improved compliance with recommended goals, both in achieving hypothermia and in the avoidance of hyperthermia. References Methods Twenty patients with a mean age of 68 ± 11 years who underwent open TAR under DHCA applied at a temperature of 20°C, with antegrade cerebral perfusion (ACP) or retrograde cerebral perfusion (RCP), were enrolled. Arterial blood gas lactate concentration (ALac), ScvO2, and P were measured at 2, 4, 6, and 10 hours after weaning from cardiopulmonary bypass (CPB). We also measured time to confi rm M6 on the Glasgow Coma Scale (GCS), and incidence of SND. 1. Nolan JP, et al.: ERC Guidelines 2010. Resuscitation 2010, 81:1219-1276. 2. HACA Study Group: N Engl J Med 2002, 346:549-556. 3. Nielsen N, et al.: N Engl J Med 2013, 369:2197-2206. 4. Nolan J: Implementation of therapeutic hypothermia. In Raising the Standard: A Compendium of Audit Recipes. 3rd edition. Edited by Colvin J, Peden C. Royal College of Anaesthetists; 2012:200-201. S182 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References Methods A retrospective review was conducted looking at SICU patients managed with a normothermia protocol, with particular 1. Badjatia N: Crit Care Med 2009, 37:S250-S255. 2. Badjatia N, et al.: Crit Care Med 2009, 37:1893-1896.
https://openalex.org/W4251331630	https://www.qeios.com/read/S8WJIZ/pdf	English	null	Alpha-tocopheryloxyacetic Acid	Definitions	2,020	cc-by	144	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Alpha-tocopheryloxyacetic Acid National Cancer Institute National Cancer Institute Source Source National Cancer Institute. Alpha-tocopheryloxyacetic Acid. NCI Thesaurus. Code C117234. Qeios ID: S8WJIZ · https://doi.org/10.32388/S8WJIZ Source National Cancer Institute. Alpha-tocopheryloxyacetic Acid. NCI Thesaurus. Code C117234. An orally bioavailable vitamin E derivative with potential antineoplastic and immunostimulating activities. Upon administration, alpha-tocopheryloxyacetic acid (alpha-TEA) induces tumor autophagy; the autophagosomes formed, which carry tumor associated antigens (TAAs), allow for increased cross-presentation of TAAs by professional antigen-presenting cells (APCs). This activates a T cell-mediated T helper type 1 (TH1) response, generates a cytotoxic T-lymphocyte (CTL) response against cancer cells, and reduces the frequency of regulatory T-cell (Treg) differentiation. In addition, alpha-TEA modulates the release of various cytokines and chemokines and induces tumor cell apoptosis. Altogether, this results in decreased tumor cell proliferation. Qeios ID: S8WJIZ · https://doi.org/10.32388/S8WJIZ 1/1
https://openalex.org/W4236820667	https://zenodo.org/records/2261471/files/article.pdf	English	null	The Mapping of Lake Chad	Geographical journal	1,908	public-domain	3,153	The Mapping of Lake Chad. The Mapping of Lake Chad. The Mapping of Lake Chad. Talbot and the other members of the expeditionconducted by Mr. Boyd Alexander. survey. Such, I am sure, is not the case. Until Mr. Boyd Alexander read his paper before the Royal Geographical Society a year ago--a paper of which the book and maps in ql~estion are merely a fuller development-I cannot recall any publication which gave as complete and truthful a map of Lake Chad as has resulted from the surveys of Mr. P. A. Talbot and the other members of the expeditionconducted by Mr. Boyd Alexander. survey. Such, I am sure, is not the case. Until Mr. Boyd Alexander read his paper before the Royal Geographical Society a year ago--a paper of which the book and maps in ql~estion are merely a fuller development-I cannot recall any publication which gave as complete and truthful a map of Lake Chad as has resulted from the surveys of Mr. P. A. Talbot and the other members of the expeditionconducted by Mr. Boyd Alexander. expedition by Boyd The Intelligence Division in England and in France, before the publication of the Boyd Alexander expedition surveys, hal no doubt realized the actual geography of Lake Chad; but that the French (for whom A. K. claims the principal merit of the existing results) have been in no hurry to make public the results of their researches is evident. I have before me now for review a most interesting work written by M. Auguste Chevalier, ' L'Afrique Centrale Francaise : Mission Chari-Lac Tchad, 1902-190~.' Tt~e publication date of this book is 1908; nevertheless, M. Chevalier and his colleagues still issue a map containing a Lake Chad of the old design, with the dotterl lines round much of the margin and showing the familiar but now incorrect version of the lake-a continuous sheet of water merely studded with islands (their own surveys having been executed farther south and east). expedition by Boyd The Intelligence Division in England and in France, before the publication of the Boyd Alexander expedition surveys, hal no doubt realized the actual geography of Lake Chad; but that the French (for whom A. K. claims the principal merit of the existing results) have been in no hurry to make public the results of their researches is evident. I have before me now for review a most interesting work written by M. The Mapping of Lake Chad. The Mapping of Lake Chad. The Mapping of Lake Chad. pp g THE letter which "A. K." has addressed to the Geographical Journal on the subject of my review of Mr. Boyd Alexander's work(dealing, amongst other things, with his expedition and survey of Lake Chad) does not seem to me to .be altogether fair, either to the reviewer or reviewed. A. K.'s initials may indicate one who is at the fountain-head of official geographical information. If that is so, he has been misled, I think, by his own facilities into imagining that the general public (from whose standpoint I reviewed Boyd Alexander's book) has been equally well informed on the subject of the progress of Lake Chad exploration and survey. pp g THE letter which "A. K." has addressed to the Geographical Journal on the subject of my review of Mr. Boyd Alexander's work(dealing, amongst other things, with his expedition and survey of Lake Chad) does not seem to me to .be altogether fair, either to the reviewer or reviewed. A. K.'s initials may indicate one who is at the fountain-head of official geographical information. If that is so, he has been misled, I think, by his own facilities into imagining that the general public (from whose standpoint I reviewed Boyd Alexander's book) has been equally well informed on the subject of the progress of Lake Chad exploration and survey. pp g THE letter which "A. K." has addressed to the Geographical Journal on the subject of my review of Mr. Boyd Alexander's work(dealing, amongst other things, with his expedition and survey of Lake Chad) does not seem to me to .be altogether fair, either to the reviewer or reviewed. A. K.'s initials may indicate one who is at the fountain-head of official geographical information. If that is so, he has been misled, I think, by his own facilities into imagining that the general public (from whose standpoint I reviewed Boyd Alexander's book) has been equally well informed on the subject of the progress of Lake Chad exploration and survey. survey. Such, I am sure, is not the case. Until Mr. Boyd Alexander read his paper before the Royal Geographical Society a year ago--a paper of which the book and maps in ql~estion are merely a fuller development-I cannot recall any publication which gave as complete and truthful a map of Lake Chad as has resulted from the surveys of Mr. P. A. The Mapping of Lake Chad. The Mapping of Lake Chad. The Mapping of Lake Chad. Auguste Chevalier, ' L'Afrique Centrale Francaise : Mission Chari-Lac Tchad, 1902-190~.' Tt~e publication date of this book is 1908; nevertheless, M. Chevalier and his colleagues still issue a map containing a Lake Chad of the old design, with the dotterl lines round much of the margin and showing the familiar but now incorrect version of the lake-a continuous sheet of water merely studded with islands (their own surveys having been executed farther south and east). expedition by Boyd The Intelligence Division in England and in France, before the publication of the Boyd Alexander expedition surveys, hal no doubt realized the actual geography of Lake Chad; but that the French (for whom A. K. claims the principal merit of the existing results) have been in no hurry to make public the results of their researches is evident. I have before me now for review a most interesting work written by M. Auguste Chevalier, ' L'Afrique Centrale Francaise : Mission Chari-Lac Tchad, 1902-190~.' Tt~e publication date of this book is 1908; nevertheless, M. Chevalier and his colleagues still issue a map containing a Lake Chad of the old design, with the dotterl lines round much of the margin and showing the familiar but now incorrect version of the lake-a continuous sheet of water merely studded with islands (their own surveys having been executed farther south and east). ) I believe, also, that I am correct in my main thesis, which was, that prior to the publication of the results of the Boyd-Alexander Expedition Lake Chad-so far as published documents were concerned--was in parts entitled to a dotted outline only, however precise may have been those surveys of portions of the shores and islands alluded to by A. K. ) I believe, also, that I am correct in my main thesis, which was, that prior to the publication of the results of the Boyd-Alexander Expedition Lake Chad-so far as published documents were concerned--was in parts entitled to a dotted outline only, however precise may have been those surveys of portions of the shores and islands alluded to by A. K. The Mapping of Lake Chad Author(s): H. H. Johnston and Boyd Alexander Source: The Geographical Journal, Vol. 31, No. 4 (Apr., 1908), pp. 452-453 Published by: The Royal Geographical Society (with the Institute of British Geographers) Stable URL: http://www.jstor.org/stable/1777864 . Accessed: 15/01/2015 03:53 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. The Royal Geographical Society (with the Institute of British Geographers) is collaborating with JSTOR to digitize, preserve and extend access to The Geographical Journal. This content downloaded from 128.235.251.160 on Thu, 15 Jan 2015 03:53:48 AM All use subject to JSTOR Terms and Conditions 452 452 452 CORRESPONDENC CORRESPONDENC CORRESPONDENC out a book only some four years ago under the title ' The Native Tribes of South- East Australia' (Macmillan, 1904). out a book only some four years ago under the title ' The Native Tribes of South- East Australia' (Macmillan, 1904). out a book only some four years ago under the title ' The Native Tribes of South- East Australia' (Macmillan, 1904). Captain 0. N. Conlan. Captain 0. N. Conlan. Captain 0. N. Conlan. p The Society has lost a Fellow of thirty years' standing in the person of Captain George Nugent Conlan, Marine Superintendent of the Pacific Steam Navigation Company. Captain Conlan had been in the service of the company for forty-seven years, and his great practical experience as a sailor was highly valued by his employers. He was much interested in the study of ocean currents, and contributed to its furtherance during his many voyages by frequently throwing over bottle- papers, some of which were recovered mally miles from the spots at which they had been dropped. His death occurred at Liverpool towards the end of last year. p The Society has lost a Fellow of thirty years' standing in the person of Captain George Nugent Conlan, Marine Superintendent of the Pacific Steam Navigation Company. Captain Conlan had been in the service of the company for forty-seven years, and his great practical experience as a sailor was highly valued by his employers. He was much interested in the study of ocean currents, and contributed to its furtherance during his many voyages by frequently throwing over bottle- papers, some of which were recovered mally miles from the spots at which they had been dropped. His death occurred at Liverpool towards the end of last year. p The Society has lost a Fellow of thirty years' standing in the person of Captain George Nugent Conlan, Marine Superintendent of the Pacific Steam Navigation Company. Captain Conlan had been in the service of the company for forty-seven years, and his great practical experience as a sailor was highly valued by his employers. He was much interested in the study of ocean currents, and contributed to its furtherance during his many voyages by frequently throwing over bottle- papers, some of which were recovered mally miles from the spots at which they had been dropped. His death occurred at Liverpool towards the end of last year. This content downloaded from 128.235.251.160 on Thu, 15 Jan 2015 03:53:48 AM All use subject to JSTOR Terms and Conditions The Mapping of Lake Chad. The Mapping of Lake Chad. The Mapping of Lake Chad. ) I believe, also, that I am correct in my main thesis, which was, that prior to the publication of the results of the Boyd-Alexander Expedition Lake Chad-so far as published documents were concerned--was in parts entitled to a dotted outline only, however precise may have been those surveys of portions of the shores and islands alluded to by A. K. H. H. JOHNSTO H. H. JOHNSTO H. H. JOHNSTO 453 453 CORRESPONDENCE. CORRESPONDENCE. I am afraid that some remarks which I have made in my book, 'From the Niger to the Nile,' regarding my determination of the size of Lake Chad in com- parison with its area as shown on former maps, have misled my kind reviewer to claim too much for the work of our expeditionin that region. I am afraid that some remarks which I have made in my book, 'From the Niger to the Nile,' regarding my determination of the size of Lake Chad in com- parison with its area as shown on former maps, have misled my kind reviewer to claim too much for the work of our expeditionin that region. p g When we were engaged upon the exploration of the lake (December, 1904, to May, 1905), of course there was no complete map more recent than Barth's, which we reconstructed, reducing the distance across the north from some 60 to 30 miles, and that across the south from 90 to 45 miles. The Mapping of Lake Chad. The Mapping of Lake Chad. The Mapping of Lake Chad. On my return home (February, 1907), I found that the French Geographical Society had published a map the year before, establishing pretty generally these facts, and I can only say now that I am sorry that I have not made this acknowledgment to them before, but my attention was engaged on the fact that, although the Flench had more or less determined the size and shape, it was my report sent home from the Shari and published in the Geograpchical Journal for November, 1905, which first established the separation of the lake into two basins; whereas Major Lenfant's map, published at the end of May, 1904, showed a clear waterway between the two parts at least 20 miles wide, so that the subsequent division by the French of the lake into two basins (though they do not go so far as I in claiming that there is no communication) was published a year after I had placed my information in the hands of our Geographica Society. p g When we were engaged upon the exploration of the lake (December, 1904, to May, 1905), of course there was no complete map more recent than Barth's, which we reconstructed, reducing the distance across the north from some 60 to 30 miles, and that across the south from 90 to 45 miles. On my return home (February, 1907), I found that the French Geographical Society had published a map the year before, establishing pretty generally these facts, and I can only say now that I am sorry that I have not made this acknowledgment to them before, but my attention was engaged on the fact that, although the Flench had more or less determined the size and shape, it was my report sent home from the Shari and published in the Geograpchical Journal for November, 1905, which first established the separation of the lake into two basins; whereas Major Lenfant's map, published at the end of May, 1904, showed a clear waterway between the two parts at least 20 miles wide, so that the subsequent division by the French of the lake into two basins (though they do not go so far as I in claiming that there is no communication) was published a year after I had placed my information in the hands of our Geographica Society. Society. The Mapping of Lake Chad. The Mapping of Lake Chad. The Mapping of Lake Chad. While making this claim for the work of the Alexander-Gosling expedition, and in consideration of the tone of "A. K.'s " letter, I think it would be as well to state what other new work was done by us uponthe lake. Society. While making this claim for the work of the Alexander-Gosling expedition, and in consideration of the tone of "A. K.'s " letter, I think it would be as well to state what other new work was done by us uponthe lake. There was- There was- There was- There was- (a) The careful mapping of the whole of the northern portion of the lake lying between the Yo mouth and Kaddai. This was published in the Geographica Journal for March, 1905. (a) The careful mapping of the whole of the northern portion of the lake lying between the Yo mouth and Kaddai. This was published in the Geographica Journal for March, 1905. , (b) Fixing by latitude the position of the Yo mouth, and also of two points on the east coast of the lake. , (b) Fixing by latitude the position of the Yo mouth, and also of two points on the east coast of the lake. (c) The astronomical determining of the position of Kaddai. (c) The astronomical determining of the position of Kaddai. g p (d) Five traverses of the lake, with the mapping and naming of many islands, and a record of soundings. g p (d) Five traverses of the lake, with the mapping and naming of many islands, and a record of soundings. g In closing, [ think I might be pardoned for remarking that when I remember those six months' work upon the lake that went to produce the map recording these things, not to mention the unique collections of birds and fish (the latter raising speculations of important geographical interest), and then read "A. K.'s" final dismissal of our labours as merely " some additional routes," I cannot help feeling that a little more generosity would have better become the modesty of his signature. g In closing, [ think I might be pardoned for remarking that when I remember those six months' work upon the lake that went to produce the map recording these things, not to mention the unique collections of birds and fish (the latter raising speculations of important geographical interest), and then read "A. This content downloaded from 128.235.251.160 on Thu, 15 Jan 2015 03:53:48 AM All use subject to JSTOR Terms and Conditions The Mapping of Lake Chad. The Mapping of Lake Chad. The Mapping of Lake Chad. K.'s" final dismissal of our labours as merely " some additional routes," I cannot help feeling that a little more generosity would have better become the modesty of his signature. y g BOYD ATERXANDE y g BOYD ATERXANDE 'Life and Voyages of Joseph Wiggins, F.R.Gh.S 'Life and Voyages of Joseph Wiggins, F.R.Gh.S I wish to draw attention to two or three mistakes in the review of the above book which appeared in the February issue of the GeographicalJournal~ I wish to draw attention to two or three mistakes in the review of the above book which appeared in the February issue of the GeographicalJournal~ pp y Geographical The writer, "C. R. M." (whose identity is evident), states, with reference to the first voyage of Captain Viggins, that he " succeeded in raising funds to buy and fit out the steamer Diana?. The captain chartered, fitted, and manned the Diana entirely at his own expense, as set forth plainly on p. 23 of the book. He neither asked fur, nor received, a penny towards the expense. He drew upon his hard- earned savings, and was under obligation to no one. pp y Geographical The writer, "C. R. M." (whose identity is evident), states, with reference to the first voyage of Captain Viggins, that he " succeeded in raising funds to buy and fit out the steamer Diana?. The captain chartered, fitted, and manned the Diana entirely at his own expense, as set forth plainly on p. 23 of the book. He neither asked fur, nor received, a penny towards the expense. He drew upon his hard- earned savings, and was under obligation to no one. g , g Further on it is asserted that the Phoenix was "hoplessly stranded." This is g , g Further on it is asserted that the Phoenix was "hoplessly stranded." This is
https://openalex.org/W2004041454	https://europepmc.org/articles/pmc3692527?pdf=render	English	null	Norcantharidin Inhibits Renal Interstitial Fibrosis by Blocking the Tubular Epithelial-Mesenchymal Transition	PloS one	2,013	cc-by	8,954	Abstract Epithelial–mesenchymal transition (EMT) is thought to contribute to the progression of renal tubulointerstitial fibrosis. Norcantharidin (NCTD) is a promising agent for inhibiting renal interstitial fibrosis. However, the molecular mechanisms of NCTD are unclear. In this study, a unilateral ureteral obstruction (UUO) rat model was established and treated with intraperitoneal NCTD (0.1 mg/kg/day). The UUO rats treated with NCTD showed a reduction in obstruction-induced upregulation of a-SMA and downregulation of E-cadherin in the rat kidney (P,0.05). Human renal proximal tubule cell lines (HK-2) stimulated with TGF-b1 were treated with different concentrations of NCTD. HK-2 cells stimulated by TGF-b1 in vitro led to downregulation of E-cadherin and increased de novo expression of a-SMA; co-treatment with NCTD attenuated all of these changes (P,0.05). NCTD reduced TGF-b1-induced expression and phosphorylation of Smad2/3 and downregulated the expression of Snail1 (P,0.05). These results suggest that NCTD antagonizes tubular EMT by inhibiting the Smad pathway. NCTD may play a critical role in preserving the normal epithelial phenotype and modulating tubular EMT. Editor: Emmanuel A Burdmann, University of Sao Paulo Medical School, Brazil Editor: Emmanuel A Burdmann, University of Sao Paulo Medical School, Brazil Received October 4, 2012; Accepted May 5, 2013; Published June 25, 2013 Received October 4, 2012; Accepted May 5, 2013; Published June 25, 2013 Copyright: 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the National Natural Science Foundation of China (Grant No.81100486), the Natural Science Foundation of Hunan Province of China (Grant No.10JJ2011), and the scientific project of the Research Center of Metabolic Syndrome in Central South University of China (Grant No.DY- 2008-02-03). The corresponding author and all the above grand funders played a role in designing and doing the experiment and writing the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yachi20101213@gmail.com Competing Interests: The authors have declared that no competing interests exist. * E-mail: yachi20101213@gmail.com . These authors contributed equally to this work. . These authors contributed equally to this work. challenged by two different groups [11,12], which conflicts with previous conclusions of Iwano et al [13]. Abstract Using cell lineage tracking techniques to track renal epithelial cells, the authors of those two groups found no evidence to suggest that epithelial cells migrate outside of the tubular basement membrane and differen- tiate into interstitial myofibroblasts in vivo. This conflicting results may be due to technical differences, underlining the complexity of the EMT process. The degree to which the EMT process contributes to kidney fibrosis remains a matter of intense debate and is likely to be context-dependent or an adaptive response to a hostile environment, chronic stress or injury. Of the numerous cytokines that regulate EMT, TGF-b1 is a key mediator that promotes myofibroblast development by inducing expression of the a-SMA phenotype. TGF-b1 is considered a key mediator in renal fibrosis and induces renal scarring by activating the downstream Smad signaling pathway [15]. Smad2 and Smad3 are critical downstream mediators responsible for the biological effects of TGF-b1. Snail1 is a key transcription factor that promotes EMT, fibroblast migration and renal fibrosis [16,17]. Manipulating downstream TGF-b1 signaling represents a viable therapeutic target for reversing EMT and renal interstitial fibrosis. We hypothesized that the effects of NCTD in ameliorating renal interstitial fibrosis may be related to inhibition or reversal of tubular EMT. Norcantharidin Inhibits Renal Interstitial Fibrosis by Blocking the Tubular Epithelial-Mesenchymal Transition Ying Li1., Yan Sun2, Fuyou Liu1., Lin Sun1, Jun Li1, Shaobin Duan1, Hong Liu1, Youming Peng1, Li Xiao1, Yuping Liu1, Yiyun Xi1, Yanhua You1, Hua Li1, Min Wang3, Shuai Wang1, Tao Hou1 1 Division of Nephrology, Second Xiangya Hospital, Central South University, Changsha, P.R. China, 2 Division of Nephrology, The first affiliated hospital, XinJiang Medical University,Uramuq, P.R. China, 3 Department of Clinical Laboratory, Second Xiangya Hospital, Central South University, P.R. China Introduction Norcantharidin (NCTD), an important derivative of canthari- din, is a specific protein phosphatase inhibitor with anti-cancer, inflammation modulation, anti-fibrosis, anti-oxidation and leuko- cytic effects [1]. Recently, our research showed that NCTD significantly alleviated renal interstitial fibrosis in rat models of protein overload nephropathy [2] and diabetic nephropathy [3]. NCTD inhibited the proliferation and expression of fibronectin (FN) in renal tubular epithelial cells stimulated by albumin in vitro [4] and decreased the expression of extracellular matrix (ECM) and TGF-b1 in HK-2 cells stimulated with high glucose [5]. As a result, NCTD is considered a promising antifibrotic drug [6]. However, the molecular mechanisms by which NCTD contributes to the inhibition of tubulointerstitial fibrosis are unclear Renal interstitial fibrosis is the final common pathway through which chronic kidney diseases progress to end-stage renal failure [7]. The process of renal interstitial fibrosis includes tubular atrophy, myofibroblast accumulation and ECM deposition. Epithelial-mesenchymal transition (EMT) is an important pathway in myofibroblast production and is a key mechanism in the pathogenesis and progression of renal interstitial fibrosis [8–10]. In EMT, the loss of epithelial cell adhesion molecules, such as epithelial (E)-cadherin, are replaced by the mesenchymal marker alpha-smooth muscle actin (a-SMA)[10,14]. However, the role of EMT in renal interstitial fibrosis in vivo has recently been In this study, we examined the effects of NCTD on renal tubular EMT in a rat model with unilateral ureteral obstruction PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66356 1 June 2013 \| Volume 8 \| Issue 6 \| e66356 NCTD Inhibits Tubular EMT Abcam, Inc., London, UK), E-cadherin (1:50, Santa Cruz, Inc., CA, USA) and TGF-b1 (1:25, Santa Cruz, Inc., CA, USA). After rinsing, the sections were incubated with HRP-labeled biotinylated goat anti-rabbit IgG at 37uC for 30 min (Abcam, Inc., London, UK) and processed using an alkaline phosphatase-streptavidin- biotin immunoperoxidase method (Maixin Biotechnological Com- pany, Shenzhen, China). The sections incubated with non- immune rabbit serum rather than the primary antibodies were used as negative controls. Nuclei were counterstained slightly with hematoxylin. All images were semi-quantitatively analyzed using the computer- assisted image-Pro Plus software, version 6.0 (Media Cybernetics, Bethesda, MD, USA). To quantitate the the positive expression area of a-SMA, E-cadherin and TGF-b1 in the tubulointerstitial compartment, 50 fields consecutively selected in the cortical areas of the kidney were examined at a magnification of 6100. Fields containing glomerular and large arteries were excluded. Western blot Analysis y The whole kidney tissues and HK-2 cells were lysed in RIPA lysis buffer and protease inhibitor PMSF at 4uC. The lysate was clarified by centrifugation at 12,000 rpm for 30 min at 4uC, and the supernatant was used for the experiments. Nuclear protein was extracted using a nuclear and cytoplasmic protein extraction kit according to the manufacturer’s instructions. The protein concentration was determined using the Bradford method. To denature the extracted proteins and expose the domain typically recognized by antibodies, a loading buffer with the anionic denaturing detergent sodium dodecyl sulfate (SDS) was used. The mixture was boiled at 95uC for 5 min. Protein was loaded on a SDS polyacrylamide gel for electrophoresis and transferred to PVDF membrane using the transfer buffer. The membrane was incubated for 2 h at room temperature under agitation with 2% BSA to prevent nonspecific background binding. The immuno- blots from the rat kidney were incubated overnight at 4uC with primary antibodies, including rabbit anti-rat a-SMA, E-cadherin Cell culture HK-2 cells from American Type Culture Collection (ATCC) were grown in DMEM supplemented with 10% FBS at 37uC in humidified 5% CO2 in air. The cells were subcultured at 80% confluence using 0.05% trypsin with 0.02% EDTA. The cells were diluted to 16106 cells/ml by mixing with medium and transferred to 6-well plates. The experiment was performed on cells at 80% confluence. The HK-2 cells were divided into the following five treatment groups: control, TGF-b1 5 ng/ml + NCTD 0 mg/ml, TGF-b1 5 ng/ml + NCTD 0.5 mg/ml, TGF-b1 5 ng/ml + NCTD 1 mg/ml and TGF-b1 5 ng/ml + NCTD 2.5 mg/ml. Ethics statement All experiments were performed in accordance with the animal experimental guidelines issued by the Animal Care and Use Committee at Xiangya Medical School of Central South University. This study was approved by the Animal Care and Use Committee of the 2nd Xiangya Hospital (protocol approval number 2008-S 062). Introduction The analysis was made by the authors without knowledge about clinical data. Values were expressed as the average optical density (AOD), which is defined as the ratio of the positive target optical density and the positive area percentage (IOD/Area). (UUO) and HK-2 cells. We explored the mechanisms by which NCTD inhibits renal tubular EMT and renal interstitial fibrosis. (UUO) and HK-2 cells. We explored the mechanisms by which NCTD inhibits renal tubular EMT and renal interstitial fibrosis. Our results suggest that NCTD may play a critical role in preserving the normal epithelial phenotype and modulating tubular EMT by inhibiting the TGF-b1/Smad pathway. Immunofluorescence staining g Cells grown on coverslips in 6-well dishes were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min at room temperature, followed by permeabilization with 0.1% TritonX-100 in PBS for 5 min. The cells were rinsed in PBS (pH 7.4) three times for 5 min. After blocking in 5% BSA and 1% TritonX-100/PBS for 30 min at room temperature, the cells on the coverslips were incubated with primary rabbit anti-human a- SMA polyclonal antibody (1:100, Abcam, Inc., London, UK) and mouse anti-human E-cadherin monoclonal antibody (1:100, Santa Cruz, Inc., California, USA) for 1 h at room temperature. After rinsing with PBS three times for 10 min with agitation, the cells were incubated with AlexaFluora 568 (red) or AlexaFluora 488 (green) conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature. The nuclei were counterstained with 49,69-diamidino-2-phenylindole dihy- drochloride. All of the images were semi-quantitatively analyzed, and the slides were mounted and viewed using a fluorescent microscope. For analysis of a-SMA and E-cadherin expression, fluorescent intensity was quantified by measuring intensity in the cells using Image-Pro Plus software, version 6.0 (Media Cyber- netics, Bethesda, MD, USA). Data were analyzed from three sections of one sample, and there were six samples for each group. Animal model Healthy 8-week-old male Sprague-Dawley rats weighing 200– 220 g (SPF, certification number62003115403) were purchased from the Animal Department at Xiangya School of Medicine, Central South University. At harvest, each rat was anesthetized by intraperitoneal injection with 10% chloralic hydras. A unilateral ureteral obstruction (UUO) model was established by surgically opening the dorsal surface of the rat with a left lateral incision. The left ureter was ligated with 4–0 silk sutures at two points and severed between the ligatures to prevent a retrograde urinary tract infection after routine skin preparation and sterilization. The incision was closed in layers. Sham-operated rats were subjected to the same anesthesia, incision and closure, but the left ureter was manipulated without ligation [18]. The total time spent in surgery was 10–15 min per rat. Respiratory rhythm and frequency were monitored during the procedure. UUO rats were treated with intraperitoneal NCTD (0.1 mg/kg/day) (n = 6). The rats in the sham group and UUO rats that were not treated with NCTD were administered with equal volumes of sterile saline (n = 6 in each group). The rats were sacrificed 14 days after surgery by exsanguination through cardiac puncture under general anesthe- sia. The abdomen of the rat was opened and both kidneys were removed into ice-cold saline. Whole blood samples were obtained from the abdominal aorta of each rat for serum creatinine measurement. June 2013 \| Volume 8 \| Issue 6 \| e66356 Results Trizol RNA extraction was used to extract the total RNA from the renal tissue. The total RNA, weighing 2.5 mg, was used for cDNA synthesis according to the instructions in the Revert Aid First Strand cDNA Synthesis Kit (Fermentas). Gene sequences were verified in Genebank, and primers were designed according to primer design principles using Primer 5 software. The primer sequences are shown in Table 1. PCR amplification of the various products was performed under the following reaction conditions: a) a-SMA mRNA: pre-degeneration at 95uC for 5 min, degener- ation at 94uC for 45 sec, annealing at 56uC for 45 sec and extension at 72uC for 45 sec, with 30 cycles total and a final extension at 72uC extension for 10 seconds terminated at 4uC; b) E-cadherin mRNA: pre-degeneration at 95uC for 3 min, degen- eration at 94uC for 30 sec, annealing at 60uC for 30 sec and extension at 72uC for 30 sec, with 34 cycles in total and a final extension at 72uC for 10 sec terminated at 4uC; for 30 sec, annealing at 58uC for 30 sec and extension at 72uC for 30 sec, with 30 cycles total and a final extension at 72uC extension for 10 seconds terminated at 4uC; and d) b-actin mRNA: pre- degeneration at 95uC for 5 min, degeneration at 94uC for 30 sec, annealing at 58uC for 30 sec and extension at 72uC for 30 sec, with 25 cycles total and a final extension at 72uC extension for 7 seconds terminated at 4uC. NCTD regulates the expression of a-SMA, E-cadherin and TGF-b1 in the kidneys of UUO rats Immunohistochemistry staining Paraffin-embedded whole kidney tissue sections were dewaxed, hydrated, treated with 5 mmol/L levamisole for blocking endog- enous alkaline phosphatase and incubated with blocking serum for 30 min at room temperature to reduce nonspecific background staining. Sections were rehydrated in PBS with 0.1% BSA for 15 min before the appropriate blocking serum was added for an additional 15 min. To detect the expression of a-SMA, E-cadherin and TGF-b1 proteins in the kidneys, sections (4 mm thick) were incubated with rabbit anti-rat a-SMA polyclonal antibody (1:50, June 2013 \| Volume 8 \| Issue 6 \| e66356 PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66356 2 NCTD Inhibits Tubular EMT and TGF-b1 (1:3000). In the immunoblots from the HK-2 cells, E- cadherin antibody (1:3000), a-SMA antibody (1:4000), rabbit anti- human Smad2/3 polyclonal antibody (1:2000, Cell Signaling, Inc., Danvers, MA, USA) and P-Smad2/3 polyclonal antibody (1:2000, Santa Cruz, Inc., CA, USA) and rat anti-human Snail 1 polyclonal antibody (1:1500, Cell Signaling, Inc., Danvers, MA, USA) were used. After several washes, the membranes were incubated with agitation for 1 h at room temperature with goat anti-rabbit horseradish peroxidase-conjugated secondary anti- body. Chemiluminescence was used to detect specific protein bands using ECL detection kits, and the results were recorded on radiograph film. Optimally exposed autoradiographs were digitally scanned and analyzed using a Kodak high-sensitivity imaging system (Carestream Health, Inc, USA). The intensity of the identified bands was quantified by densitometry. Results were normalized to b-actin and expressed as arbitrary densitometry units. and TGF-b1 (1:3000). In the immunoblots from the HK-2 cells, E- cadherin antibody (1:3000), a-SMA antibody (1:4000), rabbit anti- human Smad2/3 polyclonal antibody (1:2000, Cell Signaling, Inc., Danvers, MA, USA) and P-Smad2/3 polyclonal antibody (1:2000, Santa Cruz, Inc., CA, USA) and rat anti-human Snail 1 polyclonal antibody (1:1500, Cell Signaling, Inc., Danvers, MA, USA) were used. After several washes, the membranes were incubated with agitation for 1 h at room temperature with goat anti-rabbit horseradish peroxidase-conjugated secondary anti- body. Chemiluminescence was used to detect specific protein bands using ECL detection kits, and the results were recorded on radiograph film. Optimally exposed autoradiographs were digitally scanned and analyzed using a Kodak high-sensitivity imaging system (Carestream Health, Inc, USA). The intensity of the identified bands was quantified by densitometry. Results were normalized to b-actin and expressed as arbitrary densitometry units. gel was visualized and transillumination under UV light. Statistical methods SPSS 13.0 software was used for statistical analysis. The results are presented as Means 6 SD. Measurement data were analyzed using one-way analysis of variance (ANOVA), and the LSD t-test was used for multiple comparisons of two sample means. P,0.05 was considered statistically significant. Immunohistochemistry staining The images were obtained using a gel imaging system for pictures and Labwork 4.0 photography analysis software (Gene Company Limited, Hongkong, China) to quantify gray value of the bands observed. Light absorbance values were determined using gel image analysis software, and the semi-quantitative value was presented as a ratio between the light absorbance of each index and the light absorbance of b-actin. SYBR green, a fluorescent dye that only binds to double-stranded DNA, was used as the fluorescent probe. Fluorescence was emitted proportional to the amount of cDNA amplified by RT-PCR. NCTD regulates the expression of a-SMA, E-cadherin and TGF-b1 in the kidneys of UUO rats b1 y We investigated the effects of NCTD on the expression of a- SMA, E-cadherin and TGF-b1 in obstructive nephropathy, a well characterized and widely used model of renal interstitial fibrosis. The expression of a-SMA and TGF-b1 mRNA in the UUO group was significantly increased, while the expression of E-cadherin mRNA decreased (P,0.05). However, downregulation of a-SMA and TGF-b1 mRNA and upregulation of E-cadherin mRNA were observed in the NCTD group compared with the UUO group. The difference between the two groups was significant (P,0.05, Fig. 1a and b). Similarly, a-SMA and TGF-b1 protein expression in the obstructed kidney significantly increased and E-cadherin protein decreased based on Western blot analyses of whole kidney lysates (P,0.05). a-SMA and TGF-b1 protein expression mark- edly decreased with NCTD treatment, while E-cadherin protein increased. All the differences between these three groups had statistical significance (P,0.05, Fig. 1c and d). Immunohistochem- istry staining showed that a-SMA protein expression in the renal tubular epithelial cells and interstitium was significantly elevated in the rat kidney after obstructive injury, while expression of the E- cadherin protein decreased. TGF-b1 protein expression in renal tubular epithelial cells, stromal cells and infiltrated inflammatory cells also increased. NCTD markedly reduced the changes in these markers (Fig. 1e). These data collectively indicate that NCTD is a potent agent for reversing renal tubular EMT in UUO rats. In HK-2 cells, E-cadherin, a-SMA, and Snail 1 mRNA expression were detected by RT-PCR. PCR was followed by 40 cycles of 10 sec at 95uC, 60 sec at 60uC, 60 sec at 72uC and one cycle of 10 min at 72uC. The housekeeping gene b-actin served as a reference and was co-amplified with E-cadherin, a-SMA, and Snail 1. The primer sequences for the HK-2 cells are shown in Table 2. All of the oligonucleotide primers were designed by the Ying Jun Limited Company (Shanghai, China). After PCR amplification, 8 ml of each amplification product was electropho- resed on a 2% agarose gel at 100 V for 40 min, then the agarose TGF-b1 increases a-SMA expression in HK-2 cells We examined the suitable dose and length of exposure of TGF- b1 on a-SMA expression in HK-2 cells. a-SMA expression in HK- 2 cells following treatment with different concentrations of TGF- Table 1. RT-PCR primers for the rat kidney. Gene Upstream sequence Downstream sequence a-SMA 5’-TCC TGA CCC TGA AGT ATC CG-3’ 5’-TCT CCA GAG TCC AGCA CAA T-3’ E-cadherin 5’-GTC AAA CGG CAT CTA AAG C-3’ 5’-CAA AGA CCT CCT GGA TAA ACT-3’ TGF-b1 5’-CCG CAA CAA CGC AAT C-3’ 5’-ATG AGG AGC AGG AAG GGT -3’ b-actin 5’-ATG AGG AGC AGG AAG GGT -3’ 5’-ACCCAGGAAGGAAGGCT -3’ doi:10.1371/journal.pone.0066356.t001 Table 1. RT-PCR primers for the rat kidney. Table 1. RT-PCR primers for the rat kidney. Downstream sequence June 2013 \| Volume 8 \| Issue 6 \| e66356 3 NCTD Inhibits Tubular EMT Table 2. RT-PCR primers for the HK-2 cells. Gene Upstream sequence Downstream sequence a-SMA 5’CTG TTC CAG CCA TCC TTC ATC 3’ 5’ GCT GGC TCA AGT CAA AGT CC 3’ E-cadherin 5’TTG CAA ATT CCT GCC ATT C 3’ 5’ GCT GGC TCA AGT CAA AGT CC 3’ Smad2 5’ TTGCTGAGTGCCTAAGTGAT 3’ 5’ACAGACTGAGCCAGAGAGC 3’ Smad3 5’GGCTTTGAGGCTGTCTACCA 3’ 5’CATCTGGGTGAGGACCTTGT 3’ Snail 1 5’GAA AGG CCT TCA ACT GCA AA 3’ 5’ GTA CTT GCG CTC AGG AGG AG 3’ b-actin 5’ ACTCTTCCAGCCTTCCTTCC3’ 5’ GTACTTGCGCTCAGGAGGAG 3’ doi:10.1371/journal.pone.0066356.t002 b1 (0, 1.25, 2.5 and 5 ng/ml) for 72 h was analyzed. RT-PCR and Western blot analysis showed that the mRNA and protein levels of a-SMA were upregulated, and a dose-dependent increase was observed (P,0.05 between each concentration point) (Fig. 2a and b). a-SMA expression in HK-2 cells after TGF-b1 treatment after different periods of time was evaluated. a-SMA mRNA and protein expression in HK-2 cells were detected at 0 h, 12 h, 24 h, 48 h and 72 h after treatment with 5 ng/ml TGF-b1. There was a gradual time-dependent increase in a-SMA mRNA and protein expression (P,0.05 between each time point). (Fig. 2c and d). According to the data, HK-2 cells treated with 5 ng/ml TGF-b1 for 48 h were used in subsequent experiments. TGF-b1 stimulation. NCTD downregulated the pSmad2/3 protein after 15, 30, 60 and 120 min (P,0.05 between each time point) (Fig. 4d). These data show that NCTD is effective in inhibiting the expression and phosphorylation of Smad2/3 protein in HK-2 cells. Discussion Renal tubular EMT is an important event in the pathogenesis of tubulointerstitial fibrosis and is characterized by loss of epithelial features and acquisition of mesenchymal markers under excessive exposure to various profibrotic cytokines [22]. Thus, inhibiting myofibroblast accumulation is critical in preventing tubulointerstitial fibrosis and preserving renal function. TGF-b1 is a strong profibrotic cytokine that activates the expression of ECM components, such as collagens I and IV and fibronectin, and inhibits collagen-degrading enzymes, such as certain matrix metalloproteinases (MMPs) involved in controlling ECM homeostasis. This leads to excessive ECM accumulation and fibrosis. In addition, TGF-b1 is a well-known fibrogenic cytokine in renal disease and plays a key role in EMT. The functional role of TGF-b1 in EMT and renal fibrosis is demonstrated by blocking TGF-b1 with neutralizing TGF-b1 antibodies and antisense oligonucleotides to prevent or ameliorate renal fibrosis in vivo and in vitro [23,24]. The downstream signaling of TGF-b1 is profoundly related to a family of Smad proteins that stimulate fibrosis (Smad2 and Smad3) or inhibit fibrosis (Smad7). a-SMA -positive myofibroblasts were identified as the primary cell type responsible for interstitial matrix accumulation in fibrotic diseases, including in the kidney. Moreover, an a-SMA phenotype is considered as a useful marker for myofibroblast differentiation in tubulointerstitial fibrosis. EMT is an orchestrated, highly regulated process that TGF-b1 is a strong profibrotic cytokine that activates the expression of ECM components, such as collagens I and IV and fibronectin, and inhibits collagen-degrading enzymes, such as certain matrix metalloproteinases (MMPs) involved in controlling ECM homeostasis. This leads to excessive ECM accumulation and fibrosis. In addition, TGF-b1 is a well-known fibrogenic cytokine in renal disease and plays a key role in EMT. The functional role of TGF-b1 in EMT and renal fibrosis is demonstrated by blocking TGF-b1 with neutralizing TGF-b1 antibodies and antisense oligonucleotides to prevent or ameliorate renal fibrosis in vivo and in vitro [23,24]. The downstream signaling of TGF-b1 is profoundly related to a family of Smad proteins that stimulate fibrosis (Smad2 and Smad3) or inhibit fibrosis (Smad7). a-SMA -positive myofibroblasts were identified as the primary cell type responsible for interstitial matrix accumulation in fibrotic diseases, including in the kidney. Moreover, an a-SMA phenotype is considered as a useful marker for myofibroblast differentiation in tubulointerstitial fibrosis. EMT is an orchestrated, highly regulated process that NCTD reduces the expression of Snail 1 in HK-2 cells stimulated by TGF-b1 This study showed that all concentrations of NCTD signifi- cantly reduced Snail 1 expression in HK-2 cells after TGF-b1 treatment for 48 h compared with the control group, which was confirmed by RT-PCR and Western blot. However, the concen- tration of NCTD was inversely correlated with incremental effects on Snail 1 expression (Fig. 5a and 5b). NCTD regulates the expression of a-SMA and E-cadherin in HK-2 cells stimulated by TGF-b1 NCTD regulates the expression of a-SMA and E-cadherin in HK-2 cells stimulated by TGF-b1 Discussion After HK-2 cells were treated with 5 ng/ml TGF-b1 for 48 h, there was a marked increase in a-SMA expression and a decrease in E-cadherin expression. Different concentrations of NCTD for 48 h significantly prevented above changes of the a-SMA and E- cadherin expression in HK-2 cells. NCTD prevented the increased levels of a-SMA and the reduced E-cadherin levels in a dose- dependent manner, with the highest concentration significantly inhibiting the changes. All of these results were confirmed by RT- PCR and Western blot, which showed reduced expression of a- SMA and increased expression of E-cadherin after treatment with NCTD compared to treatment with TGF-b1 without NCTD intervention (Fig. 3a–d). Immunofluorescence results showed that a-SMA protein expression was increased in the cytoplasm and E- cadherin protein expression was decreased in the cytomembrane of HK-2 cells stimulated with TGF-b1. However, NCTD treatment obviously downregulated a-SMA protein expression and upregulated E-cadherin protein expression (Fig. 3e). These results indicate that NCTD prevented de novo expression of the myofibroblast marker a-SMA in HK-2 cells and loss of the epithelial marker E-cadherin. Interstitial fibrosis is a common pathway of progressive renal disease, leading to end-stage renal failure regardless of the etiology. Interstitial fibrosis is the strongest morphological predictor of clinical outcome and progression [19]. Fibroblasts in the renal interstitium are considered the principal source of the fibrillar matrix, and tubulointerstitial fibrosis is inevitably associated with a robust accumulation of fibroblasts [20]. In vivo studies have indicated that the majority of fibroblasts in the damaged kidney could originate from the tubular epithelium through the process of tubular EMT [21]. Renal tubular EMT is an important event in the pathogenesis of tubulointerstitial fibrosis and is characterized by loss of epithelial features and acquisition of mesenchymal markers under excessive exposure to various profibrotic cytokines [22]. Thus, inhibiting myofibroblast accumulation is critical in preventing tubulointerstitial fibrosis and preserving renal function. Interstitial fibrosis is a common pathway of progressive renal disease, leading to end-stage renal failure regardless of the etiology. Interstitial fibrosis is the strongest morphological predictor of clinical outcome and progression [19]. Fibroblasts in the renal interstitium are considered the principal source of the fibrillar matrix, and tubulointerstitial fibrosis is inevitably associated with a robust accumulation of fibroblasts [20]. In vivo studies have indicated that the majority of fibroblasts in the damaged kidney could originate from the tubular epithelium through the process of tubular EMT [21]. NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 This study showed that the expression of Smad2 and Smad3 mRNA was upregulated in HK-2 cells stimulated by TGF-b1 compared with the control group. However, NCTD significantly decreased the expression of Smad2 and Smad3 in a dose- dependent manner (Fig. 4a and b). Western blot indicated that the expression level of the Smad2/3 protein in HK-2 cells stimulated by TGF-b1 was significantly higher than the expression in control group. The NCTD intervention dose downregulated Smad2/3 protein expression (Fig. 4c). The pSmad2/3 protein in HK-2 cells stimulated by TGF-b1 increased during the first 15 minutes of exposure, then gradually declined after prolonged exposure to June 2013 \| Volume 8 \| Issue 6 \| e66356 PLOS ONE \| www.plosone.org 4 NCTD Inhibits Tubular EMT consists of loss of epithelial cell adhesion, which is characterized by the loss E-cadherin expression and increased expression of a-SMA multiple injurious pathways, to m reverse renal tubulointerstitial fibr Figure 1. NCTD regulates the expression of a-SMA, E-cadherin and TGF-b1 in a rat UUO model. (a an (a) and graphic presentation (b) show a-SMA, E-cadherin and TGF-b1 mRNA in different treatment groups. P,0.0 UUO group. (c and d) Representative Western blot (c) and quantitative data (d) for a-SMA, E-cadherin and TG groups. * P,0.05 vs. sham group and #P,0.05 vs. UUO group. (e) Immunohistochemical staining shows a-SM expression in the rat kidney of different treatment groups (6200). doi:10.1371/journal.pone.0066356.g001 Figure 1. NCTD regulates the expression of a-SMA, E-cadherin and TGF-b1 in a rat UUO model. (a and b) Representative RT-PCR results (a) and graphic presentation (b) show a-SMA, E-cadherin and TGF-b1 mRNA in different treatment groups.* P,0.05 vs. sham group and #P,0.05 vs. UUO group. (c and d) Representative Western blot (c) and quantitative data (d) for a-SMA, E-cadherin and TGF-b1 protein expression in various groups. * P,0.05 vs. sham group and #P,0.05 vs. UUO group. (e) Immunohistochemical staining shows a-SMA, E-cadherin and TGF-b1 protein expression in the rat kidney of different treatment groups (6200). doi:10.1371/journal.pone.0066356.g001 Figure 1. NCTD regulates the expression of a-SMA, E-cadherin and TGF-b1 in a rat UUO model. (a and b) Representative RT-PCR results (a) and graphic presentation (b) show a-SMA, E-cadherin and TGF-b1 mRNA in different treatment groups.* P,0.05 vs. sham group and #P,0.05 vs. UUO group. (c and d) Representative Western blot (c) and quantitative data (d) for a-SMA, E-cadherin and TGF-b1 protein expression in various groups. * P,0.05 vs. multiple injurious pathways, to more effectively arrest or even reverse renal tubulointerstitial fibrosis. NCTD, an inhibitor of protein phosphatases, including PP1 and PP2A, is the demethylated analog of cantharidin. It also possesses anticancer activity [6]. Cantharidin can inhibit migration, invasion and adhesion of highly metastatic human ovarian carcinoma cell lines by downregulating expression of the NF-kB P65 subunit and vascular endothelial growth factor (VEGF). It has been reported NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 (c and d) Representative RT-PCR (c) and Western blot results (d) show that TGF-b1 induced expression of a-SMA mRNA and protein in HK cells in a time-dependent manner. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml). Figure 2. TGF-b1 induces the expression of a-SMA in HK-2 cells. (a and b) Representative RT-PCR (a) and Western blot results (b) show that TGF-b1 induced expression of a-SMA mRNA and protein in HK-2 cells in a dose-dependent manner. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml). (c and d) Representative RT-PCR (c) and Western blot results (d) show that TGF-b1 induced expression of a-SMA mRNA and protein in HK-2 cells in a time-dependent manner. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml). doi:10.1371/journal.pone.0066356.g002 tubular epithelial cells by EMT [13]. In this study, we used a UUO rat model and HK-2 cells induced by TGF-b1 to determine the effects of NCTD on tubular EMT. The results show that NCTD decreased upregulation of a-SMA and TGF-b1, while downreg- ulation of E-cadherin was decreased in the rat kidney. NCTD also reduced a-SMA expression in vitro and increased E-cadherin expression in HK-2 cells induced by TGF-b1. However, EMT is presently a controversial subject and its existence has been recently challenged. Confirmatory studies on epithelial cells are an important source of myofibroblasts through EMT in vivo are lacking [11,12]. Our in vitro study suggests that NCTD may act by inhibiting expression of TGF-b1, thereby delaying or preventing TGF-b1 mediated EMT and renal interstitial fibrosis, but this does not constitute direct proof that this actually happens in vivo. It need to be explored in the future study to confirm the inhibitory effect of NCTD on tubular EMT. that NCTD decreased the ratio of MMP2 to TIMP2 and reduced cellular mortality, exerting anti-invasive activity in human gallbladder carcinoma cells. The anti-metastatic activity of NCTD correlates with multiple factors involved in the EMT process. Our previous findings showed that NCTD exhibited a protective function in tubular interstitial fibrosis [2–6], during which EMT plays an important role. According to our preliminary studies, NCTD is beneficial in renal tubulointerstitial fibrosis, although its underlying pharmacological mechanisms are not well understood. We speculate that NCTD may ameliorate renal fibrosis by inhibiting the EMT process. that NCTD decreased the ratio of MMP2 to TIMP2 and reduced cellular mortality, exerting anti-invasive activity in human gallbladder carcinoma cells. NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 sham group and #P,0.05 vs. UUO group. (e) Immunohistochemical staining shows a-SMA, E-cadherin and TGF-b1 protein expression in the rat kidney of different treatment groups (6200). doi:10.1371/journal.pone.0066356.g001 multiple injurious pathways, to more effectively arrest or even reverse renal tubulointerstitial fibrosis. NCTD, an inhibitor of protein phosphatases, including PP1 and PP2A, is the demethylated analog of cantharidin. It also possesses anticancer activity [6]. Cantharidin can inhibit migration, invasion and adhesion of highly metastatic human ovarian carcinoma cell lines by downregulating expression of the NF-kB P65 subunit and vascular endothelial growth factor (VEGF). It has been reported multiple injurious pathways, to more effectively arrest or even reverse renal tubulointerstitial fibrosis. consists of loss of epithelial cell adhesion, which is characterized by the loss E-cadherin expression and increased expression of a-SMA. In this study, TGF-b1 caused enhancement of a-SMA expression in dose and time-dependent manners. This supports the suggestion that TGF-b1 can induce transdifferentiation of HK-2 cells into myofibroblasts. However, few therapeutic options are currently available. There is an urgent need to discover new therapeutic agents, particularly specific antagonists targeting TGF-b1 and NCTD, an inhibitor of protein phosphatases, including PP1 and PP2A, is the demethylated analog of cantharidin. It also possesses anticancer activity [6]. Cantharidin can inhibit migration, invasion and adhesion of highly metastatic human ovarian carcinoma cell lines by downregulating expression of the NF-kB P65 subunit and vascular endothelial growth factor (VEGF). It has been reported June 2013 \| Volume 8 \| Issue 6 \| e66356 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 NCTD Inhibits Tubular EMT Figure 2. TGF-b1 induces the expression of a-SMA in HK-2 cells. (a and b) Representative RT-PCR (a) and Western blot results (b) show that TGF-b1 induced expression of a-SMA mRNA and protein in HK-2 cells in a dose-dependent manner. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml). (c and d) Representative RT-PCR (c) and Western blot results (d) show that TGF-b1 induced expression of a-SMA mRNA and protein in HK-2 cells in a time-dependent manner. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml). doi:10.1371/journal.pone.0066356.g002 NCTD Inhibits Tubular EMT Figure 2. TGF-b1 induces the expression of a-SMA in HK-2 cells. (a and b) Representative RT-PCR (a) and Western blot results (b) show th TGF-b1 induced expression of a-SMA mRNA and protein in HK-2 cells in a dose-dependent manner. * P,0.05 vs. negative control group (TGF- 0 ng/ml). NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 The anti-metastatic activity of NCTD correlates with multiple factors involved in the EMT process. Our previous findings showed that NCTD exhibited a protective function in tubular interstitial fibrosis [2–6], during which EMT plays an important role. According to our preliminary studies, NCTD is beneficial in renal tubulointerstitial fibrosis, although its underlying pharmacological mechanisms are not well understood. We speculate that NCTD may ameliorate renal fibrosis by inhibiting the EMT process. The experimental UUO rat model is widely used to study progressive renal fibrosis. The obstructed kidney after UUO exhibits significant interstitial inflammatory cell infiltration and tubulointerstitial fibrosis [25]. Matrix-producing fibroblasts are thought to convert into myofibroblasts characterized by de novo activation of a-SMA during fibrogenesis. Fibroblasts in the kidney may be derived from resident interstitial cells, mesenchymal and/ or hematopoietic stem cells, periadventitial cells or the EMT process [21]. In the UUO model of renal fibrosis, up to 36% of the cells that produce the extracellular matrix are derived from TGF-b signaling pathways are activated by a short phosphor- ylation cascade, from receptor phosphorylation to subsequent phosphorylation and activation of downstream signal transducer R-Smads (receptor-activated Smads). Upon ligand stimulation, R- Smad proteins are phosphorylated in the SXS motif by TGF-b PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66356 June 2013 \| Volume 8 \| Issue 6 \| e66356 6 NCTD Inhibits Tubular EMT Figure 3. NCTD regulates expression of a-SMA and E-cadherin in HK-2 cells stimulated by TGF-b1. (a and b) Representative RT-PC and Western blot (b) results show that NCTD inhibited the expression of a-SMA mRNA and protein in HK-2 cells stimulated by TGF-b1. * P,0.0 negative control group (TGF-b1 0 ng/ml), #P,0.05 vs. positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). (c and d) NCTD increased expre of E-cadherin mRNA and protein in HK-2 cells stimulated by TGF-b1.* P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and #P,0.05 vs. po control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). (e) NCTD (2.5 mg/ml) regulated the phenotypic changes in HK-2 cells stimulated with TG (Immunofluorescence6200). doi:10.1371/journal.pone.0066356.g003 Figure 3. NCTD regulates expression of a-SMA and E-cadherin in HK-2 cells stimulated by TGF-b1. (a and b) Representative RT-PCR (a) and Western blot (b) results show that NCTD inhibited the expression of a-SMA mRNA and protein in HK-2 cells stimulated by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml), #P,0.05 vs. NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). (c and d) NCTD increased expression of E-cadherin mRNA and protein in HK-2 cells stimulated by TGF-b1.* P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and #P,0.05 vs. positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). (e) NCTD (2.5 mg/ml) regulated the phenotypic changes in HK-2 cells stimulated with TGF-b1 (Immunofluorescence6200). doi:10.1371/journal.pone.0066356.g003 June 2013 \| Volume 8 \| Issue 6 \| e66356 7 PLOS ONE \| www.plosone.org 7 NCTD Inhibits Tubular EMT Figure 4. NCTD inhibits expression of Smad2 and Smad3 in HK-2 cells induced by TGF-b1. (a and b) NCTD inhibited expression of Smad2 and Smad3 mRNA in HK-2 cells induced by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and # P,0.05 vs. positive control (TGF-b1 5 ng/ml + NCTD 0 mg/ml). (c and d) NCTD reduced expression of Smad2/3 and p-Smad2/3 proteins in HK-2 cells induced by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and # P,0.05 vs. positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). doi:10.1371/journal.pone.0066356.g004 Figure 4. NCTD inhibits expression of Smad2 and Smad3 in HK-2 cells induced by TGF-b1. (a and b) NCTD inhibited expression of Smad2 Figure 4. NCTD inhibits expression of Smad2 and Smad3 in HK-2 cells induced by TGF-b1. (a and b) NCTD inhibited expression of Smad2 and Smad3 mRNA in HK-2 cells induced by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and # P,0.05 vs. positive control (TGF-b1 5 ng/ml + NCTD 0 mg/ml). (c and d) NCTD reduced expression of Smad2/3 and p-Smad2/3 proteins in HK-2 cells induced by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and # P,0.05 vs. positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). doi:10.1371/journal.pone.0066356.g004 Figure 5. NCTD reduces the expression of Snail 1 in HK-2 cells stimulated by TGF-b1. (a) NCTD reduced expression of Snail 1 mRNA in HK-2 cells stimulated by TGF-b1. (b) NCTD reduced expression of Snail 1 protein in HK-2 cells stimulated by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and #P,0.05 vs. positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). doi:10.1371/journal.pone.0066356.g005 Figure 5. NCTD reduces the expression of Snail 1 in HK-2 cells stimulated by TGF-b1. (a) NCTD reduced expression of Snail 1 mRNA in HK-2 cells stimulated by TGF-b1. References 1. Huang Y, Liu Q, Liu K, Yagasaki K, Zhang G (2009) Suppression of growth of highly-metastatic human breast cancer cells by norcantharidin and its mechanisms of action. Cytotechnology 59: 201–208. 1. Huang Y, Liu Q, Liu K, Yagasaki K, Zhang G (2009) Suppression of growth of highly-metastatic human breast cancer cells by norcantharidin and its mechanisms of action. Cytotechnology 59: 201–208. 12. Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, et al. (2010) Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 176: 85–97. 2. Liu FY, Li Y, Peng YM, Ye K, Li J, et al. (2008) Norcantharidin ameliorates proteinuria, associated tubulointerstitial inflammation and fibrosis in protein overload nephropathy. Am J Nephrol 28: 465–477. 13. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, et al. (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110: 341– 350. 3. Li Y, Chen Q, Liu FY, Peng YM, Hou T, et al. (2011) Norcantharidin attenuates tubulointerstitial fibrosis in rat models with diabetic nephropathy.Ren Fail 33: 233–241. 14. Nistico` P, Bissell MJ, Radisky DC (2012) Epithelial-mesenchymal transition: general principles and pathological relevance with special emphasis on the role of matrix metalloproteinases.Cold Spring HarbPerspectBiol 4. pii: a011908. doi: 10.1101/cshperspect.a011908. 4. Li Y, Liu FY, Peng YM, Zhan M, Duan SB, et al. (2011) Norcantharidin inhibits proliferation and fibronectin expression of HK-2 cells induced by albumin in vitro. Cell Biol Int 35: 1239–1241. 15. Lo´pez-Herna´ndez FJ, Lo´pez-Novoa JM (2012) Role of TGF-b in chronic kidney disease: an integration of tubular, glomerular and vascular effects.Cell Tissue Res 347: 141–154. 5. Li Y, Chen Q, Liu FY, Peng YM, Wang S, et al. (2011) Norcantharidin inhibits the expression of extracellular matrix and TGF-b1 in HK-2 cells induced by high glucose independent of calcineurin signal pathway.Lab Invest 91: 1706– 1716. 16. Ohnuki K, Umezono T, Abe M, Kobayashi T, Kato M, et al. (2012) Expression of transcription factor Snai1 and tubulointerstitialfibrosis in progressive nephropathy. J Nephrol 25: 233–239. 17. Rowe RG, Li XY, Hu Y, Saunders TL, Virtanen I, et al. (2009) Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs. J Cell Biol 184: 399–408. 6. Li Y, Ge Y, Liu FY, Peng YM, Sun L, et al. (2012) Norcantharidin, a protective therapeutic agent in renal tubulointerstitial fibrosis. Mol Cell Biochem 361:79– 83. 18. Conclusions In our study, the role of NCTD in preserving the tubular epithelial phenotype was confirmed in a rat model of tubuloin- terstitial fibrosis induced by UUO. Consistent with the in vivo results, significant changes in the cell phenotype were observed in HK-2 cells induced by TGF-b1, including downregulation of E- cadherin expression and increased a-SMA expression. We infer that treatment with NCTD may attenuate renal fibrosis and inhibit tubular EMT. The mechanisms behind the effects of NCTD on tubular EMT appear to be related to regulation of the TGF-b1/Smad pathway and expression of Snail 1. The data obtained in this study identify a new therapeutic target of NCTD for inhibiting EMT and treating related diseases. NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 After NCTD intervention for 48 h, Snail 1 expression was significantly downregulated, suggest- ing an inhibitory effect of NCTD on TGF-b1-induced Snail 1 expression in HK-2 cells. We speculate that NCTD can inhibit tubular EMT in TGF-b1-induced HK-2 cells, which may contribute to inhibition of Snail 1. However, we only find NCTD is capable of stopping the existence of events that occur downstream to binding of TGF-b1 to its receptor by blocking Smad and Snail 1 expression in vitro. All of these findings also need to be proved that NCTD exerts the same effect in vivo. It is well known that the phosphorylation cascade from receptor to Smad proteins plays an important role in the activation of TGF- b signaling. Dephosphorylation of receptors and Smad proteins also contributes to the duration and intensity of TGF-b signaling. This reversible phosphorylation provides a balance for proper functioning of signaling molecules. PP2A is a well-known protein phosphatase that associates with TGF-b receptors [38]. The regulatory function of PP2A in regulating TGF-b signaling has been demonstrated. PP2A may dephosphorylate Smad3 under hypoxic conditions [39]. NCTD has a remarkable inhibitory effect on protein phosphatases, including PP1 and PP2A. What is more important, NCTD has a stronger inhibitory effect on PP2A compared to PP1 (IC50 = 2.69 mM vs. 10.3 mM) [40]. However, whether NCTD plays an antifibrotic role by inhibiting PP2A needs to be explored in the future. In particular, determining how NCTD affects Smad expression and phosphorylation by inhibiting PP2A should be evaluated. Author Contributions Conceived and designed the experiments: YL FY-L LS YM-P. Performed the experiments: YL YS LX YP-L YY-X YH-Y. Analyzed the data: JL SB- D HL. Contributed reagents/materials/analysis tools: LS LX MW. Wrote the paper: YL SW TH. Revised the manuscript: YL FY-L. Conceived and designed the experiments: YL FY-L LS YM-P. Performed the experiments: YL YS LX YP-L YY-X YH-Y. Analyzed the data: JL SB- D HL. Contributed reagents/materials/analysis tools: LS LX MW. Wrote the paper: YL SW TH. Revised the manuscript: YL FY-L. NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 (b) NCTD reduced expression of Snail 1 protein in HK-2 cells stimulated by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and #P,0.05 vs. positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). doi:10.1371/journal.pone.0066356.g005 Figure 5. NCTD reduces the expression of Snail 1 in HK-2 cells stimulated by TGF-b1. (a) NCTD reduced expression of Snail 1 mRNA in HK-2 cells stimulated by TGF-b1. (b) NCTD reduced expression of Snail 1 protein in HK-2 cells stimulated by TGF-b1. * P,0.05 vs. negative control group (TGF-b1 0 ng/ml) and #P,0.05 vs. positive control group (TGF-b1 5 ng/ml + NCTD 0 mg/ml). doi:10.1371/journal.pone.0066356.g005 June 2013 \| Volume 8 \| Issue 6 \| e66356 PLOS ONE \| www.plosone.org 8 NCTD Inhibits Tubular EMT type I receptors, leading to the activation of a series of downstream events. The results of our in vitro experiment showed a increased expression of Smad2/3 protein and phosphorylation stimulated by TGF-b1, which was reduced by co-treatment with NCTD, providing an evidence that NCTD has an inhibitory effect on TGF-b1/Smad pathway. Snail 1, a zinc finger transcription factor, has been characterized as a key regulator of EMT. Many studies have shown that Snail 1 binds to specific DNA sequences called E- boxes in the promoter of the E-cadherin gene to repress transcription of E-cadherin [26,27]. E-cadherin is a major gene in the epithelium, which is an important determinant in maintaining the epithelial phenotype [28–31]. EMT was rapidly induced within 24 hours after UUO, and the Snail1 transcription factor was upregulated, preceding the induction of a-SMA [32]. Although activity of the Snail 1 gene is required during embryonic development to form different tissues and organs, the gene is repressed in adults to maintain epithelial integrity and homeostasis [33]. Pathological activation of Snail 1 in adult tubular epithelial cells is sufficient to induce tubulointerstitial fibrosis. Snail 1 mRNA has been shown to specifically localized to renal tubular epithelial cells in wild-type mice 7 days after UUO [34–36]. A study recently reported that overexpression of Snail 1 in the epithelial nucleus contributes to EMT. Snail 1 mRNA and protein were upregulated in the tubular cells of rat kidneys in the UUO model and human proximal tubule cells treated with TGF-b1, demonstrating that Snail 1 is involved in tubular EMT [37]. However, whether NCTD can regulate the expression of Snail 1 is unclear. NCTD inhibits the expression and phosphorylation of Smad2 and Smad3 in HK-2 cells stimulated by TGF-b1 Our experiment observed the expression of Snail 1 mRNA and protein in HK-2 cells induced by TGF-b1. After NCTD intervention for 48 h, Snail 1 expression was significantly downregulated, suggest- ing an inhibitory effect of NCTD on TGF-b1-induced Snail 1 expression in HK-2 cells. We speculate that NCTD can inhibit tubular EMT in TGF-b1-induced HK-2 cells, which may contribute to inhibition of Snail 1. However, we only find NCTD is capable of stopping the existence of events that occur downstream to binding of TGF-b1 to its receptor by blocking Smad and Snail 1 expression in vitro. All of these findings also need to be proved that NCTD exerts the same effect in vivo. type I receptors, leading to the activation of a series of downstream events. The results of our in vitro experiment showed a increased expression of Smad2/3 protein and phosphorylation stimulated by TGF-b1, which was reduced by co-treatment with NCTD, providing an evidence that NCTD has an inhibitory effect on TGF-b1/Smad pathway. Snail 1, a zinc finger transcription factor, has been characterized as a key regulator of EMT. Many studies have shown that Snail 1 binds to specific DNA sequences called E- boxes in the promoter of the E-cadherin gene to repress transcription of E-cadherin [26,27]. E-cadherin is a major gene in the epithelium, which is an important determinant in maintaining the epithelial phenotype [28–31]. EMT was rapidly induced within 24 hours after UUO, and the Snail1 transcription factor was upregulated, preceding the induction of a-SMA [32]. Although activity of the Snail 1 gene is required during embryonic development to form different tissues and organs, the gene is repressed in adults to maintain epithelial integrity and homeostasis [33]. Pathological activation of Snail 1 in adult tubular epithelial cells is sufficient to induce tubulointerstitial fibrosis. Snail 1 mRNA has been shown to specifically localized to renal tubular epithelial cells in wild-type mice 7 days after UUO [34–36]. A study recently reported that overexpression of Snail 1 in the epithelial nucleus contributes to EMT. Snail 1 mRNA and protein were upregulated in the tubular cells of rat kidneys in the UUO model and human proximal tubule cells treated with TGF-b1, demonstrating that Snail 1 is involved in tubular EMT [37]. However, whether NCTD can regulate the expression of Snail 1 is unclear. Our experiment observed the expression of Snail 1 mRNA and protein in HK-2 cells induced by TGF-b1. NCTD Inhibits Tubular EMT 22. Flier SN, Tanjore H, Kokkotou EG, Sugimoto H, Zeisberg M, et al. (2010) Identification of epithelial to mesenchymal transition as a novel source of fibroblasts in intestinal fibrosis. J Biol Chem 285: 20202–20212. 32. Lange-Sperandio B, Trautmann A, Eickelberg O, Jayachandran A, Oberle S, et al. (2007) Leukocytes induce epithelial to mesenchymal transition after unilateral ureteral obstruction in neonatal mice. Am JPathol 171: 861–871. 23. Matsuda H, Fukuda N, Ueno T, Katakawa M, Wang X, et al. (2011) Transcriptional inhibition of progressive renal disease by gene silencing pyrroleimidazole polyamide targeting of the transforming growth factor-b1 promoter. Kidney Int 79: 46–56. 33. Kokudo T, Suzuki Y, Yoshimatsu Y, Yamazaki T, Watabe T, et al. (2008) Snail is required for TGFbeta-induced endothelial-mesenchymal transition of embryonic stem cell-derived endothelial cells. J cell Sci 121: 3317–3324. 34. Boutet A, De Frutos CA, Maxwell PH, Mayol MJ, Romero J, et al. (2006) Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. EMBO J 25: 5603–5613. p y 24. Hills CE, Squires PE (2011) The role of TGF-b and epithelial-to mesenchymal transition in diabetic nephropathy.Cytokine Growth Factor Rev 22: 131–139. 25. Chevalier RL, Forbes MS, Thornhill BA (2009) Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy. Kidney Int 75: 1145– 1152. 35. Boutet A, Esteban MA, Maxwell PH, Nieto MA (2007) Reactivation of Snail genes in renal fibrosis and carcinomas: a process of reversed embryogenesis? Cell cycle 6: 638–642. 26. Rowe RG, Li XY, Hu Y, Saunders TL, Virtanen I, et al. (2009) Mesenchymal cells reactivate Snail1 expression to drive three dimensional invasion programs. J Cell Biol 184: 399–408. 36. Boutet A, Nieto MA (2008) The ‘‘Snail’’ genes and renal diseases: what we learn from organogenesis. Med Sci (Paris) 24: 238–240. 37. Yoshino J, Monkawa T, Tsuji M, Inukai M, Itoh H, et al. (2007) Snail1 is involved in the renal epithelial- mesenchymal transition. Biochem Biophys Res Commun.362: 63–68. 27. Cano A, Pe´rez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, et al. (2000) The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2: 76–83. 38. Batut J, Schmierer B, Cao J, Raftery LA, Hill CS, et al. (2008) Two highly related regulatory subunits of PP2A exert opposite effects on TGF-beta/Activin/ Nodal signalling. Development, 135: 2927–2937. p g p 28. Hills CE, Siamantouras E, Smith SW, Cockwell P, Liu KK, et al. References Liu FY, Li XZ, Peng YM, Liu H, Liu YH (2007) Arkadia-Smad7-mediated positive regulation of TGF-beta signaling in a rat model of tubulointerstitial fibrosis.Am J Nephrol 27: 176–183. 7. Liu Y (2011) Cellular and molecular mechanisms of renal fibrosis. Nat Rev Nephrol 18: 684–696. 8. Barnes JL, Glass WF 2nd (2011) Renal interstitial fibrosis: a critical evaluation of the origin of myofibroblasts.Contrib Nephrol 169: 73–93. J p 19. Zeisberg M, Duffield JS (2010) Resolved: EMT produces fibroblasts in the kidney. J Am Soc Nephrol 21: 1247–1253. 9. Kriz W, Kaissling B, Le Hir M (2011) Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy? J Clin Invest 121: 468–474. 20. Chen CZ, Raghunath M (2009) Focus on collagen: In vitro systems to study fibrogenesis and antifibrosis state of the art. Fibrogenesis Tissue Repair 2: 7 doi: 10.1186/1755–1536–2–7. 10. Liu Y (2010) New insights into epithelial- mesenchymal transition in kidney fibrosis. J Am SocNephrol 21: 212–222. 21. Grande MT, Lo´pez-Novoa JM (2009) Fibroblast activation and myofibroblast generation in obstructive nephropathy. Nat RevNephrol 5: 319–328. 11. Li L, Zepeda-Orozco D, Black R, Lin F (2010) Autophagy is a component of epithelial cell fate in obstructive uropathy. Am J Pathol 176: 1767–1778. June 2013 \| Volume 8 \| Issue 6 \| e66356 June 2013 \| Volume 8 \| Issue 6 \| e66356 PLOS ONE \| www.plosone.org 9 NCTD Inhibits Tubular EMT NCTD Inhibits Tubular EMT (2012) TGF-b modulates cell-to-cell communication in early epithelial-to-mesenchymal transition.Diabetologia 55: 812–824. 39. Heikkinen PT, Nummela M, Leivonen SK, Westermarck J, Hill CS, et al. (2010) Hypoxia-activated Smad3- specific dephosphorylation by PP2A. J Biol Chem 285: 3740–3749. g 29. Wrighton KH, Lin X, Feng XH (2009) Phospho-control of TGF-beta superfamily signaling. Cell Res 19: 8–20. p y g g 30. Galichon P, Hertig A (2011) Epithelial to mesenchymal transition as a biomarker inrenal fibrosis: are we ready for the bedside? Fibrogenesis Tissue Rep 4: 11 doi: 10.1186/1755–1536–4–11. 40. Stewart SG, Hill TA, Gilbert J, Ackland SP, Sakoff JA, et al. (2007) Synthesis and biological evaluation of norcantharidin analogues: towards PP1 selectivity. Bioorg Med Chem 15: 7301–7310. 31. Quaggin SE, Kapus A (2011) Scar wars: mapping the fate of epithelial– esenchymal- myofibroblast transition. Kidney Int 80: 41–50. PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66356 PLOS ONE \| www.plosone.org 10
https://openalex.org/W4387362456	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0291626&type=printable	English	null	An enhanced decision-making framework for predicting future trends of sharing economy	PloS one	2,023	cc-by	21,539	An enhanced decision-making framework for predicting future trends of sharing economy Qiong Wu1, Xiaoxiao Tang1, Rongjie Li2, Lei Liu3, Hui-Ling ChenID4 1 School of Marxism, Wenzhou University, Wenzhou, China, 2 Wenzhou Business College, Wenzhou, China, 3 College of Computer Science, Sichuan University, Chengdu, Sichuan, China, 4 College of Computer Science an Artificial Intelligence, Wenzhou University, Wenzhou, China 1 School of Marxism, Wenzhou University, Wenzhou, China, 2 Wenzhou Business College, Wenzhou, China, 3 College of Computer Science, Sichuan University, Chengdu, Sichuan, China, 4 College of Computer Science an Artificial Intelligence, Wenzhou University, Wenzhou, China * tony63277418@163.com (RL); chenhuiling.jlu@gmail.com (H-LC) * tony63277418@163.com (RL); chenhuiling.jlu@gmail.com (H-LC) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Wu Q, Tang X, Li R, Liu L, Chen H-L (2023) An enhanced decision-making framework for predicting future trends of sharing economy. PLoS ONE 18(10): e0291626. https://doi.org/ 10.1371/journal.pone.0291626 Editor: Salim Heddam, University 20 Aout 1955 skikda, Algeria, ALGERIA Editor: Salim Heddam, University 20 Aout 1955 skikda, Algeria, ALGERIA Received: February 24, 2023 Accepted: September 4, 2023 Published: October 5, 2023 Received: February 24, 2023 Accepted: September 4, 2023 Published: October 5, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0291626 Copyright: © 2023 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The dataset analyzed during the current study is available at https:// github.com/Hollow123e/Sharing-Economy- Dataset.git. In the year 1978, Marcus Felson, an esteemed Sociology professor at Texas State University, and Joe L. Spaeth, a professor specializing in Sociology at the University of Illinois, introduced Abstract This work aims to provide a reliable and intelligent prediction model for future trends in shar- ing economy. Moreover, it presents valuable insights for decision-making and policy devel- opment by relevant governmental bodies. Furthermore, the study introduces a predictive system that incorporates an enhanced Harris Hawk Optimization (HHO) algorithm and a K- Nearest Neighbor (KNN) forecasting framework. The method utilizes an improved simulated annealing mechanism and a Gaussian bare bone structure to improve the original HHO, termed SGHHO. To achieve optimal prediction performance and identify essential features, a refined simulated annealing mechanism is employed to mitigate the susceptibility of the original HHO algorithm to local optima. The algorithm employs a mechanism that boosts its global search ability by generating fresh solution sets at a specific likelihood. This mecha- nism dynamically adjusts the equilibrium between the exploration and exploitation phases, incorporating the Gaussian bare bone strategy. The best classification model (SGHHO- KNN) is developed to mine the key features with the improvement of both strategies. To assess the exceptional efficacy of the SGHHO algorithm, this investigation conducted a series of comparative trials employing the function set of IEEE CEC 2014. The outcomes of these experiments unequivocally demonstrate that the SGHHO algorithm outperforms the original HHO algorithm on 96.7% of the functions, substantiating its remarkable superiority. The algorithm can achieve the optimal value of the function on 67% of the tested functions and significantly outperforms other competing algorithms. In addition, the key features selected by the SGHHO-KNN model in the prediction experiment, including " Form of shar- ing economy in your region " and " Attitudes to the sharing economy ", are important for pre- dicting the future trends of the sharing economy in this study. The results of the prediction demonstrate that the proposed model achieves an accuracy rate of 99.70% and a specificity rate of 99.38%. Consequently, the SGHHO-KNN model holds great potential as a reliable tool for forecasting the forthcoming trajectory of the sharing economy. PLOS ONE PLOS ONE 1. Introduction In the year 1978, Marcus Felson, an esteemed Sociology professor at Texas State University, and Joe L. Spaeth, a professor specializing in Sociology at the University of Illinois, introduced 1 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy the notion of the sharing economy in their scholarly publication, marking its inaugural men- tion [1,2]. It is a market-based model in which the supplier of resources provides idle resources or skills to the demander through the technology platform and charges a certain fee [3]. The effective allocation of resources is achieved after integrating the technology platforms. Specifi- cally, it can combine various business models such as sharing, renting and swapping together, which is different from traditional business models and signals the emergence of a new eco- nomic model [4]. To some extent, the sharing economy model enables the optimal allocation and use of relevant resources. Unlike other economic models, the sharing economy model is an inevitable product of the internet era, as consumers are disadvantaged in the defense of their rights by businesses, dissatisfied with the asymmetry of transaction information and lack of trust in social transaction mechanisms, combined with the full spread of internet technology and continued innovation, provide the prerequisites for the birth of the sharing economy model [5]. Although the concept of "sharing economy" has been around for a long time, the academic community has not yet fully agreed on a definition of the term. Academics agree that platforms are important foundations for the existence and development of sharing econ- omy [6,7]. A quality platform enables effective exchange in the sharing economy, allowing consumers to consume higher-quality products at a lower cost. Meanwhile, the good reputa- tion of the platform is transferred to the merchant, increasing consumer trust in the merchant and effectively facilitating trading activities. Overall, the sharing economy can bring unused items back into circulation without changing ownership, enabling efficient allocation of resources [8]. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. From a certain point of view, the application of the sharing economy can achieve the Pareto optimum, which can promote the creation of benefits for the sharing platform and the partici- pating entities. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 1. Introduction Secondly, the elimination of information asymmetries can be used to achieve free market matching, so that idle resources can be utilized to the greatest extent while factor costs are effectively controlled [9]. Finally, the start-up costs of internet platforms can be effec- tively controlled under the influence of the sharing economy, and a more low-risk approach can be adopted to promote the stable operation of asset-light. Nowadays, sharing economy has been popular in many countries around the world. According to forecasts by relevant institu- tions, the global market of sharing economy for major industries will grow from $14 billion in 2014 to $335 billion by 2025. In recent years, advances in technology and the growing popular- ity of mobile communication devices and the internet have led to the widespread emergence of the sharing economy phenomenon, which has also brought a huge impact on traditional consumption models [10,11]. On the one hand: it is more adaptable to the consumption expe- rience and consumption needs of the epidemic situation and networked development; that is, sharing economy model can replace the previous intermediary model with producer-con- sumer transactions, i.e. replacing the previous employment relationship with a contractual relationship between the consumer and the platform, giving consumers a better service experi- ence. Besides, the price of products and services in the sharing economy is significantly lower than in the traditional economy, so total social demand can be significantly increased by shar- ing economy. However, on the other hand, due to its imperfect development, the implementa- tion of sharing economy involves product food testing, third-party payment supervision, i f ti it h k l i ti i i d th d it l t b di information security checks, logistics supervision and other means, and its regulatory bodies include industry and commerce, telecommunications, finance, quality inspection and public security [12,13]. And looking at the current development of the sharing economy model, its activity still lacks regulatory bodies. The regulatory bodies have not yet made a clear delinea- tion of powers and responsibilities, making the regulation of the sharing economy a phenome- non of overlapping powers and responsibilities and regulatory gaps [14,15]. 1. Introduction In addition to this, there are also many problems such as low-price dumping, monopoly agreements, unfair PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 2 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy competition and big data "killing", which are still difficult to be accepted for some users. In short, as a new trend, the acceptance of the sharing economy among the population is still unknown. Therefore, it is necessary to analyze the data to understand in detail the level of awareness and acceptance of the sharing economy. This will not only help to anticipate the future development of sharing economy and adjustment of relevant policies and regulations but will also help to further guide the benign operation of the sharing economy [2,16]. Recently, artificial intelligence technology has developed rapidly and applied successfully in predicting the future development of sharing economy. Klos et al. [17] specifically address the issue of sharing economy. Pilot work was carried out on the phenomenon of collective con- sumption of Polish consumers by means of a questionnaire to analyze the influence of sharing economy on consumers. Perles et al. [18] aimed to investigate the extent to which sharing economy affects the tourism industry. Machine learning techniques were used to solve practi- cal problems in the tourism industry to further analyze the potential impact of the sharing economy on the tourism industry. Chen et al. [19] used structural equation modeling on a questionnaire survey of 90 sharing economy companies to explore the sustainability of sharing economy. Ackermann et al. [20] examine consumer use of sharing economy platforms from a legitimacy perspective, exploring the impact of consumer attitudes and behavioral intentions towards the accommodation sector in the context of sharing economy. Cheng et al. [21] stud- ied the carbon footprint of Airbnb hosts in Sydney using peer-to-peer sharing on the Airbnb platform and analyses the notion that sharing economy facilitates the utilization of underuti- lized resources. This study contributes to the sustainability of peer-to-peer accommodation and sharing economy more generally. Ferreri et al. [22] explored the interrelationship between sharing platform economy companies and governments under the background of sharing economy, analyzing the trade-offs between corporate and public interests. Zemla et al. [23] explored the role of sharing economy in urban economic planning and analyzed the planning options made for the development and implementation of sharing economy in specific cities. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 1. Introduction Artificial intelligence technology has experienced significant advancements in the past few years, witnessing a remarkable pace of progress, and machine learning algorithms have been of great practical value in both academia and industry. With the exponential growth and intri- cacy of big data, the conventional machine learning algorithms designed for small-scale data have become inadequate for addressing the multitude of challenges presented by big data applications [24,25]. Therefore, the study of machine learning algorithms in the big data envi- ronment has become a common topic of interest for both academia and industry and has wide application to the problem of predicting the future development of the sharing economy. Kim et al. [26] used Random Forest, XGBoost and LightGBM models to predict the demand for shared bicycles and integrated the predictions into the company’s business operations to better serve the needs of customers. Wang et al. [27] constructed a multi-relational network to ana- lyze the value of cooperation in the sharing economy by using different machine learning clas- sification models and feature sets to predict consumers’ purchase behavior on the platform. Tornberg et al. [28] used a machine learning approach to classify picture profiles provided by landlords of shared rentals to assess the gap between race and gender income and to further explore the impact of sharing economy. Shokoohyar et al. [29] use multiple machine learning models (support vector machines, plain Bayes and neural networks) to predict the highest return rental strategy for a specific property in Philadelphia in the context of sharing economy based on data from 2163 properties. Nadeem et al. [30] cut through the theoretical issues of consumer participation in sharing economy platforms from a multi-dimensional perspective, using marketing and business theory literature to analyze consumers’ value co-creation orien- tation to predict future trends in sharing economy. Jiang et al. [31] conducted a study on bike- PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 3 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy sharing. A deep learning model is used to predict the most likely destination for each user to further satisfy consumer demand to explore the influence of sharing economy on people. 1. Introduction y g y Many swarm intelligence algorithms were proposed one after another in the early 1990s, such as hunger games search (HGS) [32], colony predation algorithm (CPA) [33], slime mould algorithm (SMA) [34,35], Harris hawks optimization (HHO) [36], Runge Kutta opti- mizer (RUN) [37], weighted mean of vectors (INFO) [38], and rime optimization algorithm (RIME) [39]. They have achieved exciting results on combinatorial optimization problems with NP-hard characteristics that are difficult to be handled by traditional optimization algo- rithms [40,41]. It has garnered significant attention from the academic community and has been rapidly applied in various practice environments, particularly achieving enormous suc- cess in engineering applications. For example, they have gained wide application and achieved good results in areas such as combinatorial optimization [42,43], data mining [44,45], energy scheduling [46,47], medicine [48,49] and image classification [50,51], bankruptcy prediction [52], economic emission dispatch [53], feature selection [54–58], numerical optimization [59– 61], scheduling optimization [62,63], multi-objective optimization [64], large-scale complex optimization [65], global optimization [66–70], feed-forward neural networks [71], and target tracking [72]. Therefore, to better explore the future development trend of sharing economy, this study proposes an effective prediction model for the future development trend of sharing economy. This model combines KNN with the improved Harris Hawk optimization algo- rithm, named SGHHO-KNN model. In the proposed algorithm, two novel mechanisms are introduced to the original HHO to improve its disadvantages. First, embedding an enhanced simulated annealing mechanism to address the tendency of the original HHO to fall into local optima. This mechanism generates new solution sets with a certain probability, enhancing the chance of the basic HHO escaping from the local optimum and thus strengthening the capabil- ity of the algorithm in global search. Secondly, by incorporating the principles of the Gaussian bare bone mechanism, the SGHHO algorithm effectively navigates the transition from explo- ration to exploitation phases through a refined adjustment process. This ensures both popula- tion diversity and enables the algorithm to achieve enhanced accuracy in problem-solving. Lastly, to further enhance classification performance, the SGHHO algorithm is synergistically integrated with a KNN classifier, which facilitates the evaluation of feature subsets and ulti- mately leads to improved classification outcomes. Simultaneously, a comprehensive set of opti- mization experiments was undertaken using the well-established IEEE CEC 2014 benchmark functions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 2.1. Literature review The research shows that the internal parameters in machine learning models and the feature space and sample space in big data have an important influence on the performance of classifiers. In the last decade or so, swarm intelligence algorithms have shown good benefits in solving such problems [73–76]. Furthermore, swarm intelligence algorithms exhibit notable characteristics of randomness and efficiency, encompassing a range of deterministic and stochastic techniques that find wide application in diverse optimization problems and practical scenarios, effectively har- nessing the power of collective intelligence. Examples include optimization problems [77–80], traveler problems [81], complex network problems [82,83], path planning problems [84,85], and real-time detection problems [86]. Within the scope of this investigation, a refined variation of the Harris Hawks algorithm is introduced, leveraged to forecast forthcoming patterns in the advance- ment of the sharing economy. Originating from the pioneering work of AliAsghar Heidari and SeyedaliMirjalili in 2019, the Harris Hawks Optimization (HHO) algorithm represents a biomi- metic intelligent optimization technique. The algorithm mimics the Harris Hawk predation char- acteristics and combines Levy Flights to achieve solutions to complex multidimensional problems. The algorithm is similar to common meta-heuristic algorithms, inspired by the habits of nature’s animals. It achieves global optimization algorithms by imitating the group hunting, raiding and siege strategy of the Harris Hawk. Since its introduction, the Harris Hawk algorithm has been used to solve optimization problems in a variety of fields and has achieved good results. Literature [87] introduced the HHO algorithm with chaotic search and opposition learning to perform parameter searches for PV cells and modules. The literature [88] used the HHO algo- rithm to analyze the stability of mass slope problems and to improve the accuracy of predictions. Literature [89] applied the HHO to in-vehicle location services for intelligent transportation sys- tems to reduce the delay in the delivery of emergency data in in-vehicle networks to enhance the location rate. The literature [90] used the improved adaptive Harris Hawk algorithm for obtaining the optimal queue for the 2D grey scale gradient method and used this gradient method for image processing, possessing better results compared to other multi-stage min-valorization methods. The literature [91] used HHO for industrial safety. 1. Introduction The simulation outcomes unequivocally demonstrate the remarkable superiority of the proposed algorithm over the original HHO algorithm, exhibiting a significantly higher per- formance level on 96.7% of the functions, while also exhibiting improved stability. Moreover, to thoroughly examine the key factors influencing the future trajectory of the sharing econ- omy, it is imperative to undertake comparative experiments involving SGHHO-KNN and other state-of-the-art algorithms. The empirical findings demonstrate that SGHHO-KNN out- performs conventional approaches across all four evaluated metrics, showcasing superior clas- sification accuracy and enhanced stability. Notably, the prediction outcomes reveal exceptional performance, with the proposed model achieving an impressive accuracy rate of 99.70% and a remarkable specificity rate of 99.38%. Ultimately, the primary objective of this investigation is to conduct an in-depth analysis of the influence of the sharing economy on individuals, consequently providing valuable insights into the anticipated future trajectory of this economic phenomenon. This work has made the following contributions: This work has made the following contributions: • An improved simulated annealing mechanism (ISA) and a Gaussian bare bone strategy (GB) are introduced and applied to the HHO to enhance its optimization performance. • An improved simulated annealing mechanism (ISA) and a Gaussian bare bone strategy (GB) are introduced and applied to the HHO to enhance its optimization performance. p g gy are introduced and applied to the HHO to enhance its optimization performance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 4 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy • The superior performance of SGHHO is effectively validated through comprehensive bench- mark function experiments. • The superior performance of SGHHO is effectively validated through comprehensive bench- mark function experiments. • A SGHHO-KNN-based model is developed to predict the future development of sharing economy. • A SGHHO-KNN-based model is developed to predict the future development of sharing economy. • Effective forecasting of future trends in the sharing economy is realized and relevant key fea- tures are filtered out. • Effective forecasting of future trends in the sharing economy is realized and relevant key fea- tures are filtered out. This is the organization of this study. Section 2 gives the basic principles of the HHO algo- rithm; Section 3 introduces SGHHO proposed in this paper, together with the improved simu- lated annealing mechanism and the Gaussian bare bone variation strategy introduced. 1. Introduction Section 4 introduces the SGHHO-KNN model; Section 5 evaluates the optimization performance of SGHHO on several benchmark functions; Section 6 uses the SGHHO-KNN model to predict the future development of the sharing economy. Finally, summing up the research results of this work and giving the directions of future work. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 2.1. Literature review Using HHO for augmented artificial neural networks (ANN), a hybrid model ANN-HHO model was proposed for predicting the scour depth of spillways by dam outflow water and compared with two other hybrid models ANN-GA and ANN-PSO, showing the superiority and effectiveness of the hybrid model. 5 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy 2.2. K-nearest neighbor classifier The KNN algorithm is a simple and effective classification algorithm. It is based on the idea of a nearest neighbor, i.e. the class of a new sample is determined by the nearest samples of a known class. The fundamental concept underlying the K-nearest neighbors (KNN) algorithm involves the computation of the distance between the sample under classification and all the training samples, utilizing a distance metric. By identifying the k closest neighbors to the sam- ple in question [92], a majority vote is subsequently employed to ascertain the category to which the sample belongs, based on the categories assigned to these nearest neighbors. Many research works have been done on this algorithm by scholars at home and abroad. For exam- ple, Wai Lam et al. [93] from the Chinese University of Hong Kong combined the KNN method with a linear classifier and achieved better classification results, with an accuracy of over 80% at a recall rate close to 90%. The literature [94] investigated the asymptotic nature of the K-nearest neighbor estimate of the regression function and obtained the asymptotic nor- mality of the K-nearest neighbor estimate of the regression function and the coincidence of its Bootstrap statistic. The literature [95] establishes an efficient search tree for the nearest neigh- bor algorithm and improves the query rate. In the literature [96], an iterative nearest neighbor method is proposed to solve the problem of poor classification of KNN algorithms in the envi- ronment of small sample pools. In the case where insufficient classed samples are available, the local thematic features of the samples to be classified are amplified by retrieval to obtain similar samples with sufficiently fixed classes. The literature [97] gives parallel algorithms on reconfi- gurable mesh machines (RMESH) for K nearest neighbors in Euclidean space, among others. Li et al. proposed a two-layer hierarchical combination of text classification [98]. It was dem- onstrated that the combination of support vector machine and KNN methods could make SVM noise less sensitive and improve the efficiency of KNN methods, showing better classifi- cation performance. Shi et al. [99] propose a semi-supervised classification algorithm based on rough set error, using rough set error theory to extract negative case samples. A new classifier is constructed by combining SVM, Rocchio and Naive Bayes to improve the classification accuracy. 3. Structure of HHO HHO is a heuristic algorithm put forward by Heidari et al that was enlighted by the predatory behaviors of Harris’s hawks [35]. In line with other heuristic algorithms, HHO contains explo- ration and exploitation phases. In the two different phases, Harris’s hawks produce different predatory behaviors. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 3.1. Exploration phase Harris Hawks in the exploration phase roam many locations waiting for prey to arrive. Harris’s Hawks usually consider the position of their companions and prey when selecting their perches. Two main strategies are followed during this phase, as shown in Eq (2.1). Xðt þ 1Þ ¼ XrandðtÞ r1jXrandðtÞ 2r2XðtÞj q 0:5 ðXrabbitðtÞ XmðtÞÞ r3ðLB þ r4ðUB LBÞÞ q < 0:5 Eq ð2:1Þ ( Xm tð Þ ¼ 1 N XN i¼1 XiðtÞ Eq ð2:2Þ Eq ð2:2Þ In Eq (2.1)., X(t+1) is the next updated position of the population iteration of Harris’s hawk. Xrand(t) refers to a random position in the current population. Xrabbit(t) refers to the In Eq (2.1)., X(t+1) is the next updated position of the population iteration of Harris’s hawk. Xrand(t) refers to a random position in the current population. Xrabbit(t) refers to the PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 6 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy prey’s location, i.e., the location of the current optimal solution. r1, r2, r3, r4 and q are random numbers between [0,1], respectively. UB and LB are used to control the upper boundary and lower bound, independently. In Eq (2.2), Xm(t) is the average position of individuals in the cur- rent Harris’s hawk population. where N is the population size and Xi(t) is the position of the i- th individual. 3.2. Transition factor The escape energy E of the target is designed during the exploitation phase, which determines the exploitation behavior of the Harris Hawk, as shown in Eq (2.3). E ¼ 2ð1 t=TÞ Eq ð2:3Þ El ¼ E E Eq ð2 4Þ E ¼ 2ð1 t=TÞ Eq ð2:3Þ Eq ð2:3Þ El ¼ E E0 El ¼ E E0 Eq ð2:4Þ Eq ð2:4Þ where E represents the escape energy of the target prey. E0 denotes the escape energy of target in its initial state. T represents the maximum number of iterations. t denotes the current num- ber of iterations. Harris Hawk chooses different exploitation behaviors depending on the value of E. When E1, the Harris Hawk searches for the target in a region far from the current solu- tion. When E<1, Harris Hawk searches in the neighborhood of the current solution. where E represents the escape energy of the target prey. E0 denotes the escape energy of target in its initial state. T represents the maximum number of iterations. t denotes the current num- ber of iterations. Harris Hawk chooses different exploitation behaviors depending on the value of E. When E1, the Harris Hawk searches for the target in a region far from the current solu- tion. When E<1, Harris Hawk searches in the neighborhood of the current solution. 3.3. Exploitation phase In the process of the exploitation phase, Harris’s Hawks choose the most appropriate way to attack their prey, depending on each situation. Four strategies describe the hunting behaviors of Harris’s hawk. The probability of prey escape is set to r. r<0.5 represents the state in which the prey escapes capture. r0.5 represents the state in which the prey is captured. Regardless of the state, the Harris hawk always surrounds the prey based on escape energy. When E0.5, the Harris Hawk performs a soft besiege. when E<0.5, the Harris Hawk performs a hard besiege. 3.3.1 Soft besiege. When E0.5 and r0.5, then Harris Hawk successfully captures the prey in a soft besiege state. In soft besiege, the Harris Hawk surrounds its prey and dissipates the prey’s energy. The process can be expressed as Eq (2.5). Xðt þ 1Þ ¼ DXðtÞ EljJXrand XðtÞj Eq ð2:5Þ Eq ð2:5Þ where ΔX(t) denotes the vector difference between the prey target and the current individual and can be represented as Eq (2.6). J refers to the prey escape force, as shown in Eq (2.7). DXðtÞ ¼ XrabbitðtÞ XðtÞ Eq ð2:6Þ J ¼ 2ð1 r5Þ Eq ð2:7Þ DXðtÞ ¼ XrabbitðtÞ XðtÞ Eq ð2:6Þ J ¼ 2ð1 r5Þ Eq ð2:7Þ J ¼ 2ð1 r5Þ where r5 represents a random number between [0,1]. where r5 represents a random number between [0,1]. where r5 represents a random number between [0,1]. 3.3.2 Hard besiege. When E<0.5 and r0.5, the energy of the prey target is at a low value. At this point, Harris’s hawk uses a surprise attack to approach the prey, a process that can be expressed by Eq (2.8). Xðt þ 1Þ ¼ XrabbitðtÞ EljDXðtÞj Eq ð2:8Þ Eq ð2:8Þ 3.3.3 Soft besiege with progressive rapid dives. When E0.5 and r<0.5, the energy of the prey target is at a high value. The probability of prey avoiding capture by Harris’s hawk PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 7 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy was high at this time. Harris’s hawks performed soft besiege between prey captures. The subse- quent random wandering of Levy-flight was used to simulate prey escape and Harris Hawk pursuit, a process that can be expressed by Eq (2.9). Eq (2.10) and Eq (2.11) determine the next updated position of the Harris Hawk. 4. Proposed SGHHO In the subsection, we systematically present the algorithmic implementation of SGHHO, In the subsection, we systematically present the algorithmic implementation of SGHHO, including the two novel mechanisms introduced therein. Compared to the standard HHO, the proposed SGHHO has better performance. including the two novel mechanisms introduced therein. Compared to the standard HHO, the proposed SGHHO has better performance. 3.3. Exploitation phase LF x ð Þ ¼ u s jvj 1 b ; s ¼ G 1 þ b ð Þ sin pb 2 G 1 þ b 2 b 2 b 1 2 0 B B B @ 1 C C C A 1 b Eq ð2:9Þ Eq ð2:9Þ Y ¼ XrabbitðtÞ EljJXrabbitðtÞ XðtÞj Eq ð2:10Þ Z ¼ Y þ S LFðDÞ Eq ð2:11Þ Eq ð2:11Þ where u and v are both random numbers between [0,1], β takes 1.5. D is the dimension of the problem. S vector is randomly generated. Greedy selection is then used to determine which positional update is more favorable to the optimal solution, as shown in Eq (2.12). F() calcu- lates the target fitness value. where u and v are both random numbers between [0,1], β takes 1.5. D is the dimension of the problem. S vector is randomly generated. Greedy selection is then used to determine which positional update is more favorable to the optimal solution, as shown in Eq (2.12). F() calcu- lates the target fitness value. Xðt þ 1Þ ¼ Y if FðYÞ < FðXðtÞÞ Z if FðZÞ < FðXðtÞÞ Eq ð2:12Þ ( Eq ð2:12Þ 3.3.4 Hard besiege with progressive rapid dives. When E<0.5 and r<0.5, Harris’s Hawk surrounds the low-energy prey target by forming an envelope. In the process, Harris’s hawk approaches the prey by narrowing the envelope. This process is described as Eq (2.13)–Eq (2.15). Y’ ¼ XrabbitðtÞ EljJ∗XrabbitðtÞ XmðtÞj Eq ð2:13Þ Z’ ¼ Y’ þ S LFðDÞ Eq ð2:14Þ Xðt þ 1Þ ¼ Y0 if FðY0Þ < FðXðtÞÞ Z0 if FðZ0Þ < FðXðtÞÞ Eq ð2:15Þ ( PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 4.1. Simulated annealing based HHO Simulated annealing (SA) algorithm is a random search algorithm first proposed by N. Metropolis et al [100]. Metropolis perceived a strong similarity between the solid annealing processes in the physical world and the general engineering combinatorial optimization situa- tion. SA is a generally probability-based optimization algorithm where the solid annealing pro- cess can be summarized in three parts: the heating stage, the isothermal procedure and the cooling process: 8 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy 1. Heating stage. This process is designed to increase the energy of the molecules of a solid so that the molecules within it have a more intense thermal movement, thus departing from the equilibrium state of the solid molecules. As the temperature reaches a sufficient value, the solid becomes a liquid, resulting in the disappearance of the non-uniform state in the solid system. 2. Isothermal processes. The states in the system always proceed in the direction of decreasing energy, and as the energy decreases, the system eventually enters equilibrium. 3. The cooling processes. The thermal movement of molecules is reduced, the energy of the system decreases, and a crystalline structure is formed. The main idea of the simulated annealing algorithm in a combinatorial optimization prob- lem is then shown as follows: The main idea of the simulated annealing algorithm in a combinatorial optimization prob- lem is then shown as follows: • Initialize the temperature values and solve the optimization problem. • Execute the algorithm according to the Metropolis criterion. • Execute the algorithm according to the Metropolis criterion. • Output optimal solution. In the SA algorithm, the Metropolis criterion is the assumption that the position of the pre- vious agent is X(n). After further iterative updates of the population, the agent position is updated to X(n+1). At the same time, the energy of the search agent changes from E(n) to E(n +1). The probability of receiving a search agent from X(n) to X(n+1) is p. 4.1. Simulated annealing based HHO p ¼ 1; ; Eðn þ 1Þ < EðnÞ exp Eðn þ 1Þ EðnÞ T ; E n þ 1 ð Þ E n ð Þ Eq ð3:1Þ 8 > < > : Eq ð3:1Þ where T denotes the relative temperature; when moving to the next iteration of the update, if the energy has become smaller, then this change is accepted. If the energy has increased, the search agent has moved further away from the global optimum. In this case, the algorithm does not immediately discard it but judges it by probability: a random number ε is generated on a fixed interval [0,1]. If ε<p, the transfer in this case will also be accepted, otherwise the transfer fails to proceed to the next step of the cooling process, and so on. The cooling equation for the above cooling process is: T ¼ T q Eq ð3:2Þ Eq ð3:2Þ where q denotes the cooling factor, the value of which is usually set to 0.99 [28]; T denotes the current temperature. When the temperature drops to the termination temperature Tend, the optimal value is output. In this study, chaos mapping is introduced for the simulated annealing algorithm to further enhance the performance of SA. The operation of chaos mapping is shown in detail as follows: biþ1 ¼ mbi ð1 biÞ; i ¼ 1; 2 . . . s 1 Eq ð3:3Þ Eq ð3:3Þ where μ denotes the control parameter, whose value is usually set to 4 [101]; βi represents a random value ranging in [0,1]; and s stands for the number of the entire population. The cha- otic strategy is strongly influenced by the initial conditions and can be understood as a search movement with a combination of overall ergodicity and randomness [102,103]. By effectively circumventing premature convergence of the population, this approach proficiently mitigates the occurrence thereof, expedites the convergence rate, and enhances the precision of solutions attained through the HHO algorithm. where μ denotes the control parameter, whose value is usually set to 4 [101]; βi represents a random value ranging in [0,1]; and s stands for the number of the entire population. The cha- otic strategy is strongly influenced by the initial conditions and can be understood as a search movement with a combination of overall ergodicity and randomness [102,103]. 4.2. Gaussian bare-bones Gaussian Bare Bones is widely used as a variational strategy. Wang et al [104] introduced Gaussian skeleton variation into DE by intersecting the feasible solution obtained from Gauss- ian perturbation with the solution of the original DE. In this study, enhanced Gaussian Bare Bones were designed to be applied to HHO, and the new update procedure is shown in Eq (3.4). Xi;jðt þ 1Þ ¼ Nðm; sÞ; rand < CR Xi1;jðtÞ þ r3∗ðXi2;jðtÞ Xi3;jðtÞÞ; otherwise Eq ð3:4Þ ( m ¼ ðXbest;j þ Xi;jðtÞÞ=2 Eq ð3:5Þ s ¼ b∗jXbest;j Xi;jðtÞj Eq ð3:6Þ Eq ð3:6Þ where N(μ, σ) is a Gaussian function with mean μ and variance σ. CR is the crossover factor, and its value is usually set to 0.3 [104–106]. Xi1;j; Xi2;j; Xi3;j are three random individuals in dimension j, while i6¼i16¼i26¼i3. j In the modified Gaussian Bare Bones, the new update position is generated by a Gaussian distribution. The method calculates the average of the intermediate position between the pres- ent capture and the optimal solution, utilizing the difference between the current solution and the optimal solution as a measure of variance. During the initial stages of evolution, when the disparity between the current solution and the global optimum is substantial, the population’s individuals encompass the entire search space. This stage primarily emphasizes global explora- tion. As the number of offspring increases, the deviation decreases, while the use of a linear decreasing factor b ensures that individuals in the later population will cover a smaller search area. At this point the algorithm can move more quickly into the behaviors of local exploita- tion, ensuring convergence at a later stage. 4.1. Simulated annealing based HHO By effectively circumventing premature convergence of the population, this approach proficiently mitigates the occurrence thereof, expedites the convergence rate, and enhances the precision of solutions attained through the HHO algorithm. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 9 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy 4.3. Framework of SGHHO The entire framework of SGHHO combines both of these strategies and the flow graph of its overall framework is detailed in Fig 1. First, the population needs to be set up initially and the optimal position selected as the rabbit’s position in the algorithm. After the SGHHO algorithm is updated using the core formulation of HHO, a simulated annealing strategy is introduced. Guided by the SA strategy, the iterative process of SGHHO can accept points with larger energy values, i.e., poorer solutions. As a result, the strategy possesses a better vertical search capability with a larger search range, thus effectively improving the probability of HHO find- ing a global optimum. Next, Gaussian bare bones strategy is implemented, and the process of fine-tuning the Gaussian sampling allows SGHHO to ensure diversity exploration in the first stage while focusing on exploitation in the next phase. The switch in search behavior from exploration to exploitation allows SGHHO to converge to a higher level of accuracy. Eventually, the optimal solution in the entire population is updated until it reaches the max- imum allowed number of iterations. Additionally, Algorithm 1 showcases the pseudo-code for the SGHHO method employed in this research. p y Algorithm1. The pseudocode of SGHHO. Initialize the population size N and maximum number of evaluation MaxFES; Initialize the population size N and maximum number of evaluation MaxFES; Randomly initialize the location information of population Xi(i = 1,2,. . PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 4.3. Framework of SGHHO .,N); While (termination condition is not satisfied) do 10 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Calculate the fitness value for each individual; Set Xrabbit to the current optimal solution; for (Each individual) do Updating the initial escape energy E0 and escape force J; Update E by Eq (2.3); if (\|E\| 1) then if (q < 0.5) then Updating the position according to Eq (2.1); elseif (q 0.5) then Update the location according to the random selection in Eq (2.1); end if (\|E\| < 1) then if (r 0.5 and \|E\| 0.5) then Use Eq (2.5) to update the position; elseif (r 0.5 and \|E\| < 0.5) then Use Eq (2.8) to update the position; elseif (r < 0.5 and \|E\| 0.5) then Use Eq (2.12) to update the position; elseif (r < 0.5 and \|E\| < 0.5) then Use Eq (2.15) to update the position; end if end for Calculate the fitness value for all individuals; Calculate the mutation position of the modified Gaussian bare bone strategy by Eq (2.15); Fig 1. Flowchart of SGHHO. https://doi.org/10.1371/journal.pone.0291626.g001 OS ONE An enhanced decision-making framework for predicting future trends of sharing economy Fig 1. Flowchart of SGHHO. https://doi.org/10.1371/journal.pone.0291626.g001 Fig 1. Flowchart of SGHHO. https://doi.org/10.1371/journal.pone.0291626.g001 Fig 1. Flowchart of SGHHO. https://doi.org/10.1371/journal.pone.0291626.g001 Calculate the fitness value for each individual; Set Xrabbit to the current optimal solution; 11 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy Updating search agent locations by means of the modified simu- lated annealing strategy; t = t + 1; end while return Xrabbit; return Xrabbit; 4.4. Computational complexity The evaluation of algorithmic models involves considering the time complexity of an opti- mizer, which is an important metric. In this research, the time complexity of SGHHO consists of various steps, including population initialization, updating fitness values, modifying the positions of individuals in the population, executing the simulated annealing strategy, and implementing the Gaussian bare bone strategy. In the standard SGHHO algorithm, the time complexity of these first three components is calculated as O (population initialization) = O (N); O (fitness value update) = O(TN) during T update iterations; and O (population individ- ual position update) = O(TND) during T iterations. where N denotes the assumed popula- tion size; T denotes the maximum number of iterative updates during the experiment; and D denotes the problem dimensions. The SA strategy and GB strategy are also included in the SGHHO algorithm to help SGHHO improve its performance. In particular, the time complex- ity of the SA strategy is O (SA strategy) = O(N); the time complexity of GB strategy is O (GB strategy) = O(ND). So that the overall time complexity consumed by the SGHHO algorithm is O(SGHHO) = O(N*(T+TD+2+D)). PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 5. Proposed SGHHO-KNN In this section, to enable more representative attributes to be found in the dataset and helps researchers to mine more effective information. In this paper, a simulated annealing mecha- nism and a Gaussian bare bone variation strategy for improving HHO is proposed for feature selection. Various evaluation criteria exist for different feature selection methods. In this research, a wrapped feature selection method is employed, which primarily relies on the learn- ing algorithm. The classification performance of feature selection serves as the evaluation met- ric for this method. To enhance the evaluation process, this study incorporates a KNN classifier. KNN is a non-parametric classification technique known for its ability to achieve high accuracy when dealing with unknown and non-normally distributed data. Moreover, KNN offers several advantages, including conceptual clarity and ease of implementation. Fur- thermore, the KNN approach primarily depends on a small set of neighboring samples in its classification process, rather than relying on class domain discrimination methods. This char- acteristic makes the KNN method particularly well-suited for datasets with overlapping or intersecting class domains. It is worth noting that the classification error rate of the KNN clas- sifier is closely tied to the concept of distance. In general, KNN algorithms will use distance metrics such as Euclidean and Manhattan for operations. In this study, Manhattan is used for the sample calculation. In overview, the SGHHO-KNN model is developed in this paper. The model involves three main components: primarily, in this chapter, a refined version of the HHO algorithm is intro- duced, which incorporates a simulated annealing mechanism and a Gaussian skeleton varia- tion strategy. Subsequently, the SGHHO algorithm is synergistically combined with the K-Nearest Neighbor (KNN) model, giving rise to a novel classification model referred to as SGHHO-KNN. This model utilizes a hybrid feature selection approach. Within this frame- work, the SGHHO algorithm is employed to conduct a thorough exploration of the feature space, enabling the identification and selection of the most optimal feature subset. The K-Nearest Neighbor classifier is mainly used for feature subset evaluation and for comparing PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 12 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy the classification performance after feature selection in combination with the comparison algorithm. 6. Experimental design and analysis of results To analyze the SGHHO’s performance in various aspects, comprehensive experiments are designed. In the optimization experiments, comprehensive experiments on the IEEE CEC 2014 function set had been conducted. The experimental design procedure of the work is shown as follows: firstly, experiments are designed to test the scalability of the SGHHO algo- rithm for dimensional changes; secondly, the effects of two mechanisms on the SGHHO algo- rithm are investigated; thirdly, comparison experiments are carried out between the SGHHO algorithm and 10 well-known optimization algorithms. In addition, for the metaheuristic algo- rithm setup described above, the population size is 30; the maximum number of evaluations is 300,000. Meanwhile, all competing methods were independently repeated 30 times during the testing process to avoid chance during experiments. Finally, all the above tests were conducted on Windows 10 host with 16GB RAM and 3.6GHz main frequency and coded using MATLAB R2016b. 6.1. CEC 2014 function validation In the section, IEEE CEC 2014 function set was chosen to prove each algorithm’s performance in optimization precision and convergence speed. In particular, algorithms are divided into four main types: singlet functions (F1- F3); multimodal functions (F4—F8); hybrid modal func- tions (F9- F20) and composite modal functions (F21- F30). Table 1 shows the specific data for these 30 functions. In the table, the last two columns show the type of each algorithm and the corresponding global optimum value. Furthermore, to provide a more comprehensive assess- ment of each algorithm’s performance, two metrics, namely the average value (AVG) and the standard deviation value (STD), are utilized. The experimental tables highlight the optimal val- ues for each problem in bold black font, ensuring their prominence. Additionally, non- parametric statistical tests such as the Wilcoxon signed-rank test [107] and the Friedman test [108] are employed to quantitatively evaluate the performance of SGHHO. 5. Proposed SGHHO-KNN That is, with ten-fold cross-validation, the SGHHO algorithm finds the best feature subset on the training set by internal five-fold cross-validation, and the KNN model uses this best feature subset to perform the classification task on the test set. In addition, for the experi- ments in this study, each classification experiment was run 10 times independently to ensure that the algorithm was fair and unbiased. Therefore, the performance of the algorithm was evaluated according to the average classification results and the corresponding standard devia- tion of each of the 10 independent runs. The flow chart of the SGHHO-KNN hybrid model proposed in this study is shown in Fig 2. PLOS ONE PLOS ONE Table 1. Description of the 30 IEEE CEC 2014 benchmark functions. ID Function Class Optimum F1 Rotated High Conditioned Elliptic Function Unimodal 100 F2 Rotated Bent Cigar Function Unimodal 200 F3 Rotated Discus Function Unimodal 300 F4 Shifted and Rotated Rosenbrock’s Function Multimodal 400 F5 Shifted and Rotated Ackley’s Function Multimodal 500 F6 Shifted and Rotated Weierstrass Function Multimodal 600 F7 Shifted and Rotated Griewank’s Function Multimodal 700 F8 Shifted Rastrigin’s Function Multimodal 800 F9 Shifted and Rotated Rastrigin’s Function Hybrid 900 F10 Shifted Schwefel’s Function Hybrid 1000 F11 Shifted and Rotated Schwefel’s Function Hybrid 1100 F12 Shifted and Rotated Katsuura Function Hybrid 1200 F13 Shifted and Rotated HappyCat Function Hybrid 1300 F14 Shifted and Rotated HGBat Function Hybrid 1400 F15 Shifted and Rotated Expanded Griewank’s plus Rosenbrock’s Function Hybrid 1500 F16 Shifted and Rotated Expanded Scaffer’s F6 Function Hybrid 1600 F17 Hybrid Function 1 (N = 3) Hybrid 1700 F18 Hybrid Function 2 (N = 3) Hybrid 1800 F19 Hybrid Function 3 (N = 4) Hybrid 1900 F20 Hybrid Function 4 (N = 4) Hybrid 2000 F21 Hybrid Function 5 (N = 5) Composition 2100 F22 Hybrid Function 6 (N = 5) Composition 2200 F23 Composition Function 1 (N = 5) Composition 2300 F24 Composition Function 2 (N = 3) Composition 2400 F25 Composition Function 3 (N = 3) Composition 2500 F26 Composition Function 4 (N = 5) Composition 2600 F27 Composition Function 5 (N = 5) Composition 2700 F28 Composition Function 6 (N = 5) Composition 2800 F29 Composition Function 7 (N = 3) Composition 2900 F30 Composition Function 8 (N = 3) Composition 3000 https://doi.org/10.1371/journal.pone.0291626.t001 AVG (average) and STD (standard deviation). A comprehensive overview of the results can be found in Table 2. AVG (average) and STD (standard deviation). A comprehensive overview of the results can be found in Table 2. The table provides a clear visual representation, demonstrating that the SGHHO algorithm consistently outperforms the original HHO algorithm in unimodal functions (F1-F3), irre- spective of the dimensional complexity. The AVG values achieved by the SGHHO algorithm exhibit a significant improvement compared to those of the original HHO algorithm. This superiority is evident in both low and high-dimensional problem settings. Also, SGHHO out- performs the FFA algorithm for F1 and F3 functions. https://doi.org/10.1371/journal.pone.0291626.t001 6.2. Scalability analysis of SGHHO The problem’s dimensionality corresponds to the number of factors to be optimized within the given optimization problem. Consequently, optimizing high-dimensional data allows for a more comprehensive evaluation of the algorithm’s overall performance and stability. Thus, the performance of the SGHHO algorithm can be thoroughly assessed. We use the original HHO and Farmland Fertility Algorithm (FFA) [109], which have better performance in the variable dimensionality case, as the reference objects for the experiments in this subsection. Within the experimental framework, scalability experiments are conducted using a test function set con- sisting of 30 functions from IEEE CEC 2014. The dimensions are varied, specifically set to 100, 500, and 1000, respectively. All experimental parameters remain constant, with the exception of the dimension setting (D). The metrics utilized to analyze the experimental results include PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 13 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy Fig 2. Framework structure of SGHHO-KNN. Fig 2. Framework structure of SGHHO-KNN. https://doi.org/10.1371/journal.pone.0291626.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 14 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 PLOS ONE And with the increasing complexity of the functions, the SGHHO algorithm has better optimization performance compared with the original HHO algorithm in multimodal functions (F4-F9). The implementation of the GB strategy successfully directs the HHO algorithm towards exploring regions that contain more optimal solutions, thereby enhancing the algorithm’s precision in generating solutions. The FFA, with its excessive local search capability, slightly outperforms the SGHHO on F4, F7 and F8. Further, in the mixed modal functions (F9-F20), the SGHHO algorithm still maintains its obvious superiority over FFA and HHO. Despite the variations in dimensional settings, the SGHHO algorithm demonstrates consistent performance in attaining optimal solutions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 15 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Table 2. Experimental results of scalability tests on SGHHO. PLOS ONE 100 500 1000 F Metric SGHHO FFA HHO SGHHO FFA HHO SGHHO FFA HHO F1 AVG 1.64E+06 9.44E+06 1.38E+07 1.37E+06 9.49E+06 1.43E+07 1.17E+06 9.56E+06 1.20E+07 STD 9.50E+05 4.34E+06 5.59E+06 3.93E+05 3.90E+06 6.71E+06 5.50E+05 4.13E+06 4.87E+06 F2 AVG 1.66E+04 5.86E+02 1.46E+07 2.38E+04 3.80E+02 1.38E+07 1.61E+04 7.96E+02 1.42E+07 STD 9.36E+03 1.47E+03 3.15E+06 3.43E+04 6.07E+02 3.05E+06 1.11E+04 1.66E+03 2.87E+06 F3 AVG 3.60E+02 1.91E+03 1.03E+04 3.49E+02 1.70E+03 9.48E+03 3.45E+02 1.59E+03 1.07E+04 STD 3.57E+01 1.29E+03 4.51E+03 3.56E+01 9.20E+02 3.44E+03 2.18E+01 7.38E+02 3.48E+03 F4 AVG 4.69E+02 4.60E+02 5.58E+02 4.80E+02 4.63E+02 5.55E+02 4.70E+02 4.47E+02 5.68E+02 STD 3.49E+01 3.44E+01 7.08E+01 3.87E+01 3.75E+01 4.35E+01 2.98E+01 3.64E+01 5.26E+01 F5 AVG 5.20E+02 5.21E+02 5.20E+02 5.20E+02 5.21E+02 5.20E+02 5.20E+02 5.21E+02 5.20E+02 STD 1.36E-01 4.97E-02 1.61E-01 4.80E-02 6.56E-02 1.85E-01 1.12E-01 5.95E-02 1.59E-01 F6 AVG 6.21E+02 6.18E+02 6.32E+02 6.23E+02 6.17E+02 6.30E+02 6.22E+02 6.18E+02 6.31E+02 STD 3.19E+00 4.62E+00 2.99E+00 2.92E+00 6.41E+00 4.51E+00 3.42E+00 4.60E+00 3.66E+00 F7 AVG 7.00E+02 7.00E+02 7.01E+02 7.00E+02 7.00E+02 7.01E+02 7.00E+02 7.00E+02 7.01E+02 STD 3.48E-02 1.88E-03 3.14E-02 2.80E-02 6.43E-03 2.48E-02 3.71E-02 2.57E-03 3.03E-02 F8 AVG 8.42E+02 8.19E+02 9.07E+02 8.41E+02 8.18E+02 9.14E+02 8.41E+02 8.20E+02 9.13E+02 STD 9.06E+00 4.90E+00 1.57E+01 7.32E+00 4.68E+00 1.57E+01 7.81E+00 5.99E+00 1.51E+01 F9 AVG 1.08E+03 1.05E+03 1.09E+03 1.08E+03 1.06E+03 1.09E+03 1.07E+03 1.05E+03 1.09E+03 STD 1.64E+01 2.11E+01 2.01E+01 2.16E+01 2.34E+01 2.36E+01 1.94E+01 3.06E+01 2.12E+01 F10 AVG 1.62E+03 1.50E+03 3.16E+03 1.48E+03 1.54E+03 3.05E+03 1.40E+03 1.48E+03 3.28E+03 STD 3.06E+02 5.71E+02 7.77E+02 2.71E+02 4.31E+02 7.57E+02 3.46E+02 3.05E+02 8.94E+02 F11 AVG 4.45E+03 7.60E+03 5.27E+03 4.55E+03 7.57E+03 4.98E+03 4.37E+03 7.59E+03 5.33E+03 STD 4.68E+02 4.56E+02 7.26E+02 5.97E+02 3.61E+02 9.43E+02 5.17E+02 4.72E+02 6.60E+02 F12 AVG 1.20E+03 1.20E+03 1.20E+03 1.20E+03 1.20E+03 1.20E+03 1.20E+03 1.20E+03 1.20E+03 STD 2.23E-01 3.00E-01 4.19E-01 2.14E-01 2.73E-01 4.70E-01 1.79E-01 2.66E-01 4.99E-01 F13 AVG 1.30E+03 1.30E+03 1.30E+03 1.30E+03 1.30E+03 1.30E+03 1.30E+03 1.30E+03 1.30E+03 STD 8.92E-02 5.02E-02 1.40E-01 7.31E-02 4.73E-02 1.26E-01 9.16E-02 4.37E-02 1.09E-01 F14 AVG 1.40E+03 1.40E+03 1.40E+03 1.40E+03 1.40E+03 1.40E+03 1.40E+03 1.40E+03 1.40E+03 STD 3.54E-02 3.75E-02 1.35E-01 3.74E-02 3.22E-02 5.16E-02 4.14E-02 3.86E-02 4.70E-02 F15 AVG 1.51E+03 1.52E+03 1.54E+03 1.51E+03 1.52E+03 1.54E+03 1.51E+03 1.52E+03 1.54E+03 STD 2.14E+00 1.20E+00 9.79E+00 2.82E+00 1.10E+00 9.13E+00 2.48E+00 1.13E+00 7.33E+00 F16 AVG 1.61E+03 1.61E+03 1.61E+03 1.61E+03 1.61E+03 1.61E+03 1.61E+03 1.61E+03 1.61E+03 STD 3.54E-01 2.10E-01 3.69E-01 2.68E-01 2.39E-01 4.13E-01 2.99E-01 2.79E-01 3.53E-01 F17 AVG 1.26E+05 4.95E+05 2.00E+06 1.40E+05 4.52E+05 1.79E+06 1.67E+05 4.42E+05 2.04E+06 STD 6.40E+04 2.74E+05 1.36E+06 5.57E+04 3.06E+05 1.04E+06 1.44E+05 3.05E+05 1.65E+06 F18 AVG 4.02E+03 3.30E+03 1.07E+05 4.25E+03 3.23E+03 1.50E+05 4.29E+03 3.01E+03 1.25E+05 STD 4.12E+03 2.07E+03 3.98E+04 5.27E+03 1.64E+03 2.43E+05 4.82E+03 1.65E+03 7.01E+04 F19 AVG 1.92E+03 1.91E+03 1.94E+03 1.92E+03 1.91E+03 1.93E+03 1.92E+03 1.91E+03 1.93E+03 STD 2.67E+00 1.22E+00 4.31E+01 2.84E+00 1.06E+00 2.58E+01 3.02E+00 1.28E+00 2.79E+01 F20 AVG 2.30E+03 3.84E+03 1.58E+04 2.27E+03 3.97E+03 1.74E+04 2.30E+03 3.86E+03 1.66E+04 STD 6.64E+01 7.95E+02 5.99E+03 7.00E+01 1.35E+03 8.56E+03 6.91E+01 1.06E+03 6.07E+03 F21 AVG 5.81E+04 1.45E+05 5.89E+05 6.76E+04 1.33E+05 6.04E+05 6.29E+04 1.29E+05 6.53E+05 STD 3.28E+04 9.19E+04 6.61E+05 3.41E+04 7.23E+04 4.69E+05 4.07E+04 9.05E+04 5.83E+05 F22 AVG 2.72E+03 2.39E+03 3.03E+03 2.74E+03 2.43E+03 3.09E+03 2.70E+03 2.44E+03 3.10E+03 STD 1.60E+02 9.77E+01 1.92E+02 1.70E+02 1.02E+02 2.65E+02 1.30E+02 1.13E+02 3.15E+02 F23 AVG 2.50E+03 2.62E+03 2.50E+03 2.50E+03 2.62E+03 2.50E+03 2.50E+03 2.62E+03 2.50E+03 STD 0.00E+00 1.39E-12 0.00E+00 0.00E+00 1.24E-12 0.00E+00 0.00E+00 1.49E-12 0.00E+00 (Continued) Table 2. (Continued) PLOS ONE Experimental results of scalability tests on SGHHO. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 16 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Table 2. (Continued) 100 500 1000 F Metric SGHHO FFA HHO SGHHO FFA HHO SGHHO FFA HHO F24 AVG 2.60E+03 2.62E+03 2.60E+03 2.60E+03 2.63E+03 2.60E+03 2.60E+03 2.63E+03 2.60E+03 STD 2.47E-03 1.15E+00 4.23E-04 7.39E-03 1.59E+00 7.95E-05 3.29E-03 2.71E+00 4.42E-04 F25 AVG 2.70E+03 2.71E+03 2.70E+03 2.70E+03 2.71E+03 2.70E+03 2.70E+03 2.71E+03 2.70E+03 STD 0.00E+00 1.90E+00 0.00E+00 0.00E+00 1.45E+00 0.00E+00 0.00E+00 1.88E+00 0.00E+00 F26 AVG 2.70E+03 2.70E+03 2.78E+03 2.71E+03 2.70E+03 2.76E+03 2.70E+03 2.70E+03 2.76E+03 STD 1.82E+01 6.68E-02 4.05E+01 3.04E+01 5.46E-02 4.96E+01 1.38E-01 5.45E-02 4.88E+01 F27 AVG 2.90E+03 3.12E+03 2.90E+03 2.90E+03 3.12E+03 2.90E+03 2.90E+03 3.12E+03 2.90E+03 STD 0.00E+00 1.20E+01 0.00E+00 0.00E+00 4.04E+01 0.00E+00 0.00E+00 3.42E+01 0.00E+00 F28 AVG 3.00E+03 3.65E+03 3.00E+03 3.00E+03 3.66E+03 3.00E+03 3.00E+03 3.67E+03 3.00E+03 STD 0.00E+00 5.40E+01 0.00E+00 0.00E+00 3.10E+01 0.00E+00 0.00E+00 4.45E+01 0.00E+00 F29 AVG 3.11E+03 4.53E+03 3.10E+03 3.11E+03 4.51E+03 3.10E+03 3.11E+03 4.48E+03 3.10E+03 STD 3.31E+00 4.52E+02 0.00E+00 1.47E+01 4.64E+02 0.00E+00 3.88E+00 4.84E+02 0.00E+00 F30 AVG 3.39E+03 4.78E+03 6.87E+03 3.44E+03 4.89E+03 7.90E+03 3.48E+03 4.88E+03 6.66E+03 STD 2.20E+02 3.75E+02 9.55E+03 3.01E+02 7.41E+02 7.81E+03 5.36E+02 6.23E+02 6.08E+03 https://doi.org/10.1371/journal.pone.0291626.t002 Among the composite functions (F21-F30), although there is a slight difference in the AVG value between the SGHHO algorithm and the original algorithm on the F29 function, it remains relatively small. Conversely, for all other functions, the SGHHO algorithm outper- forms the HHO algorithm by a significant margin. The results of scalability experiments indi- cate that the SGHHO algorithm exhibits notable enhancements in optimization performance and stability across 93.3% of the functions in the IEEE CEC 2014 function set when consider- ing variable dimensionality scenarios. This signifies the considerable improvements achieved by the SGHHO algorithm through the integration of SA and GB strategies. https://doi.org/10.1371/journal.pone.0291626.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 6.3. The impact of two mechanisms This subsection investigates the effect of the randomly introduced simulated annealing strat- egy and the Gaussian bare bone strategy on HHO. To enable effective verification of the role of the added mechanisms, this experiment performs all sequential sequencing of the SA and GB strategies, thus avoiding interactions between the mechanisms. The details are displayed in Table 3, where "S" and "G" denote "simulated annealing" and "Gaussian skeleton", respectively. 1 and 0 indicate that the strategy is selected and unselected, respectively. The four algorithms were compared on 30 test functions. Table 4 details the Avg and STD values obtained by the algorithms during the experiments. Similarly, the optimal values obtained under each function are marked in bold. Analysis of the table shows that SGHHO has the best solution accuracy out of the 30 functions and ranks first out of 19 functions. In addition, SGHHO is more stable than SHHO and GHHO in terms of STD values. This means that the SGHHO algorithm maxi- mizes the performance benefits of both strategies. Table 3. Experimental design of mechanism combinations. SA GB HHO 0 0 GHHO 0 1 SHHO 1 0 SGHHO 1 1 https://doi.org/10.1371/journal.pone.0291626.t003 Table 3. Experimental design of mechanism combinations. SA GB HHO 0 0 GHHO 0 1 SHHO 1 0 SGHHO 1 1 https://doi.org/10.1371/journal.pone.0291626.t003 Table 3. Experimental design of mechanism combinations. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 17 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Table 4. Optimization results of each HHO variants on IEEE CEC 2014 functions. PLOS ONE PLOS ONE Table 4. (Continued) F22 F25 F26 F27 AVG STD AVG STD AVG STD SGHHO 2.7000E+03 0.0000E+00 2.7004E+03 6.8531E-02 2.9000E+03 0.0000E+00 GHHO 2.7000E+03 0.0000E+00 2.7072E+03 2.5231E+01 3.5058E+03 2.8702E+02 SHHO 2.7000E+03 0.0000E+00 2.7004E+03 8.3865E-02 2.9000E+03 0.0000E+00 HHO 2.7000E+03 0.0000E+00 2.7768E+03 4.2799E+01 2.9000E+03 0.0000E+00 F28 F29 F30 AVG STD AVG STD AVG STD SGHHO 3.0000E+03 0.0000E+00 3.1130E+03 1.3423E+01 3.3504E+03 2.8724E+02 GHHO 3.2528E+03 6.4180E+01 3.1081E+03 3.3939E+00 3.7347E+03 3.4315E+02 SHHO 3.0000E+03 0.0000E+00 3.1214E+03 3.4282E+01 3.5270E+03 3.7261E+02 HHO 3.0000E+03 0.0000E+00 3.7090E+03 3.3357E+03 8.9845E+03 1.1111E+04 https://doi.org/10.1371/journal.pone.0291626.t004 The p-values for the Wilcoxon signed rank test at a 5% confidence level are presented in Table 5. The symbols "+/-/ = " are utilized to signify the outcomes of the comparison between SGHHO and the other methods. From the table, SGHHO outperforms HHO on 24 test func- tions; it outperforms the GHHO algorithm on 13 functions; and it outperforms the SHHO algorithm on 10 functions. The integration of the two strategies demonstrates a substantial enhancement in the performance of HHO, attaining its maximum potential in the SGHHO algorithm. Additionally, the Friedman test was employed to quantitatively assess the algo- rithm’s performance. According to the average ranking of the algorithms, the SGHHO algo- rithm secures the top position among all combinations, signifying a significant advancement in achieving optimal performance. Not only is there a significant improvement in global search, but there is also a significant breakthrough in local exploitation. Therefore, the experi- mental results in this section serve as evidence for the efficacy of the SA and GB strategies in enhancing the performance of the original HHO algorithm. 6.3. The impact of two mechanisms F1 F2 F3 AVG STD AVG STD AVG STD SGHHO 1.3004E+06 5.6615E+05 1.4639E+04 7.6388E+03 3.4932E+02 2.4514E+01 GHHO 1.2163E+07 1.2565E+07 7.5179E+03 2.4676E+04 3.1166E+02 2.3358E+01 SHHO 1.8151E+06 6.8871E+05 3.0546E+06 8.5831E+05 5.3915E+02 1.0844E+02 HHO 1.6147E+07 7.2324E+06 1.4291E+07 2.6541E+06 8.9513E+03 2.8344E+03 F4 F5 F6 AVG STD AVG STD AVG STD SGHHO 4.7760E+02 3.7443E+01 5.2005E+02 7.3211E-02 6.2164E+02 4.2301E+00 GHHO 4.9781E+02 5.0933E+01 5.2003E+02 1.2102E-01 6.2368E+02 3.2191E+00 SHHO 4.8327E+02 4.3772E+01 5.2080E+02 9.1891E-02 6.2115E+02 3.1850E+00 HHO 5.5426E+02 4.3131E+01 5.2037E+02 1.5976E-01 6.3095E+02 4.1016E+00 F7 F8 F9 AVG STD AVG STD AVG STD SGHHO 7.0005E+02 4.2950E-02 8.3916E+02 6.1689E+00 1.0734E+03 1.6391E+01 GHHO 7.0002E+02 2.6385E-02 8.6559E+02 1.4594E+01 1.0717E+03 2.6727E+01 SHHO 7.0106E+02 1.4102E-02 8.4128E+02 8.2343E+00 1.0778E+03 1.4698E+01 HHO 7.0112E+02 2.8802E-02 9.1381E+02 1.2948E+01 1.0917E+03 2.3763E+01 F10 F11 F12 AVG STD AVG STD AVG STD SGHHO 1.4347E+03 3.6411E+02 4.5081E+03 5.5811E+02 1.2005E+03 1.7728E-01 GHHO 1.9418E+03 5.2307E+02 4.7877E+03 6.1852E+02 1.2005E+03 2.2702E-01 SHHO 1.4634E+03 2.7151E+02 4.4575E+03 4.7779E+02 1.2007E+03 2.1117E-01 HHO 3.3418E+03 6.9532E+02 5.4497E+03 7.2502E+02 1.2017E+03 4.3400E-01 F13 F14 F15 AVG STD AVG STD AVG STD SGHHO 1.3004E+03 1.0094E-01 1.4002E+03 4.3851E-02 1.5113E+03 3.7681E+00 GHHO 1.3005E+03 1.1669E-01 1.4003E+03 1.5358E-01 1.5229E+03 8.9626E+00 SHHO 1.3003E+03 6.3199E-02 1.4002E+03 4.3706E-02 1.5149E+03 3.3449E+00 HHO 1.3005E+03 1.3686E-01 1.4002E+03 4.1103E-02 1.5379E+03 8.0988E+00 F16 F17 F18 AVG STD AVG STD AVG STD SGHHO 1.6118E+03 4.3066E-01 1.3856E+05 8.5489E+04 3.5586E+03 2.0875E+03 GHHO 1.6114E+03 6.4796E-01 1.2043E+06 1.3644E+06 7.4275E+03 5.6113E+03 SHHO 1.6119E+03 4.2748E-01 1.4767E+05 6.3563E+04 4.5221E+03 1.9100E+03 HHO 1.6124E+03 3.7341E-01 1.6351E+06 1.1048E+06 1.7217E+05 2.4618E+05 F19 F20 F21 AVG STD AVG STD AVG STD SGHHO 1.9156E+03 3.5495E+00 2.2927E+03 5.6645E+01 5.3399E+04 3.3235E+04 GHHO 1.9192E+03 1.3792E+01 2.4382E+03 3.8903E+02 3.3421E+05 5.1049E+05 SHHO 1.9173E+03 1.1870E+01 2.3228E+03 8.1051E+01 6.9415E+04 3.6753E+04 HHO 1.9379E+03 4.5628E+01 1.7912E+04 7.5829E+03 5.8874E+05 3.8244E+05 F22 F23 F24 AVG STD AVG STD AVG STD SGHHO 2.7440E+03 1.7939E+02 2.5000E+03 0.0000E+00 2.6000E+03 1.9498E-03 GHHO 2.7332E+03 1.7532E+02 2.5116E+03 3.5476E+01 2.6000E+03 1.6949E-04 SHHO 2.7791E+03 1.6018E+02 2.5000E+03 0.0000E+00 2.6000E+03 1.7597E-03 HHO 3.0371E+03 2.7336E+02 2.5000E+03 0.0000E+00 2.6000E+03 1.7617E-04 (Continued) Table 4. Optimization results of each HHO variants on IEEE CEC 2014 functions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 18 / 37 An enhanced decision-making framework for predicting future trends of sharing economy 6.4. Comparison with other reported well-known optimizers To verify the optimization performance of the SGHHO algorithm, 10 high-quality algorithms including BMWOA [110], CBA [111], EM [112], OBSCA [113], IGWO [114], MFO [115], ALPSO [116], CGPSO [117], SCADE [118] and HGWO [119] were selected for comparison. Furthermore, the IEEE CEC 2014 test set was utilized in the experiments to comprehensively evaluate the algorithms’ exploration and exploitation capabilities. To provide further insight into the performance comparisons, statistical tests such as the Wilcoxon rank sum test and the Friedman test were employed to assess the significance differences between SGHHO and the other algorithms. Furthermore, to allow experimental fairness, the whole algorithms were compared under the same settings, as well as each algorithm’s parameters were shown in Table 6. Table 7 shows AVG and STD values gained for all competitors on the set of functions tested. Where the optimal values obtained on each set of functions are bolded in order to allow more visual analysis of the gaps between the algorithms. The results in Table 7 demonstrate that SGHHO can reach the optimal solution of the function on 67% of the tested functions. In particular, on the unimodal function, SGHHO is only not optimal on the F2 function, second only to the ALSPSO and CBA algorithms. Compared to algorithms such as BMWOA, OBSCA, CGPSO and SCADE, the SGHHO showed strong competitiveness on the single-peak function. In addition, although the AVG values of SGHHO on the multimodal functions are not fully PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 19 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Table 5. The p-values of Wilcoxon test for HHO variants on the benchmark function. https://doi.org/10.1371/journal.pone.0291626.t006 6.4. Comparison with other reported well-known optimizers Function SGHHO GHHO SHHO HHO F1 N/A 2.1630E-05 1.1138E-03 1.7344E-06 F2 N/A 6.1564E-04 1.7344E-06 1.7344E-06 F3 N/A 1.9729E-05 1.7344E-06 1.7344E-06 F4 N/A 7.1903E-02 4.1653E-01 1.0246E-05 F5 N/A 6.1564E-04 1.7344E-06 1.7344E-06 F6 N/A 6.2683E-02 4.4052E-01 2.8786E-06 F7 N/A 4.5336E-04 1.7344E-06 1.7344E-06 F8 N/A 2.3534E-06 2.6230E-01 1.7344E-06 F9 N/A 7.4987E-01 3.0861E-01 3.8542E-03 F10 N/A 1.4839E-03 6.8836E-01 1.7344E-06 F11 N/A 1.5886E-01 6.7328E-01 4.4493E-05 F12 N/A 2.2102E-01 2.6134E-04 1.7344E-06 F13 N/A 2.8434E-05 7.9710E-01 2.4118E-04 F14 N/A 1.7344E-06 1.9152E-01 1.8910E-04 F15 N/A 3.1817E-06 2.1053E-03 1.7344E-06 F16 N/A 2.5637E-02 3.0861E-01 1.0246E-05 F17 N/A 3.8822E-06 4.2843E-01 1.7344E-06 F18 N/A 2.6134E-04 3.0010E-02 1.7344E-06 F19 N/A 1.1093E-01 9.9179E-01 4.7162E-02 F20 N/A 1.6503E-01 1.5286E-01 1.7344E-06 F21 N/A 6.1564E-04 7.5213E-02 1.9209E-06 F22 N/A 8.2901E-01 3.3886E-01 1.7423E-04 F23 N/A 2.5000E-01 1.0000E+00 1.0000E+00 F24 N/A 2.4118E-04 7.8647E-02 2.0859E-04 F25 N/A 1.0000E+00 1.0000E+00 1.0000E+00 F26 N/A 2.5967E-05 2.8948E-01 2.8786E-06 F27 N/A 1.2290E-05 1.0000E+00 1.0000E+00 F28 N/A 2.5631E-06 1.0000E+00 1.0000E+00 F29 N/A 1.2453E-02 3.1603E-02 3.1123E-05 F30 N/A 9.7110E-05 1.1079E-02 6.7328E-01 +/-/ = ~ 13/7/10 10/0/20 24/1/5 ARV 1.9689 2.4944 2.2961 3.2406 Rank 1 3 2 4 https://doi.org/10.1371/journal.pone.0291626.t005 Table 6. Parameter setting for 10 metaheuristics. Method Other parameters BMWOA b = 1; bw = 0.001; Beta = 0.1; G = MaxFES; CBA Cw = 3; fmax = 2.5 r1 = r2 = 0.5+rand(0,1); EM IndexLB = 0; LSITER = 5; delta = 0.1 OBSCA a = 1; r1 ¼ a fes∗ððaÞ=MaxFEsÞ; r2 ¼ ð2∗piÞ∗randðÞ; IGWO a ¼ 2 FEs∗ð2=MaxFEsÞ; A ¼ 2∗randð0; 1Þ∗a a; C ¼ 2∗randð0; 1Þ MFO b = 1; t = [−1, 1]; a2[−1,−2] ALCPSO ω = 0.4; c1 = c2 = 2.0; θ0 =60; T = 2 CGPSO w = 1; c1 = 2; c2 = 2 SCADE beta_min = 0.2; beta_max = 0.8; pCR = 0.8; a = 1; HGWO a2[0,−2]; beta_min = 0.2; beta_max = 0.8; pCR = 0.8; a = 1; https://doi.org/10.1371/journal.pone.0291626.t006 Table 5. The p-values of Wilcoxon test for HHO variants on the benchmark function. Table 6. Parameter setting for 10 metaheuristics. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 6.4. Comparison with other reported well-known optimizers Method Other parameters BMWOA b = 1; bw = 0.001; Beta = 0.1; G = MaxFES; CBA Cw = 3; fmax = 2.5 r1 = r2 = 0.5+rand(0,1); EM IndexLB = 0; LSITER = 5; delta = 0.1 OBSCA a = 1; r1 ¼ a fes∗ððaÞ=MaxFEsÞ; r2 ¼ ð2∗piÞ∗randðÞ; IGWO a ¼ 2 FEs∗ð2=MaxFEsÞ; A ¼ 2∗randð0; 1Þ∗a a; C ¼ 2∗randð0; 1Þ MFO b = 1; t = [−1, 1]; a2[−1,−2] ALCPSO ω = 0.4; c1 = c2 = 2.0; θ0 =60; T = 2 CGPSO w = 1; c1 = 2; c2 = 2 SCADE beta_min = 0.2; beta_max = 0.8; pCR = 0.8; a = 1; HGWO a2[0,−2]; beta_min = 0.2; beta_max = 0.8; pCR = 0.8; a = 1; https://doi.org/10.1371/journal.pone.0291626.t006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 20 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Table 7. Comparative results for SGHHO and other reported methods on the benchmark function. 6.4. Comparison with other reported well-known optimizers F1 F2 F3 F4 F5 AVG STD AVG STD AVG STD AVG STD AVG STD SGHHO 1.086E+06 4.525E+05 1.794E+04 1.095E+04 3.466E+02 2.632E+01 4.807E+02 4.176E+01 5.200E+02 4.855E-02 BMWOA 1.328E+08 6.652E+07 5.999E+08 2.419E+08 5.935E+04 1.028E+04 7.335E+02 8.664E+01 5.210E+02 9.057E-02 CBA 4.367E+06 1.501E+06 1.274E+04 8.683E+03 3.622E+03 2.818E+03 5.052E+02 4.222E+01 5.201E+02 1.542E-01 EM 2.868E+07 7.863E+06 5.761E+08 1.110E+08 1.653E+04 4.224E+03 6.235E+02 5.650E+01 5.209E+02 4.482E-02 OBSCA 4.472E+08 1.244E+08 2.451E+10 5.098E+09 5.196E+04 6.644E+03 2.273E+03 7.249E+02 5.210E+02 4.437E-02 IGWO 1.735E+07 6.680E+06 2.516E+06 1.072E+06 6.642E+03 2.756E+03 5.287E+02 2.648E+01 5.205E+02 1.276E-01 MFO 9.473E+07 8.813E+07 1.271E+10 7.749E+09 9.362E+04 5.512E+04 1.833E+03 1.816E+03 5.203E+02 1.839E-01 ALCPSO 4.340E+06 2.918E+06 2.101E+03 2.811E+03 3.908E+02 2.213E+02 5.224E+02 4.300E+01 5.208E+02 5.500E-02 CGPSO 9.345E+06 2.153E+06 1.593E+08 1.619E+07 2.287E+03 4.542E+02 4.691E+02 3.582E+01 5.210E+02 4.413E-02 SCADE 4.776E+08 9.976E+07 3.008E+10 4.162E+09 5.478E+04 6.607E+03 2.345E+03 4.389E+02 5.209E+02 7.264E-02 HGWO 1.796E+08 5.143E+07 8.579E+09 1.883E+09 6.628E+04 4.914E+03 9.308E+02 6.883E+01 5.208E+02 1.093E-01 F6 F7 F8 F9 F10 AVG STD AVG STD AVG STD AVG STD AVG STD SGHHO 6.206E+02 3.914E+00 7.001E+02 3.251E-02 8.420E+02 7.975E+00 1.075E+03 1.949E+01 1.501E+03 3.661E+02 BMWOA 6.341E+02 4.517E+00 7.057E+02 1.305E+00 9.749E+02 2.579E+01 1.132E+03 3.249E+01 5.219E+03 6.643E+02 CBA 6.396E+02 2.693E+00 7.000E+02 9.437E-03 1.010E+03 4.488E+01 1.183E+03 6.175E+01 5.551E+03 7.137E+02 EM 6.240E+02 2.478E+00 7.088E+02 2.053E+00 9.002E+02 1.453E+01 1.063E+03 1.789E+01 3.103E+03 5.394E+02 OBSCA 6.318E+02 1.320E+00 9.185E+02 3.618E+01 1.061E+03 1.842E+01 1.194E+03 1.933E+01 6.350E+03 5.035E+02 IGWO 6.199E+02 2.338E+00 7.010E+02 6.772E-02 8.845E+02 2.082E+01 1.021E+03 2.368E+01 3.348E+03 4.702E+02 MFO 6.244E+02 4.195E+00 8.363E+02 7.093E+01 9.478E+02 5.108E+01 1.121E+03 5.376E+01 4.309E+03 9.934E+02 ALCPSO 6.169E+02 2.935E+00 7.000E+02 2.268E-02 8.242E+02 1.068E+01 9.993E+02 2.213E+01 1.542E+03 3.599E+02 CGPSO 6.248E+02 2.871E+00 7.024E+02 1.693E-01 9.879E+02 2.013E+01 1.123E+03 2.248E+01 5.565E+03 6.705E+02 SCADE 6.341E+02 3.013E+00 9.135E+02 3.852E+01 1.067E+03 1.678E+01 1.207E+03 1.723E+01 7.397E+03 3.385E+02 HGWO 6.264E+02 1.716E+00 7.464E+02 1.277E+01 1.008E+03 1.311E+01 1.140E+03 1.135E+01 5.559E+03 3.283E+02 F11 F12 F13 F14 F15 AVG STD AVG STD AVG STD AVG STD AVG STD SGHHO 4.601E+03 5.183E+02 1.200E+03 1.434E-01 1.300E+03 9.774E-02 1.400E+03 4.411E-02 1.509E+03 2.665E+00 BMWOA 7.192E+03 5.139E+02 1.202E+03 4.884E-01 1.301E+03 1.220E-01 1.400E+03 1.845E-01 1.625E+03 7.009E+01 CBA 5.653E+03 7.579E+02 1.201E+03 5.669E-01 1.300E+03 1.295E-01 1.400E+03 1.858E-01 1.565E+03 1.679E+01 EM 4.839E+03 4.537E+02 1.201E+03 4.240E-01 1.300E+03 5.764E-02 1.400E+03 2.608E-02 1.531E+03 3.685E+00 OBSCA 7.284E+03 3.851E+02 1.202E+03 3.833E-01 1.304E+03 3.701E-01 1.472E+03 1.598E+01 1.481E+04 6.757E+03 IGWO 4.401E+03 7.100E+02 1.201E+03 4.191E-01 1.301E+03 1.118E-01 1.400E+03 3.082E-01 1.517E+03 5.274E+00 MFO 5.250E+03 7.927E+02 1.200E+03 2.517E-01 1.302E+03 1.398E+00 1.435E+03 2.782E+01 8.370E+04 1.388E+05 ALCPSO 4.137E+03 5.398E+02 1.201E+03 3.961E-01 1.301E+03 9.528E-02 1.401E+03 3.066E-01 1.511E+03 3.216E+00 CGPSO 5.803E+03 5.953E+02 1.202E+03 3.131E-01 1.300E+03 8.783E-02 1.400E+03 1.130E-01 1.518E+03 1.330E+00 SCADE 8.162E+03 2.648E+02 1.203E+03 3.130E-01 1.304E+03 2.754E-01 1.488E+03 1.520E+01 1.759E+04 6.057E+03 HGWO 6.535E+03 4.576E+02 1.201E+03 2.522E-01 1.302E+03 4.605E-01 1.422E+03 3.569E+00 1.934E+03 3.043E+02 F16 F17 F18 F19 F20 AVG STD AVG STD AVG STD AVG STD AVG STD SGHHO 1.612E+03 5.177E-01 1.463E+05 9.728E+04 3.516E+03 1.919E+03 1.916E+03 3.787E+00 2.290E+03 5.894E+01 BMWOA 1.613E+03 2.869E-01 6.923E+06 4.111E+06 5.517E+05 1.254E+06 1.952E+03 3.466E+01 4.331E+04 3.720E+04 CBA 1.613E+03 3.189E-01 2.449E+05 1.928E+05 8.575E+03 8.575E+03 1.941E+03 4.039E+01 3.470E+03 2.426E+03 EM 1.612E+03 4.422E-01 7.659E+05 1.916E+05 7.856E+07 6.276E+07 1.943E+03 3.484E+01 2.726E+03 1.957E+02 OBSCA 1.613E+03 1.832E-01 1.054E+07 4.426E+06 1.569E+08 1.014E+08 2.007E+03 2.367E+01 3.371E+04 1.525E+04 IGWO 1.612E+03 5.580E-01 1.043E+06 5.466E+05 2.039E+04 2.097E+04 1.924E+03 2.388E+01 3.286E+03 1.047E+03 MFO 1.613E+03 7.325E-01 3.342E+06 5.364E+06 4.243E+07 1.490E+08 1.976E+03 6.124E+01 6.043E+04 2.962E+04 (Continued) Table 7. 6.4. Comparison with other reported well-known optimizers Comparative results for SGHHO and other reported methods on the benchmark function. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 21 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Table 7. (Continued) F1 F2 F3 F4 F5 AVG STD AVG STD AVG STD AVG STD AVG STD ALCPSO 1.612E+03 4.627E-01 5.542E+05 5.301E+05 8.337E+03 6.019E+03 1.918E+03 2.354E+01 3.111E+03 6.863E+02 CGPSO 1.612E+03 4.590E-01 3.628E+05 1.930E+05 2.436E+06 6.933E+05 1.917E+03 2.717E+00 2.455E+03 1.055E+02 SCADE 1.613E+03 2.184E-01 1.534E+07 8.361E+06 1.753E+08 1.209E+08 2.013E+03 1.193E+01 2.640E+04 1.239E+04 HGWO 1.613E+03 2.712E-01 6.067E+06 2.778E+06 1.196E+08 3.500E+07 1.988E+03 1.025E+01 6.563E+04 3.160E+04 F21 F22 F23 F24 F25 AVG STD AVG STD AVG STD AVG STD AVG STD SGHHO 6.176E+04 3.141E+04 2.738E+03 1.719E+02 2.500E+03 0.000E+00 2.600E+03 4.603E-03 2.700E+03 0.000E+00 BMWOA 1.655E+06 1.812E+06 2.957E+03 2.393E+02 2.501E+03 6.967E-01 2.600E+03 1.442E-01 2.700E+03 9.742E-03 CBA 1.050E+05 7.051E+04 3.464E+03 3.632E+02 2.616E+03 2.308E-01 2.684E+03 4.089E+01 2.732E+03 1.379E+01 EM 1.685E+05 9.812E+04 2.811E+03 1.953E+02 2.572E+03 4.072E+01 2.604E+03 4.159E-01 2.701E+03 9.181E-02 OBSCA 2.434E+06 1.594E+06 3.153E+03 1.369E+02 2.680E+03 1.701E+01 2.600E+03 4.710E-04 2.700E+03 1.642E-03 IGWO 3.287E+05 2.600E+05 2.537E+03 1.604E+02 2.621E+03 2.823E+00 2.600E+03 5.320E-03 2.709E+03 1.685E+00 MFO 1.285E+06 2.537E+06 3.037E+03 2.217E+02 2.659E+03 3.606E+01 2.683E+03 2.886E+01 2.715E+03 7.177E+00 ALCPSO 8.151E+04 8.307E+04 2.682E+03 1.795E+02 2.615E+03 4.024E-02 2.638E+03 7.279E+00 2.711E+03 3.859E+00 CGPSO 1.369E+05 9.974E+04 2.919E+03 2.370E+02 2.500E+03 2.528E-03 2.600E+03 9.937E-03 2.700E+03 2.201E-05 SCADE 2.792E+06 1.574E+06 3.122E+03 1.488E+02 2.500E+03 0.000E+00 2.600E+03 1.865E-06 2.700E+03 0.000E+00 HGWO 2.214E+06 1.608E+06 3.007E+03 1.170E+02 2.522E+03 5.759E+01 2.600E+03 0.000E+00 2.700E+03 0.000E+00 F26 F27 F28 F29 F30 AVG STD AVG STD AVG STD AVG STD AVG STD SGHHO 2.704E+03 1.819E+01 2.900E+03 0.000E+00 3.000E+03 0.000E+00 3.109E+03 4.734E+00 3.500E+03 3.934E+02 BMWOA 2.701E+03 1.442E-01 2.900E+03 1.096E-01 3.000E+03 2.858E-01 9.269E+05 2.066E+06 3.734E+04 3.379E+04 CBA 2.711E+03 5.526E+01 4.043E+03 3.863E+02 5.597E+03 8.197E+02 4.368E+07 4.752E+07 2.272E+04 2.818E+04 EM 2.788E+03 3.144E+01 3.641E+03 2.867E+02 5.798E+03 1.336E+03 1.024E+07 1.936E+07 1.089E+04 3.102E+03 OBSCA 2.704E+03 5.668E-01 3.249E+03 4.240E+01 5.512E+03 3.591E+02 2.242E+07 1.211E+07 3.806E+05 1.293E+05 IGWO 2.701E+03 1.578E-01 3.109E+03 4.281E+00 3.797E+03 9.108E+01 2.356E+06 4.836E+06 2.823E+04 1.190E+04 MFO 2.703E+03 1.141E+00 3.607E+03 2.188E+02 3.914E+03 2.249E+02 2.048E+06 3.486E+06 4.859E+04 4.239E+04 ALCPSO 2.747E+03 5.066E+01 3.399E+03 2.389E+02 4.546E+03 4.293E+02 3.718E+06 7.262E+06 1.410E+04 1.036E+04 CGPSO 2.797E+03 1.820E+01 3.014E+03 2.960E+02 3.000E+03 9.194E-03 5.970E+03 3.022E+03 1.113E+04 8.882E+03 SCADE 2.707E+03 1.758E+01 3.242E+03 1.982E+02 4.906E+03 1.018E+03 1.637E+07 7.805E+06 4.419E+05 1.544E+05 HGWO 2.743E+03 4.926E+01 3.552E+03 2.675E+02 4.214E+03 2.270E+02 3.768E+06 3.657E+06 3.200E+03 1.731E-04 https://doi.org/10.1371/journal.pone.0291626.t007 optimal, there is no significant difference compared to the optimal solution for each function and the average AVG ranking is better. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 6.4. Comparison with other reported well-known optimizers On the hybrid functions, SGHHO can search for opti- mal solutions for most functions compared to other advanced algorithms. A comparison to this can be clearly seen in the F10-F20 functions. Also, using the Friedman test, this study com- pares the combined strength of SGHHO and these competing algorithms. As the data at the end of Table 8 indicates, SGHHO achieves the first overall average rank- ing in processing the CEC 2014 function set, followed by algorithms including ALCPSO, IGWO and CGPSO. Besides, according to the Wilcoxon test, the p-value of SGHHO is mostly less than 0.05 compared to other methods, which demonstrates the significant difference between the SGHHO algorithm compared to other competitors. It validates that the SGHHO algorithm, which incorporates the Gaussian Bare-Bones strategy and the simulated annealing strategy, is a more promising algorithm for different types of optimization problems, and that SGHHO significantly improves HHO’s optimization power. Fig 3 illustrates the convergence curves of SGHHO in comparison to other state-of-the-art algorithms on the CEC 2014 test suite. The convergence curves provide a more intuitive 22 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Table 8. P -values for SGHHO and other reported algorithms by Wilcoxon test on the benchmark function. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 6.4. Comparison with other reported well-known optimizers Function SGHHO BMWOA CBA EM OBSCA IGWO MFO ALCPSO CGPSO SCADE HGWO F1 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.8E-05 1.7E-06 1.7E-06 1.7E-06 F2 N/A 1.7E-06 7.2E-02 1.7E-06 1.7E-06 1.7E-06 1.7E-06 2.4E-06 1.7E-06 1.7E-06 1.7E-06 F3 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.6E-01 1.7E-06 1.7E-06 1.7E-06 F4 N/A 1.7E-06 2.3E-02 1.7E-06 1.7E-06 5.3E-05 1.7E-06 8.9E-04 3.3E-01 1.7E-06 1.7E-06 F5 N/A 1.7E-06 7.036E-01 1.7E-06 1.7E-06 1.7E-06 3.2E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 F6 N/A 1.7E-06 1.7E-06 1.2E-03 1.7E-06 3.8E-01 6.0E-03 1.2E-03 2.6E-05 1.7E-06 1.7E-06 F7 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.1E-05 1.7E-06 1.7E-06 1.7E-06 F8 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.9E-06 1.7E-06 1.4E-05 1.7E-06 1.7E-06 1.7E-06 F9 N/A 2.9E-06 1.9E-06 2.5E-02 1.7E-06 3.2E-06 2.1E-04 1.7E-06 4.3E-06 1.7E-06 1.7E-06 F10 N/A 1.7E-06 1.7E-06 1.9E-06 1.7E-06 1.7E-06 1.7E-06 9.1E-01 1.7E-06 1.7E-06 1.7E-06 F11 N/A 1.7E-06 2.2E-05 4.5E-02 1.7E-06 1.4E-01 2.3E-03 3.6E-03 2.4E-06 1.7E-06 1.7E-06 F12 N/A 1.7E-06 2.4E-06 2.4E-06 1.7E-06 2.9E-03 5.6E-01 2.6E-06 1.7E-06 1.7E-06 1.7E-06 F13 N/A 1.7E-06 1.5E-05 3.3E-04 1.7E-06 1.9E-06 1.7E-06 2.6E-06 1.1E-05 1.7E-06 1.7E-06 F14 N/A 1.7E-06 2.4E-06 9.1E-01 1.7E-06 1.4E-05 1.7E-06 1.734E-06 4.5E-04 1.7E-06 1.7E-06 F15 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 2.6E-06 1.9E-06 8.6E-02 1.7E-06 1.7E-06 1.7E-06 F16 N/A 2.3E-06 1.7E-06 2.6E-03 1.7E-06 2.2E-01 9.3E-06 4.0E-01 1.6E-01 1.7E-06 1.7E-06 F17 N/A 1.7E-06 1.2E-02 1.7E-06 1.7E-06 1.734E-06 2.8E-05 4.2E-04 3.1E-05 1.7E-06 1.7E-06 F18 N/A 1.7E-06 8.3E-04 1.7E-06 1.7E-06 8.5E-06 2.3E-06 3.8E-04 1.7E-06 1.7E-06 1.7E-06 F19 N/A 1.9E-06 6.6E-04 2.6E-02 1.7E-06 1.5E-01 2.8E-05 2.2E-02 2.8E-01 1.7E-06 1.7E-06 F20 N/A 1.7E-06 4.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 2.6E-06 2.3E-06 1.7E-06 1.7E-06 F21 N/A 1.7E-06 1.6E-03 9.3E-06 1.7E-06 1.7E-06 1.6E-05 4.9E-01 3.7E-05 1.7E-06 1.7E-06 F22 N/A 4.9E-04 2.3E-06 1.3E-01 1.7E-06 1.7E-04 5.8E-06 2.1E-01 3.6E-03 3.2E-06 4.7E-06 F23 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.0E+00 6.3E-02 F24 N/A 1.7E-06 1.7E-06 1.7E-06 8.9E-02 3.3E-04 1.7E-06 1.7E-06 1.7E-06 1.1E-05 3.8–06 F25 N/A 1.7E-06 1.7E-06 1.7E-06 6.3E-02 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.0E+00 1.0E+00 F26 N/A 7.5E-05 2.8E-02 1.7E-06 3.112E-05 3.4E-05 3.112E-05 2.163E-05 2.1E-06 2.8E-05 1.1E-05 F27 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.8E-05 2.6E-06 F28 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.8E-05 1.7E-06 F29 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.6E-04 F30 N/A 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 1.7E-06 2.6E-04 1.7E-06 1.7E-06 +/-/ = ~ 29/1/0 27/1/2 27/1/2 28/0/2 23/3/4 28/1/1 19/5/6 27/0/3 27/1/2 26/2/2 ARV 2.1461 6.8489 6.0111 5.7344 9.0017 4.5122 7.1156 3.9567 4.9189 8.7606 6.9939 Rank 1 7 6 5 11 3 9 2 4 10 8 https://doi.org/10.1371/journal.pone.0291626.t008 picture of the difference in convergence quality and optimization capability between SGHHO and the competitors. 6.4. Comparison with other reported well-known optimizers The figure clearly demonstrates that SGHHO exhibits faster convergence rates for the majority of functions. For example, F1, F10, F16, F29 and F30. This is because SGHHO utilizes the Gaussian Bare Bones strategy for individual variation and can expand the global search early in the evolutionary process. Furthermore, an observation can be made from the results of F3, F5, F18, and F21, which indicate that even though SGHHO may not exhibit the fastest early convergence, it manages to sustain its progress towards the optimal value after ALPSO, CBA, and CGPSO have reached convergence. This signifies the ability of SGHHO to effectively prevent being trapped in local optima. Also, SGHHO converges rapidly in the later evolutionary stage through SA mechanism, and eventually achieves higher preci- sion and better solutions in the search process when compared with other comparative algo- rithms. This makes algorithms such as OBSCA, ALCPSO, BMWOA and SCADE fall into premature convergence, while SGHHO can maintain the convergence trend until the global picture of the difference in convergence quality and optimization capability between SGHHO and the competitors. The figure clearly demonstrates that SGHHO exhibits faster convergence rates for the majority of functions. For example, F1, F10, F16, F29 and F30. This is because SGHHO utilizes the Gaussian Bare Bones strategy for individual variation and can expand the global search early in the evolutionary process. Furthermore, an observation can be made from the results of F3, F5, F18, and F21, which indicate that even though SGHHO may not exhibit the fastest early convergence, it manages to sustain its progress towards the optimal value after ALPSO, CBA, and CGPSO have reached convergence. This signifies the ability of SGHHO to effectively prevent being trapped in local optima. Also, SGHHO converges rapidly in the later evolutionary stage through SA mechanism, and eventually achieves higher preci- sion and better solutions in the search process when compared with other comparative algo- rithms. This makes algorithms such as OBSCA, ALCPSO, BMWOA and SCADE fall into premature convergence, while SGHHO can maintain the convergence trend until the global PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 23 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE Fig 3. Convergence curves for SGHHO and advanced algorithms on 9 selected benchmark functions. https://doi org/10 1371/journal pone 0291626 g003 Fig 3. Convergence curves for SGHHO and advanced algorithms on 9 selected benchmark functions. 6.4. Comparison with other reported well-known optimizers https://doi.org/10.1371/journal.pone.0291626.g003 Fig 3. Convergence curves for SGHHO and advanced algorithms on 9 selected benchmark functions. https://doi.org/10.1371/journal.pone.0291626.g003 ttps://doi.org/10.1371/journal.pone.0291626.g003 https://doi.org/10.1371/journal.pone.0291626.g003 optimum. SGHHO demonstrates a remarkable capability to effectively maintain a balance between exploration and exploitation throughout the search process, thereby rejuvenating the evolution of the population. This ability proves instrumental in mitigating the issue of prema- ture convergence that is commonly encountered in HHO. A comparison of the mean, standard deviation values, Wilcoxon test and convergence fig- ures leads to the conclusion that the proposed SGHHO significantly outperforms other com- peting algorithms. It can consistently maintain a leading position in function optimization problems, and the advantage of SGHHO becomes more obvious as the dimensionality of the problem increases. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 24 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy Hence, in subsequent investigations, this approach can be extended to a broader range of scenarios, such as optimization of machine learning models [120], computer-aided medical diagnosis [121,122], pathology image segmentation [123–125], image denoising [126,127], fine-grained alignment [128], cancer diagnosis [129–131], medical signals [132,133], and structured sparsity optimization [134]. 7.2. Condition configuration The experiments involved in this work were completed on a host computer with 16GB of RAM, 3.6GHz main frequency, Windows 10 and coded on MATLAB R2016b software. In addition, the experimental parameters are extremely important to the simulation and subtle changes in the parameters can affect the final experimental outcome. To ensure the fairness of the study, the proposed models were quantitatively evaluated using statistical techniques including the calculation of average values (AVG) and standard deviations (STD). The AVG represents the average predictive performance of each model, while the STD indicates the degree of variation across 10 independent runs. To further assess the effectiveness of the pro- posed models, four commonly used classification metrics were employed, namely Accuracy (ACC), Sensitivity, Specificity, and Matthew’s correlation coefficient (MCC). 7.1. Methods & data collection The sharing economy dataset is collected for this study and deals with data mainly from partic- ipants in the general public who have participated in the sharing economy. From these actual participants, 671 participants were selected for the study. The questionnaire was developed with reference to numerous scholars’ questionnaires on the sharing economy and predicted to have good reliability and validity. The questionnaire analyses 43 aspects of the respondents by examining their gender, education level, political status, age, profession, location, income level, consumption level, various forms of sharing economy and their perception of sharing economy (see Table 9). This study examines the basic situation of the public’s participation in sharing economy, explores the importance of these attributes and their inherent linkages, and builds a predictive model of future trends in the sharing economy on this basis. Since the above-mentioned questionnaires did not involve ethical issues, the review com- mittee/ethics committee of Wenzhou University granted an exemption from ethical review. All participants in the questionnaires signed a consent form. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 7.3. Experimental results and analysis of SGHHO-KNN To investigate the key factors influencing the future development of sharing economy, this experiment uses a variety of machine learning models to make predictions from a real dataset collected, including BSGHHO-KNN, BSHHO-KNN, BGHHO-KNN, BP, RF, ELM and other classification models. Meanwhile, the classification results of each model will be statistically analyzed using four indicators, namely ACC, Sensitivity, Specificity and MCC. Furthermore, the comprehensive experimental findings are disclosed in Table 10, providing detailed insights into the results obtained. And it can be intuitively found that the BSGHHO-KNN model obtained 99.70%—the highest value of accuracy in predicting the future development trend of sharing economy, while the other five models were 98.66%, 93.45%, 51.56%, 99.11% and 65.73% respectively. While the BSHHO-KNN and RF models demonstrated satisfactory accu- racy, it is worth noting that the BSGHHO-KNN model exhibited superior performance in terms of the Specificity and MCC metrics, surpassing both of them and attaining the highest PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 25 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy An enhanced decision-making framework for predicting future trends of sharing economy Table 9. Descriptions of each attribute. ID Attributes Description F1 Gender Male and female participants are represented by 1 and 2 respectively. F2 Education level The categories are classified into various levels of education, including doctoral, master’s, bachelor’s, college, and high school or below. These levels are denoted by numerical values 1, 2, 3, 4, and 5, respectively. F3 Political status The classification is categorized into four groups: members of the Communist Party, reserve party members, members of the Communist Youth League, and the general population. These categories are symbolized by the numerical values 1, 2, 3, and 4, respectively. F4 Age It is divided into under 15 years of age, 15–30 years of age, 31–35 years of age, 46–60 years of age, and 61 years of age and above denoted by 1, 2, 3, 4 and 5 respectively. F5 Professional category It is divided into law, engineering, management, education, economics, science, literature, medicine and art, indicated by 1, 2, 3, 4, 5, 6, 7, 8 and 9 respectively. F6 Region It is divided into North China, East China, Central China, South China, Northeast China, represented by 1, 2, 3, 4, 5, 6 and 7 respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 7.3. Experimental results and analysis of SGHHO-KNN F7 Form of sharing economy in your region Multiple choice types of car sharing, bicycle sharing, electric bicycle sharing and umbrella sharing. F8 Monthly consumption level Classified as below RMB1,000, RMB1,000–3,000, RMB3,001– 5,000, RMB5,001–8,000, and above RMB8,001, represented by 1, 2, 3, 4 & 5 respectively. F9 Annual household income Divided into less than $50,000, $50,000-$100,000. . .$400,000- $500,000, and over $500,000 represented by 1, 2, 3, 4, 5, 6, and 7 respectively. F10 Occupation The categories are students, teachers, doctors, civil servants, company employees, and others, represented by 1, 2, 3, 4, 5, 6, 7 and 8 respectively. F11 Awareness of the sharing economy There are five categories: not at all, basically, extremely, researched and researched in depth, represented by 1, 2, 3, 4 and 5 respectively. F12 Awareness of the sharing economy The response options are 1, 2, 3, 4 and 5 respectively. F13 How did you first learn about the sharing economy? Media coverage, shared equipment, books and articles, people around, and other are represented by 1, 2, 3, 4, and 5 respectively. F14 Whether or not you have used sharing economy services No, yes, indicated by 0, 1 respectively. F15 Frequency of use of shared products Not at all, Occasional use, Regular use, indicated by 1, 2, 3 respectively. F16 How much you pay for sharing economy products Cheap, fair, barely acceptable, expensive, indicated by 1, 2, 3, 4 respectively. F17 Willingness to learn about the sharing economy No, yes, indicated by 0, 1 respectively. F18 Whether they are excluded from using shared products and services No, yes, indicated by 0, 1 respectively. F19 Willingness to participate in the sharing economy as a provider No, yes, indicated by 0, 1 respectively. F20 Perceived most important features of the sharing economy Response options are indicated by 1, 2, 3, 4 and 5 respectively. F21 Perception of which consumer group the sharing product would be most helpful to The categories are students, teachers, doctors, civil servants, company employees, management personnel, freelancers and others, represented by 1, 2, 3, 4, 5, 6, 7 and 8 respectively. (Continued) Table 9. Descriptions of each attribute. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 26 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy Table 9. 7.3. Experimental results and analysis of SGHHO-KNN (Continued) ID Attributes Description F22 Products that have been used in the sharing sector Car-sharing, bicycle-sharing, electric bicycle-sharing, umbrella-sharing and other multiple-choice types. F23 Factors that concern you most when using shared product services Quality of service, price of service, ease of access to service, personal privacy issues, personal safety issues, other, represented by 1, 2, 3, 4, 5, 6 respectively. F24 Impact on life and work No impact, low impact, average impact, high impact, high impact, indicated by 1, 2, 3, 4 respectively. F25 Whether the sharing economy has brought convenience No, yes, indicated by 0, 1 respectively. F26 Key aspects of convenience brought about by the sharing economy Price, time, efficiency, convenience, experience, indicated by 1, 2, 3, 4, 5 respectively. F27 Whether or not the sharing economy is a nuisance No, yes, indicated by 0, 1 respectively. F28 Key aspects of distress caused by the sharing economy Answer options are indicated by 1, 2, 3, 4, 5, 6, 7 respectively. F29 Causes of distress in the sharing economy Answer options are indicated by 1, 2, 3, 4, 5, 6, 7 respectively. F30 Attitudes to the sharing economy Answer options are denoted by 1, 2, 3, 4, 5 respectively F31 The main negative aspects of the development of the sharing economy in the region Answer options are indicated by 1, 2, 3, 4, 5 F32 Positive factors for the development of the sharing economy in the region Answer options are indicated by 1, 2, 3, 4 and 5 respectively F33 Perception of the future of “sharing economy” Very unfavorable, rather unfavorable, average, rather favorable, very favorable, indicated by 1, 2, 3, 4, 5 respectively F34 Factors contributing to consumer demand for shared products Risk aversion, supply shocks, income instability, demand contraction, expectations weakening, other indicated by 1, 2, 3, 4, 5, 6 respectively F35 Main concerns about the sharing economy The available choices for answers are as follows by 1, 2, 3, 4, 5, 6, 7 respectively F36 Confidence in the sharing economy The available choices for answers are as follows by 1, 2, 3, 4, 5, 6 respectively. F37 Perceived advantages of the sharing economy The available choices for answers are as follows by 1, 2, 3, 4, 5, 6, 7 and 8 respectively. PLOS ONE From the figure: "form of sharing economy in the region" (F7), "attitude towards the sharing economy" (F30), "products in the sharing domain that have been used" (F22), "Factors of greatest concern when using shared product services" (F23), "Main aspects of distress caused by the sharing economy" (F28), "Neg- ative effects of the sharing economy on society " (F39), "Main aspects of concerns about the sharing economy" (F35) and "Any suggestions for the development of the sharing economy" Table 10. Average results of BSGHHO-KNN and other models on four indicators. Models ACC Sensitivity Specificity MCC Avg BSGHHO-KNN 99.70% 100.00% 99.38% 99.42% BSHHO-KNN 98.66% 100.00% 97.19% 97.52% BGHHO-KNN 93.45% 98.01% 88.44% 87.31% BP 51.56% 16.29% 90.00% 10.69% RF 99.11% 98.86% 99.38% 98.24% ELM 65.73% 71.50% 59.38% 31.50% https://doi.org/10.1371/journal.pone.0291626.t010 Table 11. Standard deviation values for BSGHHO-KNN and the other five models on the four indicators. Models ACC Sensitivity Specificity MCC Table 10. Average results of BSGHHO-KNN and other models on four indicators. bolded for more visual analysis of the experimental data. After analyzing the results, it can be deduced that the BSGHHO-KNN model demonstrates exceptional stability in terms of ACC, sensitivity, and MCC, surpassing other models in these aspects. In terms of specificity indica- tors, RF model gains the best value. To better compare the performance gap between the models, Fig 4 presents the histograms of AVG and STD values for the five models mentioned above. The examination of Fig 4 reveals that the BSGHHO-KNN model exhibits superior performance across all four indicators, namely ACC, Sensitivity, Specificity, and MCC, resulting in the most effective classification outcome. Following closely behind are the RF model and the BSHHO-KNN model. The figure shows that BSHHO-KNN and BGHHO-KNN both have been effective compared to the other classifiers. The experiments show that the HHO algorithm has excellent adaptability in combi- nation with the KNN classifier. In addition, the BP model performed the worst on the sharing economy dataset. The ACC value was only 51.56% and the Sensitivity was 16.29%. This sug- gests that the BP model needs to be tuned with appropriate parameters for the specific prob- lem. In addition, the BSHHO-KNN model and the BGHHO-KNN model do not perform the best on the dataset, which means that the BSGHHO algorithm can enable the KNN classifier to maximize its classification performance and thus achieve better classification results. 7.3. Experimental results and analysis of SGHHO-KNN F38 Positive impacts of the sharing economy on society The available choices for answers are as follows by 1, 2, 3, 4, 5, 6, 7 respectively F39 Negative effects of the sharing economy on society The available choices for answers are as follows by 1, 2, 3, 4 respectively F40 Overall social effects of the sharing economy The disadvantages outweigh the advantages and the advantages outweigh the disadvantages are represented by 0 and 1 respectively. F41 Ways in which people can contribute to the sharing economy Active promotion of a sharing atmosphere, acceptance of the sharing economy with an open mind, active participation, others indicated by 1, 2, 3, 4 respectively F42 Views on the future of sharing economy 0, 1 for only a temporary wave, 1 for a wider range of developments F43 Any suggestions for the development of the sharing economy Answer options are 1, 2, 3, 4, 5 and 6 respectively https://doi.org/10.1371/journal.pone.0291626.t009 classification outcomes. Based on the above four evaluation metrics it can be fully demon- strated that the BSGHHO-KNN model is feasible to be applied to the problem of predicting future trends in the sharing economy. classification outcomes. Based on the above four evaluation metrics it can be fully demon- strated that the BSGHHO-KNN model is feasible to be applied to the problem of predicting future trends in the sharing economy. Table 11 shows the standard deviation values derived from the BSGHHO-KNN model and the other five models after 10 independent experiments. The optimal values in the table are PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 27 / 37 An enhanced decision-making framework for predicting future trends of sharing economy PLOS ONE PLOS ONE Table 10. Average results of BSGHHO-KNN and other models on four indicators. Models ACC Sensitivity Specificity MCC Avg BSGHHO-KNN 99.70% 100.00% 99.38% 99.42% BSHHO-KNN 98.66% 100.00% 97.19% 97.52% BGHHO-KNN 93.45% 98.01% 88.44% 87.31% BP 51.56% 16.29% 90.00% 10.69% RF 99.11% 98.86% 99.38% 98.24% ELM 65.73% 71.50% 59.38% 31.50% https://doi.org/10.1371/journal.pone.0291626.t010 bolded for more visual analysis of the experimental data. After analyzing the results, it can be deduced that the BSGHHO-KNN model demonstrates exceptional stability in terms of ACC, sensitivity, and MCC, surpassing other models in these aspects. In terms of specificity indica- tors, RF model gains the best value. To better compare the performance gap between the models, Fig 4 presents the histograms of AVG and STD values for the five models mentioned above. The examination of Fig 4 reveals that the BSGHHO-KNN model exhibits superior performance across all four indicators, namely ACC, Sensitivity, Specificity, and MCC, resulting in the most effective classification outcome. Following closely behind are the RF model and the BSHHO-KNN model. The figure shows that BSHHO-KNN and BGHHO-KNN both have been effective compared to the other classifiers. The experiments show that the HHO algorithm has excellent adaptability in combi- nation with the KNN classifier. In addition, the BP model performed the worst on the sharing economy dataset. The ACC value was only 51.56% and the Sensitivity was 16.29%. This sug- gests that the BP model needs to be tuned with appropriate parameters for the specific prob- lem. In addition, the BSHHO-KNN model and the BGHHO-KNN model do not perform the best on the dataset, which means that the BSGHHO algorithm can enable the KNN classifier to maximize its classification performance and thus achieve better classification results. In summary, the BSGHHO-KNN model outperforms other similar methods and can be used to investigate the key factors affecting future trends in the sharing economy. In this experimental study, the proposed model successfully accomplishes the task of select- ing the optimal subset of features during the entire process. Fig 5 counts the selected times of each feature in each experiment in the form of a line graph. https://doi.org/10.1371/journal.pone.0291626.g004 https://doi.org/10.1371/journal.pone.0291626.g004 (F43) were selected most frequently. These six most common attributes appeared 10, 10, 5, 5, 5, 4, 4 and 4 times respectively. Therefore, this study concludes that these types of attributes may make a valuable contribution to predicting future trends in the sharing economy. PLOS ONE In summary, the BSGHHO-KNN model outperforms other similar methods and can be used to investigate the key factors affecting future trends in the sharing economy. In this experimental study, the proposed model successfully accomplishes the task of select- ing the optimal subset of features during the entire process. Fig 5 counts the selected times of each feature in each experiment in the form of a line graph. From the figure: "form of sharing economy in the region" (F7), "attitude towards the sharing economy" (F30), "products in the sharing domain that have been used" (F22), "Factors of greatest concern when using shared product services" (F23), "Main aspects of distress caused by the sharing economy" (F28), "Neg- ative effects of the sharing economy on society " (F39), "Main aspects of concerns about the sharing economy" (F35) and "Any suggestions for the development of the sharing economy" . Standard deviation values for BSGHHO-KNN and the other five models on the four indicators. Table 11. Standard deviation values for BSGHHO-KNN and the other five models on the four indicators. Models ACC Sensitivity Specificity MCC Std BSGHHO-KNN 9.44E-03 0.00E+00 1.98E-02 1.84E-02 BSHHO-KNN 3.75E-02 0.00E+00 7.86E-02 6.87E-02 BGHHO-KNN 3.30E-02 1.92E-02 6.76E-02 6.30E-02 BP 6.08E-02 3.16E-01 3.16E-01 1.62E-01 RF 1.26E-02 2.00E-02 1.32E-02 2.48E-02 ELM 7.55E-02 1.15E-01 1.02E-01 1.52E-01 Table 11. Standard deviation values for BSGHHO-KNN and the other five models on the four indicators. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 28 / 37 PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 28 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy Fig 4. Comparison of SGHHO_KNN with well-known classifiers. htt //d i /10 1371/j l 0291626 004 Fig 4. Comparison of SGHHO_KNN with well-known classifiers. 7.4. Discussion The emergence of the sharing economy as a novel consumption model has introduced certain challenges, yet the public’s expectations for its future development remain high. Through the analysis of the questionnaire experiment results, it becomes evident that numerous factors influence the future trajectory of the sharing economy. Among the 43 attributes considered, Fig 5. The times each feature was selected by SGHHO_KNN during the 10-fold CV process. https://doi.org/10.1371/journal.pone.0291626.g005 Fig 5. The times each feature was selected by SGHHO_KNN during the 10-fold CV process. PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 29 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy attributes F7, F22, F23, F28, F30, F35, F39, and F43 stand out as the most significant in shaping the sharing economy’s future development trend. As a model intended to cater to the entire population, it is crucial to select the appropriate type of sharing economy that effectively serves the public, thereby playing a pivotal role in its overall advancement. Attributes F22 and F23 pertain to consumer feedback regarding their experience with the sharing economy post-usage. In this context, the success of a particular type of sharing econ- omy hinges on its capacity to satisfy diverse consumer needs at a reduced cost, while also gar- nering positive experiential feedback. This attribute plays a pivotal role in driving the development of the sharing economy. When consumers have a greater choice of products and services, providers are unable to price them effectively and consumer surplus is significantly increased. On the other hand, when the price of products and services is lower than normal, market demand increases as the price decreases, so the application of sharing economy model will help to increase market demand and thus contribute to the creation of greater value for money. The F28, F35 and F39 belong to the troubles caused by sharing economy. Under this new model of sharing economy, the existing regulations and systems, etc. do not fit in with its development, thus problems such as taxation, labor security and information security arise one after another. Meanwhile, some of the current regulations are not yet effective in regulat- ing the implementation of sharing economy model, and there is even the phenomenon of shar- ing economy model being bound by the traditional model legal system, which affects the model’s value due to the lack of laws and regulations. 7.4. Discussion Therefore, under the premise of clarify- ing the development mechanism, solving the problem of fitting the environment of sharing economy model is a key factor in developing the sharing economy. As per the attributes F30 and F43, participants hold a crucial position in regulating the activities of the sharing economy through SGHHO. However, the current process of gathering participant feedback and opinions is inadequate, and there is a lack of focus on establishing targeted channels for collecting and incorporating their input. Add to this the fact that sharing platforms have yet to be effectively improved and perfected, leaving a lack of comprehensive, real-time regulation of participants. Therefore, encouraging public participation in the regula- tion of the sharing sector is a vital measure for promoting the development of sharing economy. In summary, the sharing economy exhibits positive momentum, with a wider scope of development and an increasing variety of sharing economy products. The future growth of the sharing economy relies on the support of national policies and the continuous enhancement of the management system, which serves as a political guarantee for its stable progress. Addi- tionally, the improvement of social infrastructure is a prerequisite for establishing and main- taining the sharing economy, while the enhancement of citizens’ quality and their active and respectful involvement in the sharing economy are essential for its sustainable long-term development. Despite current challenges in the sharing economy’s development, the outlook for the future remains promising. 8. Conclusions and future directions To assess the optimization prowess of SGHHO, the IEEE CEC 2014 test suite is employed for rig- orous comparative tests. The results demonstrate a substantial superiority of the SGHHO algo- rithm over the original HHO algorithm in 96.7% of the functions. This compelling evidence showcases the significant enhancements achieved by the SGHHO algorithm in optimizing multivariate problems, thanks to the integration of SA and GB strategies. At the same time, SGHHO significantly outperforms similar algorithms and achieves optimal solutions for 67% of the functions tested when compared to other superior algorithms. This highlights the strong competitiveness of SGHHO. Furthermore, by combining the SGHHO algorithm with KNN, a better subset of features can be obtained than previous methods. When compared to conven- tional machine learning approaches, the SGHHO-KNN model achieved optimal results in the evaluation metrics of ACC, sensitivity, specificity, and MCC, attaining values of 99.70%, 100.00%, 99.38%, and 99.42% respectively. The feasibility of the BSGHHO-KNN model applied to the problem of predicting future trends in the sharing economy can be fully demon- strated based on the above four evaluation indicators. In conclusion, the SGHHO-KNN approach, as proposed in this study, demonstrates its effectiveness in selecting an optimal sub- set of features from the available data, enabling accurate predictions of the development trend of the sharing economy. Al h h h SGHHO l i h h h ll f i h b li Although the SGHHO algorithm has shown excellent performance in the above applica- tions, there are still shortcomings and aspects that deserve further research. For instance, SGHHO does not offer a universal solution to all intricate optimization problems, and its parameters require careful consideration and analysis in a problem-specific context to achieve optimal performance. Furthermore, the SGHHO acts as a stochastic optimizer. It is random- ized by nature. This means that there is still the possibility that SGHHO can fall into a local optimum in other complex applications. Therefore, the SGHHO method can also be combined with the latest optimization algorithms in future research, such as the farmland fertility algo- rithm, hunger games search, etc. And furthermore, it is applied in areas such as financial risk prediction and medical data diagnosis [135]. Nowadays, along with the increasing size of data in various fields, large-scale datasets also generate a large amount of redundant, useless and noisy data. This data seriously affects the performance of learning algorithms for data analysis. 8. Conclusions and future directions Thus, feature selection has an important place in this process. It is possible to drastically reduce the size of the data while maintaining the expressiveness of the information in the original fea- ture set and thereby avoiding the combinatorial explosion problem. This is particularly true for corporate bankruptcy prediction and intelligent medical diagnosis. Therefore, the SGHHO method can be subsequently applied to the field. By combining machine learning methods to assist asset owners and medical staff in achieving intelligent decisions. 8. Conclusions and future directions In the work, we develop an effective SGHHO-KNN hybrid model to provide predictions for the future development of sharing economy. In this study, a novel variant of HHO called SGHHO is proposed. Different from other existing variants, the main contribution and inno- vation of the algorithm is the effective incorporation of an improved Gaussian bare bone strat- egy and simulated annealing mechanism. When the original HHO algorithm falls into a local optimum, the Gaussian bare-bones strategy can generate a variation factor that guides PLOS ONE \| https://doi.org/10.1371/journal.pone.0291626 October 5, 2023 30 / 37 PLOS ONE An enhanced decision-making framework for predicting future trends of sharing economy individuals to skip from the optimal solution with a higher chance of survival. The enhanced simulated annealing mechanism enhances the exploration capability of the algorithm, enabling it to conduct more comprehensive global search operations across the entire feature space. To assess the optimization prowess of SGHHO, the IEEE CEC 2014 test suite is employed for rig- orous comparative tests. The results demonstrate a substantial superiority of the SGHHO algo- rithm over the original HHO algorithm in 96.7% of the functions. This compelling evidence showcases the significant enhancements achieved by the SGHHO algorithm in optimizing multivariate problems, thanks to the integration of SA and GB strategies. At the same time, SGHHO significantly outperforms similar algorithms and achieves optimal solutions for 67% of the functions tested when compared to other superior algorithms. This highlights the strong competitiveness of SGHHO. Furthermore, by combining the SGHHO algorithm with KNN, a better subset of features can be obtained than previous methods. When compared to conven- tional machine learning approaches, the SGHHO-KNN model achieved optimal results in the evaluation metrics of ACC, sensitivity, specificity, and MCC, attaining values of 99.70%, 100.00%, 99.38%, and 99.42% respectively. The feasibility of the BSGHHO-KNN model applied to the problem of predicting future trends in the sharing economy can be fully demon- strated based on the above four evaluation indicators. In conclusion, the SGHHO-KNN approach, as proposed in this study, demonstrates its effectiveness in selecting an optimal sub- set of features from the available data, enabling accurate predictions of the development trend of the sharing economy. individuals to skip from the optimal solution with a higher chance of survival. The enhanced simulated annealing mechanism enhances the exploration capability of the algorithm, enabling it to conduct more comprehensive global search operations across the entire feature space. 1. 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https://openalex.org/W2603353782	https://www.emis.de/journals/SIGMA/2018/015/sigma18-015.pdf	English	null	Billiards and Tilting Characters for SL<sub>3</sub>	Symmetry, integrability and geometry: methods and applications	2,018	cc-by-sa	11,561	SIGMA 14 (2018), 015, 22 pages SIGMA 14 (2018), 015, 22 pages Symmetry, Integrability and Geometry: Methods and Applications Symmetry, Integrability and Geometry: Methods and Applications SIGM 1 Introduction We formulate a conjecture for the second generation characters of indecomposable tilting modu- les for SL3 in characteristic p > 2. These conjectures resulted from our attempts to understand data [25] obtained following a 9 month calculation in magma on a supercomputer at the MPIM in Bonn. These results go far beyond existing calculations and are obtained using a new algo- rithm [26]. The algorithm relies in an essential way on ideas of Libedinsky, Riche and the second author (see [15, 21]). The behaviour we observe appears highly non-trivial, which suggests that proving anything might be difficult. On the next page the reader will find a picture. This picture was obtained by analyzing the output of computer calculations for p = 5. Exactly how this picture is used to produce (second generation) tilting characters will be explained in the final section. Before reading the rest of the paper the reader is invited to consider this picture and try to discern any patterns. This paper is an attempt to explain this picture, as well as similar pictures for p = 3 and 7. This paper is a contribution to the Special Issue on the Representation Theory of the Symmetric Groups and Related Topics. The full collection is available at https://www.emis.de/journals/SIGMA/symmetric-groups- 2018.html George LUSZTIG † and Geordie WILLIAMSON ‡ † Massachusetts Institute of Technology, Cambridge, MA, USA E-mail: gyuri@math.mit.edu URL: http://www-math.mit.edu/~gyuri/ ‡ Sydney University, Sydney, NSW, Australia E-mail: g.williamson@sydney.edu.au URL: http://www.maths.usyd.edu.au/u/geordie/ Received July 18, 2017, in final form February 16, 2018; Published online February 21, 2018 https://doi.org/10.3842/SIGMA.2018.015 † Massachusetts Institute of Technology, Cambridge, MA, USA E-mail: gyuri@math.mit.edu URL: http://www-math.mit.edu/~gyuri/ Abstract. We formulate a conjecture for the second generation characters of indecompos- able tilting modules for SL3. This gives many new conjectural decomposition numbers for symmetric groups. Our conjecture can be interpreted as saying that these characters are governed by a discrete dynamical system (“billiards bouncing in alcoves”). The conjecture implies that decomposition numbers for symmetric groups display (at least) exponential growth. Key words: tilting modules; billiards; p-canonical basis; symmetric group 2010 Mathematics Subject Classiﬁcation: 20C20; 17B10; 20C30 2010 Mathematics Subject Classiﬁcation: 20C20; 17B10; 20C30 Billiards and Tilting Characters for SL3 George LUSZTIG † and Geordie WILLIAMSON ‡ 2 Generational philosophy Williamson 2 27(v),30(v2),33(v), 57(v),60(v2),63(v), 72(v4 ` v6),75(v3 ` v7),78(v4 ` v6), 35(v),65(v),80(v4 ` v6), 25(v),55(v),70(v4 ` v6), 37(v),40(v2),43(v), 55(v3),58(v4),67(v), 70(v2),73(v),82(v4 ` v6), 45(v),60(v4),75(v), 12(1),54(v4),57(v5), 23(v),47(v),50(v2), 53(v),62(v4),65(v3), 68(v4 ` v6),77(v),80(v2), 14(1),53(v3),59(v5), 16(1),61(v5), 37(v),40(v2),43(v), 52(v4),55(2v3),58(2v4), 67(v),70(v2),73(v), 82(2v4 ` 2v6), 37(v),40(v2),43(v), 52(v4),55(v3),58(v4), 67(v),70(v2),73(v), 82(2v4 ` v6), 18(1),21(v),63(v5), 66(v6), 36(v2),39(v3),45(v), 51(2v3),54(2v2),60(2v4), 66(2v2),69(2v3),75(v), 81(2v3 ` v7), 42(v2),81(v5), 45(v),51(v3),54(v2), 60(v4),66(v2),69(v3), 75(v),81(v3), 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67(v),70(v2),73(v), 73(v), 64(1), 81(v5), 75(v3),78(v4), 72(v2), 66(1), 80(v4), 74(v2), 68(1),71(v), 82(v4), 76(v2),79(v3), 73(v), 81(v3), 75(v), 72(1), 77(v),80(v2), 74(1), 82(v2), 76(1), 78(1),81(v), 82(1), Figure 1. Second generation pattern for p = 5 up to i = 81. Figure 1. 2 Generational philosophy Let G denote a split simple and simply connected algebraic group over a field k of characteristic p. We fix a Borel subgroup and maximal torus T ⊂B ⊂G. We will try to follow the notation of [24]. In particular: G. Lusztig and G. 2 Generational philosophy Second generation pattern for p = 5 up to i = 81. Billiards and Tilting Characters for SL3 3 X , X+: weights, dominant weights; Φ, Φ+: roots, positive roots; Φ∨, Φ∨ +: coroots, positive coroots; Σ, Σ∨: simple roots, simple coroots; X<p: p-restricted weights; ρ, α∨ 0 : half-sum of Φ+, highest short coroot; Wf, W: finite Weyl group, affine Weyl group; Sf, S: finite simple reflections, affine simple reflections; fW: minimal coset representatives for Wf \ W; •p, h: p-dilated dot action of W on X , Coxeter number. For every dominant weight λ ∈X+, let Lλ (resp. ∆λ, resp. Tλ) denote the simple (resp. standard, resp. indecomposable tilting) module with highest weight λ. 2.1 Generations for simple characters For fixed p and any highest weight λ ∈X+ the first author has recently defined characters [17] For fixed p and any highest weight λ ∈X+ the first author has recently defined characters [17] χ0 λ, χ1 λ, χ2 λ, . . . , χ∞ λ ∈(ZX )Wf with the following properties: with the following properties: 1) χ0 λ is the character of the simple highest weight module in characteristic 0 (i.e., χ0 λ is given by Weyl’s character formula); 1) χ0 λ is the character of the simple highest weight module in characteristic 0 (i.e., χ0 λ is given by Weyl’s character formula); 2) χ1 λ is the character of the simple highest weight module for the quantum group at a pth-root of unity; 2) χ1 λ is the character of the simple highest weight module for the quantum group at a pth-root of unity; χn λ is obtained from χn−1 λ by a formula involving Kazhdan–Lusztig polynomials; 3) χn λ is obtained from χn−1 λ by a formula involving Kazhdan–Lusztig polynomials; 4) if λ = λ0 + pλ1 + · · · + pnλn with λi ∈X<p and ⟨α∨ 0 , λn + ρ⟩≤p (a generalised Jantzen condition) then χn λ = χn+1 λ = · · · = χ∞ λ ; 4) if λ = λ0 + pλ1 + · · · + pnλn with λi ∈X<p and ⟨α∨ 0 , λn + ρ⟩≤p (a generalised Jantzen condition) then χn λ = χn+1 λ = · · · = χ∞ λ ; 5) if p is large then χ∞ λ is the character of Lλ. 5) if p is large then χ∞ λ is the character of Lλ. Remark 2.1. Recall that the Steinberg tensor product theorem has “one step” for the quantum group at a root of unity, and “infinitely many steps” for G. The characters χn λ can be thought of as the simple characters for an object with an “n step” Steinberg tensor product theorem. It is not known whether such an object exists. For sl2 such an object (for any n) has recently been proposed by Angiono [6]. It seems likely that one can combine recent work of Elias [8] with work of Riche and the second author [21] to construct such an object in type A for n = 2. 2.2 Generations for tilting characters As for simple characters, there should exist approximations to tilting characters. That is, for any p and dominant weight λ ∈X there should exist characters θ0 λ, θ1 λ, θ2 λ, . . . , θ∞ λ ∈(ZX )Wf with the following properties: with the following properties: 1) θ0 λ is the character of the simple highest weight module in characteristic 0 (i.e., θ0 λ is given by Weyl’s character formula); 1) θ0 λ is the character of the simple highest weight module in characteristic 0 (i.e., θ0 λ is given by Weyl’s character formula); 2) θ1 λ is the character of the indecomposable tilting module for the quantum group at a pth- root of unity; 2) θ1 λ is the character of the indecomposable tilting module for the quantum group at a pth- root of unity; G. Lusztig and G. Williamson 4 3) θn λ is a positive linear combination of the θn−1 µ ’s for all n ≥1; , λ + ρ⟩≤pn+1 then θn λ = θn+1 λ = · · · = θ∞ λ ; 4) if ⟨α∨ 0 , λ + ρ⟩≤pn+1 then θn λ = θn+1 λ = · · · = θ∞ λ ; 4) if ⟨α∨ 0 , λ + ρ⟩≤pn+1 then θn λ = θn+1 λ = · · · = θ∞ λ ; 4) if ⟨α∨ 0 , λ + ρ⟩≤pn+1 then θn λ = θn+1 λ = · · · = θ∞ λ ; 5) if p is large then θ∞ λ is the character of Tλ. Remark 2.2. Parts (4) and (5) for n = 1 imply Andersen’s conjecture [5], which is still open (even for p large). Remark 2.3. The characters θn λ are defined for a subset of X+ (roughly, those for which one can apply the tilting tensor product theorem) in [18]. 2.3 Our conjecture at v = 1 Let us assume that p ≥h, and consider the p-dilated action of the affine Weyl group from above. For any choice of “generation parameters” 0 ≤m ≤n ≤∞we can write θn x•p0 = X y∈W dm,n y,x · θm y•p0 (2.1) θn x•p0 = X y∈W dm,n y,x · θm y•p0 (2.1) for some dm,n y,x ∈Z≥0. (The fact that we can form such expressions follows from the linkage principle and our assumptions above.) The question of determining characters of indecomposable tilting characters is equivalent to determining the coefficients d∞,0 y,x for all y, x ∈fW. Remark 2.4. A philosophy underlying the current paper is that it might be easier to calculate the dn+1,n y,x for all n, rather than calculate the d∞,0 y,x directly. This is the case for simple characters, as explained in Section 2.1. The following is known about these coefficients: The following is known about these coefficients: 1) when G = GLn, the d∞,0 y,x are equal to decomposition numbers for symmetric groups, by work of Donkin [7] and Erdmann [10]; 1) when G = GLn, the d∞,0 y,x are equal to decomposition numbers for symmetric groups, by work of Donkin [7] and Erdmann [10]; 2) the d1,0 y,x are given as the value at 1 of certain parabolic Kazhdan–Lusztig polynomials, by work of Soergel [22, 23]; 2) the d1,0 y,x are given as the value at 1 of certain parabolic Kazhdan–Lusztig polynomials, by work of Soergel [22, 23]; 3) the d∞,1 y,x are equal to coefficients of James’ “adjustment matrix” [11]. Remark 2.5. Aside from the above, very little is known. The papers [12, 20] contain some interesting calculations for SL3 Remark 2.5. Aside from the above, very little is known. The papers [12, 20] contain some interesting calculations for SL3 In this paper we formulate a conjecture for (v-analogues of the) coefficients d2,1 y,x, when G = SL3. (Note that in the case of G = SL3 the coefficients d1,0 y,x given by Soergel’s algo- rithm are easily calculated, and may be described by closed formulas.) By property (4) of the previous section, this provides a conjecture for many d∞,1 y,x , and hence for many coefficients of the adjustment matrix (and hence decomposition numbers) for three row partitions. 2.4 Generations for the p-canonical basis , pn∞ x ∈ASv such that: such that: 1) pn1 x = nx; 2) pnn x is a Z≥0[v±1]-linear combination of the pnn−1 y ’s for all n ≥1; 3) if ⟨α∨ 0 , x •p 0 + ρ⟩≤pn+1 then pnn x = pnn+1 x = · · · = pn∞ x ; 4) if p is large then pn∞ x = pnx. 4) if p is large then pn∞ x = pnx. 2.4 Generations for the p-canonical basis Let H denote the Iwahori–Hecke algebra of the affine Weyl group W over Z v±1 . Consider the anti-spherical module (see, e.g., [21, 24]): ASv := sgnv ⊗Hf H = M x∈fW Z v±1 nx. The anti-spherical module has a canonical basis {nx \| x ∈fW} and a p-canonical basis {pnx \| x ∈ fW} [14, 21]. The anti-spherical module has a canonical basis {nx \| x ∈fW} and a p-canonical basis {pnx \| x ∈ fW} [14, 21]. Billiards and Tilting Characters for SL3 5 In [21] (see also [24, Section 2.8]) it is conjectured that if we write In [21] (see also [24, Section 2.8]) it is conjectured that if we write pnx = X pmy,xny, then the values at 1 of the coefficients pmy,x express the characters of tilting modules in terms of Weyl characters.1 Let us give a precise version of this conjecture for p ≥h: for all x, y ∈fW we have (in the notation of Section 2.3) then the values at 1 of the coefficients pmy,x express the characters of tilting modules in terms of Weyl characters.1 Let us give a precise version of this conjecture for p ≥h: for all x, y ∈fW we have (in the notation of Section 2.3) = pmy,x(1). (2.2) d∞,0 y,x = pmy,x(1). (2.2) (Recall that, by the translation principle and still under our assumption p ≥h, knowledge of the left hand side of (2.2) for all x, y ∈fW implies knowledge of the characters of all tilting modules for G.) (Recall that, by the translation principle and still under our assumption p ≥h, knowledge of the left hand side of (2.2) for all x, y ∈fW implies knowledge of the characters of all tilting modules for G.) Remark 2.6. In [21] this conjecture is proved for GLn and p > h. In [9] this conjecture is proved for GLn for general p. In [1, 2, 4] this conjecture is proved in all types for p > h. The presence of v is the shadow of a non-trivial grading on the category of tilting modules.2 It is natural to expect that the above approximations to tilting characters can also be made to respect this grading. That is, we expect that for all p > h and x ∈fW, there exist elements pn1 x, pn2 x, . . . 1See [21, Conjecture 1.7] for a precise formulation. Note that this conjecture is expected to hold without any restrictions on p. 2 p is grading is conjectural in general. In [1, 2, 4, 9, 21] its existence is established in many cases. ASv ∼ →[ASZp]⊕, 1See [21, Conjecture 1.7] for a precise formulation. Note that this conjecture is expected to hold without any restrictions on p. 2This grading is conjectural in general. In [1, 2, 4, 9, 21] its existence is established in many cases. 3 The new algorithm To ASZp are associated progressively sim- pler categories: ASZp ⇝ASQp ⇝Q ⊗R ASQp, where ⇝denotes some form of localisation. The first localisation ASQp is obtained from ASZp by inverting p. The second is the main object of study of [15]: the polynomial ring R = Qp[eα] (eα denotes the affine simple root) acts on the left on all hom spaces in ASQp, and after inverting eα one obtains Q ⊗R ASQp, where Q denotes Qp(eα). Somewhat surprisingly, this category is semi-simple [15]. (This is analogous to deformed category O, which is semi-simple for generic parameters. A big difference in the current setting is that the deformation ring R is always one dimensional.) ) The algorithm we used to calculate the p-canonical basis now proceeds in two 1. Firstly, ASQp is described as a quiver with relations, and the action of the Hecke cate- gory HZp on ASQp is described explicitly, in terms of the quiver. This is already a very non-trivial task, and is only feasible for SL2, SL3 and perhaps SL4. It is possible in these cases thanks to the localisation Q⊗R ASQp (where any calculation can be reduced to a cal- culation in matrices with entries in Q), the fact that the Kazhdan–Lusztig conjectures hold in AQp (thus one has graded dimensions for hom spaces and knows how the generators of HZp act on the Grothendieck group etc.), and the fact that the Kazhdan–Lusztig theo- ry of these anti-spherical modules in low rank is not too complicated (Kazhdan–Lusztig polynomials can be written down explicitly). 2. Secondly, via the above localisations one can describe ASZp as a Zp-lattice inside ASQp. Moreover, this lattice is the smallest lattice which contains the generating object and is stable under the action of the Hecke category HZp. One may describe this lattice explicitly and inductively via the HZp-action, using the philosophy of the light leaf basis. Details of how this is done in this setting will be contained in [26]. Remark 3.1. Recently, L.T. Jensen has done calculations which omit the first localisation and calculate the p-canonical basis using only the localisation Q ⊗R ASQp. This is a much simpler approach, and appears (much to the second author’s surprise!) not to be any slower than the approach described above. It may well be that Jensen’s modification of the algorithm ends up being the more effective. 3 The new algorithm In this section we attempt to give the reader some idea of how we perform the calculations which led to our conjecture. The second author hopes to give more details in [26]. As explained in the previous section, the main theorems of [9, 21] (see also [1, 2]) imply that it is enough to calculate the p-canonical basis {pnx \| x ∈fW} in the anti-spherical module ASv. Let H denote the diagrammatic Hecke category associated to the affine Cartan matrix of type eAn. Recall that H is built starting from a “realisation” {α∨ s }s∈S ⊂h, {αs}s∈S ⊂h∗ , where h is a free and finite rank Z-module, h∗is its dual, and the usual formulas define a rep- resentation of W. It will be important below that our realisation is chosen so that the simple roots {αs}s∈S ⊂h∗are linearly independent. where h is a free and finite rank Z-module, h∗is its dual, and the usual formulas define a rep- resentation of W. It will be important below that our realisation is chosen so that the simple roots {αs}s∈S ⊂h∗are linearly independent. The Hecke category is defined over Z. After fixing a prime p, extension of scalars yields the Hecke category HZp defined over the p-adic integers. Let ASZp denote the anti-spherical category over Zp considered in [15, 21]. It is a right HZp-module category and one has a canonical identificiation of right [HZp]⊕= H-modules: ASv ∼ →[ASZp]⊕, 1See [21, Conjecture 1.7] for a precise formulation. Note that this conjecture is expected to hold without any restrictions on p. 2This grading is conjectural in general In [1 2 4 9 21] its existence is established in many cases 1See [21, Conjecture 1.7] for a precise formulation. Note that this conjecture is expected to hold without any restrictions on p. 2 [ ] G. Lusztig and G. Williamson 6 where [−]⊕denotes the split Grothendieck group of an additive category (see [15, 21] for more details). Under this isomorphism the classes of the indecomposable self-dual objects yields the p-canonical basis in ASv. The anti-spherical category ASZp is defined by explicit generators and relations. Thus the question of determining the characters of its indecomposable objects (and hence the p-canonical basis) is a concrete question of finding idempotents in certain finite rank Zp-algebras. However these diagrammatic calculations are prohibitively difficult in all but the simplest cases. Instead we exploit the philosophy of localisation. 4.1 Overview of the algorithm We view the dominant weights X+ as the vertices of a directed graph Γ with edges λ →λ + γ if λ, λ + γ ∈X+ and γ ∈{ϖ1, ϖ2 −ϖ1, −ϖ2}: Γ = ... ... ... 0 ϖ1 ϖ2 Our algorithm consists of three steps. We begin with the labelled point 0, 0, v0 (our initial “seed”). Each step in our algorithm enlarges our multiset in a new direction, starting with seeds generated in the previous step. The first step extends our set along a wall of the dominant chamber in the direction of ϖ1 to produce a set X. The second step extends our set along the walls of the ℓ-alcoves to produce a multiset Y . Finally, the third step extends our multiset within the interior of each ℓ-alcove to produce a multiset Z. In the third step, certain labelled points arising in the second step (seeds) gives rise to two spirals inside the interior of two adjacent alcoves which move like a billiard. Finally we consider eZ := Z \ X, which is the object of our conjecture. which is the object of our conjecture. Billiards and Tilting Characters for SL3 Billiards and Tilting Characters for SL3 7 A labelled point is an element of X ×M. Labelled points will usually be denoted (µ, m) with µ ∈X and m ∈M or (µ, n, vk) with µ ∈X and n ∈Z≥0. Our goal in this section is to describe an algorithm which produces a multiset (i.e., set with multiplicities) of labelled points via an inductive procedure. Throughout this section we work entirely with multisets. All operations (union, difference, . . . ) are to be understood in the context of multisets. 4 Billiards For the rest of the paper we fix G = SL3, X = Zϖ1 ⊕Zϖ2, Σ = {α1, α2} etc. Fix ℓ≥1. (For applications to representation theory ℓwill be prime, however for the moment ℓcan be any positive integer. Soon we will assume ℓ≥3.) Elements of the set M := Z≥0 × vk \| k ∈Z will be called labels. Labels will often be denoted by an integer followed by a bracketed power of v; e.g., 51(v7). will be called labels. Labels will often be denoted by an integer followed by a bracketed power of v; e.g., 51(v7). 4.3 Step 2: dynamics on the walls From now on assume that ℓ≥3. Consider the subgraph of Γ with vertices λ ∈X+ such that ⟨λ, α∨⟩∈ℓZ for some α ∈Φ+ and edges (λ, λ′) such that ⟨λ, α∨⟩= ⟨λ′, α∨⟩∈ℓZ for some α ∈Φ+. Let Γwall denote the graph obtained by removing the gray edges and vertices as in Fig. 2. Points in X+ or Γwall such that ⟨λ, α∨⟩∈ℓZ for all α ∈Φ+ are called corner points. A point µ in X+ of Γwall is an almost corner if there exists a corner point c and an arrow c →µ in X+. + Fix a labelled point µ, n vk with µ ∈Γwall. We assume that µ is such that there is a unique edge with source µ. From µ, n vk one may obtain new labelled points as follows: 1. A rest produces the labelled point µ, (n + 3) vk+1 . 1. A rest produces the labelled point µ, (n + 3) vk+1 . 2. A small step produces (as above) the labelled point µ′, (n + 2) vk , where µ →µ′ is the unique edge in Γwall with source µ. 3. A giant leap produces either one or two new labelled points, and is only possible if µ is not a corner or almost corner point. Define d to be the direction of the unique arrow with source µ. First we proceed j < ℓ−1 steps in the graph Γwall in direction d until we reach a corner point. We then proceed for another ℓ−1 −j steps in all directions from the corner point which do not agree with the direction d. (There are either one or two such directions.) A giant leap consists of the resulting points, which are labelled by (n + 2ℓ+ 1) vk+1 (see Fig. 3). Now suppose that we are given a labelled point q = (µ, n, vk) as above (i.e., such that there is a unique edge in Γwall with source µ). We now describe a way of producing a multiset of labelled points (all with multiplicity 1) beginning with q, some of which are designated as seeds: 1. If µ is a corner point. We produce ℓnew points as follows: we take ℓ−1 small steps to produce labelled points m = m1, . . . , mℓ−1 and then rest to produce a new labelled point mℓ. 4.2 Step 1: the wall of the dominant chamber Consider the full subgraph of X+ consisting of multiples of ϖ1: Consider a labelled point m = µ, m vk such that µ belongs to this full subgraph. A small step produces the labelled point µ′, (m+2) vk where µ →µ′ is the unique edge with source µ. Consider the set X obtained by repeatedly taking small steps beginning with the labelled point 0, 0 v0 . In other words, X is the set X = kω1, 2k v0 \| k ∈Z≥0 . (4.1) X = kω1, 2k v0 \| k ∈Z≥0 . (4.1) We designate the labelled points of the form (ℓk, 2kℓ(v0)) with k > 0 as seeds. Remark 4.1. Of course we could have defined X via (4.1) directly. We prefer the inductive definition, as it is closer in spirit to the more complicated definitions which will occur in the next two steps. G. Lusztig and G. Williamson 8 Figure 2. The directed graph Γwall. Figure 2. The directed graph Γwall. 4.3 Step 2: dynamics on the walls The final labelled point mℓis designated as a seed. (See Fig. 4.) 2. If µ is an almost corner point. We produce ℓnew points as follows: let m = m1 and rest once to produce a new labelled point m2, now take ℓ−2 small steps starting with m2 to 2. If µ is an almost corner point. We produce ℓnew points as follows: let m = m1 and rest once to produce a new labelled point m2, now take ℓ−2 small steps starting with m2 to Billiards and Tilting Characters for SL3 9 µ n(vk) µ′′ (n + 2ℓ+ 1)(vk+1) µ′ (n + 2ℓ+ 1)(vk+1) or µ n(vk) µ′ (n + 2ℓ+ 1)(vk+1) Figure 3. Performing a giant leap with ℓ= 5. (Squares denote corner points.) µ n(vk) µ′ (n + 2ℓ+ 1)(vk+1) (n + 2ℓ+ 1)(vk+1) or (n + 2ℓ+ 1)(vk+1) Figure 3. Performing a giant leap with ℓ= 5. (Squares denote corner points.) 10(v0) 12(v0) 14(v0) 16(v0) 18(v0) 21(v1) 54(v4) 57(v5) 59(v5) 61(v5) 63(v5) 66(v6) Figure 4. An illustration of cases (1) and (2) when ℓ= 5. Seeds are underlined. 54(v4) 57(v5) 59(v5) 61(v5) 63(v5) 66(v6) 16(v0) 14(v0) 12(v0) Figure 4. An illustration of cases (1) and (2) when ℓ= 5. Seeds are underlined. produce ℓ−2 labelled points m3, . . . , mℓ−1 and finally rest once more to produce a final labelled point mℓ. The final labelled point mℓis designated as a seed. (See Fig. 4.) produce ℓ−2 labelled points m3, . . . , mℓ−1 and finally rest once more to produce a final labelled point mℓ. The final labelled point mℓis designated as a seed. (See Fig. 4.) 3. If µ is neither a corner nor an almost corner point. We take a giant leap to produce one or two new labelled points, each of which are designated seeds. We now iterate this process as follows. Starting with q we produce a sequence of multisets Q1, Q2, Q3, . . . , where Q1 (resp. Qi for i > 1) is obtained by applying the above procedure to q (resp. to each seed in Qi−1). We say that the union (as multisets) Q = [ i≥0 Qi Q = [ i≥0 Qi is the result of applying dynamics on the walls to the seed q. is the result of applying dynamics on the walls to the seed q. is the result of applying dynamics on the walls to the seed q. Example 4.2. Fig. 5 gives an example with ℓ= 5. Our initial seed is the unique point with label 10 v0 . We illustrate a few iterations of the above algorithm. Seeds are underlined. Note that one more iteration of the algorithm will produce the labelled point 3ϖ1 + 7ϖ2, 88 v8 with multiplicity 2. Now consider our set X from the previous step. We apply dynamicson walls to each seed in X (i.e., each labelled point of the form kℓϖ1, 2kℓ v0 with k > 0) to generate a multiset Yk. We now define a new multiset Y := X ∪ [ k>0 Yk. member (for the purposes of the next step) which elements of Y were designated seeds We also remember (for the purposes of the next step) which elements of Y were design G. Lusztig and G. Williamson 10 32(v2) 77(v7) 54(v4) 57(v5) 59(v5) 61(v5) 63(v5) 66(v6) 10(v0) 12(v0) 14(v0) 16(v0) 18(v0) 21(v1) 54(v4) 57(v5) 59(v5) 61(v5) 63(v5) 66(v6) 43(v3) 77(v7) 43(v3) 77(v7) Figure 5. Dynamics on the walls with ℓ= 5. 77(v7) 16(v0) Figure 5. Dynamics on the walls with ℓ= 5. Remark 4.3. Consider dynamics on the wall restricted to the full subgraph displayed in Fig. 6 with seed q = µ, n vk . Then 4 iterations of the above algorithm yields the following points and labels (see Fig. 6): u, u′ with label (n + 11) vk+1 , ui, u′ i with label (n + 14 + 2(i −1)) vk+2 for i = 1, 2, 3, 4, v, v′ with label (n + 23) vk+3 , w, w′ with label (n + 34) vk+4 , and then µ with label (n+45) vk+5 and multiplicity 2. (The point is that there are two directed paths leading from µ back to itself, which causes the multiplicity to double.) Repeating this algorithm 4i times leads to qi := µ, n + 45i vk+5i with multiplicity 2i. From this observation and then µ with label (n+45) vk+5 and multiplicity 2. (The point is that there are two directed paths leading from µ back to itself, which causes the multiplicity to double.) Repeating this algorithm 4i times leads to qi := µ, n + 45i vk+5i with multiplicity 2i. is the result of applying dynamics on the walls to the seed q. From this observation Billiards and Tilting Characters for SL3 11 q w u u1 u2 u3 u4 v w′ u′ u′ 1 u′ 2 u′ 3 u′ 4 v′ Figure 6. Subgraph demonstrating exponential growth of multiplicities for ℓ= 5. Figure 6. Subgraph demonstrating exponential growth of multiplicities for ℓ= 5. one deduces easily that for ℓ= 5 the set Y contains labelled points whose multiplicity grows ex- ponentially in n. Similar considerations show that the same statement about Y is true for any ℓ. one deduces easily that for ℓ= 5 the set Y contains labelled points whose multiplicity grows ex- ponentially in n. Similar considerations show that the same statement about Y is true for any ℓ. 4.4 Step 3: billiards in an alcove We now describe an algorithm which produces for each seed in Y a multiset of labelled points. We need a little more notation. We consider the dominant weights X+ as embedded in the vector space XR := X ⊗Z R, which we regard as a Euclidean space for some Wf-invariant bilinear form. An ℓ-alcove is a connected component of the complement XR \ [ α∈Φ∨ + λ \| ⟨α∨, λ⟩∈ℓZ . An ℓ-regular point is a point which belongs to some ℓ-alcove. An ℓ-alcove is dominant if it is contained in the real cone generated by X+. An ℓ-regular point is a point which belongs to some ℓ-alcove. An ℓ-alcove is dominant if it is contained in the real cone generated by X+. Now consider a seed q = (µ, m) ∈Y . We want to associate a multiset of labelled points Λq to q. If µ is a corner or almost corner, we set Λq := ∅. From now on we assume that q is neither a corner or almost corner. In this case µ belongs to the closure of two dominant ℓ-alcoves, A′ and A′′. We perform an identical procedure for both alcoves, so fix one such alcove and call it A. Let ∆denote the full subgraph of Γ consisting of points belonging to A. Then ∆has one of the following forms: ℓ+ 1 points ℓ+ 1 points 12 G. Lusztig and G. Williamson Points of ∆belong to A but not to A are called wall points. Note that our starting point µ is a wall point. Points of ∆belong to A but not to A are called wall points. Note that our starting point µ is a wall point. Variables and their initialisations: Let (µstart, mstart) := (µ, m) and let dstart denote the unique edge of ∆with source µ and target an interior point of ∆. To begin with, set Λ := {(µstart, mstart)}. In the algorithm we need the following variables, which are initialised as follows: CurrentLabel := mstart, CurrentSign := 1. CurrentLabel := mstart, CurrentSign := 1. CurrentPoint := µstart, CurrentLabel := mstart, CurrentSign := 1. CurrentDirection := dstart, CurrentDirection := dstart, CurrentSign := 1. The loop: The multiset of labelled points is obtained by repeating the following ad infinitum. Let µnew denote the point obtained by moving from CurrentPoint one step in CurrentDirection. Write CurrentLabel as n vk . 4.4 Step 3: billiards in an alcove Two possibilities may occur: 1. µnew is ℓ-regular: CurrentLabel := (n + 2) vk , CurrentPoint := µnew, add (CurrentPoint, CurrentLabel) to Λ. CurrentLabel := (n + 2) vk , Current add (CurrentPoint, CurrentLabel) to Λ. CurrentLabel := (n + 2) v , CurrentPo add (CurrentPoint, CurrentLabel) to Λ. ( ) add (CurrentPoint, CurrentLabel) to Λ. add (CurrentPoint, CurrentLabel) to Λ. (The variables CurrentDirection and CurrentSign remain unchanged.) 2. CurrentPoint is ℓ-regular and µnew is a wall point: CurrentLabel := (n + 3) vk+CurrentSign , CurrentDirection := dnew, CurrentSign := −CurrentSign, add (CurrentPoint, CurrentLabel) to Λ. 2. CurrentPoint is ℓ-regular and µnew is a wall point: add (CurrentPoint, CurrentLabel) to Λ. Here dnew denotes the vector obtained by reflecting CurrentDirection in the wall on which µnew lies. (The variable CurrentPoint remains unchanged.) Example 4.4. We illustrate the first few steps of the algorithm in some examples. In each case the starting point µstart and its label are underlined. The successive values of CurrentLabel and CurrentPoint are obtained by following the arrows. 1. A reasonably generic example with ℓ= 11: 1. A reasonably generic example with ℓ= 11: (∗) 16(v) 18(v) 20(v) 22(v) 24(v) 26(v) 28(v) 31(v2) 33(v2) 35(v2) 38(v) 40(v) 42(v) 44(v) 46(v) 48(v) 50(v) 53(v2) 55(v2) 57(v2) 60(v) 62(v) 64(v) 66(v) 68(v) 70(v) 72(v) 75(v2) 77(v2) . . . 14(v) 13 Billiards and Tilting Characters for SL3 If we focus on the point marked with an asterix () we obtain the labels: 16(v), 79 v2 , 82(v), 145 v2 , 148(v), 211 v2 , 214(v), 277 v2 , 280(v), . . . . If we focus on the point marked with an asterix () we obtain the labels: 16(v), 79 v2 , 82(v), 145 v2 , 148(v), 211 v2 , 214(v), 277 v2 , 280(v), . . . . 2. An interesting example with ℓ= 5 (we display both alcoves): 2. An interesting example with ℓ= 5 (we display both alcoves): 2. An interesting example with ℓ= 5 (we display both alcoves): 2. An interesting example with ℓ= 5 (we display both alcoves): ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ . . . ∗ ∗∗ ∗ ∗ 21(v) ∗ ∗ ∗ ∗ ∗ . . . 5 An altenative construction In this section we outline an alternative construction of the multiset eZ, which is more natural in some respects. In the previous section we regarded the dominant weights X+ as the vertices of a direc- ted graph. An important difference in the current construction is that now we consider all weights X . That is, we consider the elements of X as the vertices of a directed graph with edges λ →λ + γ if λ, λ + γ ∈X+ and γ ∈{ϖ1, ϖ2 −ϖ1, −ϖ2}. We write µ →µ′ to indicate that there is a directed edge from µ to µ′. We use similar terminology to earlier: the notions of a wall point, corner point, almost corner and ℓ-regular point extend in an obvious way to X . Note that almost corners are necessarily wall points. 4.4 Step 3: billiards in an alcove Finally, we define eZ := Z \ X. Example 4.5. In Fig. 1 we display eZ for ℓ= 5 and all labelled points µ, n vk with n ≤82. In the notation of Section 6, the labelled points corresponding to µ ∈X+ are displayed in the smaller of the two alcoves contained in Bµ. The reader is referred to [25] for further examples. 3This is equivalent in an obvious way to the usual notion of rooted tree. 4.4 Step 3: billiards in an alcove ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ In either alcove the sequence of labels obtained by following the arrows beginning at 21(v) is as follows: In either alcove the sequence of labels obtained by following the arrows beginning at 21(v) is as follows: In either alcove the sequence of labels obtained by following the arrows beginning at 21(v) is as follows: 23(v), 25(v), 27(v), 30i(v2 , 33(v), 35(v), 37(v), 40 v2 , 23(v), 25(v), 27(v), 30i(v2 , 33(v), 35(v), 37(v), 40 v2 , 43(v), 45(v), 47(v), 50 v2 , 53(v), . . . . 3. Another example with ℓ= 5 (again we display both alcoves): 3. Another example with ℓ= 5 (again we display both alcoves): ∗ . . . ∗ ∗ ∗ ∗∗ ∗ ∗ ∗ ∗ ∗∗ ∗ ∗ . . . 77(v7) In either alcove the sequence beginning at 77 v7 is 79 v7 , 81 v7 , 84 v8 , 86 v8 , 89 v7 , 91 v7 , . . . . In either alcove the sequence beginning at 77 v7 is 79 v7 , 81 v7 , 84 v8 , 86 v8 , 89 v7 , 91 v7 , . . . . 14 G. Lusztig and G. Williamson 4. Finally, an example with ℓ= 3 (again we display both alcoves): 4. Finally, an example with ℓ= 3 (again we display both alcoves): 4. Finally, an example with ℓ= 3 (again we display both alcoves): 13(v) ∗ ∗ ∗ ∗ . . . ∗ ∗ ∗ ∗ . . . The sequence at either middle vertex is: 15(v), 18(v2), 21(v), 24(v2), 27(v), . . . . The sequence at either middle vertex is: 15(v), 18(v2), 21(v), 24(v2), 27(v), . . . . Now, with q = (µ, m) as above (so that µ is neither a corner or almost corner) we apply the above algorithm to both alcoves which contain µ in their closure to produce multisets Λ′ and Λ′′. We then remove q from both, and define Λq to be the union of the resulting multisets. (Thus Λq contains infinitely many points from both alcoves, but does not contain q itself.) The above algorithm produces, for each each seed q ∈Y , a multiset Λq. We define Z to be the union Z := Y ∪ [ seeds q∈Y Λq. Finally, we define eZ := Z \ X. 5.1 Trees For us a rooted tree is what many call an “arborescence”: a directed graph with a distinguished vertex (the source) such that there is a unique directed path from the source to any other vertex.3 3This is equivalent in an obvious way to the usual notion of rooted tree. 15 Billiards and Tilting Characters for SL3 Given two rooted trees A and B, denote by A ∗B the rooted tree obtained by adjoining one copy of B to each sink in A at the source of B. That is, if t1, . . . , tk denote the sinks of A and we denote by b1, . . . , bk the sources in B ⊔B ⊔· · · ⊔B (k factors), then A ∗B := (A ⊔B ⊔B ⊔· · · ⊔B)/(ti ∼bi). This operation is associative. The image of A (resp. a copy of B) under this quotient map will be called a component of type A (resp. B). This operation is associative. The image of A (resp. a copy of B) under this quotient map will be called a component of type A (resp. B). Recall that ℓ≥3 is fixed. Consider the two rooted trees: call that ℓ≥3 is fixed. Consider the two rooted trees: I := . . . . . . . . . . . . . . . J := 0 1 2 ℓ−1 ℓ ℓ+ 1 The integers indicate the distance from the unique source in each graph. I := . . . . . . . . . . . . . . . J := 0 1 2 ℓ−1 ℓ ℓ+ 1 ℓ+ 1 The integers indicate the distance from the unique source in each graph. The integers indicate the distance from the unique source in each graph. Example 5.1. We illustrate the operation ∗with our graphs I and J. For ℓ= 3, the graph I ∗J ∗J looks as follows: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Now define: J∞:= I ∗J∗ℓ−1∗∞:= I ∗J∗ℓ−1 ∗ I ∗J∗ℓ−1 ∗ I ∗J∗ℓ−1 ∗· · · . Recall the notion of component from above. 5.2 The map Fix an almost corner λ. We will construct a map Φ = Φλ : J∞→X . It will have the following two properties: 1. If v →v′ in J∞then either Φ(v) = Φ(v′) or Φ(v) →Φ(v′) in X . 1. If v →v′ in J∞then either Φ(v) = Φ(v′) or Φ(v) →Φ(v′) in X . 2. For v ∈J∞, Φ(v) is a wall point (resp. an almost corner) if and only if v is of wall type (resp. of almost corner type). 2. For v ∈J∞, Φ(v) is a wall point (resp. an almost corner) if and only if v is of wall type (resp. of almost corner type). Because J∞is the union of components of types I or J it will suffice to define the image of Φ on the source of J∞, and then define it on each component of type I or J inductively. This is what we do now: Rule 0: Let v0 denote the source of J∞. Set Φ(v0) = λ (our fixed almost corner) Rule 1: Let I′ ⊂J∞denote a component of type I and let v′ 0 denote the source of I′. Suppose that µ := Φ(v′ 0) is defined, but that Φ is not defined on the rest of I′. We define Φ on I′ as follows: µ1 →µ1 →µ2 →. . . →µℓ−2 →µℓ−1 →µℓ−1. Here µ = µ1, µ2, . . . , µℓare uniquely determined by the requirement that µ = µ1 and µ1 →µ2 → · · · →µℓ−1 are all wall points. (The fact that µ1, . . . , µℓ−1 are well defined is a consequence of property (2) above: Φ(v′ 0) is an almost corner.) Here µ = µ1, µ2, . . . , µℓare uniquely determined by the requirement that µ = µ1 and µ1 →µ2 → · · · →µℓ−1 are all wall points. (The fact that µ1, . . . , µℓ−1 are well defined is a consequence of property (2) above: Φ(v′ 0) is an almost corner.) Rule 2: Let J′ ⊂J∞denote a component of type J and let v′ 0 denote the source of J′. Suppose that µ := Φ(v′ 0) is defined, but that Φ is not defined on all of J′. By property (2) above µ is a wall-point but is not an almost corner. corner type) in J∞are depicted as open (resp. filled) circles: corner type) in J∞are depicted as open (resp. filled) circles: corner type) in J∞are depicted as open (resp. filled) circles: yp ) ∞ p p ( p ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 Th . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Trees We say that v ∈J∞is of wall type if it lies in a component of type I or is the source in a component of type J. We say that v is of almost corner type if it is the source in a component of type I. (Thus if v ∈J∞is of almost corner type then it is also of wall type.) Recall the notion of component from above. We say that v ∈J∞is of wall type if it lies in a component of type I or is the source in a component of type J. We say that v is of almost corner type if it is the source in a component of type I. (Thus if v ∈J∞is of almost corner type then it is also of wall type.) Example 5.2. We continue Example 5.1 with ℓ= 3. The points of wall type (resp. of almost G. Lusztig and G. Williamson 16 2) if µi + di is an interior point, define (µi+1, di+1) := (µi + di, di); 5.2 The map Hence there exist two edges d′, d′′ with source µ and image an ℓ-regular point. For d ∈{d′, d′′}, define a sequence {(µi, di)}i≥0 as follows: 1) (µ0, d0) := (µ, d); 2) if µi + di is an interior point, define (µi+1, di+1) := (µi + di, di); Billiards and Tilting Characters for SL3 17 3) if µi + di is a wall point, define (µi+1, di+1) := (µi, r(di)), where r denotes the reflection in the (unique) wall containing µi + di. Denote by {(µ′ i, d′ i)}i≥0 (resp. {(µ′′ i , d′′ i ))}i≥0) the sequences associated to d = d′ (resp. d = d′′). We define Φ on J′ as follows: µ′ 0 = µ′′ 0 µ′ 1 µ′′ 1 µ′ 2 µ′′ 2 . . . . . . µ′ ℓ−1 µ′′ ℓ−1 µ′ ℓ µ′′ ℓ µ′ ℓ+1 µ′′ ℓ+1 . . . . . . µ′ ℓ−1 + d′ ℓ−1 µ′′ ℓ−1 + d′′ ℓ−1 (Note that µ′ ℓ−1 + d′ ℓ−1 and µ′′ ℓ−1 + d′′ ℓ−1 are wall points.) In order for the above definition to be well defined the we should check that properties (1) and (2) are satisfied at each step. properties (1) and (2) for wall points are immediate from the definitions. Property (2) for almost corner points follows from the following observation: suppose that J′ ⊂J∞is a component of type J, let v′ 0 denote its source, and let v′ 1, v′′ 1 denote the two sinks (so that v′ 1 and v′′ 1 are of wall type in J∞). Then if Φ(v′ 0) is of distance k ≤ℓ−1 from a corner point, then Φ(v′ 1) and Φ(v′′ 1) are of distance k −1 from a corner point. Example 5.3. We illustrate Φ on a component of type J with ℓ= 5 (the source is marked with a circle): ∗ . . . ∗ ∗ ∗ ∗∗ ∗ * * ∗ ∗ ∗ ∗∗ ∗ ∗ . . . Note the similarity to the previous section. The only difference is the arrow joining an ℓ-regular point to a wall point. Note the similarity to the previous section. The only difference is the arrow joining an ℓ-regular point to a wall point. Note the similarity to the previous section. The only difference is the arrow joining an ℓ-regular point to a wall point. 5.3 Extending Φ Then eΦ takes values in X × Z≥0 × vk, vk+1 on J′. 5.4 Pruning Consider our map Φ = Φλ : J∞→X (which depended on the choice of a fixed almost corner λ). We now “prune” our tree J∞to produce a new tree Jλ. Consider the set (which depended on the choice of a fixed almost corner λ). We now “prune” our tree J∞to produce a new tree Jλ. Consider the set Kλ := v ∈J∞ there exists v1, . . . , vm such that Φ(v1) /∈X++ and v1 →v2 →. . . →vm = v and define Jλ to be the full subgraph with vertices J∞\ Kλ. (That is, we remove all branches from J∞that contain elements which are mapped to weights which are not strictly dominant.) The restriction of Φ to Jλ defines a map and define Jλ to be the full subgraph with vertices J∞\ Kλ. (That is, we remove all branches from J∞that contain elements which are mapped to weights which are not strictly dominant.) The restriction of Φ to Jλ defines a map Φ: Jλ →X++. Restricting the extension eΦ (which depended on an additional choice of label n vk ) of Φ to J∞,++ yields a map: Restricting the extension eΦ (which depended on an additional choice of label n vk ) of Φ to J∞,++ yields a map: eΦ = eΦλ,n(vk) : Jλ →X++ × M. 5.3 Extending Φ Let v0 denote the source of J∞. For any choice of label n vk0 , we inductively extend Φ to produce a map eΦ = eΦλ,n(vk0) : J∞→X × M G. Lusztig and G. Williamson G. Lusztig and G. Williamson 18 such that eΦ(v0) = λ, n vk0 . We proceed as follows: Suppose that v →v′ in J∞and that eΦ is defined on v but not on v′, and let eΦ(v) = Φ(v), m vk . Then eΦ(v′) = Φ(v′), m′vk′ with such that eΦ(v0) = λ, n vk0 . We proceed as follows: Suppose that v →v′ in J∞and that eΦ is defined on v but not on v′, and let eΦ(v) = Φ(v), m vk . Then eΦ(v′) = Φ(v′), m′vk′ with m′ := ( m + 2 if Φ(v) ̸= Φ(v′), m + 3 if Φ(v) = Φ(v′) and and k′ :=                k if Φ(v) ̸= Φ(v′), k + 1 if Φ(v) = Φ(v′) and v →v′ belongs to a component of type I, k ± 1 if Φ(v) = Φ(v′) and v →v′ belongs to a component of type J. k′ :=                k if Φ(v) ̸= Φ(v′), k + 1 if Φ(v) = Φ(v′) and v →v′ belongs to a component of type I, k ± 1 if Φ(v) = Φ(v′) and v →v′ belongs to a component of type J. k′ :=          k + 1 ( ) ( ) v →v′ belongs to a component of type I, k ± 1 if Φ(v) = Φ(v′) and v →v′ belongs to a component of type J   k ± 1 v →v′ belongs to a component of type J. The sign ambiguity in the final case is resolved as follows: suppose that J′ ⊂J∞is a component of type J, v′ 0 is its initial vertex, and eΦ(v′ 0) = µ, m vk . Then eΦ takes values in X × Z≥0 × vk, vk+1 on J′. The sign ambiguity in the final case is resolved as follows: suppose that J′ ⊂J∞is a component of type J, v′ 0 is its initial vertex, and eΦ(v′ 0) = µ, m vk . 5.5 The alternative construction For each component of type J in J∞, if we consider the full subgraph . . . . . . . . . . . . Jint := then the restriction of eΦ to each branch of Jint produces the same set as the algorithm in Section 4.4 (“Billiards in an alcove”). ■ then the restriction of eΦ to each branch of Jint produces the same set as the algorithm in Section 4.4 (“Billiards in an alcove”). ■ then the restriction of eΦ to each branch of Jint produces the same set as the algorithm in Section 4.4 (“Billiards in an alcove”). ■ 5.5 The alternative construction For any positive integer m let λm := (ℓ−1)mϖ1 + ϖ2 and gm := λm, (2mℓ−1) v−1 . The previous constructions provide us with a rooted tree Jλm and a map eΦgm : Jλm →X++ × M. Consider the union of multisets Z′ := [ m≥1 eΦgm(v) \| v ∈Jλm . The following lemma implies that the above algorithm provides an alternative construction of eZ. The following lemma implies that the above algorithm provides an alternative construction of eZ. Billiards and Tilting Characters for SL3 19 Lemma 5.4. We have eZ = Z′ \ {gm \| m ∈Z≥0}. Lemma 5.4. We have eZ = Z′ \ {gm \| m ∈Z≥0}. Proof (sketch). Fix m≥0 and consider eΦ:= eΦgm as defined above. Also, set q= ℓmϖ1, 0 v0 and consider the set Λq defined as above. We claim that we have Λq \ {q} = eΦgm(v) \| v ∈Jℓmϖ1 \ {gm}, (5.1) Λq \ {q} = eΦgm(v) \| v ∈Jℓmϖ1 \ {gm}, (5.1) which implies the lemma. The equality (5.1) follows from the following local considerations: which implies the lemma. The equality (5.1) follows from the following local considerations: 1. The restriction of eΦ to the initial component of type I takes the same values as part (1) of “Dynamics on walls”, except for the first two values (gm and q respectively) which are removed in (5.1). 2. The restriction of eΦ to any component of type I other than the initial segment takes the same values as those produced by part (2) of “Dynamics on the walls”. 2. The restriction of eΦ to any component of type I other than the initial segment takes the same values as those produced by part (2) of “Dynamics on the walls”. 3. If v (resp. v′, v′′) denotes the source (resp. sinks) of a component of type J then eΦ(v′) and eΦ(v′′) are obtained from eΦ(v) by a giant leap, i.e., part (3) of“Dynamics on the walls”. 3. If v (resp. v′, v′′) denotes the source (resp. sinks) of a component of type J then eΦ(v′) and eΦ(v′′) are obtained from eΦ(v) by a giant leap, i.e., part (3) of“Dynamics on the walls”. ach component of type J in J∞, if we consider the full subgraph 4. For each component of type J in J∞, if we consider the full subgraph 4. 6 The conjecture Recall the notion of an ℓ-alcove in XR from above. An alcove is a 1-alcove. The alcove A0 := {λ \| 0 < ⟨α∨, λ⟩< 1 for all α∨∈Φ∨ +} is the fundamental alcove. The map x 7→xA0 gives a bijection between the affine Weyl group W and the set of alcoves. It restricts to a bijection between fW and the dominant alcoves. Given µ ∈X the open box Given µ ∈X the open box Bµ := λ ∈XR \| ⟨α∨, µ⟩< ⟨α∨, λ⟩< ⟨α∨, µ⟩+ 1 for all α∨∈Σ∨ contains exactly 2 alcoves. In this way we obtain a map W →X by sending x ∈W to the unique µ ∈X such that Bµ contains xA0. It restricts to a map contains exactly 2 alcoves. In this way we obtain a map W →X by sending x ∈W to the unique µ ∈X such that Bµ contains xA0. It restricts to a map κ: fW →X+. Let s0 denote the simple affine reflection. Consider the elements Let s0 denote the simple affine reflection. Consider the elements Let s0 denote the simple affine reflection. Consider the elements x0 := id, x1 = s0, x2 = s0s1, x3 = s0s1s2, x4 := s0s1s2s0, . . . of W. (These are the alcoves along one edge of the dominant cone.) We have κ(x2i) = κ(x2i+1) = iϖ for all i ≥0 of W. (These are the alcoves along one edge of the dominant cone.) We have κ(x2i) = κ(x2i+1) = iϖ1 for all i ≥0. We now describe a sort of inverse to κ. Recall that X++ = Z>0ϖ1 ⊕Z>0ϖ2 denotes the strictly dominant weights. Consider µ ∈X++ and let x, x′ denote the two elements of fW indexing alcoves contained in Bµ. If R(z) = {s ∈S \| zs < z} denotes the right descent set then it is easy to see that we have R(x) ⊔R(x′) = {s0, s1, s2} = S (disjoint union). R(x) ⊔R(x′) = {s0, s1, s2} = S (disjoint union). 2 20 G. Lusztig and G. Williamson It follows that for any pair µ ∈X++, s ∈S there is a unique element xs µ ∈fW such that s ∈R(xs µ) and µ = κ(xs µ). It follows that for any pair µ ∈X++, s ∈S there is a unique element xs µ ∈fW such that s ∈R(xs µ) and µ = κ(xs µ). 4However see the last sentence of Remark 2.1. Remark 6.2. Some remarks on the conjecture: Remark 6.2. Some remarks on the conjecture: 1. For an example of eZ the reader is referred to Example 4.5. 2. It is a nice exercise to compare our conjecture to the results of J.G. Jensen [12] and Par- ker [20]. For example, in Fig. 1, the results of Jensen and Parker are explained by the unique alcoves labelled 12(1), 14(1), 16(1), 18(1) and 21(v). 3. Our conjecture (in particular the definition of the set eZ) does not seem to make sense for p = 2. 3. Our conjecture (in particular the definition of the set eZ) does not seem to make sense for p = 2. 4. We have verified part (1) of the conjecture for p = 3, 5 and in many cases for p = 7 by computer. (In fact these calculations led to the conjecture.) 4. We have verified part (1) of the conjecture for p = 3, 5 and in many cases for p = 7 by computer. (In fact these calculations led to the conjecture.) 5. To determine the p-canonical basis in ASv it is enough to know the elements pnxi for all i > 0. (After exploiting the automorphism s0 7→s0, s1 7→s2, s2 7→s1 this can be deduced from a v-analogue of the fact that one can apply the tilting tensor product theorem to determine all tilting characters, provided one knows the tilting characters along the walls and in the (p −1)ρ-shift of the fundamental box, see [24, Section 1.6]. One can check by hand that one has 5. To determine the p-canonical basis in ASv it is enough to know the elements pnxi for all i > 0. (After exploiting the automorphism s0 7→s0, s1 7→s2, s2 7→s1 this can be deduced from a v-analogue of the fact that one can apply the tilting tensor product theorem to determine all tilting characters, provided one knows the tilting characters along the walls and in the (p −1)ρ-shift of the fundamental box, see [24, Section 1.6]. One can check by hand that one has pnx = nx for all x ∈{id, s0, s0s1s2s1, s0s1s2s1s0} and so the only remaining cases are pnxi for i > 1.) 6. Similarly, to know all elements pn2 x for x ∈fW it should be enough to know pn2 xi for all i. 6 The conjecture Recall our prime p from above, and consider the multiset eZ constructed in the previous section with ℓ= p. We now describe how to use eZ to define new elements in the anti-spherical module ASv. Consider the Z-linear map ϕ: Z[v] →Z v±1 given by v0 7→1 and vi 7→vi + v−i for i > 0. Set pζ0 := nx0. For any i > 0 let s ∈S denote the unique element of R(xi) and consider the element: pζi := nxi + X (µ,n(vk))∈e Z; n∈{i,i−1,i−2} ϕ vk nxsµ. pζi := nxi + X (µ n(vk))∈e Z; ϕ vk nxsµ. Conjecture 6.1. We have: Conjecture 6.1. We have: 1) pζi = pnxi for 0 ≤i < 2p(p + 1); 2) pζi = pn2 xi for all 0 ≤i. Remark 6.2. Some remarks on the conjecture: Remark 6.2. Some remarks on the conjecture: Thus our conjecture gives a formula for the pn2 x (which currently have no other rigorous definition4). 6. Similarly, to know all elements pn2 x for x ∈fW it should be enough to know pn2 xi for all i. Thus our conjecture gives a formula for the pn2 x (which currently have no other rigorous definition4). 7. Recall that the exists a bijection between two-sided cells in the affine Weyl group and nilpotent orbits in the dual group [16]. Moreover, every two sided cell intersects fW in a left cell (the canonical left cell) [19]. The “difficult” elements xi for i > 0 all lie in the left cell corresponding to the minimal nilpotent orbit of SL3. This suggests that the problem of determining tilting characters should be related to the geometry of nilpotent or- bits. Related results (connecting the p-canonical basis to coherent sheaves on the Springer resolution) may be found in [3, 4]. 7. Recall that the exists a bijection between two-sided cells in the affine Weyl group and nilpotent orbits in the dual group [16]. Moreover, every two sided cell intersects fW in a left cell (the canonical left cell) [19]. The “difficult” elements xi for i > 0 all lie in the left cell corresponding to the minimal nilpotent orbit of SL3. This suggests that the problem of determining tilting characters should be related to the geometry of nilpotent or- bits. Related results (connecting the p-canonical basis to coherent sheaves on the Springer resolution) may be found in [3, 4]. Billiards and Tilting Characters for SL3 21 8. After taking Remark 4.3 into account, our conjecture implies that the multiplicities of Weyl modules in the tilting module T3pkϖ1 grow (at least) exponentially in k. Thus our con- jecture implies that decomposition numbers for symmetric groups Sn grow exponentially in n (see [7, 10] for the connection between tilting module characters and decomposition numbers), and that the dimension of the tilting module Tkϖ1 grows exponentially in k. Neither of these statements is true for SL2 or for the quantum group of SL3 at a pth root of unity. 9. The powers of v which occur after n iterations of the algorithm of Section 4.3 are all at least n. Remark 6.2. Some remarks on the conjecture: (By contrast, the algorithm Section 4.4 only ever changes the power of v by ±1.) This implies (assuming our conjecture) that pnxi involves arbitrarily high powers of v as i grows. This in turn implies that certain structure constants for the action of hsi on the p-canonical basis involve arbitrarily high powers of v. One can use this observation (and [21, Section 1.4]) to conclude that the analogue of the a-function for the p-canonical basis for the Hecke algebra H of W is unbounded. 10. Fix m = µ, n vk ∈eZ and let x, x′ index the two alcoves contained in Bµ, chosen such that R(xn) ⊂R(x). The formula for pζi above implies that m contributes ϕ vk to the coefficient of nx (resp. nx′, nx) in pζn (resp. pζn+1, pζn+2). A key step in arriving at the multiset eZ is to observe that the elements of the p-canonical basis {pnxi} may be decom- posed into such “triples”. After a first version of this paper was written, L.T. Jensen [13] has proved that such a decomposition is always possible, as a consequence of more general results on the p-canonical basis and the star operations of Kazhdan and the first author. Finally, let us explain how to go from the p-canonical basis to a picture similar to that at the beginning of this paper (which is conjecturally described by eZ). It was this procedure (combined with heuristics as to what constitutes generation 2) that led us to our conjecture. Fix an alcove yA0 (for y ∈fW) which is not on a wall. For all i ≥0 such that pny,xi ̸= 0, we consider the polynomial obtained from pny,xi by discarding negative powers of v, and write i(f) in the alcove yA0. This produces a diagram, in which the strictly dominant alcoves are decorated by symbols of the form i(f).5 Now, for all µ ∈X++ we replace each “triple” of the form i(f), (i + 2)(f) (i + 1)(f) or i(f), (i + 2)(f) (i + 1)(f) by i(f) This is what is depicted (for a second generation version) in Fig 1 i(f), (i + 2)(f) (i + 1)(f) or i(f), (i + 2)(f) (i + 1)(f) by This is what is depicted (for a second generation version) in Fig. 1. This is what is depicted (for a second generation version) in Fig. 1. 5For an example of such a diagram for p = 5 see http://www.maths.usyd.edu.au/u/geordie/pCanA2/ p5pretriples.pdf. Acknowledgements We would like to thank the anonymous referees for their comments. 5For an example of such a diagram for p = 5 see http://www.maths.usyd.edu.au/u/geordie/pCanA2/ p5pretriples.pdf. G. Lusztig and G. Williamson 22 References [1] Achar P.N., Makisumi S., Riche S., Williamson G., Free-monodromic mixed tilting sheaves on flag varieties, arXiv:1703.05843. 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Algebra 1 (2017), 63–125, arXiv:1403.5570. lias B., Losev I., Modular representation theory in type A via Soergel bimodules, arXiv:1701.00560 [10] Erdmann K., Symmetric groups and quasi-hereditary algebras, in Finite-Dimensional Algebras and Related Topics (Ottawa, ON, 1992), NATO Adv. Sci. Inst. Ser. C Math. Phys. Sci., Vol. 424, Kluwer Acad. Publ., Dordrecht, 1994, 123–161. ] James G., The decomposition matrices of GLn(q) for n ≤10, Proc. London Math. Soc. 60 (1990) [12] Jensen J.G., On the character of some modular indecomposable tilting modules for SL3, J. Algebra 232 (2000), 397–419. [13] Jensen L.T., p-Kazhdan–Lusztig theory, Ph.D. Thesis, Max Planck Institute for Mathematics, Bonn, 2017. [14] Jensen L.T., Williamson G., The p-canonical basis for Hecke algebras, in Categorification and Higher Representation Theory, Contemp. Math., Vol. 683, Amer. Math. Soc., Providence, RI, 2017, 333–361, arXiv:1510.01556. [15] Libedinsky N., Williamson G., The anti-spherical category, arXiv:1702.00459. [16] Lusztig G., Cells in affine Weyl groups. IV, J. Fac. Sci. Univ. Tokyo Sect. IA Math. 36 (1989), 297–328. [17] Lusztig G., On the character of certain irreducible modular representations, Represent. Theo 3–8, arXiv:1407.5346. [17] Lusztig G., On the character of certain irreducible modular representations, Represent. Theory 19 (2015), 3–8, arXiv:1407.5346. [18] Lusztig G Williamson G On the character of certain tilting modules Sci China Math 61 (2018) 295 298 G., Williamson G., On the character of certain tilting modules, Sci. China Math. 61 (2018), 295–298 502.04904. ] Lusztig G., Xi N.H., Canonical left cells in affine Weyl groups, Adv. Math. 72 (1988), 284–288. References [20] Parker A., Some remarks on a result of Jensen and tilting modules for SL3(k) and q −GL3(k), arXiv:0809.2249. [21] Riche S., Williamson G., Tilting modules and the p-canonical basis, arXiv:1512.08296. 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https://openalex.org/W2072308100	http://journals.iucr.org/f/issues/2014/10/00/nj5203/nj5203.pdf	English	null	Crystallization and preliminary X-ray diffraction analyses of the redox-controlled complex of terminal oxygenase and ferredoxin components in the Rieske nonhaem iron oxygenase carbazole 1,9a-dioxygenase	Acta crystallographica. Section F, Structural biology communications	2,014	cc-by	3,949	Crystallization and preliminary X-ray diffraction analyses of the redox-controlled complex of terminal oxygenase and ferredoxin components in the Rieske nonhaem iron oxygenase carbazole 1,9a-dioxygenase Acta Crystallographica Section F Structural Biology Communications ISSN 2053-230X Acta Crystallographica Section F Structural Biology Communications ISSN 2053-230X Jun Matsuzawa,a Hiroki Aikawa,a Takashi Umeda,a Yuji Ashikawa,a,b Chiho Suzuki- Minakuchi,a Yoshiaki Kawano,c Zui Fujimoto,d Kazunori Okada,a Hisakazu Yamanee and Hideaki Nojiria* The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 A˚ and belonged to space group P21, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 A˚ , = = 90, = 100.1. The VM value is 2.85 A˚ 3 Da1, indicating a solvent content of 56.8%. aBiotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, bEducation and Research Support Section,Technology Management Division, Administration and Technology Management Center for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan, cSR Life Science Instrumentation Unit, Research Infrastructure Group, Advanced Photon Technology Division, RIKEN SPring-8 Center, RIKEN Harima Branch, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan, dBiomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan, and eDepartment of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320-0003, Japan crystallization communications crystallization communications crystallization communications Table 1 Crystallization conditions of Oxy, Fd and Oxy–Fd. NA, not applicable. CAR = carbazole. Treatment of the crystal formed Condition Space group Reduction CAR exposure Air exposure Reference Oxyox Aerobic, hanging drop, 293 K, 17–19%(v/v) MPD, 0.1 M MES pH 6.2 P213 NA NA NA Nojiri et al. (2005) Oxyred Anaerobic, hanging drop, 293 K, 30%(v/v) PEG MME 550, 0.05 M MgCl26H2O, 0.1 M HEPES pH 7.5 P65 NA NA NA Matsuzawa et al. (2013) Fdox Aerobic, hanging drop, 278 K, 17–19%(w/v) PEG MME 2000, 0.2 M (NH4)2SO4, 0.02 M MgCl26H2O, 0.1 M sodium acetate pH 4.6 P4132 NA NA NA Nam et al. (2005) Oxyox–Fdox Aerobic, hanging drop, 293 K, 1–2 mM sodium dithionite, 14%(w/v) PEG MME 2000, 0.05 M Bis-Tris pH 6.5 P21 NA NA NA Ashikawa et al. (2006) Oxyred–Fdred Yes NA NA Oxyox–Fdox–CAR NA Yes NA Oxyred–Fdred–CAR Yes Yes NA Ashikawa et al. (2012) Oxyox–Fdox–O2 Yes NA Yes Oxyox–Fdox–O2–CAR Yes Yes Yes Oxyred–Fdox Anaerobic, hanging drop, 293 K, 14%(w/v) PEG 3350, 0.1 M sodium cacodylate pH 5.7 P21 NA NA NA This study NA, not applicable. CAR = carbazole. puriﬁed as described previously (Ashikawa et al., 2005; Matsuzawa et al., 2013). Brieﬂy, histidine-tagged Oxy and Fd were expressed in Escherichia coli BL21(DE3) (Novagen, Madison, Wisconsin, USA) and puriﬁed using metal-chelation chromatography followed by gel- ﬁltration chromatography. After puriﬁcation, Oxy and Fd were subjected to ultraﬁltration and buffer-exchanged into 50 mM Tris– HCl pH 7.5 using Vivaspin 20 membranes (10 000 MWCO; Sartorius, Go¨ttingen, Germany) and Centriprep YM-10 (Millipore, Bedford, Massachusetts, USA), respectively. These protein solutions were ﬂash-frozen in liquid nitrogen and stored at 193 K. effectively, the manner of interaction between Oxy and Fd should be altered according to the redox states of the respective components. This is supported by the observation that redox-state-dependent structural change triggers the association/dissociation of the Red and Fd components of biphenyl 2,3-dioxygenase (Senda et al., 2007). However, our previously solved complex structures of Oxy and Fd, of both oxidized Oxyox–Fdox and reduced Oxyred–Fdred (Ashikawa et al., 2006), do not appear in the catalytic cycle of the CARDO reaction; however, the structures clearly revealed the binding region between the two components. 2. Methods and results 2. Methods and results crystallization communications Clariﬁcation of the manner of interaction between Oxyred and Fdox or between Oxyox and Fdred would facilitate an understanding of the redox-dependent association/dissociation mechanism between Oxy and Fd components in RO based on comparisons among the structures in the actual catalytic cycle. Here, we report crystallization and preliminary X-ray diffraction studies of the complex crystal structures of Oxyred and Fdox in CARDO. To prepare the Oxy and Fd solutions for the anaerobic crystal- lization experiments, an anaerobic chamber ﬁlled with 95% N2 and 5% H2 was used as described previously (Matsuzawa et al., 2013). Puriﬁed Oxy and Fd were put into the anaerobic chamber and incubated for several hours on ice to remove the dissolved oxygen. Oxy was then reduced using two equivalents of sodium dithionite dissolved in deoxygenated 50 mM Tris–HCl pH 7.5. Fd and reduced Oxy were separately concentrated and buffer-exchanged four times into deoxygenated 50 mM Tris using Nanosep 10K Omega devices (Pall, Port Washington, New York, USA) to eliminate the dissolved oxygen and remaining sodium dithionite (in the case of Oxy). Aliquots of the protein solutions were subsequently added to indi- vidual quartz cells sealed with butyl rubber caps and the redox states 1. Introduction Rieske nonhaem iron oxygenases (ROs) play an important role as the initial enzymes in aromatic compound catabolic pathways (Gibson & Parales, 2000; Parales & Resnick, 2006). ROs consist of two or three discrete soluble components. Two-component ROs consist of reduc- tase (Red) and terminal oxygenase (Oxy) components, whereas three-component ROs consist of Red, ferredoxin (Fd) and Oxy components. Oxy invariably contains a Rieske-type [2Fe–2S] cluster and a nonhaem iron active centre involved in dioxygen activation and it catalyzes the incorporation of molecular dioxygen into the substrate. Although there are variations in the redox-transfer machineries in both Fd and Red, these electron-transfer components transfer electrons from NAD(P)H to Oxy. Carbazole 1,9a-dioxygenase (CARDO) was originally isolated from Pseudomonas resinovorans CA10 as the initial enzyme of the carbazole-degradation pathway, and similar degradation systems have subsequently been reported in several other bacteria, for example Nocardioides aromaticivorans IC177, Novosphingobium sp. KA1 and Janthinobacterium sp. J3 (Nojiri & Omori, 2006; Nojiri, 2012; Inoue & Nojiri, 2014). The amino-acid sequences of CARDO components from CA10 are nearly identical to those of J3, and the components can replace each other. Therefore, using the CARDO components from CA10 and J3, we have evaluated the reaction cycle and the electron-transfer mechanism among components in RO, especially between Fd and Oxy, by determining the crystal structures of the oxidized forms of Fd from CA10 (Nam et al., 2005) and Oxy from J3 (Nojiri et al., 2005) and of the complex forms of Oxy from J3 and Fd from CA10 (Ashikawa et al., 2006). Recently, substrate-bound and oxygen-bound forms of the Oxy–Fd complex were determined and the catalytic cycle of Oxy was elucidated from a structural viewpoint (Ashikawa et al., 2012). Crystals of Oxy alone, Fd alone and Oxy–Fd complexes and the conditions for their formation are summarized in Table 1. Correspondence e-mail: anojiri@mail.ecc.u-tokyo.ac.jp Received 4 July 2014 Accepted 18 August 2014 Correspondence e-mail: anojiri@mail.ecc.u-tokyo.ac.jp 1406 doi:10.1107/S2053230X14018779 Electron transfer between Oxy and Fd components is proposed to proceed in three steps (Fig. 1). Firstly, Fdred associates with Oxyox (where the subscripts ‘ox’ and ‘red’ indicate the oxidized and reduced states, respectively). Next, the electron is transferred from Fdred to Oxyox and the redox states of the components are reverted. Finally, Fdox dissociates from Oxyred. To accomplish these sequential steps Acta Cryst. (2014). F70, 1406–1409 1406 doi:10.1107/S2053230X14018779 crystallization communications after 1–2 d. SDS–PAGE and Western blot analysis were performed to verify the presence of both Oxy and Fd in these crystals. The crystals obtained in this study were dissolved in 5 mM Tris–HCl. The resulting protein, Oxy and Fd solutions were subjected to SDS–PAGE and stained with Coomassie Brilliant Blue. Although there were two bands corresponding to the sizes of Oxy and Fd, possible degradation peptides derived from Oxy were also detected (Fig. 3a, lanes 1 and 3). After SDS–PAGE, the proteins were also transferred onto Sequi-Blot polyvinylidene diﬂuoride (PVDF) membranes (Bio-Rad). Oxy and Fd were detected using anti-His antibody (GE Healthcare, Buck- inghamshire, England) as the primary antibody and HRP-linked anti- mouse antibody (GE Healthcare) as the secondary antibody. Signals were visualized using the Luminescent Image Analyzer LAS-1000 Plus (Fujiﬁlm, Tokyo, Japan). The hybridization signals for possible Oxy degradation peptides were not detected, while signals corre- sponding to the positions of Fd and Oxy were clearly detected in the obtained crystal (Fig. 3b), indicating an Oxy–Fd binary complex. were conﬁrmed by measuring the absorption spectra as described previously (Matsuzawa et al., 2013). Oxy and Fd showed the peaks characteristic of the redox states of the Rieske [2Fe–2S] cluster: Oxyred, 420 and 525 nm; Oxyox, 459 and 560 nm; Fdred, 432 and 515 nm; and Fdox, 457 and 570 nm (Nam et al., 2002). Protein concentrations were estimated using a protein assay kit (Bio-Rad, Richmond, California, USA) with BSA as the standard. 2.2. Crystallization For crystallization experiments, Oxyred and Fdox were mixed in a 1:3 molar ratio and then mixed with glycerol as an additive. Total protein concentration was adjusted to 15–30 mg ml1 and the glycerol concentration was adjusted to 10%(v/v). Crystallization was performed using the hanging-drop vapour-diffusion method at 293 K. Drops consisting of 2 ml protein solution and 2 ml mother liquor were equilibrated against 400–600 ml reservoir solution. The initial crys- tallization conditions were screened using Crystal Screen, Crystal Screen 2, Crystal Screen Cryo and Index (Hampton Research, Laguna Hills, California, USA) and the Crystallization Kit for Protein Complexes (Sigma–Aldrich, St Louis, Missouri, USA). Several crys- tals were obtained using the Crystallization Kit for Protein Complexes condition No. 12 [0.1 M sodium cacodylate pH 6.5, 20% (w/v) PEG 3350]. To improve the crystallization conditions, the pH and precipitant concentration were assessed. Finally, plate-shaped crystals were obtained using 0.1 M sodium cacodylate pH 5.7, 14%(w/v) PEG 3350 (Table 1, Fig. 2). Crystal growth was observed 2.1. Purification, anaerobic procedure and protein reduction The Oxy component of CARDO from Janthinobacterium sp. J3 and the Fd component of CARDO from P. resinovorans CA10 were Figure 1 The electron-transfer reaction between the Oxy and Fd components of CARDO. Reduced and oxidized states of the components are shown in blue and red, respectively. Electrons are shown as pink spheres labelled e. The black arrow in the proposed complex state (Oxyox–Fdred) in parentheses shows electron transfer from the Rieske-type [2Fe–2S] cluster of Fdred to the Rieske-type [2Fe–2S] cluster of Oxyox. The Oxyred–Fdox complex obtained in this study is shown against a grey background. g The electron-transfer reaction between the Oxy and Fd components of CARDO. Reduced and oxidized states of the components are shown in blue and red, respectively. Electrons are shown as pink spheres labelled e. The black arrow in the proposed complex state (Oxyox–Fdred) in parentheses shows electron transfer from the Rieske-type [2Fe–2S] cluster of Fdred to the Rieske-type [2Fe–2S] cluster of Oxyox. The Oxyred–Fdox complex obtained in this study is shown against a grey background. Matsuzawa et al. Carbazole 1,9a-dioxygenase Matsuzawa et al. Carbazole 1,9a-dioxygenase Matsuzawa et al. Carbazole 1,9a-dioxygenase 1407 1407 Acta Cryst. (2014). F70, 1406–1409 1408 Matsuzawa et al. Carbazole 1,9a-dioxygenase Acta Cryst. (2014). F70, 1406–1409 2.3. Determination of the redox states of the crystals Figure 3 SDS–PAGE followed by Western blot analysis of Oxy and Fd complex crystals. (a) The dissolved crystals were veriﬁed by SDS–PAGE. Lane M, Precision Plus Protein Dual Color Standards (Bio-Rad; labelled in kDa); lane 1, Oxy solution used for crystallization; lane 2, Fd solution used for crystallization; lane 3, dissolved crystals of the complex. Approximately 3 mg protein was loaded per lane. (b) A Western blot stained with the anti-His antibody is shown; the lane numbers are the same as those in (a). Figure 3 SDS–PAGE followed by Western blot analysis of Oxy and Fd complex crystals. (a) The dissolved crystals were veriﬁed by SDS–PAGE. Lane M, Precision Plus Protein Dual Color Standards (Bio-Rad; labelled in kDa); lane 1, Oxy solution used for crystallization; lane 2, Fd solution used for crystallization; lane 3, dissolved crystals of the complex. Approximately 3 mg protein was loaded per lane. (b) A Western blot stained with the anti-His antibody is shown; the lane numbers are the same as those in (a). Figure 2 2.3. Determination of the redox states of the crystals To verify the redox state of each component in the crystals, the absorption spectrum of crystals of the binary complex was measured using a microspectrophotometer under a cryostream of nitrogen at Figure 3 SDS–PAGE followed by Western blot analysis of Oxy and Fd complex crysta The dissolved crystals were veriﬁed by SDS–PAGE. Lane M, Precision Plus Pr Dual Color Standards (Bio-Rad; labelled in kDa); lane 1, Oxy solution use crystallization; lane 2, Fd solution used for crystallization; lane 3, dissolved cr Acta Cryst (2014) F70 1406–1409 Figure 3 SDS–PAGE followed by Western blot analysis of Oxy and Fd complex crystals. (a) The dissolved crystals were veriﬁed by SDS–PAGE. Lane M, Precision Plus Protein Dual Color Standards (Bio-Rad; labelled in kDa); lane 1, Oxy solution used for crystallization; lane 2, Fd solution used for crystallization; lane 3, dissolved crystals of the complex. Approximately 3 mg protein was loaded per lane. (b) A Western blot stained with the anti-His antibody is shown; the lane numbers are the same as those in (a). Figure 2 Crystals of the binary complex between the reduced state of Oxy from Janthinobacterium sp. J3 and the oxidized state of Fd from P. resinovorans CA10. Plate-shaped crystals in the drop (a) and one piece of the crystal (b) are shown. The scale bar is 0.3 mm in length. Figure 3 SDS–PAGE followed by Western blot an The dissolved crystals were veriﬁed by SD Dual Color Standards (Bio-Rad; labelled crystallization; lane 2, Fd solution used fo of the complex. Approximately 3 mg pro blot stained with the anti-His antibody is those in (a). Figure 2 Crystals of the binary complex between the reduced state of Oxy from Janthinobacterium sp. J3 and the oxidized state of Fd from P. resinovorans CA10. Plate-shaped crystals in the drop (a) and one piece of the crystal (b) are shown. The scale bar is 0.3 mm in length. Figure 3 Figure 3 SDS–PAGE followed by Western blot analysis of Oxy and Fd complex crystals. (a) The dissolved crystals were veriﬁed by SDS–PAGE. Lane M, Precision Plus Protein Dual Color Standards (Bio-Rad; labelled in kDa); lane 1, Oxy solution used for crystallization; lane 2, Fd solution used for crystallization; lane 3, dissolved crystals of the complex. Approximately 3 mg protein was loaded per lane. (b) A Western blot stained with the anti-His antibody is shown; the lane numbers are the same as those in (a). 2.4. Data collection j M., Yamaguchi, H., Kawano, Y., Kamiya, N., Kuroda, S., Hayashi, H., Yamamoto, Y. & Tanizawa, K. (2006). Biochemistry, 45, 4105–4120. Gibson, D. T. & Parales, R. E. (2000). Curr. Opin. Biotechnol. 11, 236–243. Inoue, K. & Nojiri, H. (2014). Biodegradative Bacteria, edited by H. Nojiri, M. M., Yamaguchi, H., Kawano, Y., Kamiya, N., Kuroda, S., Hayashi, H., Yamamoto Y & Tanizawa K (2006) Biochemistry 45 4105 4120 Yamamoto, Y. & Tanizawa, K. (2006). Biochemistry, 45, 4105–4120. Gibson, D. T. & Parales, R. E. (2000). Curr. Opin. Biotechnol. 11, 236–243. Inoue, K. & Nojiri, H. (2014). Biodegradative Bacteria, edited by H. Nojiri, M. The crystals were cryocooled using liquid nitrogen in an anaerobic chamber with a mixture of 0.1 M sodium cacodylate pH 5.7, 14% PEG 3350 and 15% glycerol as a cryoprotectant. , , ( ) y, , Gibson, D. T. & Parales, R. E. (2000). Curr. Opin. Biotechnol. 11, 236–243. Gibson, D. T. & Parales, R. E. (2000). Curr. Opin. Biotechnol. 11, 236–243. Inoue, K. & Nojiri, H. (2014). Biodegradative Bacteria, edited by H. Nojiri, M. Inoue, K. & Nojiri, H. (2014). Biodegradative Bacteria, edited by H. Nojiri, M. Tsuda, M. Fukuda & Y. Kamagata, pp. 181–205. Tokyo: Springer. Matsuzawa, J., Umeda, T., Aikawa, H., Suzuki, C., Fujimoto, Z., Okada, K., g pp y p g Matsuzawa, J., Umeda, T., Aikawa, H., Suzuki, C., Fujimoto, Z., Okada, K., X-ray diffraction data were collected on a MAR225 CCD detector at 100 K using synchrotron radiation of wavelength 1.0 A˚ on BL26B2 at SPring-8 (Harima, Japan) and were processed using HKL-2000 (Otwinowski & Minor, 1997). The crystals diffracted to 2.25 A˚ resolution and belonged to space group P21, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 A˚ , = = 90, = 100.1. The data- collection and processing statistics are shown in Table 2. The struc- ture of the Oxyox–Fdox binary complex (PDB entry 2de5; Ashikawa et al., 2006) was used as a molecular-replacement model for MOLREP (Winn et al., 2011; Vagin & Teplyakov, 2010). Although previous Oxy–Fd binary-complex crystals consisted of three molecules of the Fd monomer and one molecule of the Oxy trimer, the complex structure calculated from these crystals lacked one molecule of the Fd monomer and contained only two molecules of the Fd monomer per one Oxy trimer. References Ashikawa, Y., Fujimoto, Z., Noguchi, H., Habe, H., Omori, T., Yamane, H. & Nojiri, H. (2005). Acta Cryst. F61, 577–580. Ashikawa, Y., Fujimoto, Z., Noguchi, H., Habe, H., Omori, T., Yamane, H. & Nojiri, H. (2006). Structure, 14, 1779–1789. A hik Y F ji Z U i Y I K N hi H Y H & Ashikawa, Y., Fujimoto, Z., Usami, Y., Inoue, K., Noguchi, H., Yamane, H. & Nojiri, H. (2012). BMC Struct. Biol. 12, 15. Nojiri, H. (2012). BMC Struct. Biol. 12, 15. Chiu, Y.-C., Okajima, T., Murakawa, T., Uchida, M., Taki, M., Hirota, S., Kim, j ( ) Chiu, Y.-C., Okajima, T., Murakawa, T., Uchida, M., Taki, M., Hirota, S., Kim, M., Yamaguchi, H., Kawano, Y., Kamiya, N., Kuroda, S., Hayashi, H., Figure 4 Figure 4 Figure 4 Absorption spectrum of binary-complex crystals. The blue arrow indicates the peak shoulder speciﬁc for reduced Oxy (530–540 nm) and the red arrows indicate those speciﬁc for oxidized Fd (460 and 570–590 nm). † Rmerge = P hkl P i jIiðhklÞ hIðhklÞij=P hkl P i IiðhklÞ, where Ii(hkl) is the ith observa- tion of reﬂection hkl and hI(hkl)i is the weighted average intensity for all observations of reﬂection hkl. † Rmerge = P hkl P i jIiðhklÞ hIðhklÞij=P hkl P i IiðhklÞ, where Ii(hkl) is the ith observa- tion of reﬂection hkl and hI(hkl)i is the weighted average intensity for all observations of reﬂection hkl. A full description of the structure determination followed by interpretation of the structure–function relationship will be published elsewhere. 100 K (Chiu et al., 2006). The microspectrophotometer system consisted of a deuterium tungsten halogen light (DT-MINI; Ocean Optics, Tokyo, Japan), Cassegrainian mirrors (Bunkoh-Keiki, Tokyo, Japan), an optical ﬁbre and a linear CCD array spectrometer (SD2000; Ocean Optics). The crystals showed characteristic peaks at around 460 nm and peak shoulders at 525–540 and 570–590 nm (Fig. 4). In comparison with previous results (Nam et al., 2002; Matsuzawa et al., 2013), the peak at around 460 nm and the peak shoulder at 570– 590 nm suggested that the Rieske [2Fe–2S] cluster in Fd was oxidized, because the oxidized form of Oxy shows a peak shoulder at a shorter wavelength such as 550–570 nm. On the other hand, the peak shoulder at 530–540 nm suggested that the Rieske [2Fe–2S] cluster of Oxy was reduced, because the reduced form of Fd shows a peak shoulder at a shorter wavelength such as 510–520 nm (Nam et al., 2002; Matsuzawa et al., 2013). Accordingly, the crystals were found to be composed of Oxyred and Fdox. The authors thank Dr Atsuko Yamashita of Okayama University formerly of RIKEN Harima Institute for helpful advice in data collection. This work was based on experiments performed at SPring- 8, with the approval of the Japan Synchrotron Radiation Research Institute, Harima, Japan, and at the Photon Factory, with the approval of the Photon Factory Program Advisory Committee, Tsukuba, Japan (proposal Nos. 2008G681, 2008G702, 2009G675, 2010G663 and 2013G720). Figure 2 Figure 2 Crystals of the binary complex between the reduced state of Oxy from Janthinobacterium sp. J3 and the oxidized state of Fd from P. resinovorans CA10. Plate-shaped crystals in the drop (a) and one piece of the crystal (b) are shown. The scale bar is 0.3 mm in length. Acta Cryst. (2014). F70, 1406–1409 Acta Cryst. (2014). F70, 1406–1409 crystallization communications Figure 4 Absorption spectrum of binary-complex crystals. The blue arrow indicates the peak shoulder speciﬁc for reduced Oxy (530–540 nm) and the red arrows indicate those speciﬁc for oxidized Fd (460 and 570–590 nm). Table 2 Crystal parameters and data-collection statistics. Values in parentheses are for the highest resolution shell. Space group P21 Unit-cell parameters (A˚ , ) a = 97.3, b = 81.6, c = 116.2, = = 90, = 100.1 Beamline BL26B2, SPring-8 Wavelength (A˚ ) 1.000 Rotation range per image () 0.5 Total rotation range () 360 Exposure time per image (s) 10 Crystal-to-detector distance (mm) 213.50 Resolution range (A˚ ) 50.00–2.25 (2.33–2.25) Total No. of reﬂections 579068 No. of unique reﬂections 82730 (7180) Completeness (%) 97.0 (84.7) Mosaicity () 0.54 – 0.86 Average I/(I) 42.6 (3.5) Rmerge† (%) 6.8 (38.1) Multiplicity 7.0 (5.4) Overall B factor from Wilson plot (A˚ 2) 46.9 † Rmerge = P hkl P i jIiðhklÞ hIðhklÞij=P hkl P i IiðhklÞ, where Ii(hkl) is the ith observa- tion of reﬂection hkl and hI(hkl)i is the weighted average intensity for all observations of reﬂection hkl. 2.4. Data collection After recalculation of this structure, the Matthews coefﬁcient (VM; Matthews, 1968) was found to be 2.85 A˚ 3 Da1, indicating a solvent content of 56.8%. Yamane, H. & Nojiri, H. (2013). Acta Cryst. F69, 1284–1287. 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https://openalex.org/W2914351305	https://eprints.soton.ac.uk/431927/2/Anomalous_supersymmetry.pdf	English	null	Anomalous Supersymmetry	Physical review letters	2,019	cc-by	5,247	(Received 4 April 2019; published 14 June 2019) (Received 4 April 2019; published 14 June 2019) We show that supersymmetry is anomalous in N ¼ 1 superconformal quantum field theories (SCFTs) with an anomalous R symmetry. This anomaly was originally found in holographic SCFTs at strong coupling. Here we show that this anomaly is present, in general, and demonstrate it for the massless superconformal Wess-Zumino model via a one-loop computation. The anomaly appears first in four-point functions of two supercurrents either with two R currents or with an R current and an energy-momentum tensor. In fact, the Wess-Zumino consistency conditions together with the standard R-symmetry anomaly imply the existence of the anomaly. We outline the implications of this anomaly. DOI: 10.1103/PhysRevLett.122.231602 Anomalies of symmetries play an important role in quantum field theories. If a global symmetry is anomalous, classical selection rules are not respected in the quantum theory, and classically forbidden processes may occur. This is a feature of the theory, and it is linked with observable effects. For example, the axial anomaly explains the π0 decay and leads to the resolution of the Uð1Þ problem in QCD [1,2]. On the other hand, anomalies in local (gauge) symmetries lead to inconsistencies, such as lack of unitar- ity, and they must be canceled. An important corollary is that anomalous global symmetries cannot be consistently coupled to corresponding local symmetries. Reviews on anomalies in quantum field theories may be found in Refs. [3,4]. triangle diagrams [1,2]. Here, we will carry out the analogous computation for the supersymmetry anomaly. The anomaly is associated, in particular, with anomalous one-loop contributions to four-point correlation functions between two supersymmetry currents and two R currents or an R current and an energy-momentum tensor. We will discuss the former in the free superconformal WZ model, but analogous contributions would arise in any supersym- metric theory with a (softly broken) anomalous R symmetry. Actually, as will be sketched below and is shown in detail in the companion paper [6], the WZ consistency conditions [7] together with the standard triangle anomalies imply that supersymmetry must be anomalous. Discussion of anomalies in 4d (super)conformal quan- tum field theory (QFT) has a long history. It has been known since the 1970s [8,9] that the trace of the stress tensor T μ μ is anomalous in the presence of a curved background metric gμν and background source Aμ for a chiral current J μ, and the R current is similarly anomalous. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. PHYSICAL REVIEW LETTERS 122, 231602 (2019) PHYSICAL REVIEW LETTERS 122, 231602 (2019) Editors' Suggestion Anomalous Supersymmetry Georgios Katsianis,1,2 Ioannis Papadimitriou,3 Kostas Skenderis,1,2 and Marika Taylor1,2 1STAG Research Centre, Highfield, University of Southampton, SO17 1BJ Southampton, United Kingdom 2Mathematical Sciences, Highfield, University of Southampton, SO17 1BJ Southampton, United Kingdom 3School of Physics, Korea Institute for Advanced Study, Seoul 02455, Korea Georgios Katsianis,1,2 Ioannis Papadimitriou,3 Kostas Skenderis,1,2 and Marika Taylor1,2 1STAG Research Centre, Highfield, University of Southampton, SO17 1BJ Southampton, United Kingdom 2Mathematical Sciences, Highfield, University of Southampton, SO17 1BJ Southampton, United Kingdom 3School of Physics, Korea Institute for Advanced Study, Seoul 02455, Korea (Received 4 April 2019; published 14 June 2019) (Received 4 April 2019; published 14 June 2019) Anomalies associated with correlation functions of conserved currents can be analyzed by coupling the currents to external sources, which in our case form an N ¼ 1 superconformal multiplet. As such, the anomaly we discuss here could be related to existing superspace results on anomaly candidates for D ¼ 4, N ¼ 1 supergravity theories [22–26] (in particular, in type II anomalies in Ref. [25]), though we emphasize that in our case the supergravity fields are external and thus nondynamical (off shell). The holographic results leave open the possibility that the anomaly is special to holographic theories at strong coupling. In this Letter, we show that this is not the case. One could have anticipated the anomaly based on the structure of the supersymmetric variation of the super- current, which is of the schematic form δQμ ∼γνT μνεþ Cμνρ∂νJ ρε, where Cμνρ is a tensor constructed from gamma matrices and the metric. The Ward identity for the four- point function involving two supercurrents and two R currents would then involve terms of the form A supersymmetry anomaly appears in super Yang-Mills (SYM) theory in the WZ gauge when there are gauge anomalies [27] (see also [28–30]). This anomaly is easy to understand: In the WZ gauge, supersymmetry transforma- tions require a compensating gauge transformation, and this transfers the anomaly from the gauge sector to supersym- metry. When the SYM theory is consistent at the quantum level (i.e., the gauge anomalies cancel), then supersym- metry is also nonanomalous. A supersymmetry anomaly appears in theories with gravitational anomalies [31–33], as one may anticipate based on the fact that the energy- momentum tensor and the supercurrent are part of the same supermultiplet. Indeed, this supersymmetry anomaly sits in the same multiplet as the gravitational anomaly. ∂x1 μ hQμðx1Þ ¯Qνðx2ÞJ κðx3ÞJ λðx4Þi ∼δðx1 −x2Þhδ ¯Qνðx2ÞJ κðx3ÞJ λðx4Þi þ ; ð1Þ ð1Þ where the dots denote additional terms [the exact Ward identity is given (9)]. Using the variation of the super- current, we find that the rhs contains the three-point function of three R currents, which is anomalous, and correspondingly one may anticipate (1) will be anomalous. Similarly, the same four-point function but with one of the R currents replaced by an energy-momentum tensor is expected to be anomalous, since hJ T T i is anomalous. To determine whether an anomaly appears or not, we need to carry out the computation explicitly. (Received 4 April 2019; published 14 June 2019) AdS=CFT relates the N ¼ 1 super- conformal quantum field theory (SCFT) in four dimensions to N ¼ 2 gauged supergravity in five dimensions. Starting from gauged supergravity in an asymptotically locally AdS5 spacetime and turning on sources for all super- conformal currents, one can compute the complete set of superconformal anomalies. This computation is available for holographic CFTs, which, in particular, means that the central charges should satisfy a ¼ c as N →∞[34]. Another set of studies, reviewed in Ref. [21], considers the effective action for elementary fields and examines whether it is invariant under supersymmetry including loop effects; it investigates the conservation of the supercurrent inside correlators of elementary fields and/or solves the WZ consistency conditions relevant for this setup and finds no supersymmetry anomaly. This does not contradict the results we present below: To find the anomaly, one should either put the theory on a nontrivial background or consider correlation functions of (classically) conserved currents. (To illustrate this point, consider a free fermion in a complex representation in flat spacetime. This theory has a standard axial anomaly originating from the three-point function of the axial current. However, if one looks only at correlators of elementary fields, these are nonanomalous, and the axial current inside such correlators is conserved.) Studies involving correlators of currents have also appeared, but typically only discuss three-point functions of bosonic currents. As mentioned above, the supersym- metry anomaly appears first in four-point functions involv- ing two supercurrents and two bosonic currents, and to our knowledge these have not been computed before. g y Early attempts to compute the supertrace Ward identity can be found in Refs. [36,37], but these missed contribu- tions to the anomaly involving the R-symmetry current and the Ricci tensor. Following the work of Pestun [38], there was renewed interest in supersymmetric theories on curved spacetimes and their holographic duals. The holographic anomalies for bosonic currents were computed in Ref. [39], reproducing (and correcting) known field theory results [40]. The full superconformal anomalies for the N ¼ 1 current multiplet were computed holographically in Ref. [5], while Ref. [41] obtained the superconformal anomalies in the presence of local supersymmetric scalar couplings. An analogous holographic computation relevant to two-dimensional SCFTs was reported in Ref. [42]. (Received 4 April 2019; published 14 June 2019) Moreover, there are generally mixed anomalies involving two energy-momentum tensors and a chiral current [10,11]. It has also been known since Ref. [12] that the currents sit in a supermultiplet, as do the anomalies. In particular, the trace anomaly and the R-current anomaly are in the same multiplet as the gamma trace of the supercurrent, γμQμ. The latter is an anomaly in the conservation of the special supersymmetry current, xνγνQμ. It follows that special supersymmetry (sometimes also called S supersymmetry) is anomalous. It was believed, however, that supersym- metry itself (sometimes called Q supersymmetry) is pre- served; i.e., the conservation of Qμ is nonanomalous. Anomalies in supersymmetric theories.—In this Letter, we discuss a new anomaly in four-dimensional super- symmetric quantum field theories with an anomalous R symmetry: Global supersymmetry itself is anomalous. This anomaly was discovered in the context of superconformal theories that can be realized holographically [5]. Here, we show that the same anomaly arises in the perturbation theory in the simplest supersymmetric model: the free superconformal Wess-Zumino (WZ) model. An anomaly may be detected either by putting the theory on a nontrivial background or by computing correlation functions on a flat background and checking whether the Ward identities are satisfied. The latter method was the one that led to the original discovery of anomalies via one-loop There have been extensive studies in the past regarding anomalies in supersymmetry. It was realized early on [13–18] that one cannot maintain at the quantum level simultaneously ∂μQμ ¼ 0 and γμQμ ¼ 0 and, if the model 231602-1 Published by the American Physical Society 0031-9007=19=122(23)=231602(6) PHYSICAL REVIEW LETTERS 122, 231602 (2019) conserved energy-momentum tensor. We also emphasize that we are concerned with local anomalies, not with beta functions. is a gauge theory, gauge invariance: One of the three conditions must be relaxed, and the standard choice is to have a superconformal anomaly. This is the standard superconformal anomaly mentioned above and is distinct from the anomaly discussed here. Also distinct is the Konishi anomaly [19,20], which is a superspace version of the chiral anomaly in supersymmetric gauge theories. Holographic anomalies.—The anomaly we discuss here was first computed holographically [5]. In holography, given a bulk action, one can use holographic renormaliza- tion [34,35] to compute the Ward identities and anomalies of the dual QFT. (Received 4 April 2019; published 14 June 2019) Before we turn to this, Here, we will discuss a supersymmetry anomaly in consistent QFTs (no gauge anomalies) which have a 231602-2 PHYSICAL REVIEW LETTERS 122, 231602 (2019) we discuss the consistency condition that the anomalies must satisfy. anomalies relevant for us), as well as in the context (the WZ conditions discussed in Ref. [27] are for a vector multiplet in flat space, while the anomalies in Table II are those of N ¼ 1 conformal supergravity [6]). Wess-Zumino consistency.—Let eaμ, Aμ, and ψμ denote the sources (vierbein, gauge field, and gravitino) that couple to the superconformal currents and W½e; A; ψ be the generating functional of connected graphs. We define the currents in the presence of sources (as usual) by Here we discuss only one of the WZ equations: the one obtained by considering the commutator of R symmetry (with parameter θ) with Q supersymmetry (with para- meter ε): T μ a ¼ e−1 δW δeaμ ; J μ ¼ e−1 δW δAμ ; Qμ ¼ e−1 δW δ ¯ψμ ; ð2Þ Z d4x½δεðeθARÞ −δθðeεAQÞ ¼ 0: ð5Þ ð5Þ ð2Þ Using the explicit form of AR, it is easy to see that δεAR ≠0 and the WZ consistency condition requires that AQ ≠0. This argument does not rely on the theory having conformal invariance, and thus we expect any 4d super- symmetric theory with an R-symmetry anomaly to have a corresponding anomaly in the conservation of the super- current. (This expectation has been verified in the followup paper [43].) where e ≡detðeaμÞ. In the presence of anomalies δiW ¼ Z d4xeϵiAi; ð3Þ ð3Þ where δi denotes the superconformal transformations, ϵi are the (local) parameters of the transformations, and Ai are the corresponding anomalies. The variations form an algebra, ½δi; δj ¼ fk ijδk, and using this in (3) we obtain the WZ consistency condition One may wonder whether this anomaly can be removed by adding a local counterterm Wct to the action such that Wren ¼ W þ Wct is nonanomalous, i.e., δεWren ¼ 0. Using the commutator of two supersymmetry variations, ½δε; δε0, given in Table I, we find Z d4x½δiðeϵjAjÞ −δjðeϵiAiÞ −fk ijeϵkAk ¼ 0: ð4Þ ð4Þ ðδξ þ δλ þ δθÞWren ¼ 0 ⇒ðδξ þ δλÞWren ≠0; ð6Þ since δθWren ¼ AR ≠0. It follows that if one wishes to preserve supersymmetry, Wct must break diffeomorphisms and/or local Lorentz transformations. (Received 4 April 2019; published 14 June 2019) [Note that, since AR is a genuine anomaly, it is not possible to set the rhs of the second equation in (6) to zero using a local counterterm. This implies that there are no further local counterterms that can restore diffeomeorphisms and local Lorentz invariance.] Next, we calculate this anomaly by one-loop computations within a specific model. The transformation rules and the local algebra they satisfy are derived in Ref. [6] and are given in Table I. Assuming the R-symmetry current has the standard triangle anomalies (i.e., assuming the from of AR in Table II), the WZ consistency conditions (4) may be viewed as equations to determine the remaining anomalies. This computation is presented in Ref. [6], and the results are summarized in Table II. Note, in particular, that all anomalies are given in terms of the central changes a and c. The anomalies of the bosonic currents are in agreement with the results derived in Refs. [39,40]. The supersym- metry anomaly AQ that we discuss here is related to the R- symmetry anomaly AR through the same descent equation that relates the supersymmetry anomaly discussed in Ref. [27] to the corresponding gauge anomaly. However, as noted earlier, there are important differences in the physics (in Ref. [27], the gauge anomalies must vanish for consistency of the model, while this is not so for the R Model.—Consider the massless Wess-Zumino action with one complex bosonic field ϕ and one Majorana fermionic field χ: S ¼ − Z d4x ∂μϕ∂μϕ þ 1 2 ¯χ=∂χ : ð7Þ ð7Þ The conserved currents are given in Table III. We have included improvement terms so that classically T μ μ ¼ 0, γμQμ ¼ 0 and we are dealing with an N ¼ 1 SCFT. TABLE I. Transformation rules of the current sources and their algebra, to leading order in the gravitino. All other commutators vanish, except for that of two diffeomorphisms and two local Lorentz transformations, which take a standard form. PHYSICAL REVIEW LETTERS 122, 231602 (2019) Here we discuss the former, referring to Ref. [44] for a detailed account of both cases. expressed in terms of the supersymmetry variations of the currents: δεQμ ¼ εδQμ þ ∂νεδQ0μν and idem for J μ. A similar Ward identity follows from R invariance. One-loop computation.—We now compute (9). Since the theory is free, the complete computation is one loop. The four-point function receives contributions from three classes of Feynman box diagrams, shown in Fig. 1; this computation is straightforward but tedious. Since we seek to investigate the possibility of a super- symmetry anomaly, we should not assume the existence of a supersymmetric regulator: The one-loop computation should not be done in superspace. (On the other hand, the form of anomalies respects the symmetries they break, and, thus, one may use superspace to analyze possible anomaly candidates.) We will instead do the computation in components and use the same regulator as in the original triangle anomaly computation, namely, momentum cutoff [1,2]. We will consider the four-point correlation function One may verify that (9), as well as the corresponding R- symmetry Ward identity, is (naively) satisfied by a simple shift of the loop momentum, much the same way as the triangle Ward identity is naively satisfied. Again in parallel with the triangle anomaly, (part of) the one-loop contribu- tions to the four-point function are superficially linearly divergent. This implies that there is a momentum routing ambiguity when using a momentum cutoff regulator (see, for example, Jackiw’s lectures in Ref. [3]). hQμðx1Þ ¯Qνðx2ÞJ κðx3ÞJ λðx4Þi: ð8Þ ð8Þ We proceed by taking the ∂x3 κ of (9) and subtracting from it the ∂x1 μ derivative of the corresponding R-symmetry Ward identity. By construction, the four-point functions cancel, and one is left with an identity involving three-point functions only (namely, the terms appearing on the rhs of the Ward identities). Had these three-point functions been nonanomalous, this would be an identity. However, the three-point functions involve the anomalous hJ J J i correlator, and this implies that either (9) or the corre- sponding R-symmetry Ward identity should be anomalous. PHYSICAL REVIEW LETTERS 122, 231602 (2019) PHYSICAL REVIEW LETTERS 122, 231602 (2019) TABLE II. Anomalous Ward identities and corresponding anomalies [6]. PHYSICAL REVIEW LETTERS 122, 231602 (2019) (Dμψν ≡½∂μ þ 1 4 ωab μ ðe; ψÞγab þ iγ5Aμψν −Γρ μνψρ with ωab μ ðe; ψÞ ≡ωab μ ðeÞ þ 1 4 ð ¯ψaγμψb þ ¯ψμγaψb −¯ψμγbψaÞ; ∇μ is the Levi-Civita connection; ϕμ is defined in Table I.) Weyl square: W2 ≡WμνρσWμνρσ Euler density: E ¼ RμνρσRμνρσ −4RμνRμν þ R2 Pontryagin density: P ≡˜RμνρσRμνρσ ˜Rμνρσ ≡1 2 ϵκλ μνRκλρσ Schouten tensor: Pμν ≡1 2 ðRμν −1 6 RgμνÞ Uð1ÞR field strengths: ˜Fμν ≡1 2 ϵρσ μνFρσ F2 ≡FμνFμν F ˜F ≡Fμν ˜Fμν Weyl square: W2 ≡WμνρσWμνρσ eaμT μ a þ 1 2 ¯ψμQμ ¼ AW, ∇μJ μ þ i ¯ψμγ5Qμ ¼ AR DμQμ −1 2 γaψμT μ a −3i 4 γ5ϕμJ μ ¼ AQ, γμQμ −3i 4 γ5ψμJ μ ¼ AS eaμT μ a þ 1 2 ¯ψμQμ ¼ AW, ∇μJ μ þ i ¯ψμγ5Qμ ¼ AR Weyl square: W2 ≡WμνρσWμνρσ DμQμ −1 2 γaψμT μ a −3i 4 γ5ϕμJ μ ¼ AQ, γμQμ −3i 4 γ5ψμJ μ ¼ AS Euler density: E ¼ RμνρσRμνρσ −4RμνRμν þ R2 AW ¼ ðc=16π2ÞðW2 −8 3 F2Þ −ða=16π2ÞE þ Oðψ2Þ, AR ¼ ½ð5a −3cÞ=27π2 ˜FF þ ½ðc −aÞ=24π2P Pontryagin density: P ≡˜RμνρσRμνρσ ˜Rμνρσ ≡1 2 ϵκλ μνRκλρσ AQ ¼ −½ð5a −3cÞi=9π2 ˜FμνAμγ5ϕν þ ½ða −cÞ=6π2ð∇μðAρ ˜RρσμνÞγðνψσÞ −1 4 Fμν ˜RμνρσγρψσÞ þ Oðψ3Þ Schouten tensor: Pμν ≡1 2 ðRμν −1 6 RgμνÞ AS ¼ ½ð5a −3cÞ=6π2 ˜Fμν½Dμ −ð2i=3ÞAμγ5ψν þ ðic=6π2ÞFμνðγ½σ μ δρ ν −δ½σ μ δρ ν Þγ5Dρψσ þ ½3ð2a −cÞ=4π2Pμνgμ½νγρσDρψσ þ ½ða −cÞ=8π2 × ðRμνρσγμν −1 2 Rgμνgμ½νγρσÞDρψσ þ Oðψ3Þ Uð1ÞR field strengths: ˜Fμν ≡1 2 ϵρσ μνFρσ F2 ≡FμνFμν F ˜F ≡Fμν ˜Fμν AW ¼ ðc=16π2ÞðW2 −8 3 F2Þ −ða=16π2ÞE þ Oðψ2Þ, AR ¼ ½ð5a −3cÞ=27π2 ˜FF þ ½ðc −aÞ=24π2P AQ ¼ −½ð5a −3cÞi=9π2 ˜FμνAμγ5ϕν þ ½ða −cÞ=6π2ð∇μðAρ ˜RρσμνÞγðνψσÞ −1 4 Fμν ˜RμνρσγρψσÞ þ Oðψ3Þ AW ¼ ðc=16π2ÞðW2 −8 3 F2Þ −ða=16π2ÞE þ Oðψ2Þ, AR ¼ ½ð5a −3cÞ=27π2 ˜FF þ ½ðc −aÞ=24π2P AQ ¼ −½ð5a −3cÞi=9π2 ˜FμνAμγ5ϕν þ ½ða −cÞ=6π2ð∇μðAρ ˜RρσμνÞγðνψσÞ −1 4 Fμν ˜RμνρσγρψσÞ þ Oðψ3Þ Uð1ÞR field strengths: ˜Fμν ≡1 2 ϵρσ μνFρσ F2 ≡FμνFμν F ˜F ≡Fμν ˜Fμν AS ¼ ½ð5a −3cÞ=6π2 ˜Fμν½Dμ −ð2i=3ÞAμγ5ψν þ ðic=6π2ÞFμνðγ½σ μ δρ ν −δ½σ μ δρ ν Þγ5Dρψσ þ ½3ð2a −cÞ=4π2Pμνgμ½νγρσDρψσ þ ½ða −cÞ=8π2 × ðRμνρσγμν −1 2 Rgμνgμ½νγρσÞDρψσ þ Oðψ3Þ From the form of the anomaly AQ in Table II follows that the first anomalous contribution in flat space correlators appears in four-point functions involving two supercurrents and either two R currents or an R current and an energy- momentum tensor. TABLE III. The (on-shell) energy-momentum tensor T μ a, the R-symmetry current J μ, and the supersymmetry current Qμ, for the massless superconformal WZ model in flat space. (Received 4 April 2019; published 14 June 2019) δeaμ ¼ ξλ∂λeaμ þ ea λ∂μξλ −λa bebμ þ σeaμ −1 2 ¯ψμγaε, δψμ ¼ ξλ∂λψμ þ ψλ∂μξλ −1 4 λabγabψμ þ 1 2 σψμ þ Dμε −γμη −iγ5θψμ, δAμ ¼ ξλ∂λAμ þ Aλ∂μξλ þ ð3i=4Þ ¯ϕμγ5ε −ð3i=4Þ ¯ψμγ5η þ ∂μθ, ϕμ ≡1 3 γνðDνψμ −Dμψν −ði=2Þγ5ϵρσ νμDρψσÞ ½δε; δε0 ¼ δξ þ δλ þ δθ, ξμ ¼ 1 2 ¯ε0γμε, λa b ¼ −1 2 ð¯ε0γνεÞωa νb, θ ¼ −1 2 ð¯ε0γνεÞAν ½δε; δη ¼ δσ þ δλ þ δθ, σ ¼ 1 2 ¯εη, λa b ¼ −1 2 ¯εγa bη, θ ¼ −3i 4 ¯εγ5η δeaμ ¼ ξλ∂λeaμ þ ea λ∂μξλ −λa bebμ þ σeaμ −1 2 ¯ψμγaε, δψμ ¼ ξλ∂λψμ þ ψλ∂μξλ −1 4 λabγabψμ þ 1 2 σψμ þ Dμε −γμη −iγ5θψμ, δAμ ¼ ξλ∂λAμ þ Aλ∂μξλ þ ð3i=4Þ ¯ϕμγ5ε −ð3i=4Þ ¯ψμγ5η þ ∂μθ, ϕμ ≡1 3 γνðDνψμ −Dμψν −ði=2Þγ5ϵρσ νμDρψσÞ ½δε; δε0 ¼ δξ þ δλ þ δθ, ξμ ¼ 1 2 ¯ε0γμε, λa b ¼ −1 2 ð¯ε0γνεÞωa νb, θ ¼ −1 2 ð¯ε0γνεÞAν ½δε; δη ¼ δσ þ δλ þ δθ, σ ¼ 1 2 ¯εη, λa b ¼ −1 2 ¯εγa bη, θ ¼ −3i 4 ¯εγ5η [1] S. L. Adler, Phys. Rev. 177, 2426 (1969). [2] J. S. Bell and R. Jackiw, Nuovo Cimento A 60, 47 (1969). [3] S. B. Treiman, E. Witten, R. Jackiw, and B. Zumino, Current Algebra and Anomalies (World Scientific, Singapore, 1986). [4] K. Fujikawa and H. Suzuki, Path Integrals and Quantum Anomalies (Clarendon, Oxford, 2004). [5] I. Papadimitriou, J. High Energy Phys. 07 (2017) 038. [6] I. Papadimitriou, J. High Energy Phys. 04 (2019) 040. [7] J. Wess and B. Zumino, Phys. Lett. 37B, 95 (1971). PHYSICAL REVIEW LETTERS 122, 231602 (2019) Box diagrams contributing to the four-point correlation function (8). Zigzag lines denote R currents, wavy lines denote supersymmetry currents, straight lines denote fermionic propa- gators, and dashed lines denote bosonic propagators. R-symmetry four-point function Ward identity is not anomalous, and, therefore, the supersymmetry Ward iden- tity is anomalous. This computation is the counterpart of (5) but now in terms of Feynman diagrams. One can then show that there is a momentum routing such that (i) the triangle R-symmetry anomaly is repro- duced, (ii) the four-point R-symmetry Ward identity is nonanomalous, and (iii) the supersymmetry Ward identity is anomalous, with the anomaly given in Table II and with c ¼ 2a ¼ 1=24, which are the values in our model. In addition, upon taking the gamma trace of the same four- point function, γμhQμ ¯QνJ κJ λi, one automatically repro- duces the AS anomaly given in Table II. We thank Benjamin Assel, Roberto Auzzi, Friedmann Brandt, Loriano Bonora, Davide Cassani, Cyril Closset, Camillo Imbimbo, Manthos Karydas, Heeyeon Kim, Zohar Komargodski, Dario Martelli, Sunil Mukhi, Sameer Murthy, Parameswaran Nair, Dario Rosa, Stanislav Schmidt, Ashoke Sen, and Peter West for illuminating discussions and email correspondence. K. S. and M. T. are supported in part by the Science and Technology Facilities Council (Consolidated Grant “Exploring the Limits of the Standard Model and Beyond”). This research was sup- ported in part by the National Science Foundation under Grant No. NSF PHY-1748958, and this project has received funding or support from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 690575. I. P. thanks the University of Southampton, King’s College London, and the International Center for Theoretical Physics in Trieste for hospitality and partial financial support during the completion of this work. M. T. thanks the Kavli Institute for the Physics and Mathematics of the Universe for hospitality during the completion of this work. In general, changing the momentum routing, one may move the anomaly from one conserved current to another. This would be equivalent to adding local finite counter- terms, and as argued earlier there is no choice of such counterterms that would remove the supersymmetry anomaly while preserving diffeomorphisms and local Lorentz transformations. It is also straightforward to check that the same anomaly is present in the massive WZ model as well. PHYSICAL REVIEW LETTERS 122, 231602 (2019) Assuming the form of the bosonic Ward identities is standard (i.e., given by the expressions in Table II), the Standard path integral manipulations show that this corre- lator classically satisfies the following Ward identity: −i∂x1 μ hQμ 1 ¯Qν 2J κ 3J λ 4i ¼ δð4Þðx12Þhδ ¯Qν 1J κ 3J λ 4i þ fδð4Þðx13ÞhδJ κ 1 ¯Qν 2J λ 4i −∂x1 ρ ½δð4Þðx13ÞhδJ 0ρκ 1 ¯Qν 2J λ 4i þ ð3; κÞ ↔ð4; λÞg −∂x1 ρ ½δð4Þðx12Þhδ ¯Q0νρ 1 J κ 3J λ 4i; ð9Þ ð9Þ where we have used the shorthand notation QμðxiÞ ≡Qμ i , etc., xij ≡xi −xj, and the contributions on the rhs are where we have used the shorthand notation QμðxiÞ ≡Qμ i , etc., xij ≡xi −xj, and the contributions on the rhs are TABLE III. The (on-shell) energy-momentum tensor T μ a, the R-symmetry current J μ, and the supersymmetry current Qμ, for the massless superconformal WZ model in flat space. T μ a ¼ ðημρησa þ ημσηρ a −ημ αηρσÞ∂ρϕ∂σϕ −1 3 ð∂μ∂a −ημ a∂2ÞðϕϕÞ þ 1 4 ¯χðγμ∂a þ γa∂μÞχ J μ ¼ ð2i=3Þðϕ∂μϕ −ϕ∂μϕ þ 1 4 ¯χγμγ5χÞ Qμ ¼ ð1= ﬃﬃﬃ 2 p Þð=∂ϕγμχR þ =∂ϕγμχLÞ þ ð ﬃﬃﬃ 2 p =3Þγμν∂νðϕχR þ ϕχLÞ, χR ≡1 2 ð1 −γ5Þχ. 231602-4 PHYSICAL REVIEW LETTERS 122, 231602 (2019) FIG. 1. Box diagrams contributing to the four-point correlation function (8). Zigzag lines denote R currents, wavy lines denote supersymmetry currents, straight lines denote fermionic propa- gators, and dashed lines denote bosonic propagators. quantum theory of gravity, such as string theory. However, such models may be considered in bottom-up approaches (see [46] for a recent example). Similar comments apply to bottom-up string cosmology models. This anomaly also affects supersymmetric localization computations, as has already been noted in Refs. [5,6,41,42]. However, it is possible that a suitable noncovariant local counterterm (for theories with a ¼ c, such a counterterm evaluated on supersymmetric backgrounds of the form S1 × M3, with M3 a Seifert manifold, should agree with the counterterm used in Ref. [47]) may cancel the rigid supersymmetry anomaly at the expense of breaking certain diffeomor- phisms on a given supersymmetric background. From a more formal perspective, it would be interesting to explore how the supersymmetry anomaly is captured in index theorems. It would also be interesting to investigate the existence of such an anomaly in other dimensions and/or extended supersymmetry. FIG. 1. [1] S. L. Adler, Phys. Rev. 177, 2426 (1969). [6] I. Papadimitriou, J. High Energy Phys. 04 (2019) 040. [7] J. Wess and B. Zumino, Phys. Lett. 37B, 95 (1971). PHYSICAL REVIEW LETTERS 122, 231602 (2019) [8] D. M. Capper and M. J. Duff, Nuovo Cimento A 23, 173 (1974). [29] E. Guadagnini and M. Mintchev, Nucl. Phys. B269, 543 (1986). ( ) [9] S. 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PHYSICAL REVIEW LETTERS 122, 231602 (2019) As in the case of standard triangle anomalies, adding a mass term modi- fies the Ward identities, but the anomaly remains the same. This is as expected, since the anomaly arises from the UV behavior of Feynman diagrams and the parts of the loop computation that give rise to the anomaly remain the same. Note added.—Recently, a related work [48] appeared on the arXiv. Implications of the anomaly.—Let us conclude with a few comments about the implications of this anomaly. As mentioned earlier, an important consequence is that a SQFT with such a supersymmetry anomaly cannot be coupled to dynamical supergravity. (The anomalous R symmetry alone implies that coupling to a supergravity that gauges the R symmetry is inconsistent. Here, we see that couplings to supergravity that do not gauge the R symmetry are also inconsistent.) In the context of supersymmetric model building, one does not usually work with theories with an R symmetry, anomalous or nonanomalous; nonanom- alous R symmetry is not compatible with gaugino masses (see [45]). More generally, one does not expect a theory with continuous symmetry to emerge from a consistent 231602-5 PHYSICAL REVIEW LETTERS 122, 231602 (2019) [29] E. Guadagnini and M. Mintchev, Nucl. Phys. B269, 543 (1986). PHYSICAL REVIEW LETTERS 122, 231602 (2019) 12 (2017) 107. [21] O. Piguet and K. Sibold, Renormalized supersymmetry. The perturbation theory of N ¼ 1 supersymmetric theories in flat space-time (Birkhauser, Boston, 1986), Vol. 12. [42] O. S. An, Y. H. Ko, and S.-H. Won, Phys. Rev. D 99, 106007 (2019). [43] I. Papadimitriou, arXiv:1904.00347. [22] L. Bonora, P. Pasti, and M. Tonin, Nucl. Phys. B252, 458 (1985). [44] G. Katsianis, I. Papadimitriou, K. Skenderis, and M. Taylor (to be published). [23] I. L. Buchbinder and S. M. Kuzenko, Nucl. Phys. B274, 653 (1986). [45] M. Drees, R. Godbole, and P. Roy, Theory and Phenom- enology of Sparticles: An Account of Four-Dimensional N ¼ 1 Supersymmetry in High Energy Physics (World Scientific, Hackensack, 2004). F. Brandt, Classical Quantum Gravity 11, 849 (199 25] F. Brandt, Ann. Phys. (N.Y.) 259, 357 (1997). [26] L. Bonora and S. Giaccari, J. High Energy Phys. 08 (2013) 116. [46] C. Pallis, arXiv:1812.10284. [27] H. Itoyama, V. P. Nair, and H.-c. Ren, Nucl. Phys. B262, 317 (1985). [47] P. Benetti Genolini, D. Cassani, D. Martelli, and J. Sparks, J. High Energy Phys. 02 (2017) 132. [48] O. S. An, J. U. Kang, J. C. Kim, and Y. H. Ko, J. High Energy Phys. 05 (2019) 146. [28] O. Piguet and K. Sibold, Nucl. Phys. B247, 484 (1984). 231602-6 231602-6

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